University of Groningen

The identification of cell non-autonomous roles of astrocytes in neurodegenerationLi, Yixian

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The identification of cell non-autonomous roles of

astrocytes in neurodegeneration

Yixian Li

ISBN: 978-94-034-0765-4

The research described in this thesis was performed at the Department of Cell Biology, University Medical Center Groningen, University of Groningen, the Netherlands.

Cover and layout design: ThesisExpert.nlPrinted by Gildeprint, Enschede, the Netherlands

The printing of this thesis was financially supported by the University of Groningen and University Medical Center Groningen.

Copyright © 2018 by Yixian Li. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, without prior written permission of the author.

The identification of cell non-autonomous roles of

astrocytes in neurodegeneration

PhD thesis

to obtain the degree of PhD at the

University of Groningen on the authority of the

Rector Magnificus Prof. E. Sterken and in accordance with

the decision by the College of Deans.

This thesis will be defended in public on

Monday 11 June 2018 at 16.15 hours

by

Yixian Li

born on 1 June 1986 in Beijing, China

Supervisor Prof. O.C.M. Sibon Co-supervisor Dr. P.F. Dijkers Assessment Committee Prof. B.J.L. Eggen Prof. E.M. Hol Prof. D.S. Verbeek

TABLE OF CONTENTS

Chapter 1 General Introduction

Chapter 2 A Drosophila screen elucidates roles for signaling molecules in cell non-autonomous effects of astrocytes on neurodegenerative disease

Chapter 3 Inhibition of NF-κB in astrocytes delays neurodegeneration in a cell non-autonomous manner

Chapter 4 Specific calcineurin isoforms are involved in Drosophila Toll immune signaling

Chapter 5 General discussion

Summary

Samenvatting

Acknowledgemetns

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26

54

84

108

116

120

124

128 写给亲爱的人

CHAPTER 1General Introduction

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Neurodegenerative diseases (NDs) are characterized by selective loss of neurons in the central nervous system (CNS). Some general symptoms of NDs are movement abnormalities, emotional disturbance, and memory loss1. These symptoms impair the patient’s quality of life. A group of NDs, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic lateral sclerosis (ALS) are the most common and the most costly to society2. The onset of these diseases is age-related, affecting mostly elderly and the incidence increases due to the global increase in the aging population. In 2006, worldwide 26.6 million people suffered from AD, and this number is predicted to increase to 106.2 million in 20503. The accumulation of toxic, misfolded proteins in the brain is common in all of these diseases4. Some NDs in this group are caused by mutations in known disease-causing genes and are inherited, however, most cases are sporadic. Another group of NDs, the polyQ diseases, such as Huntington’s disease (HD) and different types of Spinocerebellar ataxias (SCAs), are mostly inherited, and the onset of polyQ diseases is also age-dependent5. There is no cure for NDs, and the mechanisms behind the neurodegeneration-inducing processes need further investigation. Knowledge of these processes is necessary for the development of potential future therapies.

In NDs, neuronal death can occur through apoptosis or necrosis6,7. Apoptosis is essential for various biological processes, such as development, cell turnover, and immune responses8. However, in NDs, excessive apoptosis leads to undesired neuronal death and contributes to neurodegeneration7. A number of pathological features of NDs are able to trigger apoptosis, such as misfolded proteins, mitochondrial dysfunction9, endoplasmic reticulum (ER) stress, oxidative stress10, and neuroinflammation11. Apoptosis is characterized by the activity of proteases called caspases, which cleave proteins in the cell, resulting in fragmentation of the cell into apoptotic bodies. Necrosis can be induced by energy depletion, lack of oxygen and nutrients, and has been reported in a number of NDs associated with misfolded or aggregated proteins6. Elevation of intracellular calcium levels, as occurs in NDs, has been associated with the induction of apoptosis as well as necrosis6.

The pathogenesis of NDs is multifactorial. The accumulation of misfolded proteins, mitochondrial dysfunction, ER stress, oxidative stress, neuroinflammation and energy depletion can all contribute to the pathogenesis of various NDs. The work described in this thesis will mainly focus on neuroinflammation and misfolded proteins. These two contributory factors to NDs will be discussed in more detail in this chapter.

While considerable effort has been spent on developing therapies, most have failed in clinical trials. Therapies have focused on decreasing aggregates, for example by using antibodies targeting aggregates, as done in AD12, which looked promising

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General introduction

initially, but some of these failed in late stage, phase III clinical trials. The same is true for therapies aiming to decrease oxidative stress. Some success in AD13 but not in HD14, has been booked with therapies aimed at decreasing inflammation in the brain. Further development of therapies would benefit from further knowledge about signaling pathways and cells that contribute to NDs.

Pathogenesis

Protein misfolding

The most common age-related NDs are associated with misfolded proteins which are able to form aggregates4. This shared feature can be contributed to an age-related decline in proteostasis15 and explain why age is a common feature of these diseases. AD is characterized by the presence of two kinds of aggregates: extracellular plaques in which the major constituent is the misfolded amyloid β (Aβ) peptide, and intracellular tangles which contain tau, a microtubule-associated protein (reviewed in16). In PD patients, dopaminergic neurons are affected and show the presence of cytoplasmic inclusion bodies, which consist of misfolded α-synuclein17. PolyQ diseases, including HD and six types of SCAs, are characterized by the expansion of polyglutamine (PolyQ) repeats in specific genes. The expansion of the polyQ repeats results in misfolded proteins that form intracellular aggregates5. Accumulation of misfolded proteins in the CNS is toxic to neurons and causes neuronal loss. Misfolded proteins can acquire a toxic gain of function and accumulate in organelles, resulting in impaired cellular functions (reviewed in15). In some animal models of NDs or in ND patients it has been demonstrated that misfolded proteins accumulate in the ER (endoplasmic reticulum), an important organelle for the biosynthesis of proteins. The accumulation of cytosolic misfolded proteins in the ER can result in ER stress and the unfolded protein response18.

Misfolded or aggregated proteins that accumulate extracellularly can bind to specific receptors on cells and induced intracellular signaling, which can contribute to neuronal stress and loss. Binding of amyloid β (Aβ) peptides to the nerve growth factor (NGF)-receptor can induce apoptosis19,20.

Misfolded or aggregated proteins can also serve as DAMPs (Damage-Associated Molecular Patterns, also known as Danger-Associated Molecular Patterns). DAMPs are substances that are normally intracellularly localized, but are released upon damage of cells and constitute a variety of agents such as mitochondrial DNA, ATP, and misfolded proteins21. Activation of receptors for DAMPs, present on immune cells, results in activation of inflammation, also called ‘sterile inflammation’. Examples of receptors for DAMPs are the Toll-like receptors (TLRs). In the brain, cells that express receptors for these DAMPs are predominantly brain-resident immune

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cells22. DAMPs released from dying neurons can result in persistent inflammation, which can be detrimental to neurons23.

Neurodegeneration and neuroinflammation: the role of glia

In the CNS, neurons are surrounded by non-neuronal cells, which are called glial cells. In the human brain, glial cells and neurons are present in roughly a 1:1 ratio24. Glial cells play an important role in maintaining neuronal functions and homeostasis in the CNS and mediate innate immune responses in the brain as a result from either infection or neuronal damage25. Two types of glial cells, microglia and astrocytes, both cells modulate immune responses in the brain25,26. These cells are commonly activated in a number of age-related NDs associated with protein aggregates. One underlying cause of this activation may be related to age: microglia are more reactive to inflammatory stimuli in older individuals, resulting in an enhanced release of pro-inflammatory cytokines, suggesting general changes in microglia in aging individuals27. However, a decreased phagocytic capacity of microglia has been described in mouse models of AD, which was dependent on anti-inflammatory cytokine IL-10, suggesting changes in alterations in both pro- and anti-inflammatory signaling (reviewed in28). In gene expression studies in brains of elderly, expression of microglia-specific genes was increased, and region-specific alterations in astrocyte-specific genes were observed29. Indeed, glial-specific gene expression was found to predict age more accurately than neuron-specific genes. This finding is of particular interest, given that age is a major risk factor for aggregation-associated NDs, but also because the preclinical stage of NDs (such as AD and HD)30, occurs well before the onset of clinical symptoms31. Indeed, several studies have identified a disease- and aging-associated microglial signature32,33,34,35,36. However, the contribution of most of these genes to NDs still remains to be determined. Recent research has identified considerable heterogeneity in microglia, and identified subtypes of microglia that can restrict development of neurodegenerative disease, as shown in a mouse model for AD35. The preclinical phase in AD, but also other NDs is associated with activation of microglia and astrocytes37,38. However, activation of microglia in AD patients in the preclinical phase of disease has been associated with a protective role, whereas microglial activation in later stages was associated with a worse pathogenesis (reviewed in39). This suggests that microglia can have neuroprotective and neurotoxic roles, depending on the disease stage or the subtype of microglia.

A breakthrough that identified microglia as contributing rather than responding cells in NDs came from GWAS studies that have identified microglial genes that increase the risk for AD, such as TREM2 (triggering receptor expressed in myeloid cells 2), a cell surface protein selectively and highly expressed by microglia in the

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General introduction

brain40. Additional research placed TREM2 in a signaling network of proteins that are additional risk factors for AD41. Given that after the onset of clinical symptoms neurons are irreversibly damaged and the course of disease can be delayed but not stopped, earlier intervention may be beneficial. Possibly, targeting microglia and astrocytes will be of clinical relevance, given their activation in the presymptomatic phase of disease.

In NDs, there are elevated numbers of activated astrocytes and microglia, also termed astrogliosis or microgliosis, and they are located on sites where aggregates are present (reviewed in23). In AD, activated microglia42 and astrocytes43 are detected at surrounding sites of aggregated Aβ depositions. In PD, activated microglia and astrocytes are present in the most affected brain regions44. In SCA3, the brain regions where neurodegeneration occurs, the subthalamic nucleus and the substantia nigra, contain increased numbers of activated astrocytes and microglia45. Pro-inflammatory actions of glia include increased expression of innate immune-related receptors, activation of inflammatory signaling pathways, secretion of pro-inflammatory cytokines, and generation of free radicals, including nitric oxide (NO)23. Microglia express innate immune receptors which can be activated by pathogen-associated molecular patterns (PAMPs)46 and DAMPs47. The disease-associated, misfolded proteins in NDs can also serve as DAMPs. For example, microglia can become activated by the presence of extracellular misfolded Aβ peptides which bind to surface receptors on microglia, and this results in the release of proinflammatory factors47. In addition, astrocytes, the most abundant glial cell type in the CNS, also participate in immune responses in the CNS. Astrocytes also express many immune receptors and can be activated by immune receptor ligands, such as the AD-associated misfolded protein, Aβ48.

Astrocytes in healthy brainsAstrocytes are indispensable for neuronal survival (reviewed in49). Astrocytes contribute to neuronal homeostasis in diverse ways: they help maintain the BBB (blood brain barrier), clear cellular debris, but also provide nutrients and secrete neurotrophins. Furthermore, astrocytes are important for the development and function of synapses (reviewed in50). They can also induce synaptic pruning by releasing complement factors, resulting in the elimination of the synapse by microglia. In addition, they can regulate the balance between excitatory synapses (such as glutamatergic synapses) and inhibitory synapses (such as GABA-ergic synapses) via the release of factors that can specifically induce or inhibit their formation49. Furthermore, astrocytes can respond to neuronal activity through their expression of neurotransmitter receptors and transporters51. Neuronal activity results in the release of neurotransmitters from synapses, which can bind to

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astrocytic receptors. Astrocytes subsequently respond by a rise in intracellular calcium levels, which results in the release of calcium-dependent neurotransmitters or neuromodulators, also called gliotransmitters. These include glutamate, GABA, D-serine and ATP. Gliotransmitters contribute to neuronal function and synaptic transmission49. Glutamate release by astrocytes leads to increased intracellular calcium levels in neighbouring neurons, which can modulate neuronal activity but can also be neurotoxic52. Thus, astrocytes can directly regulate neuronal functions by releasing gliotransmitters.

Astrocytes have an important role in controlling energy supply in the brain, an organ with a very high metabolic demand, consuming around 20% of the total energy, primarily in neurons53. They establish this by modulating blood flow in the brain, and can increase blood flow to regions with high neuronal activity53. Moreover, astrocytes can store energy in the form of glycogen, providing a limited energy reserve for neurons.

Astrocytes connect blood vessels with neuronal axons and synapses50, thus they are involved in taking up energy and nutrients, such as glucose, from blood vessels for transport to neurons. For instance, glucose can be taken up from blood vessels by astrocytes and subsequently the glucose can be transformed into glycogen, which is an important energy source in the CNS. In the adult brain, glycogen is mostly present in astrocytes, and the concentration of the glycogen varies depending on the brain regions. Several studies found that glycogen levels are high in the grey matter (reviewed in54) which is consistent with the fact that synapses, which require a high energy demand, are enriched in the grey matter55.

Astrocytes are activated in responses to brain injuries due to ischemia, hypoglycemia or trauma. Compared to resting astrocytes, activated astrocytes are hypertrophic. After neuronal injury, they proliferate and form a glial scar, this structure isolates the damaged tissue56 and aids axonal regeneration57. There is evidence that activated astrocytes play a protective role after an induced injury. In a mouse model, it was demonstrated that astrocytic scars aid axonal regeneration after spinal cord injury, which was prevented by ablating astrocytic scars57. Another study performed in mice demonstrated that drug-induced ablation of activated astrocytes after spinal cord injury resulted in demyelination and loss of neurons58. However, activation of astrocytes can also be detrimental, as a result of the release of cytotoxic molecules and chronic inflammation in the brain59. For instance, activated astrocytes produce a number of pro-inflammatory cytokines, such as TNF-α, TNF-β, IL-1 and IL-660. The functions of astrocytes that are important for neuronal health may be altered once they become activated under disease-induced circumstances, which can in turn influence survival of neurons.

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General introduction

Astrocytes in NDsIn most NDs, aggregates and activated astrocytes are detected before clinical symptoms appear (reviewed in61). A marker that is commonly used to mark activated astrocytes and to discriminate them from other glia is by levels of GFAP (glial fibrillary acidic protein). While astrogliosis is correlated with the severity of for example AD62 and HD63, the contribution of astrocytes to pathogenesis is unclear.

A number of molecular triggers can activate astrocytes in NDs. For example, an increase in the amount of pro-inflammatory cytokines released from neurons and other glial cells contribute to the activation of astrocytes. Prolonged activation of astrocytes leads to increased pro-inflammatory factors produced by astrocytes, which may cause more neuronal damage. A recent report identified a subtype of astrocytes (A1 astrocytes) that are neurotoxic and which are induced by activated microglia64. These astrocytes have elevated levels of components of the complement cascade, which are harmful to synapses. A1 astrocytes are abundant in a number of NDs, including AD, PD and HD, suggesting that these astrocytes contribute to neuronal death in NDs.

As mentioned, astrocytes play a role in regulating levels of neurotransmitters. This regulation may be altered in activated astrocytes in NDs. For instance, extracellular glutamate can contribute to excitotoxicity in neurons65. It has been shown that activated astrocytes have impaired capacity to take up the extracellular glutamate, because the expression of glutamate transporters is lower or dysfunctional in these activated astrocytes. This has been shown in HD66 and AD67. Therefore, impaired capacity of astrocytes to take up extracellular glutamate may contribute to neuronal loss.

A number of studies suggest that the function of astrocytes in energy metabolism changes in NDs. For instance, after exposing to Aβ peptide, the glucose metabolism in cultured astrocytes changed, including increased glucose utilization and glycogen storage68. Moreover, there are studies which show that changes in cerebral glucose metabolism are one of the early features in AD patients69. However, the contribution of activated astrocytes to the metabolic changes in NDs is not clear yet70

Altogether, in NDs, progressive loss of neurons in the CNS can be induced by neuronal accumulation of misfolded toxic proteins, which contributes in a cell-autonomous manner to neurotoxicity. In addition, both astrocytes and microglia can cell non-autonomously contribute to neuronal homeostasis. In NDs, alterations in microglia and astrocytes importantly contribute as well. While some of the mechanisms by which these cells can have either beneficial or detrimental effects have been identified, the effect of altered expression in microglia and astrocyte-specific genes still awaits further analysis.

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Regulation of inflammation- a central role for NF-κBTranscription factors that are commonly activated in inflammatory and stress responses are members of the NF-κB (Nuclear Factor Kappa Beta) transcription factor family. Deregulation of NF-κB has been linked to a variety of disorders, including cancer, immune disorders and NF-κB is chronically activated in a variety of inflammatory diseases (reviewed in71). Furthermore, constitutive activity of NF-κB in aging has been reported (reviewed in72). NF-κB is rapidly activated in response to a number of responses, including cytokines, reactive oxygen species, calcium, neurotransmitters, DAMPs, as well as components from bacterial cell walls, such as LPS. In mammals, 5 members of this family have been identified. The modulation and specificity of their activation occur via distinct signaling pathways. However, some crosstalk between these pathways exists as well, since these transcription factors can form both homodimers and heterodimers (reviewed in73). In the brain, NF-κB can be involved in inflammation74, but also in synaptogenesis, as well as neuronal growth and survival (reviewed in75). NF-κB can be activated in neurons, microglia and astrocytes, although the stimuli involved in activation or repression of NF-κB varies depending on cell type (reviewed in76).

Activation of NF-κB commonly occurs in NDs, and has been associated with their pathogenesis. In a mouse model for ALS, NF-κB activation in microglia induces gliosis, resulting in death of motor neurons77. Elevated activation of NF-κB was found in astrocytes in HD patients as well as in HD mouse models, and this activation contributes to HD pathogenesis78. In brains of postmortem AD patients, elevation of levels of NF-κB or NF-κB activation was found (reviewed in79). NF-κB activation as well as dysregulation of calcium signaling has been shown in astrocytes of AD patients and in cultured astrocytes exposed to amyloid beta peptides, resulting in the production of pro-inflammatory cytokines (reviewed in80). In other models for neuroinflammation, astrocyte-specific inactivation of NF-κB improved clinical outcome (reviewed in81).

Therapies in NDs that targeted inflammation by using NSAIDs (non-specific anti-inflammatory drugs) have shown some promise in AD82. An inhibitor that targets NF-κB was also used as a therapy, which was successful in a model for MS (multiple sclerosis), where NF-κB activity was specifically inhibited in astrocytes but not in microglia, concomitant with attenuation of demyelination83. However, this inhibitor had no effect on HD (reviewed in14). One possible explanation for this may be that multiple NF-κB isoforms are targeted, and not just the isoform(s) that promote the inflammatory responses. Some progress has made in generating inhibitors that specifically target a specific NF-κB isoform that is associated with inflammation and neurodegeneration84. Thus, more specificity in targeting NF-κB isoforms or upstream signaling pathways that activate NF-κB, but also insight into

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General introduction

the transcriptional targets that modulate neurotoxicity in neurodegeneration may provide future options for therapeutically targeting NF-κB in neuroinflammation.

Aim of this thesis: analysis of astrocytes in neurodegenerationIt is no longer a matter of debate that neuroinflammation can have detrimental contributions in age-related NDs. Further knowledge on contributing pathways, communication between neurons, microglia and astrocytes may ultimately result in the identification of suitable therapeutic targets. In this thesis, we focus on contributions of astrocytes to neurodegeneration. The analysis is complex, given that contributions of astrocytes to neurodegeneration can be beneficial as well as detrimental.

In this thesis, we examine how astrocytic responses to neurons that express an aggregation-prone, neurodegeneration-associated protein can influence the extent of neurodegeneration (Figure 1). These so-called cell non-autonomous responses of astrocytes have not been studied extensively because of the complexity of simultaneous manipulation of gene expression in both neurons (to express an aggregation-prone protein) and astrocytes (to manipulate expression of genes that may contribute to neurodegeneration). Examining astrocytes in an in vivo model is key, given that they are altered outside their physiological context (reviewed in11). We have used the fruit fly Drosophila melanogaster as a model organism, given (1) the large conservation of genes between fly and human (2) the presence of astrocytes that are similar in function (reviewed in85) and (3) the ease of genetic manipulation and availability of genetic tools. Flies have successfully been employed as model organism for human NDs86 and have been crucial in genetic screens to identify novel players in a multitude of biological processes. In addition, analysis of a large number of genes is facilitated by the short generation time (10 days) and lifespan (60-80 days), the low costs and availability of fly lines that allow genome-wide manipulation of gene expression of conserved genes.

In this thesis, we have analyzed the cell non-autonomous contributions of astrocytes in a model for neurodegeneration. In this model, the human SCA3-associated protein containing an expanded polyQ repeat was expressed in Drosophila eyes or in neurons. Eye-specific expression of this protein results in eye degeneration and neuronal degeneration when expressed in neurons87. Employment of this SCA3 model has resulted in the identification of genes that contribute to pathogenesis in a cell-autonomous manner88. In this thesis, we have set up a Drosophila SCA3 model that allows genetic manipulation of genes in astrocytes. We carried out a candidate RNA interference screen in astrocytes to identify genes that can contribute to the degenerative SCA3 phenotype.

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Astrocyte Activated astrocyte

Neuron

Microglia Activated Microglia

Other DAMPs NeurotransmittersCytokinesMisfolded proteins

Signals from neurons

Neuron

?

Gliotransmitters

?

unidentified singals

?

Signals from activated microglia

Signals from activated astrocyte

Figure 1. A model for the aim of this thesis. Damage in neurons, as occurs in neurodegeneration, result in the activation of microglia and astrocytes. Here, we examine the signaling in astrocytes in response to expression of ND-associated misfolded proteins in neurons. The signals that contribute to the activation of astrocytes are still elusive. Some findings from the literature, show that misfolded disease-related proteins and cytokines can activate astrocytes. Other signals, such as neurotransmitters and other DAMPs have not been identified yet. Importantly, the effect on neurodegeneration of signals that are subsequently released from astrocytes will be studied. Thus, this thesis focuses on understanding the cell non-autonomous contribution of astrocytes to neurodegeneration.

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General introduction

Outline of this thesis

Chapter 2 A Drosophila screen elucidates roles for signaling molecules in cell non-autonomous effects of astrocytes on neurodegenerative disease

In this chapter, we describe the generation of a Drosophila SCA3 eye model that allows analysis of the influence of genes in astrocytes on a degenerative eye phenotype. We describe the results of a candidate RNAi screen in astrocytes, to see whether genes expressed in astrocytes can influence the degenerative SCA3 eye phenotype. We identified astrocytic genes that are enhancers as well as suppressors of SCA3, demonstrating cell non-autonomous roles of astrocytes in degeneration. We further speculate on the relevance of these genes in neurodegeneration.

Chapter 3 Inhibition of NF-κB in astrocytes delays neurodegeneration in a cell non-autonomous manner

In this chapter, we further analyze the NF-κB transcription factor Relish, a gene analogous to human NF-κB1, which was identified as an enhancer of SCA3 in the candidate RNAi screen described in chapter 2. Downregulation of Relish expression, but also of transcriptional targets of Relish in astrocytes decreased SCA3-induced eye degeneration. Relish, but not the other Drosophila NF-κB transcription factors Dif and Dorsal influenced degeneration, demonstrating specificity of NF-κB transcription factors. We further analyzed the effect of Relish on lifespan in neurons expressing a SCA3-associated polyQ protein and we examined the effect on lifespan in neurons expressing amyloid beta peptides, associated with Alzheimer’s disease. Inhibition of Relish in astrocytes extended lifespan in both models, suggesting a general cell non-autonomous role of this NF-κB pathway in astrocytes in NDs.

Chapter 4 Specific calcineurin isoforms are involved in Drosophila Toll immune signalling

In chapter 2, we identified Relish, but not NF-κB transcription factors Dif and Dorsal as an enhancer of neurodegeneration. In this chapter, we analyzed specificity of upstream signaling pathways that result in activation of Relish or Dif/Dorsal, respectively. The canonical pathways that activate Relish and Dif/Dorsal are the IMD and Toll pathway, respectively. However, additional pathways can modulate their activity. Here we analyze the different isoforms of calcium-dependent serine/threonine phosphatase, calcineurin, on activity of Relish and Dif/Dorsal. Analysis of this calcium-dependent phosphatase is also of interest in NDs, where elevation of intracellular calcium levels commonly occurs. In Drosophila there are three calcineurin catalytic subunits, and all of them in astrocytes contributed to a cell non-

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autonomous effects on SCA3 (Chapter 2). In this chapter, we demonstrate specificity of calcineurin isoforms in Relish and Dif/Dorsal activation. We investigated this in cell culture, but also in NF-κB-mediated immune activation in vivo.

Modulation of activity of calcineurin may be of relevance in regulating the activity of specific NF-κB transcription factors in NDs.

Chapter 5 General Discussion

The results presented in this thesis demonstrate that astrocytes contribute to neurodegeneration in a cell non-autonomous manner. We mainly focused on putative interactions between astrocytes and neurons. However, other types of non-neuronal cells, such as microglia, may also contribute to neurodegeneration and influence activity of astrocytes. In this chapter, the involvement of microglia in ND, and the interactions between astrocytes and microglia in neurodegeneration are discussed.

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39. Sarlus, H. & Heneka, M. T. Microglia in Alzheimer’s disease. Journal of Clinical Investigation 127, 3240–3249 (2017).

40. Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015).

41. Sims, R. et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat. Genet. 49, 1373–1384 (2017).

42. Perry, V. H., Nicoll, J. A. R. & Holmes, C. Microglia in neurodegenerative disease. Nat. Publ. Gr. 6, 193–20117 (2010).

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Immune Receptor to Clear Amyloid β1-42 and Delay the Cognitive Decline in a Mouse Model of Alzheimer’s Disease. J. Neurosci. 28, 5784–5793 (2008).

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51. Cervetto, C. et al. A2A-D2 receptor–receptor interaction modulates gliotransmitter release from striatal astrocyte processes. J. Neurochem. 140, 268–279 (2017).

52. Bezzi, P. et al. Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391, 281–285 (1998).

53. Nortley, R. & Attwell, D. Control of brain energy supply by astrocytes. Current Opinion in Neurobiology 47, 80–85 (2017).

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63. Myers, R. H. et al. Decreased neuronal and increased oligodendroglial densities in Huntington’s Disease caudate nucleus. J. Neuropathol. Exp. Neurol. 50, 729–742 (1991).

64. Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017).

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65. Salińska, E., Danysz, W. & Łazarewicz, J. W. The role of excitotoxicity in neurodegeneration. Folia Neuropathologica 43, 322–339 (2005).

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67. Lauderback, C. M. et al. The glial glutamate transporter, GLT-1, is oxidatively modified by 4-hydroxy-2-nonenal in the Alzheimer’s disease brain: The role of Abeta1-42. J. Neurochem. 78, 413–416 (2001).

68. Allaman, I. et al. Amyloid-β Aggregates Cause Alterations of Astrocytic Metabolic Phenotype: Impact on Neuronal Viability. J. Neurosci. 30, 3326–3338 (2010).

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CHAPTER 2A Drosophila screen elucidates roles for signaling

molecules in cell non-autonomous effects of as-

trocytes on neurodegenerative disease

Li-YX, Sibon-OCM, Dijkers-PF

Manuscript in preparation

ABSTRACTMost protein aggregation-associated neurodegenerative diseases are associated with activation of astrocytes. Astrocytes are activated in early stages of these diseases, however, their contribution to pathogenesis is unclear. Cellular stress or damage in neurons (cell-autonomous contribution) is associated with neurodegeneration. In addition, cell non-autonomous contributions of non-neuronal cells may also contribute to neurodegeneration.

Here, we established a Drosophila (fruit fly) model to analyze whether astrocytes can contribute to neurodegeneration in a cell non-autonomous manner. In a candidate RNAi screen targeting astrocytes in a fly model for neurodegeneration, we identified genes that could non-autonomously affect tissue degeneration. We examined these genes in a Drosophila model for Spinocerebellar Ataxia-3 (SCA3, also known as Machado Joseph Disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN-3 gene. In this model, a biologically relevant, truncated part of the ATXN-3 gene containing an expanded polyQ stretch (SCA3polyQ78) was expressed in cells in eyes, including photoreceptors but excluding astrocytes. Simultaneously, candidate genes were exclusively downregulated in astrocytes. We identified both enhancers and suppressors of SCA3polyQ78-induced eye degeneration, strongly demonstrating that astrocytic functioning can contribute to neurodegeneration.

Our data point to novel mechanisms of cell non-autonomous contributions to neurodegeneration via astrocytes. We speculate about mechanistic contributions of several candidate genes.

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INTRODUCTION Glia are non-neuronal cells in the central nervous system (CNS) and astrocytes form a sub-class of glial cells. In mammals, among all types of cells in the CNS, astrocytes are the most abundant. They are present in the entire CNS and envelop synapses, and are involved in maintaining neurotransmitter homeostasis, synaptic function, energy metabolism and inflammation in the CNS1. A number of studies point out that astrocyte dysfunction can cause damage to neurons and contribute to disease development, such as stroke and epilepsy2. Also, neurodegenerative diseases (NDs) are associated with changes in activation of astrocytes2.

A feature of most age-related NDs, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington’s disease (HD), and different types of Spinocerebellar ataxias (SCAs) is the accumulation of misfolded or aggregated proteins3. Neuronal loss in NDs is associated with neuronal accumulation of toxic misfolded proteins. These neuronal misfolded proteins contribute to neuronal death or damage in a cell-autonomous manner. However, cell non-autonomous contributions from non-neuronal cells, such as astrocytes, may also contribute to neuronal damage or influence neuronal functioning. In reactive astrocytes that are associated with NDs or neuronal damage, the physiological functions can be altered, which consequently could lead to a further increase in neuronal damage (cell non-autonomous contribution). Astrocytes are activated in most if not all NDs4. However, the signals that mediated activation of astrocytes, as well as which signaling events in astrocytes may modulate neuronal functioning in neurodegeneration remain to be identified.

Astrocytes can respond to a variety of signaling molecules that are released from other cells. These signaling molecules are amongst others DAMPs (danger or damage-associated molecular patterns), which are released from damaged or dying cells or can be cytokines or neurotransmitters5. Receptors for the DAMPs are PRRs (pattern recognition receptors), which are expressed on astrocytes and binding of DAMPs to PRRs in astrocytes could contribute to their activation (reviewed in6). Astrocytes can be stimulated by mitochondrial DNA (a DAMP) to produce pro-inflammatory cytokines (reviewed in7). In NDs, misfolded or aggregated proteins, can be released from the damaged or dying neurons and can also act as DAMPs and stimulate astrocytes. It has indeed been shown that clearance of aggregates mediated by astrocytes can occur in NDs, however the mechanisms behind this are not known8. Here we will focus on the signaling in astrocytes that is triggered by neurons that express aggregation-prone proteins. Misfolded or aggregated proteins function as ligands in one class of PRRs, Toll-like receptors (TLRs)9. Upon binding of the ligand to the TLR, intracellular signaling cascades are initiated,

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

leading to increased pro-inflammatory cytokines synthesis. However, it remains to be determined whether PRRs in astrocytes contribute to neurodegeneration. Besides PRRs, stimulation of pro-inflammatory cytokine receptors on astrocytes also contributes to their activation. Interleukin 1 beta (IL-1β) can stimulate astrocytes to produce the pro-inflammatory cytokines interleukin 6 (IL-6)10 and TNFα11. Thus, increased pro-inflammatory cytokine production in neurodegenerative diseases can also lead to activation of astrocytes, resulting in further cytokine production12.

In a healthy individual, astrocytes are maintained in a quiescent state. In NDs, this quiescent state is disrupted by alterations in signals (reviewed in6). In general, the receptors that are engaged by astrocytes to become activated and to contribute to NDs, such as individual PPRs and cytokine receptors, remain to be identified. Other transmembrane proteins present at the plasma membrane of astrocytes may play a role in their activation as well. A previous study showed that knocking out integrin subunit β1 specifically in astrocytes in a mouse model resulted in activation of astrocytes13. This suggests that integrins are necessary to keep astrocytes in a resting state. Integrins are transmembrane proteins, which consist of an α subunit and a β subunit heterodimer. They are involved in cell adhesion and signaling between cells as well as in cell migration. However, it is unclear how alterations in integrin signaling can result in the activation of astrocytes.

In NDs, there are a number of functional changes in astrocytes, illustrated by the observation that the capacity of maintaining neurotransmitter homeostasis in astrocytes can be altered in NDs14. Altered homeostasis of neurotransmitters is harmful to neurons14 and contributes to neurotoxicity. Levels of the excitatory neurotransmitters, such as glutamate, are elevated and are toxic to neurons15. In a healthy individual, astrocytes efficiently take up the extracellular glutamate by glutamate transporters, known as excitatory amino acid transporters (EAATs)16. In NDs, activated astrocytes are less efficient in clearing excessive extracellular glutamate, which consequently causes neuronal damage17. Not only the homeostasis of excitatory neurotransmitters but also of inhibitory neurotransmitters, such as gamma-aminobutyric acid (GABA), is altered in astrocytes18,19. Changes in both uptake, as well as in release of GABA in reactive astrocytes have been reported4. In AD patients as well as in an AD mouse model, astrocytes have elevated intracellular levels of GABA18. This suggests that changes in GABA homeostasis in astrocytes may be associated with NDs. In an AD mouse model, reactive astrocytes release excessive levels of GABA, which contribute to impaired learning ability and memory20. These defects are fully restored upon suppression of GABA synthesis or release in astrocytes. However, the molecular mechanisms of GABA homeostasis regulation in astrocytes have not been fully elucidated yet.

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Besides changes in the regulation of neurotransmitter levels, calcium homeostasis in astrocytes is also altered in NDs. Alterations in calcium homeostasis in astrocytes will affect calcium-dependent intracellular signalling1. Enhanced calcium-induced signaling in astrocytes has been observed in AD21 as well as in ALS22 models. Calcium can activate downstream signaling via the calcium/calmodulin-dependent serine-threonine phosphatase, calcineurin (reviewed in23). This results in the activation of calcineurin-dependent transcription factor, NFAT (Nuclear Factor of Activated T cells)23. The importance of calcineurin signaling in activation of astrocytes has been demonstrated (reviewed in24). Calcineurin activity is upregulated in aging and AD models25. Moreover, there is an NFAT binding site in the promoter of glutamate transporter (EAAT2)26, although the direct regulation has not been studied yet. This may indicate a potential regulation of calcineurin and glutamate homeostasis in astrocytes.

PRRs and cytokine receptors can promote intracellular signaling to activate the transcription factor NF-κB to produce pro-inflammatory cytokines. In NDs, the NF-κB is activated in astrocytes4, suggesting that this transcription factor may contribute as well. However, which molecules in astrocytes are important for the regulation of NF-κB signaling have not been fully elucidated. To what extent NF-κB, calcineurin signaling, neurotransmitter homeostasis, and calcium homeostasis contribute to NDs is also not well understood.

Intracellular signaling in astrocytes results in the secretion of molecules that are secreted by astrocytes to signal to neurons. For instance, altered calcium homeostasis in astrocytes results in changes in the release of gliotransmitters, such as glutamate, secreted by glia required for glia-neuron communication27. Dysregulation of gliotransmitter secretion can cause neuronal damage. For instance, excessive levels of glutamate were released from astrocytes in a calcium-dependent manner when astrocytes were exposed to amyloid beta peptides, resulting in synaptic damage28. Furthermore, activation of calcineurin results in astrocytic inflammatory responses, through which the secreted neurotoxic factors can also cause neuronal damage. Therefore, it is important to understand which molecules are involved in releasing signals from astrocytes to neurons in NDs.

Thus, in NDs, astrocytes can be activated as a result of neuronal signaling. Consequently, the activated astrocytes can signal to neurons. Currently, it is unclear how cell non-autonomous signaling from astrocytes to neurons contributes to NDs. Earlier work has demonstrated that expression of aggregation-prone proteins in astrocytes can cell non-autonomously influence neuronal viability29 (reviewed in6). However, it is unclear whether signaling in astrocytes can influence neuronal viability when aggregation-prone proteins are expressed specifically in neurons.

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

We investigated if and how astrocytes contribute to neurodegenerative diseases in a cell non-autonomous manner, within the context of an intact animal. Examining astrocytes in an in vivo model is key, given that their morphology and activity changes when taken outside their physiological context (reviewed in30). For this, we conducted a dedicated RNAi screen to selectively knock down individual genes in astrocytes in a Drosophila melanogaster (fruit fly) model of the polyQ disease SCA3. In SCA3, the ATXN-3 gene contains an expanded CAG repeat (coding for glutamine) that leads to the expression of a misfolded aggregation-prone ATXN-3 polyglutamine protein. These misfolded polyQ-containing proteins accumulate intracellularly, resulting in neuronal damage and activation of astrocytes (reviewed in31). In SCA3, the stretch of polyglutamine repeats is in the range of 62 to 86 glutamines32. Activated astrocytes were found in SCA3 patients33, suggesting potential contributions of astrocytes in the pathogenesis of SCA3.

To independently manipulate neurons and astrocytes, Drosophila melanogaster is a suitable model organism. Drosophila has been successfully used as an organism for genetic screens for over a century, which has yielded fundamental insights in biology and in human health. More than half of the Drosophila genes have orthologs in human, and nearly 75% of disease-associated genes in humans have orthologs in Drosophila34. Moreover, many physiological processes are conserved from fly to human. To gain insight into human diseases using Drosophila, either the ortholog of the disease-causing gene can be mutated in Drosophila, or alternatively, a human disease-causing gene can be expressed in Drosophila. Expression of human amyloid beta peptides, associated with Alzheimer’s disease, causes neurodegeneration and shortening of lifespan in Drosophila35. Similarly, expression of a biologically relevant part of the ATXN-3 gene, containing an expanded polyQ stretch, SCA3polyQ78, associated with SCA3, resulted in neurodegeneration36. As a model for neurodegeneration, the Drosophila eye was used in this study: eye-specific expression of genes associated with neurodegeneration can also cause eye degeneration. Expression of SCA3polyQ78 in the Drosophila eye results in an easily screenable phenotype36. An advantage of this approach is that the eye can easily and quickly be screened, and does not require time-consuming procedures such as analysis of lifespan. To assess the relevance of cell non-autonomous contributions of astrocytes to a neurodegenerative disease associated with aggregation, SCA3, we expressed SCA3polyQ78 specifically in Drosophila eyes and simultaneously downregulated expression of candidate genes exclusively in astrocytes. The availability of fly lines that express RNAi constructs and genetic tools allow specific down-regulation of candidate genes in astrocytes. We carried out a candidate RNAi screen of genes that are putatively involved in recognizing signals from neurons (receptors), intracellular signaling or genes that encode putative signaling molecules that can signal to neurons (such as neuropeptides).

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Similar to mammals, astrocytes are important for neuronal functioning in Drosophila31. Drosophila astrocytes share structural similarities with mammalian astrocytes, such as a branched appearance37. The distribution of Drosophila astrocytes is also comparable with mammalian astrocytes, as they connect with the blood-brain barrier and fill in the spaces between neurons37. Similar to mammalian astrocytes, they play an important role in sensing as well as clearing of glutamate (reviewed in31). Some conserved genes in Drosophila astrocytes have been identified. For example, glutamate transporters (EAATs) are expressed in Drosophila astrocytes. Drosophila EAAT1 is orthologous to the mammalian EAATs, GLAST and GLT-138. Similar to vertebrates, there are also inhibitory neurotransmitters in the Drosophila CNS, such as GABA. GABA-A receptors have orthologs in Drosophila: ligand-gated chloride channel homolog 3 (Lcch3), Resistant to dieldrin (Rdl) and Glycine receptor (Grd)39. Drosophila astrocytes express NF-κB genes and calcineurin genes40, however, their functions have not been examined in Drosophila astrocytes. Some aspects of astrocytic functioning are not conserved. For example, adult astrocytes in Drosophila do not contribute to clearance of degenerating neurons41.

We performed a candidate screen to investigate whether RNAi-mediated downregulation of genes in astrocytes could influence the extent of degeneration in eyes expressing SCA3polyQ78. Identification of enhancers or suppressors will demonstrate cell non-autonomous involvement of astrocytes and shed light on the relevant signaling molecules in astrocytes. This setup allows screening of a large number of genes (around 160) in a short time frame.

We analyzed putative involvement of genes in astrocytes in the recognition of signals from SCA3poly78-expressing eyes, intracellular signaling and genes involved in generation of signals from astrocytes (gliotransmitters or neuropeptides) that could influence the extent of SCA3poly78-induced degeneration. Analysis of genes may provide answers to the following questions:

1. What are the signals from degenerating neurons that signal to astrocytes?

2. Which intracellular signaling pathways in astrocytes contribute to polyQ disease?

3. What are the signaling molecules released by astrocytes that influence neurodegeneration?

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

RESULTS AND DISCUSSION

Generating SCA3polyQ27 and SCA3polyQ78 in the Q systemPrevious studies have shown that Drosophila eyes are a suitable model to study SCA336,42. Expressing a biologically relevant, truncated fragment of the SCA3 disease-causing protein of the human ATXN-3 containing the expanded polyglutamine stretch, SCA3polyQ78, specifically in Drosophila eyes, resulted in a degenerative eye phenotype43. This distinct phenotype is easily screenable for modifiers. Similar to ATXN-3 containing an expanded polyQ stretch in SCA3 in humans, SCA3polyQ78 in Drosophila forms aggregates. The degenerative eye phenotype, as well as the extent of SCA3polyQ78 aggregation, can be used to screen for modifiers and thus identify genes that are relevant in SCA3. Such screens have been successfully done and yielded novel insight into SCA342. However, these screens were performed to identify cell-autonomous modifiers of SCA3, identifying genes that are also expressed in the same cells of the eye as SCA3polyQ78 protein.

Tissue-specific gene expression in Drosophila has been established by using a binary expression system that was derived from yeast, UAS-GAL444. The UAS-GAL4 consists of two components: the GAL4 transcription factor and the UAS promoter. GAL4 binds to the UAS promoter to activate the expression of genes under the control of GAL4-specific UAS (upstream activating sequence). Tissue-specific expression of GAL4 in Drosophila has no effect, and a gene under control of the UAS promoter (UAS-gene) is not expressed in the absence of GAL4. However, the combined presence of both GAL4 and UAS-gene results in expression of the gene in the tissues that express GAL4. One advantage is that a gene that would be toxic when ubiquitously expressed can be analyzed in a tissue that is not essential for Drosophila viability, such as eyes or wings.

To specifically express SCA3polyQ78 in the Drosophila eye, we used the Q system. The Q system also consists of two components: the transcription factor QF2 and the QUAS sequence, which is the promoter sequence for QF2. QF2 activates the expression of genes under the control of QUAS. This system has recently been employed in Drosophila and its functioning independent of UAS-GAL4 has been established45. We used the Q system to express human SCA3polyQ78 in Drosophila eyes and the UAS-GAL4 system to express RNAi constructs in astrocytes.

We express SCA3polyQ78 in the Drosophila eye and analyze whether astrocytes can contribute to the degenerative phenotype. We analyzed the involvement of specific genes in astrocytes in SCA3polyQ78-induced eye degeneration. For this, we downregulated expression of individual genes in astrocytes, using RNAi constructs.

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Thus, to enable simultaneous modulation of gene expression (expression of SCA3polyQ78 in eyes and RNAi constructs in astrocytes), we combined the Q system (to express SCA3polyQ78 specifically in eyes) with the UAS-GAL4 system to modulate gene expression in astrocytes.

We used a fly line that expressed QF2 under the control of an eye-specific promoter GMR, resulting in a QUAS-dependent expression that was exclusively restricted to the eye (Figure 1a). As the Q system has not been used before to express human ATXN3 in Drosophila tissue, we first compared expression levels of a truncated fragment of human ATXN3 containing either a non-pathogenic glutamine stretch of 27 glutamines (SCA3polyQ27) or a pathogenic length of 78 glutamine repeats

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Figure 1. The Q system was used to express truncated human ATXN-3 (SCA3) protein containing different lengths of polyglutamine (polyQ) repeats in Drosophila eyes. (a) To express human ATXN-3 in Drosophila eyes we used the Q system, using an eye-specific QF2, GMR-QF2 to express QUAS-SCA3polyQ27 or QUAS-SCA3polyQ78. (b) The expression levels and extent of aggregation of HA-tagged SCA3polyQ27 and SCA3polyQ78 were analyzed on Western blot. Tubulin was used as a loading control. Figures represent two-time experiments. (c) Eye phenotypes of SCA3polyQ27 or SCA3polyQ78 expression. SCA3polyQ78-induced phenotypes are depigmentation and necrotic spots (‘necrotic’). The arrow points at a necrotic spot. Figures represent at least three experiments.(d) Quantification of the eyes that have a normal appearance, display depigmentation or necrotic spots as shown in (c). n=3.Genotypes in (b), (c) and (d): control, GMR-QF2/+. SCA3polyQ27, GMR-QF2/+; QUAS-SCA3polyQ27/+. SCA3polyQ78, GMR-QF2/+; QUAS-SCA3polyQ78/+.

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

(SCA3polyQ78). Also, we tested whether expression of SCA3polyQ78 (but not SCA3polyQ27)

could induce a degenerative eye phenotype.

We checked expression levels and extent of aggregation of SCA3polyQ27 and SCA3polyQ78 by analyzing head lysates of flies expressing SCA3 in the eyes on Western blot. Expression levels of SCA3polyQ27 and SCA3polyQ78 were comparable (Figure 1b), but only expression of SCA3polyQ78 resulted in the formation of insoluble aggregates of SCA3polyQ78 (Figure 1b). Expression of SCA3polyQ27 in Drosophila eyes did not induce eye degeneration (Figure 1c), whereas expression of pathogenic SCA3polyQ78 in Drosophila eyes resulted in degeneration, as shown by loss of pigmentation (Depigmentation, mild degeneration) or loss of pigmentation together with the presence of necrotic patches (Necrotic, severe degeneration) (Figure 1c, arrow). When we expressed SCA3polyQ78 in Drosophila eyes, about 20% of the eyes displayed severe degeneration while the rest of the eyes were mildly degenerated (Figure 1d). No degeneration was observed in control eyes or in eyes expressing SCA3polyQ27. We used the fraction of eyes displaying necrotic spots to quantify the extent of degeneration. The observation that SCA3polyQ78 formed aggregates and induced degeneration whereas no degeneration or aggregation was seen with SCA3polyQ27, demonstrates that protein misfolding or aggregation accounts for the degeneration, as shown before42.

Thus, the Q system we used to express SCA3polyQ78 can be used as a tool to study and quantify misfolded protein-associated degeneration.

Simultaneous and independent modulation of gene expression in eyes and astrocytesCombining two binary expression systems allows independent manipulation of gene expression in different tissues. To study the effect of astrocytes on SCA3polyQ78-induced eye degeneration, we used the Q system to express SCA3polyQ78, and the UAS-GAL4 system to modulate gene expression in astrocytes. In this study, we used GAL4 expressed under control of the astrocyte-specific promoter alrm41 to specifically modulate expression of UAS constructs in astrocytes. We visualized localization of astrocytes in the Drosophila brain by combining alrm-GAL4 with UAS-RFP allowing the expression of RFP in astrocytes. As shown before46, in the adult fly brain astrocytes are present in the lamina and medulla region of the brain where the axons of photoreceptors terminate (Figure 2). The lamina region is a superficial layer and the medulla region is the deeper layer47. This localization indicates possible interactions between the photoreceptors and astrocytes48. This also may indicate that astrocytes contribute to the functioning of photoreceptors, a hypothesis that can be further tested in the presented screen.

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CHAPTER 2

A screen to elucidate possible cell non-autonomous roles of astrocytes in SCA3To investigate whether genes in astrocytes can cell non-autonomously contribute to SCA3polyQ78-induced degeneration, a candidate RNAi screen in astrocytes was carried out in the SCA3polyQ78 eye model.

As Figure 3a shows, the Q system was used to express SCA3polyQ78 in Drosophila eyes, and the UAS-GAL4 system was used to knock down individual genes specifically in astrocytes by expressing different UAS-RNAi constructs. This way, we can independently manipulate gene expression in the eye and in astrocytes. Indeed, in pupae, expression of RFP (via UAS-GAL4) in astrocytes and GFP (via the Q system) in the developing eyes shows that there is no overlap in expression (Figure 3b). In our candidate RNAi screen, RNAi constructs targeting a set of 156 selected genes were expressed exclusively in astrocytes in flies expressing SCA3polyQ78 exclusively in the eyes, and the extent of eye degeneration was analyzed. We quantified and compared the extent of degeneration (necrotic phenotype, Figure 1c) in fly eyes expressing SCA3polyQ78 to fly eyes expressing SCA3polyQ78 together with an RNAi construct in astrocytes. We selected genes (Table 1) potentially involved in (1) receiving signals from neurons, such as DAMPs, neuropeptides and neurotransmitters. (2) intracellular signalling pathways in astrocytes, such as immune signaling pathways, nuclear factor kappa B (NF-κB) and (3) signaling of molecules that might be released by astrocytes.

brain medulla

laminalamina astrocytes

astrocytes>RFP

Figure 2

DAPI

Figure 2. The localization of astrocytes in the fly brain. The representative figure to show the localization of astrocytes in lamina and medulla in the adult fly brain was visualized by expressing RFP specifically in astrocytes (red), using the UAS-GAL4 system. Nuclei were stained with DAPI (blue). Genotype: alrm-GAL4/UAS-RFP.

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

A gene was identified as an enhancer gene of SCA3polyQ78-induced degeneration, when astrocyte-specific downregulation of this gene resulted in a reduction of more than 40% of the SCA3 polyQ78 severe degenerative eye phenotype. A suppressor gene was identified when its downregulation resulted in an increase of more than 60% of the SCA3polyQ78 severe degenerative phenotype. In total, 156 genes were tested in our screen, resulting in the identification of 19 enhancers and 25 suppressors (Table 1). Additional experiments need to be done to confirm the identified enhancers or suppressors. Independent RNAi lines targeting a different part of the target candidate should be used to exclude off-target effects. In addition, the efficiency of the RNAi-mediated knock down needs to be investigated by qPCR to confirm efficient knockdown of the gene. The identification of suppressors and enhancers indicate that astrocytes make cell non-autonomous contributions to the SCA3polyQ78 eye phenotype.

To decide which genes would be of interest to further investigate, we grouped the identified enhancer or suppressor genes that have similar functions or belong to the same signaling pathway in astrocytes. One example is a group of genes that belong to an NF-κB (Relish) signaling pathway (Table 1, indicated in green). We showed that the transcription factor Relish acted as an enhancer of SCA3polyQ78-induced degeneration. PGRP-LE49,50, which can activate Relish was also enhancers. Moreover, downregulation of other genes in the Relish pathway (such as Dredd) showed similar effects on the SCA3 phenotype. In chapter 3, further experiments

astrocytes

UAS gene X

alrm GAL4

GAL4

eyes

QUAS SCA3polyQ78

GMR QF2

QF2

cell non-autonomous

Figure 3

RFP (astrocytes)

GFP (eyes)

a b

Figure 3. Genetic setup of candidate RNAi screen to study the cell non-autonomous roles of astrocytes in SCA3. (a) Two binary expression systems were combined in the screen to allow independent regulation of expression in eyes and astrocytes. The Q system was used to express SCA3polyQ78 in fly eyes. To knock down individual genes in astrocytes, the UAS-GAL4 system was used. Different UAS-RNAi constructs were expressed specifically in astrocytes using alrm-GAL4. (b) Independent expression of the QUAS-QF system (green, eyes) and the UAS-GAL4 system (red, astrocytes) in the late pupa. Genotype, GMR-QF2/+; alrm-GAL4/UAS-RFP; QUAS-mCD8-GFP/+. The figure is representative of two independent experiments.

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220%180%120%

60%20%

0%-20%-40%

-100%lethal

Rel i sh pathway genesTol l pathway genes

enhancers

suppressorsgene VDRC stockCG17336, Lcch3 212,48% 109606-KKCG6378, SPARC 168,48% 100566-KKCG14358, CCHa1 156,06% 104974-KKCG8394, VGAT 141,03% 103586-KKCG18176, defl 117,46% 20604-GDCG16827, ItgaPS4 109,47% 109783-KKCG3143, foxo 103,78% 107786-KKCG3408 101,19% 36306-GDCG3022, GABA-B-R3 91,30% 108036-KKCG13758, Pdfr 85,75% 106381-KKCG6357 82,35% 8782-GDCG10997, Clic 81,09% 105975-KKCG7121, Tehao 80,14% 109705-KKCG7665, Lgr1 77,92% 104877-KKCG10233, rtp 76,06% 109000-KKCG3173, IntS1 74,74% 25825-GDCG5195, atk 74,74% 100110-KKCG4845, psidin 73,67% 103558-KKCG5372, ItgaPS5 73,21% 100120-KKCG14375, CCHa2 73,21% 102257-KKCG4641,nwk 71,63% 102133-KKCG17262, cnir 70,19% 104009-KKCG5528, Toll-9 66,80% 109635-KKCG8639, Cirl 63,66% 100749-KKCG8909 62,50% 108629-KKCG8250, Alk 58,89% 107083-KKCG9681, PGRP-SB1 58,00% 101298-KKCG7449, hbs 55,33% 105913-KKCG1411, CRMP 55,33% 101510-KKCG43119, Ect4 54,79% 102044-KKCG11335, lox 53,95% 107435-KKCG8784, PK2-R1 52,80% 103822-KKCG4604, Glaz 52,65% 107433-KKCG7105, Proc 52,00% 102488-KKCG42611, mgl 50,00% 105071-KKCG10342, NPF 49,00% 108772-KKCG31221 48,37% 103017-KKCG15274, GABA-B-R1 47,15% 105863-KKCG7250, Toll-6 46,77% 27103-GDCG2872, AstA-R1 46,70% 101395-KKCG34399, Nox 46,27% 100753-KKCG10698, CrzR 45,90% 108506-KKCG9453, Spn42Da 44,36% 106306-KKCG13480, LK 44,00% 14091-GDCG7250, Toll-6 42,69% 928-GDCG6531, wgn 41,70% 9152-GDCG18870 40,09% 100135-KKCG5490, Tl 38,64% 100078-KKCG11303, TM4SF 36,76% 8847-GDCG6438, amon 36,00% 110788-KKCG17800, Dscam1 33,73% 108835-KKCG7446, Grd 33,14% 5329-GDCG2736 32,95% 102672-KKCG6692, Cp1 32,62% 110619-KKCG8434, lbk 32,44% 106679-KKCG31094, LpR1 32,41% 106364-KKCG32540, CCKLR-17D3 32,05% 102039 KKCG6456, Mip 31,00% 106076-KKCG13984 29,91% 101831-KKCG42613 29,26% 102823-KKCG5811, Rya-R 26,80% 103973-KKCG6794, Dif 25,97% 100537-KKCG7395, sNPF-R 25,90% 9379-GDCG1147, NPFR 25,40% 9605-GDCG11217, CanB2 25,08% 104370-KKCG3131, Duox 24,36% 2593-GDCG10537, Rdl 24,26% 100429-KKCG33126, Nlaz 24,26% 101321-KKCG30340 24,10% 100088-KKCG34370 22,97% 100162-KKCG6072, Sra 22,75% 107573-KKCG4280, crq 22,54% 45883-GDCG34385, dpr12 21,57% 44741-GDCG13633, AstA 20,50% 103215-KKCG33950, trol 20,00% 110494-KKCG7586, Mcr 19,17% 100197 KKCG14734, Tk 19,00% 103662-KKCG4636, SCAR 18,93% 21908-GD

Signalinggene VDRC stockCG4167, Hsp67Ba 18,74% 104341-KKCG7000, snmp1 17,00% 104210-KKCG4096 16,04% 109025-KKCG13061, Nplp3 16,00% 105584-KKCG40733, RYa 15,48% 109264-KKCG7887, TkR99D 15,48% 43329-GDCG3302, Crz 14,10% 102204-KKCG4168 13,91% 100080-KKCG3441, Nplp1 13,00% 14035-GDCG4432, PGRP-LC 12,80% 101633-KKCG14593, CCHa2-R 10,74% 100290-KKCG7228, pes 9,55% 100391-KKCG31094, LpR1 8,73% 106364-KKCG4821, teq 8,33% 15362-GDCG6667, dl 7,23% 45996-GDCG31619, nolo 6,12% 104736-KKCG6817, foi 6,05% 10102-GDCG1804, Kek6 2,52% 109681-KKCG12079, ND-30 1,67% 103412-KKCG7285, AstC-R1 1,30% 110739-KKCG1857, nec 0,17% 108366-KKCG13419, Burs -1,00% 111063-KKCG4437, PGRP-LF -3,68% 108313-KKCG3048, Traf4 -4,02% 110766-KKCG14162, dpr6 -5,38% 103521-KKCG12004 -5,52% 101732-KKCG12919, egr -8,27% 108814-KKCG13422, GNBP-like3 -8,82% 107358-KKCG33087, LRP1 -10,78% 109605-KKCG4545, SerT -11,71% 11346-GDCG10823, SIFaR -11,80% 1783-GDCG14575, CapaR -12,41% 105556-KKCG9623, if -13,39% 100770-KKCG1618, comt -13,80% 105552-KKCG7285, AstC-R1 -14,41% 13560-GDCG8942, NimC1 -15,18% 105799-KKCG5008, GNBP3 -17,65% 37256-GDCG12489, dnr1 -20,49% 106453-KKCG9918, PK1-R -20,75% 101115-KKCG33717, PGRP-LD -21,00% 51023-GDCG10590, TM9SF3 -21,85% 110679-KKCG8795, PK2-R2 -23,10% 100927-KKCG7486, Dredd -23,20% 104726-KKCG7052, Tep2 -23,67% 106997-KKCG1632 -24,42% 106107-KKCG6134, spz -24,69% 105017-KKCG8743, Trpml -25,44% 108088-KKCG6440, Ms -27,00% 108760-KKCG15520, capa -28,00% 41124-GDCG11372, galectin -30,71% 107054-KKCG11992, Rel -30,75% 49414-GDCG14746, PGRP-SC1a -33,00% 43201-GDCG6890, Tollo -33,90% 27099-GDCG14919, AstC -34,00% 102735-KKCG1358 -34,35% 101453-KKCG6515,TkR86C -37,64% 107090-KKCG33696, CNMaR -40,00% 101076-KKCG7509 -40,55% 51584-GDCG30106, CCHa1-R -40,56% 103055-KKCG9819, CanA-14F #2 -41,69%CG30040, jeb -43,48% 103047-KKCG43119, Ect4 -44,57% 105369-KKCG14928, SPZ-4 -45,35% 7679-GDCG8329 -45,83% 101603-KKCG8896, 18w -46,49% 963-GDCG31092, LpR2 -48,30% 107597-KKCG6890, Tollo -49,84% 9431-GDCG1771, mew -50,51% 109608-KKCG8995, PGRP-LE -51,14% 108199-KKCG2086, drpr -51,37% 4833-GDCG11051, Nplp2 -54,00% 15305-GDCG11992, Rel -56,70% 49413-GDCG11709, PGRP-SA -57,00% 5594-GDCG6706, GABA-B-R2 -63,53% 1785-GDCG42610, Fhos -63,53% 34035-GDCG4099, Sr-CI 110014-KKCG1732, Gat 106638-KKCG6378, SPARC 16678-GD

Signaling

37

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

confirmed that inhibition of these NF-κB pathway-associated genes in astrocytes delays neurodegeneration. These results underscore the relevance of astrocytes in the progression of SCA3polyQ78-induced eye degeneration. Astrocytes in Drosophila share structural and functional similarities with mammalian astrocytes: amongst others, they provide trophic support to neurons and are involved in neurotransmitter recycling (reviewed in51). Thus, genes that we identified in our screen may be relevant for mammalian astrocytes as well.

Putative ways in which signaling from astrocytes can contribute to SCA3polyQ78-induced eye degenerationThe candidate genes that we screened to study the astrocytes in SCA3 include (1) receptors that receive signals from neurons, (2) molecules that are involved in intercellular signaling and (3) molecules that can be secreted from the astrocytes. In this section, we will speculate how some genes can influence the extent of SCA3polyQ78-induced degeneration.

Integrins

Our screen identified genes in astrocytes (mew and if) that encode conserved integrin subunits and can modulate SCA3polyQ78-induced eye degeneration. mew was identified as an enhancer of SCA3, because RNAi of mew reduced the degenerative eye phenotype. Downregulation of another integrin gene (if) in astrocytes showed similar effects (Table 1). Integrins have widespread roles, such as cell growth, migration and inflammation. They predominantly interact with components of the extracellular matrix (ECM), but also with some cell surface proteins and microorganisms52. In our SCA3 model, integrins may also play a role in the targeting of astrocytes to the eye (Figure 4a). In eyes expressing SCA3polyQ78

but not in control eyes astrocytes are present (Figure 4). Whether integrins are involved in targeting astrocytes to SCA3polyQ78-expressing eyes still needs additional investigation.

Table 1. The results of the candidate RNAi screen to identify genes in astrocytes that contribute to SCA3polyQ78-induced eye degeneration. The effect of downregulating specific genes in astrocytes on SCA3polyQ78-induced eye degeneration is shown for each gene. The extent of eye degeneration was quantified as follows: the percentage of severe eye degeneration phenotype in eyes expressing SCA3polyQ78 was set as 100% and compared to the percentage of severe eye degeneration upon downregulation of individual genes in astrocytes. An increased percentage of severe eye degeneration upon downregulation of a gene was indicated in orange (suppressor genes), and a decreased percentage of severe eye degeneration by downregulation of a gene was indicated in purple (enhancer genes). The darker the shade of the color of the enhancer or the suppressor, the larger the effect. The genes belonging to the Toll-Dif/Dorsal pathway are indicated in grey. Genes belonging to the IMD-Relish pathway are indicated in light green. Suppressor genes are marked in orange and enhancer genes are marked in purple. At least 80 eyes were counted for each condition. UAS-RNAi lines were randomly numbered and tested blindly by two people. Data present one time experiment.

38

CHAPTER 2

Integrins appear as heterodimers, and consist of two type I transmembrane proteins, an α subunit and a β subunit. In Drosophila, five α integrin subunits and two β integrin subunits have been identified. Our screen identified a conserved Drosophila integrin alpha subunit, multiple edematous wings (mew), also known as alpha PS1, as an enhancer of SCA3 (Table 1): downregulation of mew in astrocytes reduced the SCA3polyQ78-induced eye degeneration. Vertebrate orthologs of mew are the subunits α3, α6 and α753. In addition, downregulation of another conserved alpha subunit, inflated (if), also reduced the SCA3 polyQ78-induced eye degeneration, but to a lesser extent. If is orthologous to vertebrate α5, α8, αv and αIIβ53. In contrast, integrin α subunits ItgaPS4 and ItgaPS5 were identified as SCA3 suppressors, however, they have no ortholog in mammals. These data indicate cell non-autonomous contributions of α integrin subunits in astrocytes in SCA3, why some integrins are suppressors of SCA3 and other are enhancers is currently not clear.

There is some evidence that integrin signaling in astrocytes may play a role in neurodegeneration. In cultured primary rat astrocytes, stimulation with pro-inflammatory cytokine TNF-α increases rat astrocytic αv integrin expression54. Increased expression of αv integrin in astrocytes was also observed in a rat model of experimental autoimmune encephalomyelitis (EAE)54. Inhibiting αv integrin activity can alleviate symptoms of neurodegeneration: rat hippocampal slices incubated with beta-amyloid peptides display synaptic dysfunction, which was alleviated by addition of either a specific antagonist or antibody targeting αv integrin55. However,

Figure 4

RFP (astrocytes) RFP (astrocytes)

control SCA3polyQ78

RFP

Tubulin

25

55

Mw(kD)

contro

l

SCA3polyQ

78

SCA3polyQ

78a b

Figure 4. Eye-specific expression of SCA3polyQ78 results in the presence of astrocytes in the eye. (a) In flies expressing SCA3polyQ78 in the eye, astrocytes were present in the eye, shown by the presence of astrocyte-specific expressed RFP. This astrocytes-specific RFP was not observed in the eyes of control flies in which the SCA3polyQ78 was not expressed . Figures are representative of at least three independent experiments.(b) Western blot showed that control and SCA3polyQ78-expressing flies were expressing equal levels of RFP (right). The figure of western blot represents two experiments. Genotypes in (a) and (b): control, GMR-QF2/+; alrm-GAL4::UAS-myr-RFP/+. SCA3polyQ78: GMR-QF2/+; alrm-GAL4::UAS-myr-RFP/ QUAS-SCA3polyQ78.

39

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

a specific role of αv integrin in astrocytes in this study remains to be determined. Given that the αv integrin subunit can play a role in migration, phagocytosis and secretion of cytokines, it warrants further investigation in astrocytes.

Our results are in line with an enhancing role for αv integrin in neurodegeneration, as downregulation of αv integrin Drosophila ortholog if in astrocytes reduced the SCA3 phenotype. Together with the demonstration that another conserved Drosophila integrin alpha subunit (mew) was identified as an enhancer of SCA3, both Drosophila integrin alpha subunits, mew and if could be good candidates to investigate further to understand contributions of integrins in astrocytes to neurodegeneration.

GABA signaling

Another group of receptors that would be of interest to investigate further is the GABA receptors that respond to GABA, the main inhibitory compound in the CNS. Upon activation of the ionotropic GABA-A receptor with γ-aminobutyric acid (GABA), the receptor conducts chloride ions through its pore, thereby hyperpolarizing the neurons, thus having an inhibitory effect on neurotransmission in neurons (reviewed in56).

We identified ligand-gated chloride channel homolog 3 (Lcch3), an ortholog of the mammalian inotropic GABA-A receptor subunit, as a suppressor of SCA3 (Table 1). Drosophila expresses three orthologs of the mammalian ionotropic GABA-A receptor subunit: Lcch3, Resistant to dieldrin (Rdl) and Glycine receptor (Grd). Downregulation of each of them in astrocytes showed the enhanced severity of the SCA3polyQ78-induced phenotype to a varying extent (Table 1). The similar effects of three GABA-A receptor genes on SCA3 may indicate that the GABA-A receptor in astrocytes plays a role in SCA3.

While studies examining astrocytic GABA-A receptor function in neurodegeneration are limited, prior research suggested that the astrocytic GABA-A receptor is involved in signaling between astrocytes and neurons. Addition of GABA to isolated hippocampal rat astrocytes or addition of a GABA-A receptor agonist resulted in an inward chloride current57. Furthermore, GABA resulted in an increase of intracellular calcium, possibly via voltage-gated channels. While the role of GABA signaling in astrocytes needs to be investigated further, it could indicate a means by which astrocytes can respond to inhibitory GABA signaling in degenerating neurons. Expression of the GABA-A receptor can be induced by exposure to proinflammatory cytokine IL-6, which can be secreted by glial cells after brain inflammation or injury. In a culture of rat astrocytes, GABA-A receptor expression is upregulated after stimulation with IL-658. While these studies point at involvement of astrocytic GABA-A receptor activation and function during ND, its exact role remains to be demonstrated.

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Receptors for DAMPs or cytokines on astrocytes

We did not find conclusive effects of astrocyte-specific downregulation of receptors, which either recognize DAMPs or cytokines.

CalcineurinAn important target of calcium-induced signaling is the serine/threonine phosphatase calcineurin. We examined calcineurin in our SCA3 model, given that calcium signaling plays an important role in glia and aberrant signaling has been associated with neurodegeneration24. Previous work showed that astrocytic calcineurin activity is associated with changes in morphology as well as neuroinflammation (reviewed in24). Calcineurin consists of a catalytic subunit A and a regulatory subunit B, both of which are required to respond to calcium.

There are three genes in Drosophila encoding the A subunit (CanA1, CanA-14F and Pp2B-14D). CanA-14F and Pp2B-14D are homologous to each other and probably arose as a result of gene duplication. There are two genes encoding the B subunit (CanB and CanB2). CanA-14F was identified as an enhancer of SCA3 (Table 1), because downregulation of CanA-14F in astrocytes reduced the SCA3polyQ78-induced eye degeneration. Other calcineurin genes were not tested in our screen. Therefore, we tested whether the other isoforms of the calcineurin catalytic subunit could similarly contribute to SCA3 (Figure 5). Knockdown of CanA1 in astrocytes also reduced the SCA3polyQ78-induced eye degeneration. Similar results were found with downregulation of Pp2B-14D or CanA-14F, but the effects were less strong. Moreover, downregulation of both Pp2B-14D and CanA-14F attenuated the SCA3 phenotype. However, a gain of function or overexpression construct of a gene should have the opposite effect to the reduction of function by RNAi and provides additional evidence for involvement of a gene in SCA3. Indeed, expression of the active Pp2B-14D construct in astrocytes enhanced the SCA3polyQ78-induced eye phenotype (Figure 5). Together, these data support a role for calcineurin in astrocytes as an enhancer of SCA3.

Our earlier studies provide evidence for a putative downstream target by which calcineurin can enhance activation of astrocytes: calcineurin activity can contribute to activation of NF-κB transcription factors (chapter 4)59. There is specificity of calcineurin with specific isoforms activating specific NF-κB transcription factor: the activity of CanA1 contributes to activation of NF-κB transcription factor Relish59, activation of homologous Pp2B-14D/CanA14F can result in activation of NF-κB transcription factor Dif/Dorsal (chapter 4). However, we cannot exclude other calcineurin targets that account for their effects on SCA3. The effect of calcineurin on NF-κB is of particular interest, given that we also identified NF-κB transcription

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

factor Relish as an enhancer of SCA3.

Dysregulation of calcium signaling in astrocytes occurs in a mouse model of Huntington’s disease, with astrocytes displaying spontaneous calcium signals, which may be associated with calcineurin activation60. Disturbances in calcium signaling and the resulting calcineurin activation by cleavage can occur in the brains of AD patients61. The cleavage of calcineurin occurs through the activation of calpain, a calcium-dependent protease62. We have preliminary evidence that suggests that calcineurin is activated in our SCA3 model: expression of SCA3polyQ78 in the eye resulted in the cleavage of calcineurin in lysates of fly heads (data not shown).

Other studies showed that calcineurin-dependent signaling in astrocytes contributes to their activation. Overexpression of calcineurin in astrocytes resulted in their activation and increased the expression of pro-inflammatory genes63. Our data are in line with a study, which showed that application of calcineurin inhibitor targeting astrocytes in the hippocampus attenuated synaptic dysfunction in AD mouse

0

10

20

30

40

% o

f nec

rotic

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deg

ener

atio

n -

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D RNAi

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D:Can

A-14F R

NAi

CanA 1

RNAi

Figure 5

activ

e Pp2

B-14D

CanA-14

F RNAi

SCA3polyQ78

Figure 5. The effect of calcineurin in astrocytes on SCA3polyQ78-induced eye degeneration. The fraction of necrotic eyes in SCA3polyQ78-expressing eyes was compared to SCA3polyQ78 eyes expressing RNAi or overexpression constructs in astrocytes that target specific calcineurin isoforms. For each condition, at least 40 flies were examined. n=1. Genotypes: - (the SCA3polyQ78 control), GMR-QF2/+; QUAS-SCA3polyQ78::alrm-GAL4/+. CanA1 RNAi, GMR-QF2/+; QUAS-SCA3polyQ78::alrm-GAL4/+; UAS-CanA1 RNAi/+. Pp2B-14D RNAi, GMR-QF2/+; QUAS-SCA3polyQ78::alrm-GAL4/UAS-Pp2B-14D RNAi. CanA-14F RNAi, GMR-QF2/+; QUAS-SCA3polyQ78::alrm-GAL4/UAS-CanA-14F RNAi. Pp2B-14B:CanA-14F RNAi, GMR-QF2/+;QUAS-SCA3polyQ78::alrm-GAL4/UAS-CanA-14F RNAi::UAS-Pp2B-14D RNAi. Active Pp2B-14D, GMR-QF2/+; QUAS-SCA3polyQ78::alrm-GAL4/UAS-∆Pp2B-14D59.

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model64. However, it is still a matter of debate whether the activation of calcineurin in astrocytes play a detrimental or beneficial role in NDs (reviewed in24).

Our current understanding of the interactions between astrocytes and neurons, which could possibly influence neuronal health are summarized in a model (Figure 6). Neuronal expression of an aggregation-prone protein results in a number of responses, including the release of neurotransmitters, pro-inflammatory cytokines and DAMPs. Aggregates can serve as DAMPs as well. Receptors for DAMPs include PGRPs (Peptidoglycan Recognition Receptors), and TLRs (Toll-like Receptors); however, integrins have been identified as receptors for DAMPs as well65.

In our screen, we identified putative cell non-autonomous actions of astrocytes, which may contribute to neurodegeneration. Our setup of the screen is complementary to prior research where a misfolded, neurodegeneration-associated protein was expressed specifically in either neurons or astrocytes or ubiquitously in the brain (including neurons and astrocytes). Specific expression of aggregation-prone proteins in astrocytes also had detrimental effects on neurons29,67. Expression of misfolded, aggregation-prone proteins in astrocytes was also found in patients suffering from neurodegenerative diseases68,69. This expression may be detrimental to astrocytes (cell-autonomous) or alternatively, the resulting change in signaling in astrocytes may be detrimental to neurons (cell non-autonomous signaling). Our data are complementary to this research: we identify putative signaling events in astrocytes induced upon expression of misfolded proteins in neurons. Signals derived from cells (neurons or photoreceptors) are received by astrocytes. Subsequent signaling induced in astrocytes then results in signals that signal back to neurons (e.g. neuropeptides) or alternatively, affect levels of neurotransmitters (e.g. glutamate) that both influence neuronal functioning. In Drosophila, neuronal expression of SCA3polyQ78 resulted in alteration of expression of genes in the head70, suggesting the involvement of these genes in the responses to SCA3polyQ78. However, how gene expression in astrocytes is altered still needs further investigation. Previous studies have identified genes of interest that are expressed in SCA3 Drosophila models, including NF-κB, ligands for receptors acting upstream of NF-κB, as well as transcriptional targets of NF-κB70,71. We also observed downregulation of EAAT1, a glutamate transporter (prelim. data), suggesting alterations in glutamate levels.

The effect of genes that were identified in our SCA3 eye model can be tested in flies expressing SCA3polyQ78 in neurons to verify whether these genes in astrocytes also have similar effects when SCA3polyQ78 is expressed in neurons. In chapter 3, we indeed verify that a subset of genes in astrocytes we tested in our neuronal model also modulated the effects of neuronal SCA3. This underscores the relevance of our SCA3 eye model for neurodegenerative diseases.

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Cell non-autonomous effects of astrocytes on neurodegenerative disease in Drosophila

Calciumhomeostasis

Neurons (expressing misfolded proteins)

NF-kB

astrocytes

Calcineurinsignalling

pro-inflammatorycytokines

pro-inflammatorycytokines

DAMPstoxic levels of

neurotransmitters:Glutamate, GABA

enhanced release of gliotransmitters:

Glutamate, GABA

neurotransmitterreceptors/transporters

gliotransmittersreceptors/transporters

cytokinereceptors

pattern recognitionreceptors

integrins

Figure 6

Figure 6. Model for molecules and signaling pathways that may be involved in neuron-astrocyte interactions in NDs. Neurons that express misfolded proteins as occurs in NDs can release DAMPs, pro-inflammatory cytokines or elevated levels of neurotransmitters. These molecules are recognized by different receptors on astrocytes, resulting in the activation of intracellular signaling pathways. This includes elevation of intracellular calcium and activation of calcineurin, as well as activation of NF-κB signaling, which can occur downstream of calcineurin59,66. The activation of intercellular signaling results in the generation of molecules that are released from astrocytes, such as pro-inflammatory cytokines and gliotransmitters which may have negative effects on neurons.

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MATERIALS AND METHODS

Drosophila strainsAll flies were raised at 25 degrees and cultured on standard fly food. GMR-QF2 (stock number 59283), alrm-GAL4 (stock number 67031) were obtained from Bloomington Drosophila stock center (BDSC, Bloomington, Indiana, U.S.A.). Information on additional fly lines can be found in the materials and methods section in chapter 3. For the screen, all the RNAi fly stocks we used were mentioned in the screen table (Table 1) and were obtained from Vienna Drosophila Research Center (VDRC) or Bloomington. RNAi lines targeting calcineurin genes as well as a fly line expressing active calcineurin have been described previously59,66. Generation of QUAS-SCA3polyQ27 and QUAS-SCA3polyQ78 are described in chapter 3). All the transgenic fly lines were used were made in the w1118 background.

GeneticsTo independently and simultaneously manipulate gene expression in eyes or astrocytes, we used the QF-QUAS system to express constructs in neurons or in eyes and UAS-GAL4 to manipulate gene expression in astrocytes (using alrm-GAL4).

To screen for involvement of astrocyte-associated genes in the SCA3polyQ78-induced eye degeneration we used the following fly line: GMR-QF2/(Y); QUAS-SCA3polyQ78:: alrm-GAL4/CyO-tub-QS. In this line, expression of SCA3polyQ78 is suppressed by QF2 suppressor QS72. To analyze the effect of gene expression in astrocytes on the SCA3 eye phenotype, we crossed this line to different UAS constructs. To quantify eye degeneration by analyzing levels of mCD8-GFP, we used the line GMR-QF2/(Y); QUAS-SCA3polyQ78 /CyO-tub-QS: alrm-GAL4: QUAS-mCD8-GFP, and crossed them to UAS lines or w1118 flies. The fly line gmr-QF2/(Y); QUAS-SCA3polyQ78:: alrm-GAL4/CyO-tub-QS was crossed to individual UAS-RNAi lines to obtain astrocyte-specific knockdown of the indicated genes (genotype of the cross GMR-QF2/+; QUAS-SCA3polyQ78: alrm-GAL4/+ together with UAS-RNAi/+). To prevent bias, UAS-RNAi lines were randomly numbered and tested blindly.

Analysis of eye degenerationAt least 40 2-day old flies were collected for each fly line that was examined. The degenerative eye phenotypes of the flies were examined under a dissecting microscope and two persons scored them independently. To prevent bias, UAS-RNAi lines were randomly numbered and tested blindly. The numbers of the mild

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and severe SCA3 eye phenotype were recorded and the fraction of each eye phenotype was calculated.

Immunofluorescence stainingFly heads were fixed with 3.7% formaldehyde (Sigma Aldrich) for 15 min, washed 3 times in PBS. Then brains were dissected in PBS, and fixed again in 3.7% formaldehyde for 10 min, followed by washing 5 times with PBS-T (0.1% Triton-X-100). Fly brains were next blocked with 2% BSA /0.1% Triton-X-100 in PBS for 1 h. After washing 3 times with PBS-T, brains were incubated with DAPI (1:100 in 2% BSA PBS-T) for 40 min and washed with PBS-T 3 times. Brains were mounted on the microscope slide. Fluorescent images were obtained with a confocal microscope, SP8 (Leica DMI 6000).

Western BlottingFor each condition, at least 30 two-day-old flies were collected and frozen in liquid nitrogen. Flies were decapitated by vortexing. Fly heads were lysed in the laemili buffer by sonification. An equal amount of samples were loaded on 12.5% SDS-PAGE gels. The antibody was used were rat anti-HA-Peroxidase (1:1000, Roche Diagnostics GmbH, Germany), mouse anti-RFP (1:1000, Chromotek 6G6) and mouse anti-alpha tubulin (1:2000, Sigma T5138). The membranes were detected in Chemi DocTM Touch (Bio-Rad). The intensity of the bands was analyzed by using Image J.

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CHAPTER 3Inhibition of NF-κB in astrocytes delays neurode-

generation in a cell non-autonomous manner

Li-YX, Sibon, O.C.M., Dijkers, P.F. *

Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands

*Corresponding author; p.f.dijkers@umcg.nlManuscript submitted

ABSTRACTMost neurodegenerative diseases associated with protein aggregation are hallmarked by activation of astrocytes, yet their contribution to pathogenesis is unclear. One long-standing question is whether the responses in astrocytes in neurodegenerative diseases are due to cellular stress or damage in astrocytes (cell-autonomous responses) and/or because of responses to cellular stress or damage in neurons (cell non-autonomous responses).

Previously, we identified genes in astrocytes that affected neurodegeneration in a cell non-autonomous manner in a candidate RNAi screen in Drosophila (Chapter 2). Here, we examined these genes in detail in a Drosophila model for Spinocerebellar Ataxia-3 (SCA3, also known as Machado Joseph Disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN-3 gene. The conserved NF-κB transcription factor Relish, was identified as a modifier gene of SCA3poly78-induced degeneration, demonstrating that astrocytic functioning influences the rate of neurodegeneration (Chapter 2). In this Chapter, several of these disease-modifying genes belong to the Relish pathway were further studied. Selective inhibition of Relish signaling exclusively in astrocytes extended the lifespan of flies expressing SCA3polyQ78 exclusively in neurons.

Importantly, inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer’s disease. Our data provide direct evidence for cell non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases.

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INTRODUCTIONA hallmark of many age-related neurodegenerative diseases is the presence of protein aggregates, which precedes the onset of clinical symptoms. Genetic profiling of transcriptional changes in neurodegenerative diseases has identified glial genes whose altered expression could potentially contribute to pathogenesis1,2. Besides this, another characteristic in most, if not all neurodegenerative diseases associated with protein aggregation is the activation of astrocytes3. If and how astrocytes contribute to the disease progression has remained largely elusive. Astrocytes have important functions in neuronal homeostasis, but it is unclear whether dysfunction to maintain homeostasis or toxic gain of function in astrocytes can contribute to neurodegenerative diseases.

The presence of aggregates in astrocytes can result in their activation (cell-autonomous signaling), and subsequent signaling to neurons4, reviewed in5. In chapter 2 we showed that signals from aggregate-expressing neurons can modulate the intercellular signaling of astrocytes, which in turn can influence disease pathogenesis (cell non-autonomous signaling).

Astrocytes are involved in essential CNS functions, including metabolic functions, regulating levels of neurotransmitters, maintaining the blood-brain barrier, as well as immune defense, thus contributing to neuronal homeostasis6. Astrocytes are activated early in neurodegenerative diseases, at times that may precede the appearance of aggregates5. This only occurs in specific areas of the brain, i.e. those that are primarily affected by the respective diseases, such as for example the striatum in Huntington’s Disease (HD), the pons in SCA3, and the cortex in Alzheimer’s Disease (AD)7,8. Dysfunction of astrocytes can contribute to pathogenesis in a mouse model for HD, and therefore astrocytes have been suggested as potential therapeutic targets9. In this model, the HD-associated protein was amongst others expressed in the brain, including neurons, microglia and astrocytes. Thus, in this model both cell-autonomous as well as cell non-autonomous of astrocytes contribute to pathogenesis. There is ample evidence of cell-autonomous activation of astrocytes in the pathogenesis of neurodegenerative diseases (reviewed in5,10).

We showed that signaling from aggregate-expressing neurons can influence signaling in astrocytes which subsequently can modulate neurodegeneration (Chapter 2). We also demonstrated that astrocytes can contribute to neurodegenerative diseases in a cell non-autonomous manner, within the context of an intact animal. Examining astrocytes in an in vivo model is key, given that their morphology and activity changes when taken outside their physiological context (reviewed in11). For

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this, we conducted a dedicated RNAi screen (Chapter 2) to selectively knock down individual genes in astrocytes in a Drosophila melanogaster model of the polyQ disease SCA3. In SCA3, the ATXN-3 gene contains an expanded CAG repeat (coding for glutamine) that leads to the expression of a misfolded aggregation-prone ATXN-3 polyglutamine protein. These misfolded polyQ-containing proteins accumulate intracellularly, resulting in neuronal damage and activation of astrocytes12. Several enhancer and suppressor genes in astrocytes that contributed to neurodegeneration in a cell non-autonomous manner were identified, and their putative contributions were discussed in Chapter 2. Here, we further analyze one of the identified enhancers of SCA3 in Drosophila, the conserved NF-κB gene Relish. We examine the potential mechanism by which Relish in astrocytes can influence neurodegeneration. For this, we tested whether modulation of Relish expression could influence levels of SCA3polyQ78 aggregates. We tested the effect of Relish target genes, antimicrobial peptides, on degeneration in our SCA3 model. Finally, we examined whether modulation of Relish in astrocytes could influence the lifespan of neurons expressing SCA3polyQ78 aggregates. To see whether we could extend our effects with Relish to another neurodegenerative disease, we used a Drosophila model for Alzheimer’s disease, that expresses human amyloid beta peptides specifically in neurons.

METHODS

Drosophila strainsAll fly lines were maintained at 25°C on standard fly food unless indicated otherwise. The following stocks were obtained from the Bloomington Drosophila stock center (BDSC, Bloomington, Indiana, U.S.A.): UAS-MJD.tr-Q27 (8149), UAS-MJD.tr-Q78 (8150), QUAS-mCD8-GFP (30003), UAS-myr-RFP (7118), two tub-QS lines (52112 and 30024), alrm-GAL4 (number 67031); GMR-GAL4 (1104); daughterless-GAL4 (8641); UAS-Relish RNAi#2 (33661); Relish E20 mutant (9457). The following stocks were obtained from the Vienna Drosophila Research Center (VDRC): UAS-Relish RNAi #1 (49414-GD), UAS-Attacin A RNAi (50320-GD), UAS-Cecropin A (42859-GD). UAS-GFP-Relish has been described13. GMR-QF2 (BDSC #59283) nSyb-QF2 (BDSC 51956), the CyO-tub-QS balancer as well as the pQUAST vector were a gift from C. Potter (Baltimore, MD, U.S.A.). UAS-necrotic-Abeta42 flies were a gift from D. Crowther (Cambridge University, Cambridge, United Kingdom) and have been described previously14. All flies were backcrossed to w1118 flies for at least six generations. We generated QUAST-SCA3polyQ78 and QUAST-SCA3polyQ27 transgenic flies by amplifying SCA3polyQ78

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(MJD.tr-Q78) or SCA3polyQ27 (MJD.tr-Q27) from gDNA from stock #8150 or #8149, using the following UAS-specific primers: 5’- ATAGGGAATTGGGAATTCGTT-3’ and 5’- CAATTATGTCACACCACAGAA-3’ and cloned into pUAST using EcoRI and XbaI. We generated pQUAST-Aβ42 by amplifying from gDNA from UAS-necrotic-Abeta42 flies using 5’-cgaattcaacATGgcgagcaaagtctcgatc-3’ and 5’-ctctagaTTACGCAATCACCACGCCGC-3’ and cloned into pQUAST using EcoRI and XbaI. Constructs were verified by sequencing. Transgenic fly lines were generated in the w1118 background, using BestGene (Chino Hills, CA, U.S.A.).

GeneticsTo independently and simultaneously manipulate gene expression in either eyes or neurons or astrocytes, we used the QF-QUAS system to express constructs in neurons or in eyes and UAS-GAL4 to manipulate gene expression in astrocytes (using alrm-GAL4). For cell-autonomous effects of genes in eyes, we used UAS-GAL4 (as described in Chapter 2). We suppressed QF-dependent expression by expressing QS under control of the tubulin promoter. A schematic drawing of the genetic systems that were used is shown in Chapter 2 and Suppl. Fig. 5b. To analyze astrocyte-associated genes in the SCA3polyQ78-induced eye degeneration we used the following fly line: gmr-QF2/(Y); QUAS-SCA3polyQ78:: alrm-GAL4/CyO-tub-QS. In this line, expression of SCA3polyQ78 is suppressed. To analyze genes in astrocytes on the SCA3polyQ78 eye phenotype, we crossed this line to different UAS constructs. To quantify eye degeneration by analyzing levels of mCD8-GFP, we used the line gmr-QF2/(Y); QUAS-SCA3polyQ78/CyO-tub-QS: alrm-GAL4: QUAS-mCD8-GFP, and crossed them to UAS lines or w1118 flies.

To analyze the effect of SCA3polyQ78 in neurons and a possible effect of astrocytes on the SCA3polyQ78-induced pathogenesis, we used the following line: tub-QS/(y); QUAS-SCA3polyQ78: alrm-GAL4; nsyb-QF2: tub-QS. We used two copies of tub-QS, since 1 copy was not able to suppress the expression of SCA3polyQ78. We supplemented adult flies with quinic acid to suppress QS and allow expression of SCA3polyQ78 only in adulthood.

Analysis of eye degenerationFor each condition, we calculated the fraction of the eyes that showed an SCA3polyQ78-induced necrotic degenerative phenotype as shown previously15. For each line we analyzed, we scored the eyes of at least 40 flies. The results were average of at least three independent experiments -/+SEM. Statistical analysis was performed using the Student T test. *p<0.05; ** p<0.01.

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Real-time quantification PCR (RT-qPCR)We collected 120 fly heads per point to isolate total RNA using the RNeasy Mini Kit (Qiagen). To collect the heads, flies were frozen in liquid nitrogen and decapitated by vortexing. Fly heads were collected using a mesh. M-MLV reverse transcriptase (Invitrogen, 28025-013) was used to transcribe the same quantity of RNA into cDNA. Relative quantification of the gene expression level was determined in CFX ConnectTM (Bio-Rad) by using iQTM SYBR® Green Supermix (Bio-Rad Laboratories, Inc.). For all the samples, gene expression levels were normalized to a housekeeping gene, ribosomal protein 49 (RP49).

Primer pairs used for QPCR:

RP49: 5’-CCGCTTCAAGGGACAGTATC-3’/5’-GACAATCTCCTTGCGCTTCT-3’IM1:5’-TGCCCAGTGCACTCAGTATC-3’/5’-GATCACATTTCCTGGATCGG-3’IM2: 5’-AAATACTGCAATGTGCACGG-3’/5’-ATGGTGCTTTGGATTTGAGG-3’AttC: 5’-CCAATGGCTTCAAGTTCGAT-3’/5’-AGGGTCCACTTGTCCACTTG-3’AttA: 5’-ACAAGCATCCTAATCGTGGC-3’/5’-GGTCAGATCCAAACGAGCAT-3’DptA: 5’-ACCGCAGTACCCACTCAATC-3’/5’-ACTTTCCAGCTCGGTTCTGA-3’CecA: 5’-GAACTTCTACAACATCTTCGT-3’/5’-TCCCAGTCCCTGGATTGT-3’Relish: 5’-AGCAGTGGCGCACTAAAGTT-3’ /5’-GATGGCTGACCATTCGTTTT-3’Results are average of at least three independent experiments -/+ SEM. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01.

Western blottingFor examining SCA3polyQ78 levels and aggregation and its effect on eye viability (using mCD8-GFP), two-day-old flies were used. At least 30 fly heads per genotype were collected, lysed in Laemli buffer by sonification and boiled at 95°C for 5 min. Samples were separated on 12.5% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Fisher Scientific). After blocking in 5% (w/v) non-fat dried milk in PBST for 1h, the membrane was incubated with primary antibody overnight at 4 degrees. Antibodies that were used: rat anti-HA-Peroxidase (Roche Diagnostics GmbH, Germany), rabbit anti-GFP (A11122; Invitrogen,) 6E10 Ab was purchased from Covenance and mouse anti-alpha tubulin (Sigma T5138). After incubation with secondary antibody, ECL (Amersham) signal was detected in ChemiDocTM Touch (Bio-Rad). The intensity of the bands was analyzed by using Image J (National Institutes of Health, USA). Quantification of western blots was of three independent experiments, statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01.

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LifespansTo determine the lifespan of flies, a maximum of 20 flies were kept in a vial. At least 80 flies per condition were analyzed. Every two days flies were transferred into new vials containing fresh food. The following fly lines were used to determine the effect of genes in astrocytes on survival: (1) tub-QS/(y); QUAS-SCA3polyQ78:: alrm-GAL4/CyO; nSyb-QF:: tub-QS/TM6B (SCA3 line) and (2) tub-QS/(y); alrm-GAL4/CyO; nSyb-QF: tub-QS/TM6B (control line). These lines were used to test the effect of astrocyte-specific genes on flies that express SCA3polyQ78 in adulthood. Expression of SCA3polyQ78 was induced in adult flies by inhibiting QS through supplementing the food with quinic acid. Quinic acid was used in a concentration of 300mg/ml with a fixed PH of 7. 250 µl of quinic acid solution was put on top of the food to cover the entire surface and used when the solution was completely absorbed into the food 16. Lifespan curves were analyzed in GraphPad Prism (GraphPad Software, San Diego, CA, USA), statistical significance was analyzed by Log-rank (Mantel-cox) test.

Climbing assayFor each cross, 5 vials of flies, 20 flies per vial, were analyzed. To determine the mobility of flies we analyzed their climbing ability. For this, the vial was divided into four compartments, with the lowest compartment numbered with 1 (slow climbers) and the highest with number 3 (fast climbers). Flies were tapped down to the bottom of vials and were allowed 10 seconds to climb up. The number of flies in each compartment was scored. The average score of each cross at each time point was determined by the total score divided by the total number of flies. The climbing test was repeated 3 times for each group, and the average of the 3 times was shown.

Drosophila brain dissection and microscopyDrosophila heads were collected in ice-cold PBS, fixed in 3.7% formaldehyde solution (0.1 Triton x-100 in PBS) for 15 min and washed 5 times in PBS. Next Drosophila brains were dissected in PBS and stained with DAPI solution (0.2mg/ml, 2% BSA and 0.1% Triton x-100 in PBS) at room temperature. After washing with PBS for 3 times, the brains were mounted in 80% glycerol on slides and imaged by a Leica SP8 Confocal Microscope.

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RESULTSExpression of a biologically relevant, truncated form of the human ATXN3 gene containing an expansion of the glutamine region (trSCA3 Q78, containing 78 glutamines, hereafter referred to as SCA3polyQ78) in Drosophila eyes resulted in progressive degeneration of photoreceptor cells17, accompanied by eye depigmentation (mild phenotype) as well as depigmentation accompanied by black necrotic spots (severe phenotype; arrow, Fig. 1a, left). No such effects were seen with SCA3 containing a non-pathogenic length of repeats (SCA3polyQ27, data shown in Chapter 2)17. Degeneration can be quantified by examining the external structure of the eye to determine the fraction of eyes containing necrotic spots (Fig. 1a, right). As an alternative way of analysis, we coexpressed membrane-targeted mCD8-GFP18 with SCA3polyQ78. The severity of eye degeneration was inversely correlated with GFP fluorescence intensity (Fig. 1b, left) and decrease of mCD8-GFP expression levels, as measured in lysates of fly heads (Fig. 1b, right). Both the

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Fig. 1 Quantification of the degenerative eye phenotype induced by expression of SCA3polyQ78 (a) (Left) Expression of SCA3polyQ78 in the Drosophila eye resulted in loss of pigment (“depigmented”), as well as the presence of necrotic patches (“necrotic”, indicated with an arrow). (Right) The fraction of eyes containing necrotic patches was used as a quantitative measure for degeneration. n=3. Genotypes in (a): control, GMR-QF2/+. SCA3polyQ78, GMR-QF2/+; QUAS-SCA3polyQ78/+.(b) Expression of mCD8-GFP in the eye to analyze SCA3polyQ78-induced degeneration. (Left) GFP fluorescence of a representative control eye compared to eyes coexpressing SCA3polyQ78. (Right) The extent of degeneration can be quantified by analyzing the GFP levels in fly eyes on Western blot. Tubulin was used as an equal loading control. Figures of western blot represent for 3 experiments. Genotypes in (b): control, GMR-QF2/+; QUAS-mCD8-GFP/+. SCA3polyQ78, GMR-QF2/+; QUAS-SCA3polyQ78/+; QUAS-mCD8-GFP/+.

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fraction of degeneration and GFP levels were used as readouts to determine the effect of genes in astrocytes on degeneration.

One of the genes identified from the RNAi candidate screen (Chapter 2) was NF-κB transcription factor Relish (orthologous to mammalian NF-κB1). Interestingly, RNAi targeting Relish, but also RNAi targeting several other genes in the Relish pathway, suppressed the SCA3 phenotype. In Drosophila, there are two independent NF-κB pathways, activating transcription factors Relish or Dif/Dorsal respectively. We examined the expression of anti-microbial peptides (AMPs) in our SCA3 model, transcriptional NF-κB targets which help fight infection19. In inflammatory responses, these peptides are considered to aid in activating and recruiting immune cells (reviewed in20). Both NF-κB pathways are activated by eye-specific expression of SCA3polyQ78. AMPs specific for Relish (CecA and DptA) and Dif/Dorsal (IM1 and IM2), are upregulated (Fig. 2a) and shown in 21. Interestingly, while downregulation of Relish attenuated eye degeneration induced by SCA3polyQ78 (Chapter 2 Table 1, indicated in green), targeting genes of the Dif/Dorsal (Chapter 2 Table 1, indicated in grey) did not influence eye degeneration, suggesting the differential involvement of Relish and Dif/Dorsal signaling.

We further tested the activation of Relish in astrocytes upon the expression of SCA3polyQ78 in the eye. GFP under control of the promoter of a Relish-dependent gene was co-expressed with RFP specifically in the astrocytes (suppl. Fig. 1). GFP partly colocalized with RFP in astrocytes, indicating that the activation of Relish occurred in astrocytes. In control eyes, no Relish-dependent GFP signal was present. Thus, the Relish activation in astrocytes can occur via a cell non-autonomous stimulation. We further investigated this astrocyte-specific, cell non-autonomous contribution to degeneration.

We first confirmed the results of our screen by using two independent Relish RNAi constructs (for the efficacy of knockdown, see Suppl. Fig. 2), which both decreased the SCA3polyQ78-induced degeneration (Fig. 2b). Inversely, the opposite effect was seen upon overexpression of Relish (Fig. 2b). In addition, in flies heterozygous for Relish, degeneration was attenuated, similar to astrocyte-specific Relish RNAi (Fig. 2b). Importantly, when coexpressing SCA3polyQ78 and constructs targeting Relish in the eyes, thus assessing the cell-autonomous contribution of Relish, eye degeneration was not attenuated (Fig. 2b), demonstrating that effects of Relish are cell non-autonomous, mediated via astrocytes.

The protective or enhancing effects of the astrocyte-specific modulation of Relish levels were confirmed (Fig. 2c) when membrane-targeted mCD8-GFP was used as readout for degeneration18. Astrocyte-specific Relish RNAi and Relish heterozygous flies showed a similar increase in GFP levels compared to the SCA3polyQ78 expressing

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controls, suggesting that astrocyte-specific Relish signaling modulates SCA3polyQ78-induced degeneration. The effect of Relish on GFP levels is specifically related to SCA3polyQ78-induced degeneration, as in control flies GFP levels were similar, irrespective of Relish expression (Fig. 2d). Together, these experiments demonstrate that in our SCA3 model NF-κB signaling pathways are activated and that Relish signaling in astrocytes enhances SCA3polyQ78-induced degeneration.

In the brain, Relish is predominantly activated in glia22,23. We analyzed the contribution of astrocytes to overall Relish signaling in the fly head. For this, we examined levels of Relish-dependent AMPs of flies expressing SCA3polyQ78 in the eyes together with either Relish RNAi or Relish overexpression constructs targeted to astrocytes, as well as of SCA3polyQ78 flies heterozygous for Relish. Expression of Relish RNAi

Fig. 2 Effect of NF-κB transcription factor Relish in astrocytes on SCA3polyQ78-induced degeneration (a) Eye-specific expression of SCA3polyQ78 resulted in NF-κB activation in the head. Heads of control flies or flies expressing SCA3polyQ78 in eyes were analyzed for expression of Relish target gene (CecA, DptA) or Dif/Dorsal target genes (IM-1, IM-2 ). Data are representative of at least three independent experiments -/+SEM. Statistical analysis was performed using the Student T test. * p<0.05.Genotypes in (a): control, GMR-QF2/+. SCA3polyQ78, GMR-QF2/+; QUAS-SCA3polyQ78 /+.(b) Effect of Relish on SCA3polyQ78-induced degeneration. Top: representative images of fly eyes expressing SCA3polyQ78 (-) or SCA3polyQ78 together with either astrocyte-specific expression of two independent RNAi constructs targeting Relish (Relish RNAi#1 and Relish RNAi#2), a Relish overexpression construct (Relish overexpression) or flies heterozygous for Relish (Relish -/+). Bottom, left: quantification of the fraction of degeneration in (b) top; Bottom, right: effect of cell-autonomous modulation of Relish (eyes). At least 80 eyes were counted per genotype, data represent three independent experiments -/+SEM. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01; *** p<0.001.Genotypes in (b) Top and Bottom left: -, GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-Gal4/+. Relish RNAi #1, GMR-QF2/+; QUAS-SCA3polyQ78::alrm-Gal4/UAS-Relish RNAi#1. Relish RNAi #2, GMR-QF2/+; QUAS- SCA3polyQ78:: alrm-Gal4/UAS-Relish RNAi #2. Relish overexpression, GMR-QF2/+; QUAS- SCA3polyQ78:: alrm-Gal4/UAS-Relish. Relish-/+, GMR-QF2/+; QUAS- SCA3polyQ78:: alrm-Gal4; Relish E20/+.Genotypes in (b) Bottom right: -, GMR-GAL4::UAS-SCA3polyQ78/+. Relish RNAi #1, GMR-GAL4::UAS-SCA3polyQ78/UAS-Relish RNAi#1. Relish RNAi #2, GMR-GAL4::UAS-SCA3polyQ78/UAS-Relish RNAi#2. Relish overexpression, GMR-GAL4::UAS-SCA3polyQ78/UAS-Relish.(c) Effect of Relish signaling in astrocytes on SCA3polyQ78-induced degeneration by analyzing membrane-targeted mCD8-GFP. Left: levels of GFP in lysates of fly heads of flies expressing SCA3polyQ78 in eyes together with mCD8-GFP. SCA3polyQ78 flies (-) were compared to flies co-expressing Relish RNAi constructs (Relish RNAi#1 and Relish RNAi#2) in astrocytes, flies heterozygous for Relish or flies overexpressing Relish in astrocytes. Tubulin was used as a control for equal loading. Right: quantification of western blots. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01; *** p<0.001. n=3.Genotypes in (c): -, GMR-QF2/+; QUAS-SCA3polyQ78/+; QUAS-mCD8-GFP/+. Relish RNAi #1, GMR-QF2/+; QUAS-SCA3polyQ78::alrm-GAL4/UAS-Relish RNAi#1;QUAS-mCD8-GFP/+. Relish RNAi #2, GMR-QF2/+; QUAS- SCA3polyQ78:: alrm-GAL4/UAS-Relish RNAi #2; QUAS-mCD8-GFP. Relish overexpression, GMR-QF2/+; QUAS- SCA3polyQ78:: alrm-GAL4/UAS-Relish;QUAS-mCD8-GFP. Relish-/+, GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-GAL4; Relish E20/+;QUAS-mCD8-GFP.(d) Modulating Relish expression in astrocytes does not affect levels of mCD8-GFP in the eyes. Lysates of fly heads expressing eye-specific mCD8-GFP but not SCA3polyQ78 (control) were compared to fly heads expressing eye-specific mCD8-GFP together with astrocyte-specific knockdown or overexpression of Relish or flies heterozygous for Relish (Relish -/+). Figures of western blot represent two independent experiments.Genotypes in (d): control, GMR-QF2/+;alrm-GAL4/+;QUAS-mCD8-GFP/+. Relish RNAi #1, GMR-QF2/+;alrm-GAL4/UAS-Relish RNAi#1;QUAS-mCD8-GFP/+. Relish RNAi #2, GMR-QF2/+;alrm-GAL4/UAS-Relish RNAi #2; QUAS-mCD8-GFP. Relish overexpression, GMR-QF2/+;alrm-GAL4/UAS-Relish;QUAS-mCD8-GFP. Relish-/+, GMR-QF2/+;alrm-GAL4; Relish E20/+;QUAS-mCD8-GFP.

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Fig. 3 Relish signaling in astrocytes influences SCA3polyQ78-induced gene expression but not the extent of SCA3 aggregation. (a) Quantification of activation of Relish in the head in flies expressing SCA3polyQ78 in the eyes and the effect of modulating Relish levels in astrocytes or Relish heterozygosity. Expression of Relish target genes (DptA or AttC) was determined in heads of control flies (control) and flies expressing SCA3polyQ78 in the eyes. The effect of Relish RNAi targeted to astrocytes (Relish RNAi#1 and Relish RNAi#2), Relish overexpression or heterozygosity for Relish (Relish -/+) in flies expressing eye-specific SCA3polyQ78 was determined. Quantifications are of three independent experiments -/+SEM. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01; n.s.: not significant.(b) Lysates of fly heads described in (a) were analyzed on Western blot to determine levels of soluble and aggregated HA-tagged SCA3polyQ78. (c) Quantification of the soluble/insoluble ratio of SCA3polyQ78 of three independent experiments. n.s.: not significant, compared to SCA3polyQ78 (-). Quantifications are of three independent experiments -/+SEM. Statistical analysis was performed using the Student T test.Genotypes in (a), (b) and (c): control, GMR-QF2/+; for other genotypes, see Fig. 2b Top.

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Fig. 4 Relish target genes influence SCA3polyQ78-induced degeneration but not SCA3 aggregation. (a) Representative images of flies expressing SCA3polyQ78 in the eyes were compared to flies coexpressing RNAi constructs in astrocytes targeting Relish target genes AttA or CecA. Genotypes (a) -: GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-GAL4/+. AttA RNAi, GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-GAL4/UAS-AttA RNAi. CecA RNAi, GMR-QF2/+;QUAS-SCA3polyQ78: alrm-GAL4/+; UAS-CecA RNAi/+.(b) Quantification of the fraction of degeneration of eyes shown in (a). Data represent 3 independent experiments -/+SEM. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01; *** p<0.001.(c) Quantification of the effect of Relish target genes in astrocytes on SCA3-induced degeneration by using membrane-targeted mCD8-GFP. Lysates of fly heads expressing mCD8-GFP and SCA3polyQ78 in eyes were analyzed on western blot, and compared to flies coexpressing RNAi constructs targeting AttA or CecA in astrocytes.

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constructs in astrocytes of SCA3polyQ78 flies resulted in decreased expression of Relish-dependent AMPs DptA and AttC in the head to levels comparable to control (Fig. 3a), whereas overexpression of Relish enhanced their expression. Flies heterozygous for Relish expressed levels of Relish target genes comparable to those expressing Relish RNAi constructs in the astrocytes. Dif/Dorsal-specific target genes were unaffected by modulating Relish levels in astrocytes (Suppl. Fig. 3). These data highlight the importance of Relish signaling in astrocytes for Relish-dependent immune signaling in the head.

The presence of aggregated or misfolded proteins is toxic to neurons24. Possibly, astrocytes could have an effect on aggregation. To see whether Relish-specific signaling in astrocytes could have an effect on SCA3polyQ78-induced aggregation in the eyes, we collected heads of flies expressing SCA3polyQ78 two days after eclosion and analyzed the cell extract. Modulating Relish expression in astrocytes or heterozygosity for Relish did not affect levels or solubility of SCA3polyQ78 (Fig. 3b, 3c). This suggests that the effect of Relish on SCA3polyQ78-induced degeneration is not the result of either a clearance of SCA3polyQ78 aggregates or of a shift of the balance of soluble-insoluble SCA3polyQ78 towards more soluble SCA3polyQ78.

To see whether Relish-specific AMPs have an effect on SCA3polyQ78-induced eye degeneration, we expressed RNAi constructs targeting two Relish-dependent AMPs, AttA and CecA (efficacy of knockdown in Suppl. Fig. 4a). Similar to decreasing Relish activity, astrocyte-specific downregulation of these AMPs attenuated the SCA3polyQ78 eye phenotype (Fig. 4a; quantification of SCA3polyQ78-induced eye degeneration in Fig. 4b). We next analyzed SCA3polyQ78-induced degeneration by coexpressing mCD8-GFP and examining the effect of knockdown of Relish-specific AMPs. Levels of mCD8-GFP in SCA3polyQ78-expressing fly eyes were increased upon knockdown of AMPs, demonstrating partial rescue (Fig. 4c). In the absence of SCA3polyQ78, no effects of AMPs on mCD8-GFP levels were found. (Suppl. Fig. 4b). The extent of SCA3polyQ78 aggregation was not altered upon astrocyte-specific downregulation of AMPs (Fig. 4e; quantification of three independent experiments in Fig. 4f), similar

Genotype in (c): -, GMR-QF2/+; QUAS- SCA3polyQ78/+; QUAS-mCD8-GFP/+. AttA RNAi, GMR-QF2/+;alrm-GAL4/UAS-AttA RNAi;QUAS-mCD8-GFP/+. CecA RNAi, GMR-QF2/+;alrm-GAL4/+; UAS-CecA RNAi/+.(d) Quantification of at least three independent experiments shown in (c). Data represent 3 independent experiments -/+SEM. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01.(e) Analysis of the effect of Relish target genes in astrocytes on aggregation of SCA3polyQ78 expressed in eyes. Head lysates of flies expressing HA-tagged SCA3polyQ78 in the eyes were compared to flies coexpressing constructs targeting AttA or CecA in astrocytes. Genotypes in (e): control: GMR-QF2/+; alrm-GAL4/+. -: GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-GAL4/+. AttA RNAi, GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-GAL4/UAS-AttA RNAi. CecA RNAi, GMR-QF2/+;QUAS-SCA3polyQ78: alrm-GAL4/+; UAS-CecA RNAi/+.(f) Quantification of soluble/insoluble ratio of three independent experiments shown in (e). Statistical analysis was performed using the Student T test. n.s.: not significant.

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to modulating Relish levels in astrocytes. These data show the importance for Relish-specific AMPs in degeneration in our SCA3 model, independently of SCA3 aggregation.

We next tested whether the observed effects of the Relish pathway on (short term) degeneration also translates into a (long term) survival benefit. Given that inhibition of Relish in astrocytes attenuates SCA3polyQ78-induced eye degeneration, astrocyte-specific inhibition may also enhance lifespan in neurons expressing SCA3polyQ78. We expressed SCA3polyQ78 pan-neuronally (not in astrocytes) and examined effects of modulation of Relish expression specifically in astrocytes (specificity of expression shown in Suppl. Fig. 5a), using two independent binary expression systems (Suppl. Fig. 5b). To avoid potential detrimental effects of SCA3polyQ78 during development, we expressed SCA3polyQ78 in adult flies only, using the inducible “Q system”, in which the neuronally expressed transcription factor (QF2) that induces SCA3polyQ78

expression is coexpressed with QF-suppressor QS16 (Suppl. Fig. 5b). Expression of SCA3polyQ78 is suppressed during development (Fig. 5a). Feeding quinic acid to flies suppresses QS, thus alleviating inhibition of QF2 and allowing transcription of SCA3polyQ78, leading to the expression of SCA3polyQ78. As expected, the extent of SCA3 aggregation in these flies increased over time (Fig. 5a) and the lifespan of SCA3polyQ78-expressing flies was significantly shortened (Fig. 5b). Quinic acid by itself did not affect lifespan (Suppl. Fig. 6), excluding non-specific effects. Neuronally expressed SCA3polyQ78 resulted in an elevation of Relish-dependent gene expression (Fig. 5b, bottom), similar to expression of SCA3polyQ78 in the eyes (Fig. 2a).

We next wished to examine whether modulating Relish levels in astrocytes in adult flies could have an effect on the lifespan of SCA3 flies, expressing SCA3polyQ78

pan-neuronally. For this, levels of Relish were modulated in adult flies to exclude effects on development. Hereto, we used the transcription factor (GAL4) to drive the expression of the Relish constructs. At a lower temperature (18°C), activity of GAL4 is low and Relish RNAi constructs do not decrease Relish expression (Suppl. Fig. 7). Four days after eclosion, we induced expression of SCA3polyQ78 by adding quinic acid to the food. Flies were shifted to 25°C to induce expression of the Relish constructs in astrocytes. Importantly, reducing Relish expression in astrocytes extended the lifespan of SCA3 flies, whereas overexpression shortened the lifespan (Fig. 5c). In control flies without expression of SCA3polyQ78, modulating Relish expression in astrocytes had no effect on lifespan (not shown), indicating that the effects are linked to SCA3polyQ78-mediated degeneration of neurons. In parallel, SCA3polyQ78-related effects on motor function, measured as climbing ability (Suppl. Fig. 8), were partially alleviated by knockdown of Relish in astrocytes. No effects of Relish in astrocytes on total brain SCA3 aggregation load could be detected (Fig.

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5d), similar to the results observed with modulating Relish in astrocytes together with expression of SCA3polyQ78 in eyes.

The effects of Relish modulation on SCA3polyQ78-related degeneration were not related to alterations of the levels of SCA3polyQ78 aggregates (Fig. 3b and Fig. 5d). Therefore, we hypothesized that modulation of the Relish pathway in astrocytes could be more generic and relevant to a neurodegenerative disease associated with the presence of extraneuronal aggregates such as in Alzheimer’s disease. For this, we turned to an established Drosophila model of Alzheimer’s disease14, in which the Amyloid beta (Abeta42; Aβ42) peptides are fused to a secretion signal peptide. When expressed in neurons, these Aβ42 peptides are secreted and form extracellular aggregates in flies, reminiscent of the plaques in human AD25. In this AD fly model, flies displayed progressive neurodegeneration, with mobility and memory deficits and reduced lifespan14,26. In line with our hypothesis, the lifespan of flies expressing Aβ42 in neurons was shortened, but extended when Relish expression was suppressed in astrocytes, using two independent RNAi lines targeting Relish (Fig. 5e). This, together with the data on SCA3, demonstrates that modulating Relish signaling in astrocytes has beneficial effects in neurodegeneration, irrespective of the aggregation process that initiates it.

Our data show that NF-κB responses in astrocytes can modulate the rate of progression of neurodegeneration in a cell non-autonomous manner. Astrocyte activation occurs in many neurodegenerative diseases, and for the first time evidence is provided that, by modulating astrocyte-specific gene expression, symptoms induced by specifically expressing a neurodegeneration-inducing protein in neurons, can be attenuated.

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Fig. 5 Analysis of SCA3polyQ78 in neurons and the effect of Relish in astrocytes on neuronal expression of SCA3polyQ78 or Abeta42 (a) Inducible expression of SCA3polyQ78 in neurons can be suppressed during development. Control larvae or larvae expressing inducible SCA3polyQ78 were cultured on food with and without quinic acid. Flies expressing inducible SCA3polyQ78 were cultured on food with quinic acid for the times indicated to induce SCA3polyQ78 expression. Levels and aggregation of HA-tagged SCA3polyQ78 were determined in lysates of larvae or fly heads on Western blot. Tubulin was used to verify equal loading. Genotypes in (a): no SCA3polyQ78: tub-QS/+; alrm-GAL4/+; nSyb-QF:: tub-QS/+. SCA3polyQ78, tub-QS/+; QUAS-SCA3polyQ78::alrm-GAL4; nSyb-QF::tub-QS/+.(b) (top) Control flies or 2-day old flies expressing inducible SCA3polyQ78 were cultured on food containing quinic acid and the percentage of dead flies over time was determined. (bottom) Heads of 15-days old control flies or flies expressing SCA3polyQ78 in neurons were analyzed for expression of Relish target genes AttC, DptA and CecA. Genotypes in (b): Control: tub-QS/+; alrm-GAL4/+; nSyb-QF:: tub-QS/+. SCA3polyQ78, tub-QS/+; QUAS-SCA3polyQ78::alrm-GAL4; nSyb-QF::tub-QS/+.(c) Analysis of Relish constructs expressed specifically in astrocytes on lifespan of SCA3 flies. Flies expressing SCA3polyQ78 in neurons induced as in (b) were compared to SCA3 flies co-expressing astrocyte-targeted Relish RNAi (Relish RNAi#1 or #2) or overexpressing Relish. To induce changes in Relish expression only in adult flies, crosses were kept at 18°C. Two-day-old adult flies were shifted to 25°C to allow expression of Relish constructs in astrocytes. The fraction of dead flies over time was determined. Genotypes in (c): -, tub-QS/+;alrm-GAL4/+;nSyb-QF::tub-QS/+. SCA3polyQ78, tub-QS/+;alrm-GAL4::QUAS-SCA3polyQ78/+;nSyb-QF::tub-QS/+; Relish RNAi or Relish overexpression, as in control but with a copy of UAS-Relish RNAi #1 or #2 or UAS-GFP-Relish. (d) Head lysates of 15-day old flies as in (c) were analyzed for SCA3polyQ78 on Western blot. Tubulin was used as a control for equal loading. Genotypes were same as in (c).(e) Relish signaling in astrocytes modulates lifespan in an Alzheimer model. The lifespan of control flies and flies expressing Abeta42 in neurons were compared to flies coexpressing Abeta42 in neurons and Relish constructs in astrocytes. Genotypes in (e): -, alrm-GAL4/+; nSyb-QF2. Aβ42: alrm-GAL4:: QUAS-Abeta42/+; nSyb-QF2/+. Relish RNAi (#1 or #2), alrm-GAL4:: QUAS-Abeta42/UAS-Relish RNAi #1 or #2); nSyb-QF2/+. Relish overexpression, alrm-GAL4:: QUAS-Abeta42/UAS-GFP-Relish; nSyb-QF2/+.

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DISCUSSIONWhile astrocytes become reactive during the course of a number of age-related neurodegenerative diseases associated with aggregates5, their contribution to pathogenesis is unclear. Several reports have suggested that during the course of disease astrocytes gain neurotoxic properties (reviewed in5). However, direct evidence and potential mechanisms for how astrocytes affect progression of disease have yet been lacking, in part due to the fact that in most studies disease-causing genes are expressed in both neuronal and non-neuronal cells in the brain. Earlier work has demonstrated that expression of aggregation-prone proteins in astrocytes can cell non-autonomously influence neuronal viability4, reviewed in5. We previously demonstrated that signaling in astrocytes can influence neuronal viability when aggregation-prone proteins are expressed specifically in neurons.

We identified several astrocyte-specific enhancers and suppressors of the SCA3-related neurodegeneration (Chapter 2) and several of these genes that were identified as enhancers, were involved in a conserved NF-κB pathway (Chapter 2). We investigated how NF-κB (Relish) signaling in astrocytes can cell non-autonomously influence neurons that express aggregation-prone proteins. Downregulation of the Drosophila NF-κB transcription factor Relish in astrocytes not only delayed neurodegeneration (as shown by improved mobility of flies) and extended lifespan of flies expressing SCA3polyQ78 in neurons (Fig. 5c), but also extended lifespan in flies expressing neuronal Aβ42 peptides (Fig. 5e). We did not see any detrimental effects of modulating Relish expression in astrocytes, underscoring the efficacy of using this pathway as a target to inhibit the detrimental effects of SCA3.

Our data thus provide direct evidence for earlier suggestions that astrocytes are not only activated in response to neurodegeneration (as e.g. demonstrated by elevation of GFAP, Glial fibrillary acidic protein, a marker for astrocyte activation)4 but indeed can modulate disease progression. A toxic gain of function in astrocytes was also demonstrated in mice expressing human aggregation-prone TAR DNA-binding protein 43 (TDP-43) in neurons27, resulting in alterations in protein expression in astrocytes. Depletion of an upregulated, astrocyte-specific factor reduced neurotoxicity, underscoring the importance of astrocytes to neurotoxicity. Our findings that genes in the NF-κB pathway are key mediators of such a response extends the observation that astrocyte-specific, transient expression of IKK (IkB kinase 2), resulted in neurodegeneration in mice27.

Whereas the latter study supported the involvement of astrocyte-specific NF-κB signaling in the modulation of neurodegeneration, it did not demonstrate whether activation of NF-κB in astrocytes in neurodegeneration is due to cell-autonomous

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signaling, expression of a disease-associated protein in astrocytes, or due to cell non-autonomous signals derived from degenerating neurons. Our data now demonstrate that signals from neurons expressing aggregation-prone proteins suffice to induce cell non-autonomous NF-κB activation in astrocytes. This NF-κB activation occurred in astrocytes, suggested in Figure 3a and supported by the observation that in flies expressing SCA3polyQ78 in eyes together with a construct where GFP was under control of NF-κB-dependent promoters of AttA, GFP expression occurred in astrocytes (suppl. Fig. 1). Together, these data are suggestive for the existence and importance of intercellular signaling as an important determinant for the speed of neurodegeneration.

The nature of the neuronal signals that trigger NF-κB activation in astrocytes remains to be elucidated. Possibly, damage-associated molecular patterns (DAMPs), including SCA3polyQ78 aggregates, released from neurons can result in activation of NF-κB in astrocytes. It is unlikely that the Relish canonical pathway, headed by PGRP-LC (reviewed in19) accounts for Relish activation, since modulation of PGRP-LC did not affect the SCA3polyQ78 eye phenotype (Chapter 2). Previous reports have demonstrated Relish activation independent of PGRP-LC, either by neuropeptides or nitric oxide28,29, which may account for Relish activation in our model.

Our data do provide insight in the cell non-autonomous signals from astrocytes that play a role in the modulation of neuronal survival. They show how Relish-specific AMPs derived from astrocytes mediated to a significant extent the detrimental effects on SCA3polyQ78-induced degeneration (Figure 4). This is in line with findings that Relish signaling in glia increases with aging, that elevated AMPs are associated with accelerated neurodegeneration23, and that expression of AMPs in the Drosophila brain is sufficient to induce neurodegeneration30.

Whereas we show that astrocytes can directly modulate neuronal health, we found this not to be associated with alteration in the burden of protein aggregates in the brain. However, decreasing the levels of misfolded or aggregated proteins has been suggested as a therapeutic strategy31. Furthermore, reduction of oxidative damage can decrease degeneration32. Thus, a combination of therapeutic strategies targeting aggregates, astrocyte-mediated neurotoxicity as well as oxidative damage may have optimal effects in alleviating aggregates-associated neurodegeneration.

Targeting NF-κB signaling in astrocytes to alleviate neurodegeneration may be more broadly applicable: the lifespan of flies expressing Aβ peptides, present in patients with AD, was extended upon astrocyte-specific inhibition of NF-κB (Fig. 5e). Thus, despite intracellular localization of aggregates in SCA3 and extracellular localization of Aβ peptides in AD, astrocyte-specific NF-κB inhibition extended lifespan in fly models for both diseases. This suggests that targeting NF-κB in astrocytes may be

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beneficial in other aggregates-associated neurodegenerative diseases.

Altogether, these findings highlight astrocytes as important mediators in neurodegenerative diseases and provide putative therapeutic potential.

AUTHORS’ CONTRIBUTIONSY.X.L. and P.F.D. designed the research plan, performed the work and analyzed the data, O.C.M.S. provided feedback and editorial comments on the manuscript.

CONFLICTS OF INTERESTThe authors declare no conflict of interest.

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SUPPLEMENTARY FIGURES

Li_Dijkers_Suppl._Figure 1

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Supplementary Fig. 1: Eye-specific expression of SCA3polyQ78 results in the activation of Relish in astrocytes.(Top) Representative picture of a control eye expressing GFP under the control of the Attacin promoter (att-GFP). Astrocytes expressed RFP pictures were taken from live eyes. (Bottom) In flies with eye-specific SCA3polyQ78 expression, astrocytes-specific expressing PFR is localized with a Relish-dependent reporter construct, Att-GFP. Higher magnification showing RFP-expressing astrocytes express GFP in the ommatidia (arrows). Genotypes: control, GMR-QF2/+; alrm-GAL4::UAS-my-RFP/Att-GFP. SCA3polyQ78, GMR-QF2/+; alrm-GAL4::UAS-my-RFP::QUAS-SCA3polyQ78.

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Supplementary Fig. 5: Genetic setup to inducibly express SCA3polyQ78 in neurons and simultaneously modulate gene expression in astrocytes. (a) UAS-GAL4 and QUAS-QF system function independently. QUAS-QF2-driven GFP expression specifically in neurons (green); UAS-GAL4-driven expression of mry-RFP specifically in astrocytes (red). Nuclei are stained with Hoechst (blue). Genotype: alrm-GAL4/UAS-myr-RFP; nSyb-QF2/QUAS-mCD8-GFP. (b) Genetic setup for inducibly expressing SCA3polyQ78 in neurons and simultaneously modulating gene expression in astrocytes. QUAS-QF2 was used to express SCA3polyQ78; UAS-GAL4 was used to modulate expression in astrocytes. Neuronally expressed QF2 (expressed under control of the pan-neuronal nSyb promoter) is suppressed by QS (expressed under control of the tubulin promoter). This suppression is alleviated by quinic acid, resulting in expression of SCA3polyQ78. For details on the fly lines used, see experimental procedures.

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CHAPTER 4Specific calcineurin isoforms are involved in

Drosophila Toll immune signaling

Yixian Li* and Pascale F. Dijkers* §

* Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Manuscript published in Journal of ImmunologyReference: J Immunol. 2015; 194(1): 168-76

ABSTRACTAs excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism in dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-κB transcription factors Dorsal/Dif, was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-κB transcription factor Relish, these pathways constitute a central immune signaling network. Inputs in these pathways contribute to specific and appropriate responses to microbial insults. Relish activity during infection is modulated by Ca2+-dependent serine/threonine phosphatase calcineurin, an important target of immunosuppressants in transplantation biology. Only one of three Drosophila calcineurin isoforms, CanA1, acts on Relish during infection. However, whether there is a role for calcineurin in Dorsal/ Dif immune signaling is not known. Here, we demonstrate involvement of specific calcineurin isoforms, Pp2B-14D/CanA-14F, in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference (RNAi) against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform CanA1 decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNAi attenuated the Dorsal/Dif-dependent response to infection, without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling, and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-κB signaling.

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INTRODUCTIONWhile higher organisms have acquired adaptive immunity, most metazoans rely on the innate immune system to successfully fight off microbes. Depending on the type of immune insult, immune responses can be local or systemic, and control systems are in check to maintain these. Excessive or inadequate responses can be detrimental to the organism. Insight into the pathways regulating innate immunity is important for identifying how dysregulation causes disease, and could provide potential targets for therapeutic intervention.

Drosophila has been instrumental for the genetic dissection of immune defenses after infection to identify the involved pathways, which turned out to be evolutionarily conserved1. Two central pathways in Drosophila involved in the response to infection are the TNFR (Tumor Necrosis Factor Receptor)-related IMD (immunodeficiency) pathway and the Toll pathway, known as TLR (Toll-Like Receptor) in mammals. Signaling through IMD or Toll results in the activation of NF-κB transcription factors, which have a key role in mediating inflammatory gene expression. Important transcriptional targets are antimicrobial peptides (AMPs), which help to eliminate infections.

Recognition of PAMPs (pathogen-associated molecular patterns) on microbes, such as peptidoglycan (PGN), occurs through pattern recognition receptors (PRRs). While in mammals TLRs can serve as PRR and directly bind to PAMPs, Toll in Drosophila does not. Instead, Toll gets activated upon binding of a cytokine, spätzle. Spätzle gets cleaved downstream of a protease cascade, which gets activated upon binding of PAMPs (Lys-type PGNs on bacteria or b-glucans on fungi) to specific PRRs. Signaling downstream of Toll promotes activation of NF-κB transcription factors Dorsal and related transcription factor Dif. In contrast, PRRs that act upstream of IMD mediates responses to diaminopimalic (DAP)-type PGNs, mostly found on gram-negative bacteria. IMD signaling results in activation of NF-κB transcription factor Relish. AMPs downstream of Relish are more effective against gram-negative bacteria, whereas the Dorsal/Dif-specific AMPs help fight infections with gram-positive bacteria or fungi1. In addition to this humoral response to infection, the cellular response by hemocytes (blood cells) also plays a central defending role, by phagocytosing the microbes. The Toll pathway is also involved in this cellular response, as activation of this pathway promotes proliferation of hemocytes2.

Besides the canonical pathways that mediate recognition of microbes to activation of NF-κB transcription factors, there are other pathways that modulate their activity. For example, activity of the Ras/MAPK pathway negatively regulates IMD-Relish

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signaling3. Signaling by the small molecule nitric oxide (NO) can promote Relish activity independently of IMD4. Activation of Relish by NO occurs through the calcium-dependent serine/threonine phosphatase calcineurin5. This phosphatase constitutes a major target for immunosuppression and prevention of graft rejection in mammals. While the target of calcineurin involved in graft rejection, NFAT (Nuclear factor activated in T cells) is not present in Drosophila, calcineurin can also regulate the distantly related NF-κB in mammals6,7.

Calcineurin acts as a heterodimer, consisting of a catalytic subunit A, and a regulatory subunit B. In Drosophila, there are 3 catalytic subunits: CanA1, and the related and functionally homologous Pp2B-14D and CanA-14F, which are next to each other on the chromosome and probably arose by gene duplication8. There are 2 B subunits, CanB and CanB2. CanB is mostly expressed in the brain, CanB2 is ubiquitously expressed (Flyatlas), and therefore CanB2 probably mediates signaling of the catalytic A subunits in immune tissues. Only CanA1 influences Relish activity in response to infection or to NO signaling, independently of IMD4,5, indicating that calcineurin is not part of the canonical IMD-Relish signaling cascade. Differences in protein sequences may explain why CanA1 is sensitive to NO and Pp2B-14D/CanA-14F are not, but the structural base for this difference is not known. While treatment with a calcium-mobilizing drug can alter the mobility of Dorsal on SDS-PAGE, possibly via calcineurin9, a role for calcineurin in Dorsal/Dif-mediated immunity has never been explored (see Fig. 1A).

Here, we examine a potential role for calcineurin in Dorsal/Dif-mediated immunity. We show that in cell culture, specific isoforms of the calcineurin catalytic subunit, Pp2B-14D and CanA-14F, can mediate nuclear translocation of GFP-tagged Dorsal (GFP-Dorsal). Co-immunnoprecipitation of Pp-2B-14D and GFP-Dorsal suggests that Dorsal may be a direct target for calcineurin. Toll-dependent activation of Dorsal/Dif was attenuated after pharmacologically inhibiting calcineurin or with RNAi against Pp2B-14D/CanA-14F. In vivo, flies expressing Pp2B-14D/CanA-14F RNAi constructs displayed a decrease in the Toll-dependent immune response and decreased viability after infection with gram-positive bacteria. Expression of active Pp2B-14D was sufficient to induce Dorsal/Dif-dependent expression of LacZ in hemocytes. Together, these data demonstrate involvement of specific calcineurin isoforms Dorsal/Dif-mediated immunity. Thus, specific calcineurin isoforms can modulate activity of either Relish or Dorsal/Dif in immunity, thus providing an additional means of regulation of immunity in Drosophila.

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MATERIALS AND METHODS

Cells, flies, reagents, antibodiesDrosophila S2 cells were cultured in Schneider’s medium (Gibco, San Diego, California, U.S.A.) supplemented with 10% heat-inactivated fetal calf serum, penicillin, and streptomycin. Cells expressing EGFR-Toll10 were a generous gift from the Wasserman lab. Transient expression in S2 cells was done using Cellfectin (Invitrogen, Carlsbad, California, U.S.A.).

Daughterless-Gal4, hml-Gal4 and cg-GAL4 fly lines were obtained from the stock center (Bloomington, Indiana, U.S.A.). The srp-Gal4 line was a gift from B. Lemaitre; the Dorsal reporter D4/hsp7011 was a gift from U. Banerjee (UCLA, Los Angeles, U.S.A.). RNAi lines for SERCA, CanA-14F and Pp2B-14D were obtained from VDRC (Vienna, Austria). Transgenic fly lines were made in the w1118 background, using Bestgene (Chino Hills, CA, U.S.A.).

Drosophila Pp2B-14D or DPp2B-14D, which is truncated at aa 470, cloned in frame with a C-terminal HA tag into pUAST or pAc5/V5HisB. A plasmid containing Dorsal was a gift from T. Yp, and was subsequently subcloned into pAc5/V5HisB containing GFP at the N-terminus using NotI and XbaI, thus generating GFP-Dorsal. The constructs for GFP-Relish12, DCanA15 GFP-VIVIT13 and Dorsal-specific luciferase construct (353-Luciferase)14 have been described previously. TK-renilla, was from Promega (WI, U.S.A.). All constructs were verified by sequencing.

GFP antibody was obtained from Life Technologies (Carlsbad, CA, U.S.A.); GFP-Trap from ChromoTek (Germany); HA monoclonal antibody (HA11) from BabCo (Richmond, CA, U.S.A.), Dorsal mAb from Developmental Studies Hybridoma Bank (Iowa city, IA, U.S.A.). FK506 (tacrolimus) was from USP Reference Standard (Rockville, MD). SNAP and thapsigargin were from Calbiochem (San Diego, CA, U.S.A.). Human EGF from Cellsciences (Canton, MA, U.S.A.). YM-5843 was from Sigma.

Primers:

Pp2B-14D/CanA-14F: 5’-GACTTCCTGCAGAACAACAACC-3’/5’CATCCGTTCGTTGACGGCATCC-3’ and 5’-GGAGAAGACGATGATCGATA-3’/5’-GTGCGTGTAGAAGTCCGAGT-3’; CanA1: 5’-GGAGCGCATGGTCGATGA/5’-GGTTCTTCCGATACATGCGA-3’ and 5’-ATCCTCGCACATCCACTCC/5’-TCAGGCACTGAAATAAATTGGC-3’;IM1: 5’-TGCCCAGTGCACTCAGTATC-3’/5’-GATCACATTTCCTGGATCGG-3’;

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IM2: 5’-AAATACTGCAATGTGCACGG /5’-ATGGTGCTTTGGATTTGAGG; Drosomycin:5’-GTACTTGTTCGCCCTCTTCG-3’/5’-GATTTAGCATCCTTCGCACC-3’; Diptericin: 5’-ACCGCAGTACCCACTCAATC-3’/5’-ACTTTCCAGCTCGGTTCTGA-3’; Attacin: 5’-GCTTCGCAAAATAAACTGG-3’/5’-TCCCGTGAGATCCAAGGTAG-3’ RP49: 5’-CCGCTTCAAGGGACAGTATC-3’/5’-GACAATCTCCTTGCGCTTCT-3’, and also 21-mers of the full coding region of RP49. Dorsal mutagenesis primers: S3A mutant: 5’-ccgcagccgctgGcgccTGcAGCcaactacaaccacaac/5’gttgtggttgtagttgGCTgCAggcgCcagcggctgcgg-3’; S3D mutant: 5’-ccgcagccgctgGATccaGACGAcaactacaaccacaac-3’/5’-gttgtggttgtagttgTCGTCtggATCcagcggctgcgg-3’.

RNAi, nuclear translocation experiments and fluorescenceRNAi experiments were carried out as described in15, using a Promega T7 Ribomax kit to generate dsRNA. As control dsRNA, either LacZ or hMAZ dsRNA was used. Transient expression in S2 cells was carried out using Fugene transfection reagent (Promega) or PEI (polyethylenimine), using pAc5/V5HisB expression vector. Cells were transfected 3 days after adding dsRNA, and analyzed 40h later. Experimental procedures for preparing and analysis of the cells displaying nuclear GFP (GFP-Rel or GFP-Dl) have been described previously5. At least 300 cells were counted per sample for GFP localization. Images were taken on an inverted microscope (DM 1RB; Leica) equipped with a spinning disk confocal unit (CSU10; Yokagowa), 40× Plan Fluotar 0.7 NA objective (Leica), a camera (Orca AG; Hamamatsu Photonics), and Volocity 4 acquisition software (PerkinElmer). Samples were randomized prior to counting. Data represent the average of at least three independent experiments.

Immunoprecipitation and Western blot analysis For pull down of GFP or GFP-tagged proteins, cells were lysed in a buffer containing 50mM HEPES pH 7.4, 0.5% NP40; 10% glycerol, 60mM KCl, 2mM CaCl2, supplemented with protease and phosphatase inhibitors. GFP pull down using GFP-Trap was performed according to manufacturer’s protocol. Analysis by western blot and preparation of lysates for GFP-Dorsal mobility have been described previously5.

Luciferase assays Cells expressing EGFR-Toll were transfected with appropriate plasmids. 6h after transfection 20-hydroxy ecdysone (1 mM) was added to the cells to sensitize them

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to immune induction16. EGF (0.2 ng/ml) was added overnight 30h after transfection. In case of pharmacological inhibition of calcineurin, inhibitors were added 30 min. prior to adding EGF. Luciferase activity was measured with the Dual Luciferase Assay System Kit (Promega, WI. U.S.A.), and samples were normalized with TK-Renilla. Data represent the average of at least three independent experiments.

InfectionsInfections of larvae were performed by dipping a needle into a M. luteus or Ecc-15 bacterial solution of 200 OD and pricking the tail of a larva. For analysis of fluorescence, drs-GFP larvae, expressing GFP under control of the drosomycin promoter, were used. Transgenes were ubiquitously expressed using daughterless-Gal4. Larvae were harvested 20h post infection and analyzed for GFP fluorescence (in the case of drs-GFP larvae) or RNA was harvested using the Rneasy Mini Kit (Qiagen) for Real Time quantitative PCR analysis (RT-qPCR) (15 larvae/point). RT-qPCR was performed using IQTM SYBR Green Supermix (Bio-Rad Laboratories, Inc.), and analyzed on MyiQTM and IQTM 5 Real-time PCR detection system (Bio-Rad Laboratories, Inc.) Data represent the ratio of the detected mRNA levels normalized to RP49 mRNA levels as control. For survival experiments of adult flies, 3-4 day old flies (40 flies/point) were infected with M. luteus by septic injury, maintained at 29°C and counted every 2 days. Survival experiments were repeated three times.

Analysis of hemocytesFlies expressing active Pp2B-14D, a SERCA RNAi construct or control flies (w1118) were crossed to either hml-Gal4 or srp-Gal4. For the analysis of beta-galactosidase activity, hemocytes of third instar larvae were bled onto chilled coverslips coated with Concanavalin A, fixed with 4% formaldehyde and analyzed for beta-galactosidase activity as done previously5.

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RESULTS

Calcineurin-dependent translocation of GFP-Relish and GFP-DorsalWhile calcineurin isoform CanA1 is a downstream target of nitric oxide (NO) in Relish signaling5, a possible involvement of NO or calcineurin in Toll-Dorsal/Dif (Dorsal-related Immune Factor) immune signaling has never been explored (Fig. 1A). There are indications for calcineurin involvement in Dorsal signaling: elevation of calcium results in a change in Dorsal phosphomobility9 and Dorsal nuclear translocation17. Changes in mobility of Dorsal were prevented by pretreatment with a serine-threonine phosphatase inhibitor18. Furthermore, glutamate-dependent decrease of Dorsal levels in synaptic boutons at the neuromuscular junction was prevented by inhibition of calcineurin19. However, whether calcineurin is involved in Dorsal/Dif-dependent immunity and if there is a preference for a specific calcineurin isoform is unknown. A suitable way to identify players involved in Relish signaling is through examination of the nuclear translocation of GFP-tagged Relish (GFP-Rel)5,20, so we used the same assay for Dorsal signaling. To see whether NO, calcium, or calcineurin are involved in Dorsal signaling, nuclear translocation of GFP-Dorsal (GFP-Dl) was examined in Drosophila Schneider cells (S2 cells) and compared to that of GFP-Relish (GFP-Rel). As demonstrated before, a similar percentage of cells displaying nuclear GFP-Rel was observed after treatment with NO donor S-nitroso-N-acetylpenicillamine (SNAP) or after treatment with thapsigargin (Fig. 1B left). Thapsigargin promotes a rise in cytoplasmic calcium by inhibiting transporter SERCA, responsible for the sequestration of Ca2+ in the endoplasmic reticulum21. In contrast, a much smaller percentage of cells displayed nuclear GFP-Dl (Fig. 1B, right) after NO treatment, while thapsigargin-treated GFP-Dl cells showed a percentage similar to that of nuclear GFP-Rel (Fig 1B, left). To compare involvement of calcineurin in nuclear translocation of Dorsal and Relish, cells were treated with calcineurin inhibitor FK506 (or cyclosporin A, Suppl. Fig. 1B) prior to treatment with SNAP or thapsigargin. As shown before5, FK506 treatment inhibited GFP-Rel translocation induced by both NO and thapsigargin. In cells expressing GFP-Dl, calcium-induced nuclear translocation was also largely abrogated by FK506. Altogether, these data suggest that the calcineurin isoform(s) activated upon increase of cytosolic calcium preferentially act on Dorsal.

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Specific calcineurin isoforms involved in Dorsal translocationNext, we tested which isoforms of the catalytic calcineurin subunit, CanA1, Pp2B-14D and/or CanA-14F, mediate nuclear translocation of Dorsal. The highly homologous Pp2B-14D and CanA-14F are next to each other on the chromosome and probably arose because of gene duplication. They were shown to be functionally redundant22. Therefore, we studied Pp2B-14D/CanA-14F together. The different isoforms differ at the N- or C-terminus, CanA1 differs from Pp2B-14D and CanA-14F throughout the protein. We examined a role for CanA1, or Pp2B-14D/CanA-14F in Dorsal signaling by RNAi, using 2 independent, non-overlapping dsRNAs to downregulate their expression. Homology between these isoforms and the regions of the dsRNAs targeting the different isoforms are shown in Suppl. Fig. 1A. For Pp2B-14D/CanA-14F we used dsRNAs that target both isoforms. We verified specific knockdown

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Figure 1. A Involvement of calcineurin in immune activation by Relish or Dorsal/Dif.Activation of Relish during infection can be modulated by NO and its downstream target calcineurin (isoform CanA1). The other calcineurin isoforms, Pp2B-14D and CanA-14F, are homologous and are activated downstream of calcium. Whether any of these isoforms are involved in Dorsal/Dif immune signaling is not known.Figure 1. B Effects of nitric oxide and calcium on nuclear localization of GFP-Dorsal and GFP-Relish. S2 cells transiently expressing GFP-Relish (left) or GFP-Dorsal (right) were either left untreated (con; white bars), treated with NO donor SNAP (2 mM) for 3h (NO, grey bars) or thapsigargin (1 mM) for 1h (thap., black bars) in the presence or absence of FK506 (1 nM, pretreatment for 0.5h). At least 300 cells/point were counted. Data represent the average of at least three independent experiments -/+ the SEM. Statistical analysis was performed using the Student T test. * p<0.05; ** p<0.01; *** p<0.001.

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of calcineurin isoforms using RT-PCR, RNAi against Pp2B-14D/CanA-14F did not affect mRNA levels of CanA1 and vice versa (Fig. 2A, top).

We examined the effect of RNAi against the different calcineurin isoforms on thapsigargin-induced GFP-Dl translocation. Both dsRNAs targeting Pp2B-14D/CanA-14F inhibited GFP-Dl translocation, whereas no effect on thapsigargin-induced translocation was seen with CanA1 RNAi (Fig. 2A, middle). With CanA1 RNAi, NO-induced translocation of GFP-Rel was inhibited (not shown, 5), showing that CanA1 RNAi is sufficient to inhibit CanA1 activity. Quantification of these results is shown below (Fig. 2A, bottom).

A serine-rich region in Dorsal is homologous to that in NFAT, and these serines in NFAT are targets of calcineurin. Dephosphorylation of these serine residues promotes nuclear translocation of NFAT, concomitant with a mobility shift of NFAT on Western blot23. To see whether Dorsal mobility is affected upon calcineurin activation, and which calcineurin isoforms may be involved, we examined changes in GFP-Dl mobility on SDS-PAGE. Treatment with thapsigargin promoted a downward mobility shift, and this was inhibited by calcineurin inhibitor FK506 (Fig 2A, bottom). When cells were treated with 2 different non-overlapping dsRNAs against Pp2B-14D/CanA-14F or CanA1, RNAi against Pp2B-14D/CanA-14F but not against CanA1 was able to interfere with the thapsigargin-induced GFP-Dorsal mobility shift (Fig. 2A, bottom). These data show that calcineurin isoforms Pp2B-14D/CanA-14F mediate thapsigargin-induced GFP-Dorsal translocation, possibly by dephosphorylating Dorsal.

To see whether a gain-of-function of calcineurin is sufficient to promote nuclear translocation of GFP-Dl, we expressed constitutively active DPp2B-14D (DPp2B), which lacks the autoinhibitory domain24. Indeed, expression of DPp2B-14D resulted in an increase in nuclear GFP-Dl (Fig. 2B, top). Quantification of these results is shown in Fig. 2B (bottom). To see whether a gain-of-function of calcineurin can alter the mobility of Dorsal on Western blot, we expressed DPp2B. Indeed, upon expression of DPp2B, GFP-Dl was shifted downwards, possibly suggesting a decrease in phospho-content of Dorsal. (Fig. 2B, bottom).

We next tested whether calcineurin can mediate activity of Dorsal and Dorsal-related transcription factor Dif downstream of Toll. For this purpose, we used S2 cells stably expressing an EGFR-Toll chimeric fusion construct, consisting of the extracellular and transmembrane region of the human Epidermal Growth Factor (EGF) and the intracellular region of Drosophila Toll. Addition of EGF results in activation of Dorsal/Dif10. To examine Dorsal/Dif activity, a luciferase construct under the control of a Dorsal/Dif-specific promoter was used14. Luciferase induced by EGF was largely inhibited upon addition of calcineurin inhibitor FK506 (Fig. 2C), suggesting that Toll

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activation promotes calcineurin activity involved in Dorsal/Dif signaling. Expression of active Pp2B-14D (DPp2B) but not of active CanA1 (DCanA1) was sufficient to induce luciferase, showing specificity of distinct calcineurin isoforms for inducing Dorsal/Dif activity. The luciferase induction by expression of active Pp2B-14D is lower than by EGF addition, possibly suggesting that additional components of the Toll pathway are required for optimal Dorsal/Dif transcriptional activity. We also tested whether calcineurin RNAi could interfere with Dorsal/Dif activity. We found that two independent dsRNAs targeting Pp2B-14D/CanA-14F attenuated luciferase induction, whereas no effect was found with two independent dsRNAs targeting CanA1. RNAi was not as efficient in EGFR-Toll cells as in regular S2 cells (not shown), possibly explaining the difference between pharmacological inhibition of calcineurin and RNAi against calcineurin. Activation of the Toll pathway results in phosphorylation and a concomitant increase in phosphomobility of Dorsal18. In EGFR-Toll cells, EGF treatment also resulted in an increase in phosphorylation of endogenous Dorsal (Fig. 2C, bottom). However upon pharmacological inhibition of calcineurin, this phosphomobility was no longer seen with EGF. This may indicate that calcineurin activity is necessary for kinases in the Toll pathway to phosphorylate Dorsal.

To see whether calcineurin can act on the Toll pathway via direct interaction with Dorsal, we examined whether Dorsal can interact with Pp2B-14D. We also tested interactions of Pp2B-14D with GFP as a negative control and with GFP-VIVIT as a positive control. GFP-VIVIT is GFP fused to a peptide that contains the optimal binding site for calcineurin13. No interaction was found between GFP and HA-tagged Pp2B-14D, whereas GFP-VIVIT bound HA-Pp2B-14D quite well (Fig. 2D). GFP-Dorsal also co-precipitated Pp2B-14D, with a stoichiometry that appeared comparable to that of GFP-VIVIT and Pp2B-14D. This indicates that calcineurin may bind to and dephosphorylate Dorsal.

Dorsal contains putative consensus calcineurin serines in a serine-rich region (SRR), homologous to that of NFAT, which gets dephosphorylated by calcineurin to subsequently translocate to the nucleus23, presumably by exposing the NLS (nuclear localization signal). These serines in the SRR of Dorsal are different from the serines in the Rel homology region (S79, S103, S213, S312 and S317) that get phosphorylated by activation of the Toll pathway25. In GFP-Dorsal we mutated three serines in the SRR (S404, S413 and S414) to either alanine (phosphodefective) or aspartic acid (phosphomimetic), indicated in Fig. 2E, top. We examined the effect of these mutations on mobility of Dorsal, responsiveness to thapsigargin or Dorsal-dependent luciferase activity. Mutations of the serines to alanine (S3A) resulted in a downward shift of Dorsal on Western blot, whereas the aspartic acid mutant (S3D) displayed an upward shift (Fig. 2E, middle). Treatment with thapsigargin resulted in

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a further downward shift of S3A, however, the downward shift with the S3D mutant was less than that of the wildtype (wt) GFP-Dorsal.

We also examined nuclear localization of these mutants: a significant fraction of S3A was present in the nucleus and this fraction was increased with thapsigargin treatment, but not as much as the control (Fig. 2E, bottom). In contrast, the S3D mutant was cytoplasmic and thapsigargin treatment promoted nuclear translocation but the total fraction was lower than that of wildtype Dorsal. This indicates that phosphorylation of these serines are involved in mediating nuclear localization of Dorsal. However, since the response the S3A and S3D mutants to thapsigargin was attenuated but not inhibited, it indicates that additional serines are involved in mediating the response to calcium. This has been shown for NFAT as well23. When we examined the effect of expressing GFP-Dorsal, its S3A or S3D mutant and examined their effect on Dorsal-dependent luciferase activity, we found that the increase in activity with GFP-Dorsal was further elevated in the S3A but not in the S3D mutant. This indicates that serines in the SRR of Dorsal are involved in mediating its transcriptional activity. These findings indicate that the serines in the SRR are target serines for calcineurin, and contribute to nuclear localization and activation of Dorsal.

Together, these data suggest that calcineurin can act on the Toll pathway by binding and subsequent dephosphorylation of Dorsal allowing nuclear translocation.

Specific calcineurin isoforms mediate Dorsal/Dif activity in vivoNext, we investigated involvement of calcineurin isoforms Pp2B-14D/CanA-14F in Dorsal/Dif-mediated immune responses in vivo. We used Drosophila larvae, as Dorsal mediates immune responses in larvae but not in adults, where Dif alone is required26. This way, we could extend our S2 cell data, in which we used a Dorsal construct. We tested 2 independent Pp2B-14D/CanA-14F RNAi foldback constructs27, which specifically downregulated Pp2B-14D/CanA-14F mRNA without affecting CanA1 mRNA (Fig. 3A, top). We infected larvae with gram-positive bacteria Micrococcus luteus (M. luteus) to induce the Toll pathway, using septic injury instead of natural infection (oral ingestion), as there was a high variability in oral bacterial intake in the larval population. We examined third instar larvae expressing GFP under control of the Dorsal/Dif-dependent drosomycin promoter (drs-GFP larvae). Following infection, GFP expression was induced, but this induction was lower when either Pp2B-14D/CanA-14F RNAi construct was expressed (Fig 3A).

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To verify whether expression of endogenous Toll-dependent genes was also dependent on calcineurin, we examined drosomycin, IM1 and IM2 by RT-qPCR. Expression of all these genes was upregulated after infection (Fig. 3A, bottom), and this was attenuated by downregulation of Pp2B-14D/CanA-14F expression. We also tested the whether Pp2B-14D/CanA-14F RNAi could affect the Relish-dependent response to infection with gram-negative bacteria Erwinia carotavora carotavora 15 (Ecc15). No significant changes were found on levels of the Relish-specific AMPs diptericin (Dipt) and attacin A (AttA) using RT-qPCR upon downregulation of

Figure 2. Distinct calcineurin isoforms are involved in Dorsal/Dif signaling. (A) Top: RNAi-mediated knockdown of calcineurin isoforms. S2 cells were treated with dsRNA targeting either CanA1 or 2 different regions of Pp2B-14D/CanA-14F (14D/14F-1, 14D/14F-2) and analyzed for transcript knockdown by RT-PCR. Equal input was verified by RP49 expression. Middle: Fluorescent images of S2 cells treated with dsRNA as in (A) prior to transfection with GFP-Dl and treatment with thapsigargin (1 mM, 1h). Bottom left: analysis of the percentage of cells displaying nuclear GFP-Dl. At least 300 cells/point were counted. Data represent the average of at least three independent experiments -/+ the SEM. Statistical analysis was performed using the Student T test. *** p<0.001. Bottom right, top blot: cells expressing GFP-Dorsal were either left untreated (control) or treated with thapsigargin (thap. 1 mM) in the presence or absence of FK506 (1 nM) and analyzed on western blot for differences in GFP mobility. Bottom right, bottom blot: S2 cells were treated with dsRNA targeting either CanA1 or 2 different regions of Pp2B-14D/CanA-14F (14D/14F-1, 14D/14F-2) for 4 days prior to transfection with GFP-Dl, treatment with thapsigargin (thap. 1 mM, 1h) and analysis on Western blot. (B) Top: fluorescent images of S2 cells transfected with GFP-Dl together with empty vector (control) or active Pp2B-14D (DPp2B). Bottom: quantification of the percentage of cells expressing nuclear GFP-Dl. At least 300 cells/point were counted. Data represent the average of at least 3 different experiments -/+ SEM. Statistical analysis was performed using the Student T test *** p<0.001. Bottom right: GFP-Dl-expressing cells with or without HA-tagged active Pp2B-14D (DPp2B-HA) were analyzed for differences in GFP mobility and HA expression. (C) Top left: EGFR-Toll-expressing S2 cells were transfected with a luciferase construct under control of a Dorsal/Dif-specific promoter together with either empty vector or active Pp2B-14D (DPp2B) or active CanA1 (DCanA1). 30h after transfection cells were left untreated (control), or incubated overnight with EGF (0.2 mg/ml) in the presence or absence of FK506 (1 nM) prior to analysis of luciferase activity. Top right: EGFR-Toll-expressing S2 cells were treated with two independent dsRNAs targeting either Pp2B-14D/CanA-14F (14D/14F-1 or 14D/14F-2) or CanA1 (CanA1-1 or CanA1-2) for 4 days prior to transfecting them with Dif/Dorsal-dependent Luciferase, treatment with EGF (0.2 mg/ml) and analysis for luciferase activity. Fold induction is calculated by comparing individual transfectants with and without EGF. Data represent the average of at least 3 different experiments -/+ SEM. Statistical analysis was performed using the Student T test, * p<0.05. Bottom: S2 cells expressing EGFR-Toll were left untreated or treated with FK506 (1 nM) prior to treatment with EGF (0.5 mg/ml) for 30 min and analysis on Western blot for mobility of endogenous Dorsal. (D) Association of GFP-Dorsal with Pp2B-14D. S2 cells expressing Pp2B-14D-HA together with either GFP, GFP-VIVIT or GFP-Dorsal were lysed, GFP was immmunoprecipitated and analyzed on Western blot for either GFP expression or HA expression to see whether Pp2B-14D-HA coprecipitated. Expression of Pp2B-14D-HA was verified by analyzing whole cell lysates (WCL) for HA expression. (E) Top: Cartoon of Dorsal. Putative calcineurin target serines are located in the Serine-Rich Region (SRR) adjacent to the NLS and 3 were mutated to either alanine (S3A) or aspartic acid (S3D). Serines that are phosphorylated upon Toll activation are located in the Rel homology region. Middle left: Mutating of 3 target calcineurin serines into alanine (S3A) or (S3D) affects mobility of GFP-Dorsal. Middle right: S2 cells expressing GFP-Dorsal or its S3A or S3D mutant were left untreated or treated with thapsigargin (thap. 1 mM, 1h) prior to analysis for mobility on Western blot or analysis for the percentage of cells displaying nuclear localization of GFP (bottom left). Bottom right: S2 cells were transfected with a luciferase construct under control of a Dorsal/Dif-specific promoter together with either empty vector (vector control), GFP-Dorsal (wt) or serine mutants (S3A or S3D) and luciferase activity was analyzed. Data represent the average of at least 3 different experiments -/+ SEM. Statistical analysis was performed using the Student T test, * p<0.05; *** p<0.001.

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Pp2B-14D/CanA-14F expression, arguing against a role for these isoforms in Relish signaling.

To examine the consequences of downregulating Pp2B-14D/CanA-14F expression on viability after infection we examined survival of these flies after septic injury with M. luteus. While no effect on viability was found with flies on control infected compared to uninfected flies, Pp2B-14D/CanA-14F RNAi decreased viability after infection but not in uninfected flies (Fig. 3B). This demonstrates that Pp2B-14D/CanA-14F are important mediators in the Toll pathway.

To test sufficiency for calcineurin in activating Dorsal/Dif in vivo, we generated flies containing an HA-tagged, active Pp2B-14D transgene (DPp2B-HA) and examined Drosophila hemocytes, in which Toll signaling is known to play a role2. Expression of this transgene was verified (Fig. 3C, top). To visualize Dorsal/Dif activity, we examined induction of a beta-galactosidase transgene under control of a Dorsal/Dif-specific promoter, D4/hsp70-LacZ11, containing binding sites specific for Dorsal/Dif14. While little beta-galactosidase activity was seen in control hemocytes (Fig. 3C, top), a significant fraction of hemocytes expressing active Pp2B-14D displayed beta-galactosidase activity. Similar observations were made when other fly lines expressing this transgene were used, as well as by using other Gal4 lines with expression in hemocytes (not shown). This shows sufficiency of calcineurin in inducing Dorsal/Dif activity in vivo.

We also investigated whether elevations in cytoplasmic calcium could similarly induce Dorsal/Dif-dependent beta-galactosidase by expressing a SERCA RNAi foldback transgene. Inhibition of SERCA in S2 cells promoted calcineurin-dependent translocation of Dorsal (Fig. 1B), mediated by isoforms Pp2B-14D/CanA-14F (Fig. 2). Efficacy of this SERCA transgene was tested by RT-PCR in larvae ubiquitously expressing the SERCA RNAi construct (Fig. 3C, bottom). We then expressed the SERCA RNAi transgene in the hemocytes and also observed Dorsal/Dif-dependent induction of LacZ. Similar results were found when a SERCA RNAi construct was used against a different region of SERCA (not shown). This suggests that elevation of calcium (and subsequent induction of calcineurin activity) can promote Dorsal/Dif activity, although we cannot exclude calcium-independent effects of SERCA knockdown such as ER stress.

Thus, specific calcineurin isoforms are an important input for modulating the activity of either Relish or Dorsal/Dif without cross-activation between these NF-κB pathways. Specific inputs of calcineurin help to achieve appropriate and specific immune activation.

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Figure 3. Specific calcineurin isoforms promote activity of Dif/Dorsal in vivo. (A) Top left: third instar control larvae or larvae ubiquitously expressing 2 independent RNAi constructs targeting Pp2B-14D/CanA-14F (14D/14F RNAi1 or 14D/14F RNAi2) were analyzed for expression of Pp2B-14D/CanA-14F or CanA1 respectively by RT-PCR. Expression of RP49 was used as a control for equal input. Top right: fluorescent images of uninfected third instar drs-GFP larvae (uninf.) or infected third instar drs-GFP larvae (inf.) and drs-GFP larvae ubiquitously expressing Pp2B-14D/CanA-14F RNAi constructs (RNAi1- inf. and RNAi2- inf.) infected with gram-positive bacteria M. luteus by septic injury. Bottom: control larvae or larvae expressing Pp2B-14D/CanA-14F RNAi were infected with M. luteus or gram-negative bacteria Ecc-15 by septic injury and analyzed for expression of Dorsal/Dif-dependent genes Drs, IM1, and IM2 or Relish-dependent genes AttA or Dipt by RT-qPCR. Expression of RP49 was used as a control. Data are average of at least 3 independent experiments -/+SEM. Statistical analysis was performed using the Student T test. * p<0.05. (B) 4 day old male control flies (cg-Gal4/+) or male flies expressing constructs targeting Pp2B-14D/CanA-14F using cg-Gal4, which expresses in the fatbody and in hemocytes, were left untreated (control uninfected: open squares; RNA uninfected: open triangles) or infected with M. luteus (control infected: closed squares; RNAi infected: closed triangles) and their survival was analyzed. Survival curves of control flies -/+ infection and RNAi flies – infection were not significantly different from each other, whereas the curve of the infected RNAi flies was (p<0.0001, log-rank test). Data are representative of at least 3 independent experiments. (C) Induction of Dorsal/Dif-dependent LacZ in hemocytes. Top, left: Western blot showing expression of active HA-tagged Pp2B-14D (DPp2B-HA) third instar larvae, using cg-Gal4. Top, right: DPp2B-HA expression in larval hemocytes induces expression of Dorsal/Dif-dependent beta-galactosidase, D4/Hsp70-LacZ. Hemocytes from D4/Hsp70-LacZ third instar control larvae or D4/Hsp70-LacZ larvae expressing DPp2B-HA were isolated and analyzed for beta-galactosidase activity. Bottom, left: Efficacy of SERCA RNAi knockdown. RT-PCR of larvae ubiquitously expressing a SERCA RNAi construct, using daughterless-Gal4. Bottom, right: SERCA RNAi induces Dorsal/Dif-dependent beta-galactosidase expression. Hemocytes of third D4/Hsp70-LacZ instar larvae without or with SERCA RNAi stained for beta-galactosidase activity. Data are representative of at least 3 independent experiments.

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DISCUSSIONWe show that activity of the two distinct pathways of the NF-κB family in Drosophila, IMD- Relish and Toll- Dorsal/Dif, can be modulated by calcium-dependent serine/threonine phosphatase, calcineurin. However, specificity of calcineurin activity in these pathways is achieved by distinct isoforms of the catalytic calcineurin subunit A interacting with each pathway. Calcineurin isoform CanA1 modulates activity of Relish, whereas the functionally homologous and related Pp2B-14D and CanA-14F mediate activity of Dorsal/Dif.

Previous work showed that activity of CanA1 modulates activity of Relish during infection5, and that CanA1 acts downstream of NO. When we set out to analyze whether NO could also be involved in Dorsal/Dif signaling we found that nuclear translocation of GFP-Dorsal by NO was much smaller compared to that of GFP-Relish. However, treatment with a drug that promotes elevation of intracellular calcium and subsequent activation of calcineurin resulted in much higher levels of GFP-Dorsal nuclear translocation. This suggests that calcineurin can mediate Dorsal signaling, but via a different isoform than CanA1. Subsequent RNAi experiments showed that the calcineurin isoforms responsible for GFP-Dorsal translocation were the functionally homologous and related Pp2B-14D and CanA-14F. We show here that RNAi targeting Pp2B-14D/CanA-14F downregulates the Dorsal/Dif-mediated response to gram-positive bacteria, whereas the Relish-dependent response to gram-negative bacteria was unaffected (Fig. 3A). This decrease in response by Pp2B-14D/CanA-14F RNAi was accompanied with a decrease in survival (Fig. 3B). This indicates that in vivo Pp2B-14D/CanA-14F are important in mediating Toll-dependent signaling, and do not interact with IMD signaling.

This is the first report implying calcineurin in Toll-mediated immune signaling, and demonstrating involvement of specific calcineurin isoforms in this pathway. Thus, calcineurin acts in both IMD and Toll signaling, and specificity of calcineurin in these pathways is acquired by specific isoforms acting on Dorsal/Dif or Relish. Involvement of calcineurin in Drosophila NF-κB immune signaling is summarized in a model (Fig. 4). Only CanA1 can act downstream of NO, and modulates Relish signaling in response to infection or to NO 5. NO is generated during infection with gram-negative bacteria4. Pp2B-14D and CanA-14F are activated in response to calcium elevation, and can promote nuclear translocation and activation of Dorsal in cell culture downstream of Toll activation (Fig. 2A-C). In vivo, RNAi against Pp2B-14D/CanA-14F is sufficient to dampen the Dorsal/Dif-dependent response to infection with gram-positive bacteria, concomitant with a decrease in survival. These findings suggest that activation of Toll results in mobilization of calcium and subsequent activation of Pp2B-14D/CanA-14F.

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Does calcineurin contribute to Toll signaling by directly acting on Dorsal? Pp2B-14D does co-precipitate with Dorsal. There is a putative calcineurin docking site (PxIxIT)13 in Dorsal (aa 47-52: PYVKIT). However, mutation of this site did not abrogate association between Dorsal and Pp2B-14D (not shown). Recent analysis of docking sites on calcineurin substrate demonstrated that the calcineurin docking site is a bit more versatile28, so additional sites in Dorsal (such as aa 15-20; PAVDGQ) may serve as a calcineurin docking site. While we do not know whether the interaction between Dorsal and calcineurin is direct, it may allow calcineurin to dephosphorylate serine residues on Dorsal, which is the main residue of Dorsal that gets phosphorylated25. To examine whether Dorsal is a target for calcineurin, we mutated target phosphorylation sites for calcineurin on Dorsal in a serine-rich region (SRR), which is adjacent to the NLS (Fig. 2E), homologous to those on NFAT. Action of calcineurin on NFAT results in unmasking an NLS, resulting in nuclear translocation of NFAT 23. Similarly, Relish also contains calcineurin target sites surrounding the NLS. Mutation of serines to alanine of putative calcineurin target sites on Relish yielded a constitutively nuclear protein5. Mutation of serines in the SRR of Dorsal demonstrated involvement of these serines in mediating nuclear

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Figure 4. Model for involvement of specific calcineurin isoforms in Drosophila NF-κB signaling. Specific isoforms of the catalytic calcineurin subunit A mediate activity of either Relish or Dorsal/Dif. In the IMD pathway, a specific calcineurin isoform, CanA1, gets activated upon generation of NO during infection, and promotes nuclear localization of Relish, independently of IMD. In the absence of CanA1, Relish-dependent responses to infection are attenuated. In contrast, activation of Dorsal/Dif downstream of Toll is dependent on 2 homologous calcineurin isoforms, Pp2B-14D and CanA-14F. Activity of these calcineurin isoforms induces nuclear localization and contributes to activation of Dorsal. In the absence of Pp2B-14D/CanA-14F, responses to infection with gram-positive bacteria are attenuated and viability is decreased, demonstrating the importance for these isoforms in Dorsal/Dif activity. Since Pp2B-14D/CanA-14F are dependent on an increase in cytoplasmic calcium for their activation, we infer that activation of the Toll pathway is accompanied with calcium mobilization, possibly via the CRAC calcium channel (see suppl. Fig. 1B).

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localization of Dorsal and contribution to its activity (Fig. 2E). Previous work in Drosophila showed that Toll-dependent phosphorylation occurs in the N-terminal Rel Homology Domain, and that 6 serines in this domain are phosphorylated25. However, these are not calcineurin consensus sites. Our data and work on NFAT29 suggest that calcineurin acts on residues in a serine-rich region. We also show that pharmacological inhibition of calcineurin inhibits Toll-dependent phosphorylation of Dorsal and Dorsal/Dif activity (Fig. 2C). What are the events that contribute to the phosphorylation state of Dorsal after Toll activation? A model explaining our observations could be that upon Toll activation, calcineurin gets activated and dephosphorylates Dorsal on serine residues (in the SRR). Calcineurin activity appears required for Toll-dependent kinases to phosphorylate Dorsal on different serines in the Rel homology domain to fully activate Dorsal. While we only examined mobility of Dorsal and not with Dif in response to calcineurin activity, Dif will probably also be subject to regulation by calcineurin, since blocking calcineurin in cells attenuated Dif/Dorsal-dependent transcription.

Why NO only signals through CanA1 and not through Pp2B-14D/CanA-14F is not known. While the catalytic domains of these subunits are highly conserved (suppl. Fig. 1A), the N- and C-termini of CanA1 are rich in serine and threonine, possibly allowing for posttranslational modification downstream of NO. Differences in calcineurin sequence may also account for the differences in substrate specificity. While the Pp2B-14D and CanA-14F are functionally redundant22, there may still be differences in their substrate specificity, as they differ in their N- and C-terminus (supp. Fig. 1A). In mammals, the catalytic A subunits of calcineurin are also most variable in their N- and C-termini. A proline-rich region in the N-terminus of the catalytic calcineurin subunit CanA beta, is involved in recognition of NFAT30. The different isoforms appear to have different distinct targets in vivo, with CanA beta being the main regulator of NFAT activity (reviewed in 31). Isoform-specific inhibitors could be able to circumvent the significant side effects occurring with the current immunosuppressing drugs that target all calcineurin isoforms.

The calcium channel that mediates influx of calcium that promotes activation of NFAT is called CRAC (Ca2+ release-activated Ca2+ channel). Patients with mutations in this channel are severely immunocompromised. Interestingly, the identity of this channel, Orai1, was first identified in a Drosophila RNAi screen in S2 cells expressing human NFAT tested for mediators of nuclear translocation 32, suggesting evolutionary conservation of calcium and calcineurin signaling. Dysregulation of calcium homeostasis, resulting in intracellular calcium elevation and subsequent calcineurin activation may result in aberrant Dorsal/Dif activation. This is supported by data showing that DPp2B-expressing larvae as well as SERCA RNAi-expressing larvae had increased levels of beta-galactosidase under control of a Dorsal/Dif-

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dependent promoter (Fig. 3B). Moreover, the number of hemocytes was increased in these larvae (not shown). An increase in hemocyte numbers may also be mediated by Dorsal/Dif, since constitutive activation of the Toll pathway results in overproliferation of hemocytes2. Preliminary research suggests that the channel involved in calcium increase resulting in nuclear translocation of Dorsal as well as Dorsal/Dif-dependent transcription may indeed be Orai1. Pharmacological inhibition of CRAC, as well RNAi against Orai1, inhibited Dorsal/Dif-dependent translocation in response to thapsigargin (Suppl. Fig. 1B, left). Moreover, pharmacological inhibition of this channel or Orai RNAi, attenuated Dorsal/Dif-dependent induction of luciferase downstream of Toll (Suppl. Fig. 1B, right), suggesting putative involvement of this channel in Dif/Dorsal-dependent immunity.

Altogether, calcineurin is an important phosphatase involved in specific immune pathways in both flies and mammals. Specifically targeting the isoforms involved in these particular pathways as well as targeting phosphorylation sites of calcineurin substrates in these pathways may be a way to modulate immune activation.

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ACKNOWLEDGEMENTSWe thank the Bloomington and the VDRC stock center for Drosophila stocks, Scott Lindsay from the Wasserman lab for the EGFR-Toll cells and the Toll-specific luciferase constructs, P.H. O’Farrell for valuable comments and suggestions, and O.C.M. Sibon, S. Bergink and B. Wertheim for critically reading the manuscript.

FOOTNOTES This work was supported by a Rosalind Franklin Fellowship to P.F.D.

*Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. §Correspondence should be addressed to Dr. P.F. Dijkers, Antonius Deusinglaan 1, 9712AV Groningen, The Netherlands. Email: p.f.dijkers@umcg.nl

Abbreviations used in this work: AMPs, antimicrobial peptides; CanA1, calcineurin A1; CanA-14F, calcineurin A at 14F; Dif, Dorsal-related immune factor; Dl, Dipt, Diptericin; Dorsal; Drs; Drosomycin; EGF, epidermal growth factor; EGFR, EGF receptor; IM, immune-induced molecule; IMD, immunodeficiency; NFAT, nuclear factor activated in T cells; Pp2B-14D, protein phosphatase at 14D; RNAi, RNA interference; RT-qPCR RT quantitative PCR; SERCA, sarco/endoplasmic reticulum calcium ATPase; thap., thapsigargin; UAS, upstream activating sequence;

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(B) Putative involvement of CRAC in Toll signaling. Left: S2 cells were treated with dsRNA targeting LacZ or Orai for 3 days prior to transfection with GFP-Dl. 36h after transfection cells were either left untreated (con), treated with thapsigargin (1 μΜ, 1h; black bars) with or without pretreatment of CsA (2.5 nM) and analysed for nuclear localization of GFP. Data represent average of at least three independent experiments -/+ SEM. Right: EGFR-Toll-expressing S2 cells were treated with dsRNA targeting LacZ or Orai for 3 days prior to transfection with with a luciferase construct under control of a Dorsal/Dif-specific promoter. 30h after transfection cells were left untreated (con), treated with EGF overnight (EGF; 0.2 μg/ml, black bars), or pretreated with YM-5843 (low: 10 μM; high 20 μM) for 30 min. prior to overnight incubation with EGF and subsequent analysis of luciferase activity. Data represent the average of at least three independent experiments -/+ SEM.

(A) Alignment of the Drosophila calcineurin catalytic isoforms. Amino acids that are different are indicated in grey. Underlined amino acids: catalytic domain; in Italics: binding region to regulatory subunit CanB. Differences exist between the N- and C-terminus of all three isoforms; CanA1 is different in DNA and protein sequence throughout the protein.

+++ : Pp2B-14D/CanA-14F RNAi-2 : Pp2B-14D/CanA-14F RNAi-1 ^^^ : CanA1 RNAi-1 is also directed against the 3’ UTR ^^^ : CanA1 RNAi-2

CanA1 -----------------------------------KMQYTKTRERMVDDVPLPPTHKLTM Pp2B-14D -AAAGNNSDNSS------PTTGTGTGASTGK-LHGGHTAVNTKERVVDSVPFPPSHKLTL CanA-14F GTAAGSGSGGAAGSAGTQQQGQGGTGTSSGPSSPTKRSTISTKERVIDSVAFPPSRKLTC .*:**::*.* :**::*** CanA1 SEVYDDPKTGKPNFDALRQHFLLEGRIEEAVALRIITEGAALLREEKNMIDVEAPITVCG Pp2B-14D AEVFD-QRTGKPNHELLKQHFILEGRIEEAPALKIIQDGAALLRQEKTMIDIEAPVTVCG CanA-14F ADVFD-ARTGKPQHDVLKQHFILEGRIEESAALRIIQEGATLLRTEKTMIDIEAPVTVCG ::*:* :****:.: *:***:*******: **:** :**:*** **.***:***:****

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CanA1 DIHGQFFDLVKLFEVGGPPATTRYLFLGDYVDRGYFSIECVLYLWSLKITYPTTLSLLRG Pp2B-14D DIHGQFYDLMKLFEVGGSPASTKYLFLGDYVDRGYFSIECVLYLWSLKITYPQTLFLLRG CanA-14F DIHGQFYDLMKLFEIGGSPATTKYLFLGDYVDRGYFSIECVLYLWSLKITYPQTLFLLRG ******:**:****:** **:*:***************************** ** **** ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ CanA1 NHECRHLTEYFTFKQECIIKYSESIYDACMEAFDCLPLAALLNQQFLCIHGGLSPEIFTL Pp2B-14D NHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCVHGGLSPEIHEL CanA-14F NHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCVHGGLSPEIHEL ***************** ***** :*****:**********:******:********. * ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ CanA1 DDIKTLNRFREPPAYGPMCDLLWSDPLEDFGNEKTNEFFSHNSVRGCSYFFSYSACCEFL Pp2B-14D EDIRRLDRFKEPPAFGPMCDLLWSDPLEDFGNEKNSDFYTHNSVRGCSYFYSYAACCDFL CanA-14F EDIRRLDRFKEPPAFGPMCDLLWSDPLEDFGNEKNSDFYTHNSVRGCSYFYSYAACCDFL :**: *:**:****:*******************..:*::**********:**:***:** +++++++++++++++++++++++++++++++++++++++++ CanA1 QKNNLLSIVRAHEAQDAGYRMYRKNQVTGFPSLITIFSAPNYLDVYNNKAAVLKYENNV M Pp2B-14D QNNNLLSIIRAHEAQDAGYRMYRKSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNV M CanA-14F QNNNLLSIIRAHEAQDAGYRMYRKSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNV M *:******:***************.*.******************************** * +++++++++++++++++++++++++++++++++++++++ + CanA1 NIRQFNCSPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNILNICS DDELVAGPDDELEEEL Pp2B-14D NIRQFNCSPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLNICS DDELMTEESEEP---- CanA-14F NIRQFNCSPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLNICS DDELMTEESEEP---- **************************************:***** ****:: .:* ++++++++++++++++++++++++++++++++++++++++++++++++++++++++ CanA1 RKKIVLVPANASNNNNNNNTPSKPASMSALRKEIIRNKIRAIGKMSRVFSILREESESVL Pp2B-14D ----------------------LSDDEAALRKEVIRNKIRAIGKMARVFSVLREESESVL CanA-14F ----------------------LSDDEAALRKEVIRNKIRAIGKMARVFSVLREESESVL . :*****:***********:****:********* ++++++++++++++++++++++++++++++++++++++ CanA1 QLKGLTPTGALPVGALSGGRDSLKEALQGLTASSHIHSFAEAKGLDAVNERMPPRRPLLM Pp2B-14D QLKGLTPTGALPLGALSGGKQSLKNAMQGFSPNHKITSFAEAKGLDAVNERMPPRRDQPP CanA-14F QLKGLTPTGALPLGALSGGKQSLKNAMQGFSPNHKITSFAEAKGLDAVNERMPPRRDATP ************:******::***:*:**:: . :* ******************* ++++++++++++++++++++++++++++++++++++++++++++++++++++ CanA1 SASSSSITTVTRSSSSSSNNNNNNSNTSSTTTTKDISNTSSNDTATVTKTSRTTVKSATT Pp2B-14D TPSEDPNQHSQQGGKNGAGHG--------------------------------------- CanA-14F SPAEEGQKSLSAAAAAAANANANSING--------------------------------- : :.. .. .: CanA1 SNVRAGFTAKKFP Pp2B-14D ------------- CanA-14F -------------

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CHAPTER 5General discussion

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The onset of the most common neurodegenerative diseases (NDs) is age-related, thus the world widely increased aging population enhanced the prevalence of NDs1. There is still no cure for NDs. Neuronal dysfunction and loss are related to the neuronal accumulation of toxic misfolded proteins, resulting in cell-autonomous neurodegeneration2. However, other cell types in the central nervous system (CNS), such as astrocytes and microglia, can also have effects on neurodegeneration, and influence neurons in a cell non-autonomous manner3.

Astrocytes are involved in maintaining neurotransmitter homeostasis, synaptic function, energy metabolism and inflammation in the CNS4,5,6. Astrocyte engages different molecules to exert these functions. For instance, they clear excessive extracellular glutamate, which is a neurotransmitter via the glutamate transporters (EAATs)7. For the regulation of immune responses in astrocytes, the NF-κB pathway plays a role8. However, astrocytes are activated in most NDs and can contribute to neurotoxicity, leading to neuronal damage and death9.

To obtain insight into cell non-autonomous contributions of astrocytes to neurodegeneration, we performed an RNAi candidate screen in a Drosophila ND model for Spinocerebellar ataxia type 3 (SCA3), targeting genes in astrocytes. We identified genes in astrocytes that may contribute to neurodegeneration in a cell non-autonomous manner. This highlights important cell non-autonomous roles for astrocytes in neurodegeneration. Our results suggest involvement of integrins and GABA-A receptors in astrocytes in neurodegeneration (Figure 1). We also identified intracellular signaling proteins in the RNAi screen, such as calcineurin, a calcium-dependent phosphatase as well as an NF-κB transcription factor (Relish). Upon further exploration of the involvement of Relish in astrocytes, we showed that transcriptional targets of Relish, AMPs, contribute to neurodegeneration in SCA3 (Figure 1). In Chapter 3 we showed that expression the SCA3-associated protein in fly eyes or neurons resulted in an increased levels of NF-κB-dependent target genes in fly heads compared to control flies. Preliminary data show that activation of NF-κB (Relish) occurs in astrocytes.

While we identified candidate genes in astrocytes that can make a cell non-autonomous contribution to neurodegeneration, it still remains to be determined how astrocytes and neurons can mutually influence each other’s activity. One means that would provide insight into this would be to examine how gene expression in both astrocytes and neurons is altered upon expression of an aggregation-prone protein in neurons or astrocytes. This would provide further insight into the signaling that occurs between astrocytes and neurons in NDs, but may also result in the identification of candidate genes (in astrocytes but also in neurons) that can make contributions in NDs.

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Astrocyte

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Figure 1. A model for the cell non-autonomous role of astrocytes in neurodegeneration. Our data suggest that neurons expressing diseased-associated misfolded proteins can result in the activation of astrocytes. Activated astrocytes are normally hypertrophic. Candidate molecules that can be released from neurons and signal to astrocytes include misfolded proteins (that can act as DAMPs), other DAMPs, cytokines, neurotransmitter and signals that remain to be identified (‘unknown signals’). We identified receptors in astrocytes, such as integrins and GABA-A receptors, which may contribute to neurodegeneration. We also identified intracellular signaling proteins such as NF-κB (Relish) and calcineurin. Activity of Relish and Relish-dependent target genes in astrocytes may have detrimental effects on neurons.

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For future directions, using our SCA3 Drosophila model, we propose to examine gene expression in astrocytes and neurons. This will shed further light on the signaling events that occur in response to neurodegeneration and may result in the identification of genes that can be manipulated to attenuate or delay neurodegeneration. For this purpose, we will compare gene expression in Drosophila neurons that express GFP or GFP together with either SCA3polyQ78 or SCA3polyQ27. Simultaneously, RFP will be expressed in astrocytes. Neurons and astrocytes can be sorted by FACS and gene expression profiles can be determined, as has successfully been done before in Drosophila brains10. Genes whose expression is altered in neurons expressing SCA3polyQ78 (and GFP) compared to the neurons only expressing GFP or expressing SCA3polyQ27 (and GFP) will be identified. Moreover, the corresponding gene expression signature of these samples in RFP-expressing astrocytes will provide insight in the signaling events that occur in astrocytes following neuronal expression of SCA3polyQ78. These data will provide insight into signaling in both astrocytes and neurons in response to neuron-specific expression of a disease-associated, misfolded protein. It would be of additional value to perform a similar experiment when expressing amyloid beta peptides in neurons, which are secreted, in contrast to SCA3polyQ78, which predominantly stays intracellular. Such an experiment would reveal potential differences, or similarities in signaling events between neurons and astrocytes that express an aggregation-prone protein that is extracellular or remains intracellular.

To further advance our understanding in the detrimental effect of NF-κB signaling in astrocytes on SCA3polyQ78-expressing neurons (Chapter 3), we could compare gene expression in control astrocytes to astrocytes in which expression of NF-κB is downregulated. Alteration of gene expression in these astrocytes may provide insight of the neurotoxic effects of NF-κB signaling in astrocytes.

In addition to astrocytes, microglia can contribute in neurodegenerative diseases11. Activation of microglia results in changes in their morphology, ability of phagocytosis, and inflammatory responses (reviewed in12), which can influence neuronal functioning. For instance, damaged neurons can activate microglia to produce an excessive amount of pro-inflammatory cytokines, which have been shown to be toxic to neurons (reviewed in11,13). While gene expression profiling in microglia in NDs have been determined14,15, the cell non-autonomous contribution of these genes to neurodegeneration still need to be determined. Similarly to what we did for astrocytes, our Drosophila SCA3 model can be used for studying the interaction between microglia (called ensheathing glia in Drosophila) and neurons that expressing disease-related misfolded proteins, using microglia-specific downregulation of genes. In order to downregulate individual genes in ensheathing glia, the ensheathing glia-specific driver line, mz0709-GAL4 can be used16.

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Using a similar experimental setup as described above, sorting out astrocytes and neurons, signaling events between microglia and neurons can be identified as well. Given the ease of genetic manipulation of flies and the reagents we already generated, establishing these fly lines will not be very time-consuming. Together, these experiments will shed light in the interplay between neurons, astrocytes and microglia. Whether altered gene expression of specific genes in either microglia or astrocytes is relevant for the pathogenesis of NDs can be subsequently established by testing knockdown of these genes specifically in microglia or astrocytes. This can initially be done in the SCA3 eye model, and can subsequently tested in lifespans in flies expressing an aggregation-prone protein (SCA3polyQ78 or amyloid

beta peptides) in neurons.

Gene profiling in microglia in humans that suffer from NDs has been done14 and while a large number of genes have been identified, the relevance of these in the pathogenesis remains to be established. Comparison of these genes to the genes that we identified in the experiments proposed above would yield (1) insight into conserved genes that have altered expression in NDs (2) candidate genes that can be tested in our SCA3 or Alzheimer fly model. This would narrow down the number of genes of interest that can be further analyzed in other animal models like mice.

While beyond the scope of this thesis, similar experiments can also be done upon expression of a disease-associated, misfolded protein in either astrocytes or microglia and examine the resulting changes of gene expression in neurons, astrocytes and microglia. As aggregation-prone proteins in NDs are also found in astrocytes17 and microglia18, it would shed light on another aspect of NDs. The comprehensive studies for the specific contribution of microglia and astrocytes might benefit to develop a target-specific therapy for neurodegeneration.

Together, these future directions will open exciting avenues to explore, and hopefully advance the field of NDs.

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REFERENCES:1. Brookmeyer, R., Johnson, E., Ziegler-Graham, K.

& Arrighi, H. M. Forecasting the global burden of Alzheimer’s disease. Alzheimer’s Dement. 3, 186–191 (2007).

2. Taylor, J. P. Toxic Proteins in Neurodegenerative Disease. Science (80-. ). 296, 1991–1995 (2002).

3. Ransohoff, R. M. How neuroinflammation contributes to neurodegeneration. Science (80-. ). 353, 777–83 (2016).

4. Chung, W. S., Allen, N. J. & Eroglu, C. Astrocytes control synapse formation, function, and elimination. Cold Spring Harb. Perspect. Biol. 7, (2015).

5. Cervetto, C. et al. A2A-D2 receptor–receptor interaction modulates gliotransmitter release from striatal astrocyte processes. J. Neurochem. 140, 268–279 (2017).

6. Anderson, M. A. et al. Astrocyte scar formation aids central nervous system axon regeneration. Nature 532, 195–200 (2016).

7. Zagami, C. J., O’Shea, R. D., Lau, C. L., Cheema, S. S. & Beart, P. M. Regulation of glutamate transporters in astrocytes: Evidence for a relationship between transporter expression and astrocytic phenotype. Neurotox. Res. 7, 143–149 (2005).

8. Kaltschmidt, B., Widera, D. & Kaltschmidt, C. Signaling via NF-κB in the nervous system. Biochim. Biophys. Acta - Mol. Cell Res. 1745, 287–299 (2005).

9. Ben Haim, L., Carrillo-de Sauvage, M.-A., Ceyzériat, K. & Escartin, C. Elusive roles for reactive astrocytes in neurodegenerative diseases. Front. Cell. Neurosci. 9, (2015).

10. Berger, C. et al. FACS Purification and Transcriptome Analysis of Drosophila Neural Stem Cells Reveals a Role for Klumpfuss in Self-Renewal. Cell Rep. 2, 407–418 (2012).

11. Lull, M. E. & Block, M. L. Microglial Activation and Chronic Neurodegeneration. Neurotherapeutics 7, 354–365 (2010).

12. Lee, Y. B., Nagai, A. & Kim, S. U. Cytokines, chemokines, and cytokine receptors in human microglia. J. Neurosci. Res. 69, 94–103 (2002).

13. Block, M. L., Zecca, L. & Hong, J. S. Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nature Reviews Neuroscience 8, 57–69 (2007).

14. Holtman, I. R. et al. Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis. Acta Neuropathol. Commun. 3, 31 (2015).

15. Chiu, I. M. et al. A neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model. Cell Rep. 4, 385–401 (2013).

16. Doherty, J., Logan, M. A., Tasdemir, O. E. & Freeman, M. R. Ensheathing Glia Function as Phagocytes in the Adult Drosophila Brain. J. Neurosci. 29, 4768–4781 (2009).

17. Phatnani, H. & Maniatis, T. Astrocytes in Neurodegenerative Disease. Cold Spring Harb. Perspect. Biol. 7, a020628 (2015).

18. Lee, J. S. & Lee, S.-J. Mechanism of Anti-α-Synuclein Immunotherapy. J. Mov. Disord. 9, 14–19 (2016).

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ENGLISH SUMMARY

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Aging is a natural process for all living organisms. One consequence of aging is a decline in organ function, including the brain. Aging is a major risk factor for neurodegenerative diseases. Nowadays, the lifespan of the human population increases, resulting in an increased prevalence of neurodegenerative diseases. The pathogenesis of age-related neurodegenerative diseases is complex and multifactorial. A common feature of these diseases is the accumulation of misfolded or aggregated proteins in the brain. This is considered one of the contributory factors in neurodegenerative diseases. Normally, misfolded proteins can be broken down and removed, but in neurodegenerative diseases they accumulate and form aggregates that are toxic to neurons. Specific misfolded proteins are associated with specific neurodegenerative diseases. For example, beta amyloid peptides form the so-called plaques in Alzheimer’s disease.

Neuronal function and health are supported by non-neuronal cells, glia. In the brain, there are different types of glial cells, including astrocytes and microglia. Astrocytes are star-shaped cells and are the most abundant glial cells. They have multiple roles in the brain and are involved in maintaining levels of neuronally released chemical signals, energy metabolism, inflammation as well as the repair of injuries. Activation of astrocytes can occur though signals from neurons and microglia, and this helps the astrocytes to support the well-being of neurons. However, chronically activated astrocytes are found in almost all neurodegenerative diseases and can contribute to their pathogenesis. It is now clear that this activation state during disease is distinct from activation in a healthy individual. The mechanisms by which astrocytes can influence neurodegeneration are not fully understood yet. Knowledge about these may ultimately help in the identification and development of therapeutic targets.

In this thesis, the aim is to understand how astrocytes can influence neurodegeneration. For this, we examined the effect of astrocytes on neurotoxicity of a specific misfolded protein that is associated with a neurodegenerative disease. One misfolded protein we used causes the inherited neurodegenerative disease SCA3 (Spinocerebellar Ataxia 3). We also used plaque-forming beta amyloid peptides, associated with Alzheimer’s disease. As we cannot use humans as experimental models, we used an animal model that also has astrocytes, and in which neurodegeneration occurs upon the neuronal presence of human misfolded proteins. The model organism we used is the fruit fly, also known as Drosophila melanogaster. Although seemingly quite different from humans, 50% of fly genes are conserved in humans, and their astrocytes are functionally similar. Flies have a short lifespan (60-80 days), allowing quick analysis of the effect of a certain treatment. Moreover, the fruit fly can also be used to examine the effect of a large number of genes in specific cells in a short time frame. Research in flies has been very important for human biology and has resulted in the identification of the functions of genes associated with cancer,

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neurodegeneration, and many other diseases.

We tested the effect of more than 150 conserved genes in a genetic screen in astrocytes on neurodegeneration by inhibiting individual genes. We identified 46 genes that could enhance or inhibit neurodegeneration. This shows that astrocytes are important modulators of neurodegeneration. When the activity of an enhancer gene, called NF-κB, which causes inflammation, was inhibited in astrocytes, flies lived significantly longer and were also more active, indicative of higher “quality of life”. Similar effects were found in “Alzheimer’s disease flies”, where human beta amyloid peptides were present in the brains. While inhibiting NF-κB in astrocytes is not a cure for neurodegeneration, it extends life and appears to enhance the quality of life. NF-κB-activated inflammation in the brain, however, did not influence the amount of aggregates. Possibly, a future therapy that targets genes in astrocytes can be combined with a therapy that targets aggregates for optimal effects.

So far, we only solved a small part of this puzzle and additional questions remain and arise. For example, what is the effect of other genes that we identified on lifespan? And more mechanistically: what molecular pathways in astrocytes influence neurodegeneration, and how? Will these observations in flies also be true in mammals? It will be of interest to reproduce our findings in mouse models, which more closely reflect humans.

All in all, the work done in this thesis underscores the importance of astrocytes in neurodegenerative diseases and provides insight into how they are influential in the pathogenesis of these diseases.

NEDERLANDSE SAMENVATTING

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Veroudering is een natuurlijk proces voor alle levende organismen. Eén gevolg van veroudering is een verminderd functioneren van organen, inclusief de hersenen. Veroudering is een belangrijke risicofactor voor neurodegeneratieve ziektes, zoals de ziekte van Alzheimer. Tegenwoordig worden mensen steeds ouder, hetgeen resulteert in een toename van de prevalentie van neurodegeneratieve ziektes. De pathogenese van leeftijdsgerelateerde neurodegeneratieve ziektes is complex en meerdere factoren dragen er aan bij. Een belangrijk kenmerk van deze ziektes is de ophoping in de hersenen van verkeerd gevouwen of geaggregeerde eiwitten, wat bij zou kunnen dragen aan het ziektebeeld. Normaliter worden verkeerd gevouwen eiwitten afgebroken en verwijderd, maar in neurodegeneratieve ziektes hopen ze op en zijn ze toxisch voor neuronen. Een voorbeeld zijn bèta amyloïde peptiden, die de zogenaamde plaques vormen bij de ziekte van Alzheimer.

Het functioneren en welzijn van neuronen wordt ondersteund door niet-neuronale cellen, glia. Er zijn verschillende typen glia in de hersenen, waaronder astrocyten en microglia. Astrocyten zijn stervormige cellen en zijn het meest voorkomende type glia cel. Ze vervullen meerdere functies in de hersenen, zoals de regulatie van de hoeveelheid chemische signalen die door neuronen worden uitgescheiden, energie metabolisme, ontstekingsreacties en het repareren van neuronale schade. Astrocyten kunnen worden geactiveerd door signalen van neuronen en microglia, en dit helpt de astrocyten om het welzijn van neuronen te ondersteunen. Chronisch geactiveerde astrocyten worden in bijna alle neurodegeneratieve ziektes gevonden en kunnen bijdragen aan hun pathogenese. Het is nu duidelijk dat de staat van activatie in deze astrocyten anders is dan die van astrocyten in een gezond individu. Kennis over deze veranderde staat van activatie zou uiteindelijk bij kunnen dragen aan de identificatie en ontwikkeling van therapeutische targets.

In dit proefschrift is gekeken naar hoe astrocyten neurodegeneratie kunnen beïnvloeden. Hiervoor hebben we effecten van astrocyten onderzocht op neuronen waarin toxische, verkeerd gevouwen eiwitten zaten die geassocieerd zijn met een neurodegeneratieve ziekte. Eén zo’n eiwit dat we gebruikt hebben veroorzaakt SCA3 (Spinocerebellar Ataxia 3). We hebben ook de plaque-vormende bèta amyloïde peptiden gebruikt, geassocieerd met de ziekte van Alzheimer. Aangezien we geen mensen kunnen gebruiken als experimenteel ziektemodel, hebben we een diermodel gebruikt dat ook astrocyten heeft. De aanwezigheid van verkeerd gevouwen humane eiwitten in neuronen veroorzaakt ook in dit diermodel neurodegeneratie. Het diermodel dat we hebben gebruikt is de fruitvlieg, ook bekend als Drosophila melanogaster. Alhoewel dit insect op het eerste gezicht niet lijkt op een mens zijn 50% van de genen in de fruitvlieg vergelijkbaar met die van de mens en zijn hun astrocyten vergelijkbaar in functie. Vliegen hebben een korte levensduur (60-80 dagen), waardoor effecten van een bepaalde behandeling snel te analyseren zijn.

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Verder kun je in korte tijd de effecten van een grote hoeveelheid genen bekijken in specifieke cellen (zoals astrocyten). Onderzoek met fruitvliegen was en is heel belangrijk voor humane biologie en heeft geresulteerd in de identificatie van de functies van genen in kanker, neurodegeneratie en vele andere ziektes.

We hebben het effect op neurodegeneratie bekeken van 150 genen in astrocyten op neurodegeneratie door activiteit van elk gen apart te remmen in een “genetische screen”. Dit heeft geresulteerd in de identificatie van 46 genen die neurodegeneratie kunnen bevorderen dan wel remmen. Als bijvoorbeeld de activiteit van een gen, NF-κB, dat betrokken is bij ontstekingsreacties, geremd wordt in astrocyten, leefden de vliegen significant langer en waren ook actiever, een indicatie van een hogere kwaliteit van leven. We vonden vergelijkbare effecten in “Alzheimer vliegen” waar humane bèta amyloïde peptiden aanwezig waren in de vliegenhersenen.

Alhoewel het remmen van NF-κB in astrocyten geen genezing is voor neurodegeneratieve ziektes, werkt het levensverlengend en verhoogt het ook de kwaliteit van leven. NF-κB heeft echter geen invloed op de hoeveelheid toxische eiwitten in de hersenen. Therapieën die de hoeveelheid van deze eiwitten in de hersenen verminderen zijn tot nu toe niet effectief gebleken, misschien doordat de andere processen, zoals ontstekingsreacties in de hersenen, ook belangrijk zijn van de pathogenese van neurodegeneratieve ziekten. Mogelijk kan in de toekomst een therapie die genactiviteit in astrocyten beïnvloedt gecombineerd worden met een therapie die de hoeveelheid toxische eiwitten in de hersenen vermindert voor optimale effecten.

Tot nu toe hebben we slechts een klein stukje van de puzzel opgelost en blijven er nog veel vragen over. Bijvoorbeeld: wat is het effect van de andere genen die we gevonden hebben op levensduur in neurodegeneratieve ziektes? En meer mechanistisch: welke signaleringroutes van andere genen in astrocyten beïnvloeden neurodegeneratie? Kloppen onze bevindingen in vliegen ook in muizenmodellen, dieren die dichter bij de mens staan?

Het werk dat is uitgevoerd in dit proefschrift onderstreept het belang van astrocyten in neurodegeneratieve ziektes en geeft inzicht in hoe ze bij kunnen dragen aan de pathogenese van deze ziektes.

ACKNOWLEDGEMENTS

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I spent my late 20s doing a PhD, which is an important training to be creative and independent. I wish to thank all the people that appeared in my life during these wonderful years.

First and foremost, I would like to express my sincere gratitude to my supervisor Pascale Dijkers. She was the one who gave me the opportunity to start the project and has been supportive during the whole project. She guided me to learn Drosophila genetics and many other techniques. She taught me how to do a better design for experiments. Also, she helped me to improve my writing and presentation skills. I am grateful to have her as my supervisor.

I want to thank Ody Sibon who, over the years, always listened and gave good suggestions during all the meetings we had. She spent a lot of time to help me complete my thesis.

I would like to acknowledge the members of the reading committee, Professor B.J.L Eggen, Professor E. M. Hol, and Professor D. S. Verbeek for carefully reading and evaluating this thesis.

Francesco and Wondwossen, no matter how many times I say thank you and no matter how good my English is to express my deep gratitude to you, it will never be enough! I am the only child in my family, therefore I have never known how it feels to have brothers or sisters. However, if I had brothers, they would be exactly like the two of you. You were always there to help me with my problems. You always gave me good feedback on my project, you always helped me to figure out technical problems in my experiments. And, you always cheer me up. The stickers (with strange faces, crying faces, smiley faces, and “be happy”) that you put on my desk, I will keep them with me wherever I go from here.

It was great to share an office with Anita, Liza, Jan, Fra, and Wondy. I also feel lucky to work together with Roald, Nico, Balaji, Madina, Yuyi, and Hein. The atmosphere that we had, made me feel free to discuss everything. I often bothered Anita, Jan and Roald with translations of my Dutch documents. I got suggestions from Nico and Hein to improve protocols. I enjoyed the dinner that we had at Nico’s place and the sushi night. I want to thank Madina, via whom I got to know Midas, who did a super nice design for my thesis cover.

I would like to thank dear Bas, who helped me with performing the screen and was always kind and patient.

The 5th floor is really a nice place to work, I enjoyed every moment I spent here. Everyone is nice and friendly. I would especially like to thank all of the following people.

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Dear Harrie, I enjoyed every conversation we had, not only because of your critical comments, but also the encouragement you gave me. I wish I could have had more discussions with you.

I would like to thank Steven who gave me nice suggestions after my presentations and helped me wrestle with the confocal early in the morning.

I need to thank Despina, Matteo, Els, Eduardo, Malanie, Vaishali, Wouter and Suzanne. When I joined Lab meeting in your group, I always got good feedback.

Dear Despina, I probably did something super brilliant in my last life to have, as a reward, you as my friend in this life. You helped me a lot with solving problems with the protocol of the soluble/insoluble fraction experiment. Also, thank you so much for helping me to improve writing and presentations. The time that we were trying to finish the thesis and writing together in many different places (UMCG Library, the room on the 10th floor, Zernike study room, Central Library and Coffee Company) will always be a cherished part in my memory. Because of you, writing was not boring at all.

I would like to thank all the people in Rob’s group, Peter, Cecilia, Daisy, Paola, Lara, Vivian, Julie and Yi. I had a great time with you inside and outside the lab. Thank you all for helping me doing new experiments, introducing me to new techniques and comforting me when I was upset. Peter, you are a special one, I feel I can talk to you about everything. Thank you for not getting annoyed by my “belly-harass”. Cecilia, you are a beautiful one, always ready to help people. Daisy, your hard-working style has been an inspiration for me. I am lucky to have you as a true friend. I enjoyed the dinners we had.

I would like to thank Bart, Marianne, Ellie, Jeanette, Maria, Mirjam, Anne, and Hette. All the things that you helped me in the lab saved my experiments. Bart, thank you for being at my wedding, and always reminding me for the anniversary. Marianne, thank you for teaching me how to run a better blot. I like your voice; it always comforted me whenever I felt a bit sad. Also, I really enjoyed that we did run 4 miles together, twice! You inspired me to practice more!

I would like to thank Klaas, who helped me to learn how to use the confocal. Also I want to thank everyone in the DCB for all the suggestions and helpful comments I received during the DCB meetings.

From Tango, I understand the importance of being patient to concentrate on understanding what is happening and then make a movement. This can not only be applied to dance but also to do a PhD. Many thanks to dear Inez, Rob, Frank and Koen, who brought Tango into my life. You are always nice and kind. I had a lot of fun with all of you.

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亲爱的爸爸和妈妈： 慈母手中线，游子身上衣。 这些年，母亲凡见到的好看的好玩的东西，都会想着给我寄来一份。衣食用品，不一而足。母亲教的菜谱，足够编辑成珍藏版家常菜食谱集。面茶，酱胗，豆泡汤。粽子，米酒，麻豆腐。各式菜肴，应有尽有。 慈父教导，应须饱经术。 这些年，和父亲一起读的书，摆满了整个书架。父亲把从前家人的纸质相片变成电子版，放在我的硬盘。书和照片都是思乡游子的慰藉。 无论去哪里开会，总能收到父母的祝福：望一切顺利。出门旅行，总能收到父母的叮咛：注意安全。大小节日的传统食单，从数九到数伏的加减衣服，从没少过父母的提醒。让游子不管走到哪里，都有一份此心安处是吾乡的从容。 游子寸草心，难报三春晖。 亲爱的英剑： 谢谢你的执子之手，有彼此偕老。 亲爱的潇潇和思涵： 一起上学的日子，已经差不多是二十年前的事。当年的玩伴成了好友，好友不知不觉成了亲人。不论时隔多久，我们都能无缝连接地谈天说地，每次再见面都是我的期待和幸福。 谢谢你们，常去看我父母。 谢谢你们，坚守着我们的情谊。 亲爱的王小姐： 谢谢你，一次次地陪我加班实验，不管是周末，还是通宵。谢谢你，让我们一起学了 Argentina Tango,并一直跳着。更谢谢你，对我所有的包容。 没遇见你的话，我至今认不全五十音图，看不下来日本语初级教材，不会用微信，不能自己剪刘海，也不知道有种好喝的茶叫苦荞。 这个城市，有你在的时候，觉得很大。总有我们还没去过的咖啡馆，酒馆和各种商店。你搬走了，这个城市就变小了。之前没去过的地方，最终也没有去过。 谢谢你，让我们的关系适应所有距离和时差。不管进近，都彼此信任。

私たちの物语はまだ続いていきますね。

亲爱的翠峰姐： 我收到的明信片，大部分是你寄来的。 有你爬了五个小时的挪威布道石，有让我和你去西西里的科隆教堂，有你觉得我也该去看看的纽约，有祝我读博顺利的里斯本，还有很多你说不好也不坏的地方。谢谢你，时常地惦念和问候。谢谢你，不进万里来看我毕业。能做你的妇女之友是我的幸福。

亲爱的郢颖儿： 终于有机会跟你说，你是我的动力。实验做烦了，只要看见你还在努力，就总能马上振作起来。来加班，看见你已经放好的自行车，就觉得自己还可以更努力。谢谢你，存在本身，让我受益匪浅。谢谢你，总毫不犹豫的帮我解决各种问题。谢谢你，教了我很多东西。谢谢你，逢年过节喊我去包饺子。帄颖，祝文章一直高产，能学能玩的继续开心快乐。 亲爱的小余哥： 谢谢你，愿意跟我谈实验聊生活。谢谢你，从不计较我的各种调侃。谢谢你，对羽毛球的热情，让我们有机会一起打球，一起加入俱乐部，一起有了更多的回忆。祝，文章发得顺利，球艺精迚神速，人生处处都有 high five。 亲爱的小红姐： 四年来，一起说的最多的大概是实验相关。 偶尔问候,“喝点热水”和“吃点水果”，就成了独特的温暖。 谢谢你，在实验室的帮忙和不尚虚华的友谊。 亲爱的小吴怡： 凉雨知秋，相见恨晚。 谢谢你，督促我完成各种计划。让我终于看完了人生里第一本英文书。谢谢你，愿意跟我分享喜怒哀乐，也愿意听我说些有用的和没用的建议。谢谢你，时常体贴地叫我一起吃饭，陪我各种溜达。可爱又温柔的小姑娘，祝你一切都好。顺利毕业。

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亲爱的郢颖儿： 终于有机会跟你说，你是我的动力。实验做烦了，只要看见你还在努力，就总能马上振作起来。来加班，看见你已经放好的自行车，就觉得自己还可以更努力。谢谢你，存在本身，让我受益匪浅。谢谢你，总毫不犹豫的帮我解决各种问题。谢谢你，教了我很多东西。谢谢你，逢年过节喊我去包饺子。帄颖，祝文章一直高产，能学能玩的继续开心快乐。 亲爱的小余哥： 谢谢你，愿意跟我谈实验聊生活。谢谢你，从不计较我的各种调侃。谢谢你，对羽毛球的热情，让我们有机会一起打球，一起加入俱乐部，一起有了更多的回忆。祝，文章发得顺利，球艺精迚神速，人生处处都有 high five。 亲爱的小红姐： 四年来，一起说的最多的大概是实验相关。 偶尔问候,“喝点热水”和“吃点水果”，就成了独特的温暖。 谢谢你，在实验室的帮忙和不尚虚华的友谊。 亲爱的小吴怡： 凉雨知秋，相见恨晚。 谢谢你，督促我完成各种计划。让我终于看完了人生里第一本英文书。谢谢你，愿意跟我分享喜怒哀乐，也愿意听我说些有用的和没用的建议。谢谢你，时常体贴地叫我一起吃饭，陪我各种溜达。可爱又温柔的小姑娘，祝你一切都好。顺利毕业。

毅娴 二零一八年 五月

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