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Analysis of Alzheimer disease pathology and Nurr1 in AD miceSarah Metcalfe2; Rebecca Courtney1; Gary E. Landreth1

1Alzheimer’s Disease Research Laboratory, Department of Neuroscience, Case Western Reserve University, Cleveland, OH 44106 2Department of Biology, St. Bonaventure University, St. Bonaventure, NY 14778

ABSTRACTAlzheimer disease (AD) is a neurodegenerative disease characterized by neuronal loss,impaired cognitive function, memory loss, and personality changes. AD pathogenesis includesthe development of plaques formed from amyloid-β (Aβ) protein and neurofibrillary tanglescomposed of intracellular tau protein . The accumulation of plaques correlates with increasedbrain inflammation as shown by activation of microglia and astrocytes. One of the first brainareas to exhibit Alzheimer pathology is the hippocampus, which plays a critical role in long-term memory consolidation and experiences profound neuronal death during the progressionof AD. Hippocampal-dependent memory formation is facilitated by the NR4A subfamily ofnuclear receptors which have broad neuroprotective roles (Hawk and Abel 2011). The NR4Areceptors have been found to be dysregulated in the brains of AD patients and mice withamyloid pathology (Skerrett et al. 2014), but the time course and cellular localization of thesechanges in expression remains largely unstudied.

To study the relationship between NR4A expression and neuronal death in AD we utilized theB6-5XFAD mouse model, which experiences severe amyloidosis and neuronal death. Ourstudy focused on the subiculum, part of the main output pathway from the hippocampus tothe cortex, where we observed neuronal death to occur by 4 months of age. We observedamyloid plaques and microgliosis in the subiculum of B6-5XFAD mice and saw an increase inplaque area with age. We focused on the Nurr1 (NR4A2) nuclear receptor which has alteredexpression with pathology progression in AD mice and has been shown to play aneuroprotective role. We saw Nurr1 colocalized with neurons in the subiculum, anddetermined that overall hippocampal Nurr1 transcript and protein is decreased in B6-5XFADmice by 4 months of age. Our study indicates that genetic manipulation of Nurr1 using viralvectors will be useful if targeted to neurons in mice younger than 4 months of age. A long-term goal of our study is to both overexpress and knock down neuronal levels of Nurr1 in thesubiculum, to determine its value as a potential novel therapeutic target.

INTRODUCTION

• Plaques are caused by aggregation of extracellular Aβ leading to the formation ofdeposits in the brain. Aβ is formed through specific processing of thetransmembrane amyloid precursor protein (APP) and has been shown to haveneurotoxic properties disrupting crosstalk between neurons.

• The 5XFAD mouse model has 5 mutations expressed in neurons, including 3 in APPand 2 in PS1 (a catalytic subunit of one APP secretase). This AD model producesamyloid deposits at an earlier age than other models, and these mice experienceneuronal loss and hippocampal based memory impairments.

• The hippocampus is part of the limbic system and involved with memoryformation and storage. Its function includes consolidation of new memories,emotional responses, navigation, and spatial orientation. The subiculum is themain output for the hippocampus.

• NR4A receptors, especially Nurr1, are important for hippocampal function andhave neuroprotective roles.

• We explore Nurr1 expression in the hippocampus and in specific cell type in5XFAD mice. Along with this we look at inflammation, plaque formation, andneuronal loss in the hippocampus to determine the relationship between Nurr1expression and AD pathology.

REFERENCES & ACKNOWLEDGMENTS

• Hawk, J. D., & Abel, T. (2011). Brain Research Bulletin, 85, 21-29. http://dx.doi.org/10.1016/j.brainresbull.2011.02.001

• Saijo, K. et al. (2009). Cell, 137(1), 47-59.http://dx.doi.org/10.1016/j.cell.2009.01.038

• Skerret, R., Malm, T., & Landreth, G. (2014). Neurobiology of Disease, 72, 104116.http://dx.doi.org/10.1016/j.nbd.2014.05.019

Thank you to SURP for the opportunity to have this experience. Thank you also to theLandreth lab for allowing me to work alongside them. This work was funded by theNIH R01 AG030482 and NIH Grant F31 AG 046055.

RESULTS RESULTS

CONCLUSIONS• As 5XFAD mice age the plaque area in the subiculum tends toward an increase (p=0.07)

showing AD pathology progression. Microgliosis develops by 4 months, and does not appear to increase further by 10 months of age (Fig 1).

• The number of neurons in the subiculum of 5XFAD mice is significantly less than in WT counterparts by 4 months of age (Fig 2). This is probably due to neuronal death, since at 2 months There was no significant neuronal loss between 4m and 10m in the 5XFAD mice, showing that all significant loss in the subiculum occurs by 4m of age in this mouse model.

• Overall Nurr1 protein and transcript levels are decreased in the hippocampi of 5XFAD mice compared to the non-transgenic control by 4 months of age (Fig 3). This indicates that changes in Nurr1 expression in the 5XFAD are following a similar time course to neuronal death in this model.

• While it is known that Nurr1 can play anti-inflammatory roles in microglia and astrocytes (Saijo et al. 2009), we find that Nurr1 is mainly expressed in neurons in the subiculum of both wild-type and transgenic mice (Fig 4). We find it likely that Nurr1 is playing a neuronal role during the development of AD pathology in these mice.

Figure 2. Neuronal deathoccurs in the subiculum by 4Min 5XFAD mice. Neuron counts in

4M and 10M subiculum of WT andtransgenic mice. Neurons wereimmunolabeled with NeuN andquantified (n=4-6/group). Thenumber of neurons significantlydecreased in the 4M transgenicsubiculum compared to WT of thesame age. There was no significantneuronal loss between 4M transgenicand 10M transgenic mice (*p≤0.05).

Figure 1. 5XFAD mice experience microgliosis and increases in plaquearea during aging. Immunostaining of plaques and microglia in the subiculum of

4M (a) and 10M (b) mice. Amyloid-β plaques and microglia are increased in thesubiculum of transgenic mice (10X magnification). (c) Microglial area analysis of 4Mand 10M WT and 5XFAD subiculum labeled using Iba1 and analyzed using ImageProPlus (n=4-6/group). Percent microglial area increased from WT to transgenic but didnot significantly change with age (*p≤0.05) (d) Senile plaque area in 4M and 10M5XFAD subiculum labeled with 6e10 and analyzed using ImagePro Plus (n=4-6/group).Plaque area trend increases with brain age (p=.07).

(a) 4M B65XFAD

(b) 10M B65XFAD

Iba1

Iba1

6e10

6e10

Merged

Merged

(c) (d)

Figure 3. Nurr1 transcripts and protein decrease in the hippocampusin 5XFAD mice. (a) qPCR of RNA from 4M and 10M animals including 5XFAD and

non-transgenic homogenized hippocampus (n=4-6). (b) Analyzed Nurr1 western of 4Mand 10M WT and 5XFAD protein from hippocampal homogenates (n=4-6).

Figure 4. Nurr1 colocalizes with neurons in 4M hippocampus.Immunolabeling in the subiculum with Nurr1 and NeuN along with a DAPI counterstain.Includes 4M transgenic (a) and WT (b) mice (10X magnification).

Nurr1

Nurr1

NeuN

NeuN

MergedDAPI

MergedDAPI

(a) B65XFAD

(b) WT

(b) (a)