actin's role in signaling light-induced phase shifts in the mammalian

ACTIN’S ROLE IN SIGNALING LIGHT-INDUCED PHASE SHIFTS IN THE MAMMALIAN CIRCADIAN CLOCK

BY

JENNIFER MARIE ARNOLD

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Integrative Physiology

in the Graduate College of the University of Illinois at Urbana-Champaign, 2012

Urbana, Illinois

Doctoral Committee: Professor Martha Gillette, Chair Professor Rhanor Gillette Professor Edward Roy Professor Lori Raetzman Professor William Brieher

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ABSTRACT

The actin cytoskeleton is a critical part of intracellular structure, responsible for the general

shape and integrity of all cells. New insights into the dynamic nature of the cytoskeleton

describe additional roles for actin, including synaptic plasticity in the hippocampus, regulation of

hormone release in endocrine cells, and as a signaling component involved in transcription. My

research focused on the hypothesis that changes in actin state contribute to light-induced changes

in the state of the circadian clock in the mammalian brain. In early night, the cells of the

mammalian circadian clock, in the SCN, initiate a series of signaling events to translate

environmental light into transcriptional changes which induce a delay of clock state. This

signaling pathway, mediated by glutamate, involves activation of NMDA receptors, nitric oxide

synthesis, and opening of ryanodine receptors with subsequent influx of calcium from

intracellular stores. Activation of this cascade results in up-regulation of the clock genes, Period

1 and Period 2. This induction is mediated in part via CRE elements, through activation of the

transcription factor, CREB. Connecting upstream signaling processes with the end result of

transcriptional changes was a target of this study.

Based on responses to glutamate in vitro, I hypothesized that activation of the early night

light signaling pathway results in a rapid and transient decrease in actin polymerization, which is

required for the phase delay to occur. I first characterized actin state around a day-night cycle, in

order to determine whether sensitivity to actin depolymerization changes over this period. To

this end, I showed that polymerized actin peaks in early night, but this oscillation in actin state is

abolished in mice rendered genetically arrhythmic, the BMAL1 -/- mice. Secondly, I measured

actin state after light exposure, and found that brief light pulses induced a breakdown of

polymerized actin. By injecting the actin-stabilizing chemical jasplakinolide directly into the

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mouse SCN, I demonstrated a requirement for actin breakdown in mediating the phase delay,

while conversely I induced a delay by injecting the actin depolymerzing agent latrunculin A.

Thirdly, I measured induction of potential downstream effectors upon actin depolymerization, to

analyze the effect of actin dynamics on transcription. Breaking down the cytoskeleton with

latrunculin A resulted in activation of the MAP kinase component PERK, as well as a potential

activation of PCREB. The importance of actin dynamics on CRE-mediated transcription was

demonstrated using electrophysiological recordings, in which inhibition of CRE-mediated

transcription abolished the effects of latrunculin A on clock state. Finally, I showed that actin

depolymerization induced the clock gene Period 2, providing a potential link between

cytoplasmic signaling events that facilitate actin remodeling and nuclear mechanisms of

transcription. This work, together with previous in vitro studies, places actin dynamics firmly

within the light signaling pathway. This not only contributes to knowledge about circadian cell

signaling, but provides a new important role for actin in mediating cellular physiological

processes.

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ACKNOWLEDGEMENTS

First of all, I would like to thank my advisor, Martha Gillette, for her support during my graduate studies. I am grateful for the opportunity to work in a lab environment which allowed me to work and develop into an independent, free-thinking scientist. Her enthusiasm for the field of circadian rhythms is clear, and she definitely infected me with that same interest. I would also like to thank the members of my committee, past and present, including Rhanor Gillette, Lori Raetzman, Bill Brieher, Ed Roy, and Paul Gold. Their attention and advice has been very helpful during my final years of work.

The members of the Gillette lab have been amazing. I felt welcome in the lab from the first day of my rotation, and that never changed during my graduate work. Special thanks to Jennifer Mitchell and Karen Weis, without whom I would not have gotten through many of my experiments, or many of my days. Thanks to the other members of the lab, including Alex Wang, Harry Rosenberg, Anika Jain, Raj Iyer, Sam Irving, Chris Liu, James Chu, Mia Yu, and Olivia Cangellaris, for their advice and friendship. I wish them all luck in their future careers. I would also like to acknowledge past members of the lab, including Sabra Abbott, Patty Kandalepas, and Sheue-Houy Tyan, who provided me with advice and ideas while they were in lab and also afterwards.

I am fortunate to have an amazing group of friends in my life. Pam Monahan, Kjirsten Walt, Shawna Smith, and Jennifer Laprise have been there for me from the beginning, providing distractions from the daily stresses of graduate school as well as great times around Champaign and beyond.

Most importantly, I am here because of the support and love of my family. My parents, sisters Samantha and Kimberly, and brother Brian have supported me through this long journey, and I will always be grateful that they are here for me. I love you!

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TABLE OF CONTENTS List of abbreviations ....................................................................................................................vi Chapter 1: INTRODUCTION ................................................................................................1 Statement of problem and hypothesis ...................................................................17 Figures ...................................................................................................................18 Chapter 2: THE STATE OF THE ACTIN CYTOSKELETON OF THE SCN AROUND THE CLOCK ............................................................................................................23 Abstract .................................................................................................................24 Introduction ...........................................................................................................24 Materials and Methods ..........................................................................................27 Results ...................................................................................................................30 Discussion .............................................................................................................31 Figures ...................................................................................................................34 Chapter 3: LIGHT EXPOSURE ALTERS THE CYTOSKELETON IN THE SCN ......38 Abstract .................................................................................................................39 Introduction ...........................................................................................................39 Materials and Methods ..........................................................................................41 Results ...................................................................................................................47 Discussion .............................................................................................................49 Figures ...................................................................................................................54 Chapter 4: ACTIN CHANGES IN THE SCN ENGAGE DOWNSTREAM EFFECTORS OF CLOCK STATE ..........................................................................................59 Abstract .................................................................................................................60 Introduction ...........................................................................................................60 Materials and Methods ..........................................................................................63 Results ...................................................................................................................66 Discussion .............................................................................................................68 Figures ...................................................................................................................70 Chapter 5: SUMMARY AND CONCLUSIONS .................................................................74 Figure ....................................................................................................................81 Appendix LIGHT PULSE LENGTH DETERMINES ROBUSTNESS OF THE DELAY OF CLOCK PHASE ..........................................................................................82 Introduction ...........................................................................................................83 Materials and Methods ..........................................................................................83 Results ...................................................................................................................85 Discussion .............................................................................................................86 Figures ...................................................................................................................88 References ....................................................................................................................................93

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LIST OF ABBREVIATIONS ACh acetylcholine aCSF artificial cerebrospinal fluid AMPA 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid Arc activity-regulated cytoskeleton-associated protein Arp2/3 actin-related proteins 2 & 3 AVP arginine vasopressin BMAL1 brain and muscle Arnt-like protein 1 Ca2+ calcium CAMKI/II Ca2+/calmodulin-dependent protein kinase I/II cAMP cyclic adenosine 3’, 5’- monophosphate CAMS circadian activity monitoring system cGMP cyclic guanosine monophospate CKIε casein kinase Iε CLOCK mammalian Clock gene product CPP 3-(2-Carboxypiperazine-4-yl)-propyl-1-phosphonic acid CRE cyclic AMP response element CREB cyclic AMP response element binding protein CRY mammalian Cryptochrome gene product CT circadian time Cyto B/D cytochalasin B/D DAPI 4’,6’-diamidino-2-phenylindole DD/D:D total darkness (dark:dark cycle) DMSO dimethyl sulfoxide DNase I deoxyribonuclease I EBSS Earle’s balanced salt solution ERK extracellular signal-related kinase F-actin filamentous actin G-actin globular actin GABA gamma-aminobutyric acid GFAP glial fibrillary acidic protein Glu glutamate GSK3β glycogen synthase kinase 3-beta i.p. intraperitoneal Jasp jasplakinolide KO knockout Lat A/B latrunculin A/B L:D light/dark cycle LP light pulse LTP long-term potentiation MAPK mitogen activated protein kinase MEK mitogen activated protein kinase/extracellular signal-related kinase kinase MSK mitogen- and stress-activated protein kinase NMDA N-methyl-D-aspartate NO nitric oxide

vii

NOS nitric oxide synthase NPF nucleation-promoting factor Nrf2 NF-E2-related factor 2 ODN oligodeoxynucleotide PER Period gene product PER2::LUC fused Period gene product and luciferase gene product PKA cyclic AMP-dependent protein kinase PKG cyclic GMP-dependent protein kinase PKC protein kinase C RHT retinohypothalamic tract RSK 90-kDa ribosomal S6 kinase RyR ryanodine receptor SCN suprachiasmatic nucleus SON supraoptic nucleus TPA 12-o-tetradecanoylphorbol 13-acetate VASP vasodilator-stimulated phosphoprotein VIP vasoactive intestinal peptide WT wild type ZT zeitgeber time

1

CHAPTER ONE

INTRODUCTION

2

Biological rhythms

The daily 24-hour oscillation in light and dark has provided life on Earth with defined

periods of time to carry out their basic daily functions. By anticipating these daily cycles,

organisms gained distinct evolutionary advantages in activities, such feeding, reproduction, or

rest. This has resulted in a highly conserved daily biological rhythm in most known higher

organisms.

The first scientific study of daily oscillations was undertaken by Jean Jacques d’Ortous

de Mairan in 1729. He observed that the daily opening and closing of a heliotrope plant’s leaves

continued even when the plant was moved to a location receiving no sun. Many experiments

followed, providing evidence of an endogenous clock and its “free-running” nature, two essential

properties of clocks that would eventually become foundations of the circadian field 1.

Some of the first modern studies of rhythms in animals were carried out by Colin

Pittendrigh. Using Drosophila, he found that flies emerge from their pupa at a specific time of

day, regardless of environmental temperature, a factor that had been thought to drive rhythms.

He, together with Serge Daan, then defined many properties of activity rhythms in rodents that

are still valid and in use today 2-4. More recently, significant advances in the understanding of

the genetic structure that underlies circadian rhythms have been made using the Drosophila and

mouse models, allowing new molecular studies to further knowledge of the inner workings of

biological clocks 5.

Mammalian circadian rhythms are generated in the hypothalamic suprachiasmatic nucleus

One of the first discoveries into the biology and physiology of biological rhythms was

elucidating that these rhythms are centrally generated by a specific site in the body. By

removing or manipulating organs and systems within rats, Curt Richter found that the central

3

rhythm generator was located in the brain 6. He then undertook an extensive lesioning study of

the brain and was able to localize the ventral hypothalamus as the only location in the body that

resulted in a loss of circadian rhythmicity in rats when removed 7. This followed a line of

evidence suggesting that the ventral hypothalamus was involved in regulating sleep 8, 9.

In 1972, a series of detailed ablation studies was undertaken that suggested that the

suprachiasmatic nucleus (SCN) is the hypothalamic area involved in regulating timing. When

this region was electrically ablated in rats, oscillations in adrenal corticosterone levels were

abolished 10. Additionally, complete ablation of the SCN also abolished rhythms in running-

wheel activity and drinking behavior 11. In both of these studies, animals that received

transection of the optic nerve or ablation of only part of the SCN retained these rhythms. More

definitive evidence was provided by a study in 1984, in which fetal tissue of SCN-containing

hypothalamus was transplanted into the third ventricle of an SCN-ablated rat, restoring drinking

rhythms to that rat 12. In 1990, fetal SCN tissue from a wild type hamster was transplanted into a

SCN-ablated hamster with an altered circadian period, restoring rhythms to the wild-type

phenotype of the SCN donor 13. These studies led to the conclusion that the hypothalamic SCN

is the central pacemaker in mammals.

The SCN are paired nuclei located directly dorsal to the optic chiasm, and directly lateral

to the third ventricle (Fig 1.1). This is an ideal location for a central clock. The SCN receives

inputs from many hypothalamic regions, as well as a substantial input from the retina. Retinal

inputs provide information to the SCN about environmental light. Direct retinal projections

innervate the SCN via the retinohypothalamic tract (RHT). Indirectly, the SCN receives visual

input from the retina by projections from secondary visual processing areas, the intergeniculate

leaflet and pretectal area. Other regions shown to have direct input to the SCN include the

4

median raphe nucleus, several hypothalamic regions, the basal forebrain, limbic cortex, septal

area, and brainstem. The SCN projects to several hypothalamic regions, including the preoptic

area, subparaventricular zone, dorsomedial hypothalamus, and posterior hypothalamus, as well

as to regions in the basal forebrain, thalamus, and midbrain 14, 15.

SCN neurons are some of the smallest in the brain, and lack the long processes seen in

other neuronal cell types, such as hippocampal cells 16. Within the SCN, there are distinct

regions that participate in different aspects of timekeeping. The two main divisions define the

anatomical location of the neurons – the ventral and medial region and the more dorsal and

lateral region of the nuclei. The cells of the ventromedial region appear to receive most of the

visual input as well as input from the dorsal raphe. This suggests that it is this ventromedial

region that is responsible for receiving and integrating input signals, which are relayed to the

laterodorsal SCN area. The SCN, then, modulates its outputs based on this input from the center

14, 15. These divisions are, therefore, both anatomical and functional.

Endogenous circadian rhythms are generated by neurons in the SCN

As originally observed by Curt Richter, circadian rhythms in wheel-running activity

persist in constant conditions with no time cues. Endogenous rhythms have even been shown to

exist when the SCN is isolated from other brain regions. In 1979, an experiment to isolate the

SCN neurons from the rest of the brain showed that this “hypothalamic island” maintained a

rhythm in neuronal activity, while tissue outside of this island did not 17. Evidence for

autonomous generation of the rhythm was furthered by three independent studies in 1982, in

which thin slices of hypothalamus containing the SCN of rats were incubated in a brain slice

chamber. When extracellular recordings were made of SCN cells, all three groups found an

oscillation in the spontaneous firing rate, with a peak in single-unit activity occurring in the

5

donor animal’s subjective day 18-20. These initial, short-term (< 24 h) studies of oscillatory

activity were extended to multiple cycles of up to 3 days, demonstrating that the free-running

period of the SCN in vitro was ~24 h 21. David Welsh and colleagues furthered these studies

using patch clamping and multielectrode plates to record multiple cycles of electrical activity

from dispersed embryonic SCN cells. They found that individual cells are capable of

independently maintaining an endogenous rhythm 22. This implies that the endogenous rhythm is

generated within the cells of the SCN itself.

Specialized techniques have been developed to investigate circadian rhythms in behavior.

Behavioral activity assays take advantage of a natural tendency for most rodents to run on a

wheel. Upon entering the active period of their circadian cycle, rodents immediately begin

running, and continue to do so, stopping only briefly to eat, drink and groom, until the onset of

rest. This behavior of consolidated activity and rest continues even under constant conditions.

Hamsters, rats, and mice are nocturnal, and so this active period is in the subjective night.

Because they begin running so precisely at the beginning of the active cycle, this onset in wheel-

running can be used as a marker of phase in constant dark conditions, and is designated as

Circadian Time (CT) 12. If the time of onset in activity changes, it indicates a shift in the

underlying clock mechanism of the animal 23.

This behavioral approach is in contrast to that of electrophysiology, which allows the

study of isolated SCN tissue or even individual cells. The spontaneously-firing neurons of the

SCN can be measured using patch-clamp techniques 24, 25, electrodes placed close to cells to

record single-cell (Green and Gillette 1982) or multi-cell activity 26, or by using large multi-unit

electrode plates to measure several cells simultaneously 22, 27. The three studies mentioned above

from 1982 recorded single units with extracellular electrodes and found that the spontaneous

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firing rate of SCN cells changes around the day-night cycle, and that the peak of firing rate

occurs in the mid-day. A shift in this peak time, like a shift on onset of wheel-running, indicates

a shift in the state of the circadian clock 28.

A molecular feedback loop drives cellular endogenous rhythms

Since these founding studies, the molecular processes that drive endogenous rhythms

have been elucidated. The endogenous clock is generated in the cells in mammals by clock

genes: Clock 29, Bmal1 30-32, two Period genes, Per1 33, 34 and Per2 35-37, and two Cryptochrome

genes 38, 39. The clock is driven by a transcriptional-translational feedback loop in which the

positive elements, BMAL1 and CLOCK, serve as transcription factors for a variety of rhythmic

genes 40, and also drive the expression of the negative elements, the PERIOD (PER 1/2) and

CRYPTOCHROME (CRY 1/2) proteins 30, 38, 40. The PERs and CRYs in turn downregulate the

positive elements after a delay of many hours because they are sequestered to the cytoplasm due

to a variety of post-translational modifications, including phosphorylation 41-43, ubiquitination 44,

45, and sumoylation 46. Some kinases that are responsible for modifying the clock genes include

casein kinase 1ε (CK1ε) 42, 47 and glycogen synthase kinase-3β (GSK3β) 48, 49. Once activated,

these negative elements dimerize and repress BMAL1 and CLOCK 38, 40. The entire process

undergoes an approximately 24-hour cycle, and therefore determines the endogenous, free-

running period in an animal (Fig 1.2). Because this rhythm is nearly 24 hours, it is termed

circadian (circa, about; dian, day).

An important feature of circadian rhythms is that they are able to coordinate an organism

to its immediate environment, a process known as entrainment. This ability to adapt to changing

conditions involves a phenomenon known as a phase shift. Any number of environmental

conditions, such as food availability or certain chemicals, are able to entrain rhythms, but the

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most important of these entraining zeitgebers (zeit, time; geber, giver) is light. Environmental

light information, which is transmitted to the cells of the SCN through the eyes and RHT, is used

by the clock to synchronize an organism’s rhythm to the time of day, according to the presence

or absence of light. This aligns the not-exactly 24-hour endogenous clock with a 24-hour light-

dark (L:D) cycle 50.

When referring to the time of day under an entrained condition, such as a 12-h light – 12-

h dark cycle, zeitgeber time is used. Zeitgeber time (ZT) 0 refers to the start of the day cycle,

while ZT 12 refers to the start of night. In contrast, circadian time (CT) is used when referring to

free-running or endogenous conditions. CT 0-12 refers to the subjective day while CT 12-0

refers to subjective night. The ability of the clock to entrain so readily to a zeitgeber is due to the

clock’s ability to adjust its phase in response to an inappropriately timed signal. A phase shift

occurs when the circadian rhythm changes, and this can occur in either direction. If a change

shifts the clock forward in time, it is known as a phase advance, while a change that shifts the

clock backwards is known as a phase delay. These alterations can help align an endogenous

clock during daily or seasonal entrainment, but also allow for larger adjustments of the clock,

such as those occurring in humans during travel or due to daylight savings time 50.

The endogenous rhythm can be reset

As mentioned above, the clock is sensitive to light and changes its phase as a result of

exposure to light. Interestingly, the phase shift that occurs in response to light is different

depending on the timing of light exposure. In 1984, a group exposed rats in constant darkness

(D:D) to light at all different times around a circadian day and then measured the phase shifts in

the animals’ wheel-running behaviors 51. The results were expressed as a phase-response curve,

which plots changes in phase against circadian time, with advances designated as positive

8

changes and delays designated as negative changes in phase. These data show us that mammals

respond to light only during subjective night. The nighttime response is biphasic, in that light

exposure in the first half of the night causes phase delays in behavior, while light in the second

half of the night causes phase advances 51, 52. After this finding, the search was on to identify

how light information signals to the SCN.

Glutamate mediates light signaling in the SCN

Several neurotransmitters can be measured in the SCN, such as acetylcholine (ACh),

serotonin, melatonin, GABA, and excitatory amino acids such as glutamate (Glu) and aspartate,

as well as a variety of neuropeptides, including arginine vasopressin (AVP), vasoactive intestinal

polypeptide (VIP), neuropeptide Y, and several others 53. Serotonin and ACh, long known to

affect SCN rhythms, were not likely to be involved in light signaling, as treatment with serotonin

or its agonists induces shifts in the clock unlike those seen with light exposure 54-56, while ACh

depletion or inhibition does not block the light-induced phase changes in wheel-running or the

evoked field potentials in SCN neurons after electrical stimulation 57-59.

Evidence for the excitatory amino acids as mediators of the light response was mixed.

They had been shown to be released at the SCN after optic nerve stimulation 60, and general

excitatory amino acid blockage was found to inhibit the electrical response of SCN cells after

optic nerve stimulation 61. However, direct injection of Glu into the third ventricle, near the

SCN, of hamsters did not induce light-like responses in wheel-running behavior, but instead

induced advances during subjective day and few delays during subjective night 62. Despite this

contradictory finding, studies continued to look at Glu and aspartate relating to light responses.

When MK-801 or 3-(2-Carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP), two

antagonists specific to the excitatory amino acid receptor N-methyl-D-aspartate (NMDA), but

9

not to the other excitatory amino acid receptors, such as kainate and AMPA receptors, were

injected into hamsters or mice intraperitoneally. They blocked the light-induced phase shifts in

wheel-running behavior both in early and late night 63, 64. Direct NMDA application to an SCN-

containing slice, which activates NMDA receptors in vitro, induced light-like phase shifts in

electrical activity, whereas blocking NMDA receptors in these slices blocked these NMDA-

induced phase shifts 65. An anatomical study undertaken during this time found both Glu and

aspartate immunoreactivity in presynaptic terminals in the SCN, but found much more Glu,

suggesting that Glu is the excitatory amino acid responsible for these effects 66.

Conclusive evidence for Glu mediating the light-induced phase response came from

studies using Glu, rather than NMDA, to stimulate the light signaling pathway. Direct

application of Glu onto in vitro brain slices induced delays in spontaneous peak firing rate in

early night, advances in this peak in late night, and elicited no effects during the daytime 23, 67.

When Glu was applied to slices at many points around the circadian cycle, a phase response

curve in peak firing rate matching that seen for light exposure shifts in wheel-running behavior

was observed 23, 51. NMDA antagonists applied with Glu completely blocked these shifts,

showing that NMDA receptors are required for Glu’s action in shifting the clock 23. Also in this

study, they found that application of agents that increase nitric oxide (NO) induce phase shifts

with the same phase response curve as Glu and NMDA. This study concluded that Glu is the

neurotransmitter involved in mediating phase shifts due to light exposure, and also presented a

possible downstream pathway for this Glu signaling 23.

In support of these findings, Watanabe et al. found that application in early night of L-

arginine, a precursor of NO, induced phase delays in firing rate similarly to Glu and NMDA.

Additionally, blockade of NO synthesis by L-NAME inhibited NMDA-induced delays in the

10

early night, adding support that NMDA acts through NO in the SCN 68. This was further

supported in vivo, when L-NAME injection into the third ventricle blocked light-induced delays

in the early night and advances in the late night in wheel-running activity in hamsters 69.

Since these findings, many components of the light signaling pathway have been

identified. Cyclic GMP (cGMP), stimulated by NO, also affects the clock in nighttime only.

cGMP was found to induce advances in neuronal firing rate in SCN slices, but the mechanism

underlying this was not known at the time 70. Several studies followed the initial NO findings

that connected NO synthesis with cGMP and, ultimately, with phase shifting the clock. This

pathway, which involves activation of guanylyl cyclase and subsequent production of cGMP,

thereby activating cGMP-dependent protein kinase (PKG), is only required in late night. PKG

inhibitor application blocks the Glu- or light-induced phase advances seen in late night, but has

no affect on Glu or light effects in early night 71, 72.

There is divergence of the signaling pathways activated by light and glutamate in early

and late night (Fig 1.4). Light signals in early night require intracellular calcium (Cai2+)

signaling, as application of thapsigargin, which depletes Cai2+ , inhibits the Glu-induced delay in

firing rate in SCN slices 71, 73. This Cai2+ was found to be released from ryanodine receptors

(RyRs), which are integral membrane proteins found on endoplasmic reticulum that can mediate

release of Cai2+ 74. Activation of these receptors by caffeine induces Glu-like delays in the early

night, while inhibition of RyRs by dantrolene or ruthenium red blocks the Glu-induced delay 71.

In addition to the primary elements of the light signaling pathway that have been

described, several other signaling components also play a role. Because Ca2+ influx results from

activation of NMDA receptors, the Ca2+/calmodulin-dependent protein kinase (CAMK), cAMP-

dependent protein kinase (PKA), and protein kinase C (PKC) pathways have all been implicated

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to play important roles. In support of this, CAMK and PKC are both activated in the SCN after

exposure to light in the night 75-77. Levels of the PKA activator, cAMP, are also increased in the

SCN after light exposure at night 78. PKA has been shown to have clock-shifting effects in the

daytime domain, but in the night, application of cAMP has no effect on the clock when applied

alone. It is only when cAMP is applied with Glu that the Glu-induced delay is enhanced in

magnitude. Additionally, blockage of the cAMP/PKA pathway using the inhibitor KT5720

results in complete blockage of the Glu-induced delay 78. This suggests a modulatory role for

PKA in the early night, but not a role in the primary signaling pathway.

CAMKII is another molecule that responds to Ca2+ fluxes and, therefore, has been

studied in the SCN. Brief light pulses in the night result in activation of CAMKII 75, 77, while

inhibition of CAMK in the SCN results in attenuated phase delays after light pulse 77, 79, 80 and

Glu 81 induction by about 50-60%. Calmodulin antagonists, which prevent calmodulin from

activating CAM kinases, also attenuate the Glu-induced delay by the same amount 80. These

findings suggest that CAMK plays a role in mediating the phase delay after light, although it is

not the sole regulator.

Similar to CAMKII, PKC activity is rapidly induced in the SCN by light exposure in the

night. Interestingly, inhibition of PKC amplifies, rather than blocks, the light-induced delay in

rhythms. This inhibition also does not block the rapid induction of the immediate early genes,

JunB and c-Fos, seen with light exposure, which provides further support that PKC is not part of

the primary pathway for light signaling, but rather modulates the light response 76.

The mitogen-activated protein kinase (MAPK) pathway forms another important

component of light-induced signaling in the SCN. Light or Glu treatment in the night induces

phosphorylation of extracellular-signal regulated kinases (ERKs) 82. Specific inhibition of

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MAPK/ERK Kinase (MEK) significantly attenuates light-induced delays in behavior by about

50%, indicating that MAPK signaling components are important for light signaling 78, 83. The

Obrietan group found that MAPK lies downstream of the Glu-induced Ca2+ influx, as inhibition

of MEK does not inhibit the Ca2+ influx induced by Glu treatment 84. Additionally, CAMKII lies

upstream of MAPK signaling, as CAMKII inhibition attenuates the light-induced rise in PERK at

night 83.

ERK activates additional kinases. Specific downstream kinase targets of ERK activation

include both the 90-kDa ribosomal S6 kinases (RSKs) and mitogen- and stress-activated protein

kinases (MSKs), which show increases in activity upon light exposure at night 85, 86, but their

involvement in downstream events required for phase shifting is still unclear. This body of

evidence places the ERK pathway downstream of Glu-induced Ca2+ influx, but further work is

needed to fully integrate this kinase system into the light signaling pathway.

Resetting the clock involves transcriptional activation

Downstream from activation of the signaling elements described above, light exposure at

night in the SCN causes transcriptional changes. cAMP response element-binding protein

(CREB) is phosphorylated within 5 min of light exposure 87. Phosphorylated CREB (PCREB)

induction is mediated by the light and Glu signaling pathway, in that Glu or SNAP, a NO donor,

application to SCN slices in either early or late night induces PCREB, while NMDA receptor

inhibitors or NO production inhibitors applied with Glu block this induction 88. Inhibition of the

ERK kinase, MEK, blocks PCREB induction, as well 82, 84. Evidence that CREB mediates

transcription was acquired using an oligodeoxynucleotide (ODN) containing the CRE

(Ca2+/cAMP response element) sequence that sequesters CRE-binding proteins to act as a CRE-

decoy 89. Preincubation of an SCN brain slice with the CRE-decoy inhibits the Glu-induced

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phase advance. Additionally, injection of CRE-decoy into the SCN of mice blocks a light-

induced advance in wheel-running activity 90. The effects of blocking CRE-mediated

transcription have not been studied in the early night.

Light-induced transcriptional changes also include changes in clock gene expression.

When a light pulse is applied to mice in the early night, mRNA expression for both Per1 and

Per2 is induced, however Per1 induction is rapid, within 30 min, while Per2 is much slower,

around 180 min after light pulse 36. On the other hand, light pulses in late night induce only Per1

35. In 2002, a study was undertaken to examine a potential link between PCREB and clock gene

induction. This study found that the promoter regions of both Per1 and Per2 contain CRE

sequences, and that both are able to bind to CREB in vitro. Additionally, in vitro activation of

the cAMP or MAPK pathways causes activation of the Per1 promoter, and somewhat smaller

activation of the Per2 promoter 91. These results suggest that activation of the light signaling

pathway in early night induces transcriptional changes via kinases that activate CRE sites, but a

direct connection between the ryanodine-induced Ca2+ flux and these transcriptional changes has

not been made. This gap in knowledge is the subject of my research.

Actin is a mediator of cellular neuroplasticity

In order to identify potential candidates for further signaling components, other CNS

systems that exhibit changes in state were examined. The most well studied example of neuronal

plasticity is that involved in learning and memory formation, found in the cerebral cortex and

hippocampus. In the cortex and hippocampus, Glu is the primary excitatory neurotransmitter

conveying signals to the neurons that undergo long-term state changes. This Glu activates

NMDA receptors 92, as well as AMPA receptors 93, inducing a signaling pathway containing

several elements in common with those of the SCN light signaling pathways, including Ca2+

14

influxes 94-97, activation of NOS and production of NO 98-101, and activation of kinases (PKA,

PKG, and the MAPK pathway) 98, 99, 101-105. In hippocampal neurons, transcriptional changes

involve regulation by several factors, including PCREB 106, 107. Many genes are upregulated by

activation of this signaling pathway, including Arc (activity-regulated cytoskeleton-associated

gene) 108, 109. Arc also has been found to be upregulated in the SCN after a light pulse at night

110. With these features in common, further consideration of the hippocampal system is

warranted.

The process by which the neurons in the hippocampus undergo long-term state changes is

known as long-term potentiation (LTP). This is thought to be an important aspect of memory

formation. During LTP, post-synaptic responses are strengthened after stimulation by a

presynaptic neuron, leading to long-lasting signal transmission 111. In vitro, this can be initiated

by tetanic stimulation to an input region of the hippocampus, such as cells of the entorhinal

cortex, which synapse with granular cells of the dentate gyrus. LTP also can be initiated by

NMDA receptor activation 92. The dendritic spines in these neurons are rich in cytoskeletal actin

filaments 112-114, and change shape as a result of LTP-induced NMDA and/or AMPA receptor

activation 93. LTP and changing actin state were suspected to be linked after it was found that

blocking actin rearrangements using two chemicals, cytochalasin D (Cyto D) or latrunculin B,

decreased LTP in hippocampal neurons 115, 116. Further, stimulation of the entorhinal cortex,

which initiates LTP in the dentate gyrus molecular layer, caused an increase in F-actin in this

specific region only, as measured by rhodamine-conjugated phalloidin staining and quantitation

of F- and G-actin after differential separation by centrifugation and immunoblotting 117.

Delivering latrunculin A (Lat A), which prevents actin rearrangements, into the dentate gyrus of

live anesthetized rats inhibited LTP. This inhibition did not affect the induction phase of LTP,

15

but was only seen in later stages 117. These findings also have been supported in vivo. When Lat

A or Cyto D were injected after fear conditioning into dorsal hippocampus or the amygdala of

mice or rats, the freezing response, which demonstrates memory of the fear conditioning, was

diminished 118, 119. These findings imply that actin changes are necessary for long-term

maintenance of learning. Impairment after injecting actin-stabilizing chemicals into the

amygdala demonstrates that not just the hippocampus but other regions involved in learning and

memory consolidation require actin remodeling.

Similarities of the light signaling pathway in the SCN to that of the learning and memory

system suggest a potential involvement of actin rearrangements in Glu-induced state changes in

the SCN. Actin is a ubiquitous molecule found in all eukaryotic cells. It exists in two forms:

monomeric actin (globular or G-actin) molecules and polymeric actin (filamentous or F-actin)

molecules (Fig 1.5). In living cells, there is a dynamic shift between the two forms, with

constant turnover of G-actin from the F-actin strands. New actin monomers are added to one end

of the strand, termed the “barbed” end, while monomers or small oligomers are removed from

the opposite end of the strand, termed the “pointed end” 120. These actin strands form the

microfilament component of the cytoskeleton and play roles in many cellular processes.

Cytoskeletal functions include cell support and structure, as well as providing a scaffold for

intracellular and membranous cell components to bind 121. Another important role for actin is

cell migration, such as that seen in macrophages and during development 122.

More recently, actin has been suggested to be involved in signaling. In a 2002 study,

actin was found bound to the transcription factor Nrf2 in rat hepatoma cells, until a specific

stimulus, oxidative stress, resulted in F-actin depolymerization and release of this bound

transcription factor. As a result of being released from actin filaments, Nrf2 translocates to the

16

nucleus and regulates expression of genes initiating a stress response 123. The same group then

found that actin-Nrf2 interactions are mediated through Keap1, an actin-binding protein which

senses oxidative stress and releases bound Nrf2, thereby allowing Nrf2 to translocate to the

nucleus 124. This indicates that the actin cytoskeleton may be involved not only in structural

changes, but in internal cellular signaling, as well.

Another role for actin has been demonstrated in the vasopressin (AVP) and oxytocin

secreting cells of the supraoptic nucleus (SON) of the hypothalamus, as well as in insulin-

secreting β-cells of the pancreas. In both types of endocrine cells, secretion of peptide hormone

seems to require rapid and transient actin depolymerization 125, 126. Stabilization of actin

filaments using the chemical jasplakinolide (Jasp) prevents oxytocin or AVP release in SON

cells 125, while application of actin depolymerizing agents, such as Lat A and Cyto B 125-128,

results in release of peptide stores. For these and other secretory cells, such as catecholamine-

secreting chromaffin cells, rapid breakdown of the cortical actin, which forms a meshwork under

the plasma membrane, is thought to allow for hormone-filled vesicles to fuse to the membrane

and release their contents 125, 128, 129. Elucidating the mechanisms for this actin-induced vesicle

release continues.

Preliminary findings from our lab support a role for actin in signaling. When Glu is

applied to SCN slices in vitro, an approximately 25% decrease in the F-actin:G-actin ratio

occurs, indicating a depolymerization. Additionally, when SCN slices are incubated with the

actin-stabilizing chemical, jasplakinolide, Glu-induced delays in firing rhythms are inhibited,

whereas chemicals that induce a decrease in F-actin, Cyto D or Lat A, induce a delay directly,

without Glu stimulation 130. These findings provide the first evidence that actin changes in the

SCN may be a required component of the long-term state changes that are phase delays.

17

Statement of problem and hypothesis

The preliminary results presented above have allowed me to hypothesize that actin

rearrangements are a necessary component of the light signaling pathway in the SCN in the early

night. In order to test this hypothesis, I have undertaken the following specific aims: 1) Examine

the actin state in the SCN at various points in the day-night cycle, in order to determine if there

are differences in sensitivity to changes in actin state. 2) Examine actin state in response to light

exposure in early night, and determine whether changes are required for a phase delay to occur.

3) Evaluate how actin changes mediate transcriptional changes by examining PCREB and clock

gene induction. The outcome of this study characterizes actin’s role in the response to light,

defining more clearly the early night light signaling pathway, and also extending the potential

role for actin in signal transduction and transcriptional activation.

18

Figures

Fig. 1.1 The suprachiasmatic nucleus is in the ventral hypothalamus of mammals. A. 10x DAPI-stained image of mouse SCN. B. 4x cresyl-violet stained image of mouse SCN. Scale bar = 200 µm.

19

Fig. 1.2 A transcriptional-translational feedback loop drives cellular rhythms. The positive clock elements, CLOCK and BMAL1 (Brain and Muscle Arnt-like protein 1), drive transcription of the negative elements, the Period (Per) and Cryptochrome (Cry) genes. These are translated and feed back to inhibit CLOCK and BMAL1. Phosphorylation by kinases, such as casein kinase 1ε (CK1ε), is one component controlling PERIOD and CRYPTOCHROME subcellular location. Redrawn from Gallego and Virshup 2007.

20

Fig. 1.3 The effect of light on circadian rhythms is time-of-day specific. In animals in constant conditions, including constant darkness, the circadian clock continues to keep near-24-h time, circadian time. During subjective day, exposure to light induces no change in phase of the clock. During early subjective night, light induces a delay in the clock, while in late subjective night, light induces an advance in the clock. Redrawn from Summer et al. 1984.

21

Fig 1.4 Early night light signaling pathway in the SCN. NMDA receptor activation by Glu induces a series of steps required for phase delay in the early night. RHT = retinohypothalamic tract, Glu = glutamate, NMDA-R = N-methyl-D-aspartate receptor, Ca2+ = calcium (i denotes intracellular), NOS = nitric oxide synthase, NO = nitric oxide, Ry-R = ryanodine receptor, CAMK = Ca2+/calmodulin-dependent protein kinase. PKC = protein kinase C, PKA = cAMP-dependent protein kinase, MEK = MAPK/ERK kinase, ERK1/2 = extracellular-signal regulated kinases 1 & 2, PCREB = phosphorylated cAMP response-element binding protein, Per1/2 = Period 1 and Period 2. Redrawn from Gillette and Mitchell 2002.

22

Fig. 1.5 The actin cytoskeleton is a dynamic structure that exists in two states. Addition of monomeric G-actin molecules to the barbed end of a growing F-actin filament is in dynamic equilibrium with removal of G-actin from the opposite pole, the pointed end, of F-actin. This process is ATP-dependent. (Korn et al. 1987)

23

CHAPTER TWO

THE STATE OF THE ACTIN CYTOSKELETON OF THE SCN AROUND THE CLOCK

24

Abstract

The actin cytoskeleton plays a critical role in diverse cellular functions, including cell migration,

scaffolding, formation of new memories, and even signaling. The dynamic nature of actin is

highly regulated by a variety of signaling components, which act on actin-binding proteins to

modify actin filaments for specific functions in the cell. Many of these regulatory signaling

molecules also have been shown to play a role in light signaling to the central circadian clock.

Many of these signaling components oscillate in a circadian fashion, resulting in specific

temporal windows of sensitivity for these molecules to act on the cells of the clock. In this study

I examined actin polymerization state around the day-night cycle in order to determine if actin

state also displays specific windows of sensitivity to change. Additionally, I examined actin

state in an arrhythmic mouse model lacking a functional molecular clock, in order to assess

whether oscillations in actin state are endogenously driven by the circadian clock. I found that

F-actin levels in the SCN oscillate, peaking in early night. This oscillation is abolished in

arrhythmic mice. These findings support the notion that the circadian clock regulates actin

dynamics, and support a potential role for actin in signaling within these cells.

Introduction

Actin is a ubiquitous protein that plays a role in a wide variety of cellular processes.

While originally identified in its role in contractile muscle tissue, actin forms the main structural

component of microfilaments, which serve as a critical scaffold and flexible support structure

present in all eukaryotic cells. Actin protein monomers, termed “globular (G-) actin”, assemble

into long strands, termed “filamentous (F-) actin,” which are the functional form of the protein.

This assembly is ATP-dependent and occurs faster on one end of the growing filament, termed

25

the “barbed” end. Assembly on the barbed end and disassembly on the opposite pole of the

filament, the “pointed” end, lead to a dynamic turnover of the filament 131.

Many types of actin-binding proteins are known to play roles in regulating

assembly/disassembly of actin. The Rho GTPase family, which includes Rho, Rac, and Cdc42,

receive and transmit signals from cell surface receptors to cytoskeletal effectors which then

affect actin state 132. G-actin binding proteins include profilin, ADF/cofilin, and thymosin-β4,

among others. These proteins bind and sequester G-actin, and therefore regulate availability of

monomers for filament assembly and disassembly 131. The Arp2/3 complex, along with a group

of proteins known as nucleation-promoting factors (NPFs), are responsible for the nucleation of

the first actin monomers, which will ultimately form a new F-actin strand 133, 134. Members of

the Ena/VASP family of proteins play a role in strand elongation in several important cellular

functions, including cancer cell migration 131, 133, while formins regulate assembly of F-actin

networks that are unbranched, such as those seen in filopodia 135. The spectrin family of proteins

mediates crosslinking of filaments to other F-actin polymers, and also to organelles and the

plasma membrane 136. These few examples of actin-binding proteins illustrate the variety of

factors that regulate the dynamic assembly and disassembly of actin filaments.

Actin polymerization state can be changed as part of several important physiological

functions. Actin rearrangements are crucial to migratory cell movement, such as that seen in

neutrophils or invasive cancer cells 122. In order to move, chemoattractant chemicals bind to cell

membrane receptors, which signal effector molecules in the cell, often the Rho-GTPase family.

Actin polymerization at the leading edge of the cell and depolymerization at the back of the cell,

together with the formation of new adhesions at the front of the cell and breaking of adhesions at

the back of the cell, contribute to the cell moving in the direction of the chemical signal 122, 137.

26

In the brain, actin-rich dendritic spines on neurons of the hippocampus and cortex change shape

as a result of stimulation during long-term potentiation, and it is the rearrangement of the actin

filaments that is responsible 115, 116. A further function for actin is in signaling. In rat hepatoma

cells, oxidative stress can induce cytoskeletal breakdown. This allows the transcription factor

Nrf2 to be released from actin and translocated to the nucleus, where it upregulates expression of

genes involved in the oxidative stress response 123.

In all of these systems, actin changes are regulated through various cell signaling

components. Calcium (Ca2+) fluxes are a common consequence of signaling pathway activation,

and often lead to changes in the cytoskeleton 121, 138. For example, the Ca2+-sensitive calmodulin

kinase (CAMK) II is one signaling molecule that is associated with rearrangment of the

cytoskeleton 139, 140. This kinase is involved in a variety of cellular responses to a stimulus,

including long-term potentiation in hippocampal cells 141-143 and oxidative stress response in

vascular cells 144, 145. Additionally, actin changes are often associated with activation of MAP

kinase signaling components, as in the hippocampus, where actin rearrangements and the ERK

pathway are important for long-term potentiation 104, 115, 116 and memory formation 118, 119, 146.

Preliminary evidence from our lab and new findings presented in chapter 3 of this

proposal indicate a role for actin in circadian plasticity. In the early subjective night, light

induces a series of signaling events in the cells of the central circadian clock, the suprachiasmatic

nucleus (SCN), resulting in a delay of clock state. Transient actin depolymerization has been

found to be a necessary component of this signaling pathway, which include extracellular Ca2+

influxes and activation of CAMKII 75, 77, production of nitric oxide with subsequent activation of

ryanodine receptors 71, and activation of the MAP kinase ERK pathway 82-84.

27

Many of these signaling components have been found to oscillate in a circadian fashion.

For example, the phosphorylated form of ERK, PERK, peaks in the middle of the day, around

circadian time 8 (CT 8, 8 hours from the beginning of subjective day) 82, 147, 148, while the

phosphorylated, active form of CAMKII peaks around CT 4 75. In addition to these components,

a wide variety of other clock components required for a delay in the clock also oscillate,

including PCREB, which peaks in late night (CT 18-22) 149, and transcription of the clock genes

Period 1 and Period 2, with peak mRNA occurring in the second half of the day 33-36.

In this study, I evaluated actin polymerization state in the SCN around the day-night

cycle in order to determine whether this state oscillates in a circadian fashion. Preliminary

evidence from our lab using a centrifugation-based F- and G-actin separation indicates a

circadian oscillation in rat SCN with peak actin polymerization occurring over the day-night

transition, from CT 10-14. I found that F-actin state in mouse SCN oscillates as well, with a

peak in the early night. Additionally, I utilized a transgenic mouse that lacks a functional central

clock in order to examine whether the oscillation in actin state is clock-driven, and found that

actin state oscillations are abolished in these mice. These findings about the oscillating state of

actin in the SCN provide clues to its role in clock signaling, which will be explored in further

chapters of this dissertation.

Materials and Methods

Animals and circadian time

Male C57Bl/6 mice 6-12 weeks of age, obtained from Jackson Laboratories (Bar Harbor,

ME) were used for this study. Mice heterozygous for the clock gene BMAL1 (BMAL1 +/-)

were obtained from Jackson Laboratories and bred to produce BMAL1 -/- mice. This genotype

28

was determined by PCR (Fig. 2.1 A). BMAL1 -/- mice lack circadian rhythms under constant

conditions (Fig. 2.1 B) 150. The BMAL1 -/- mice were placed in total darkness (D:D) for three

days, and tissue collection occurred at the same circadian timepoints as the wild type controls

and labeled as “approximately circadian time __”, as previously described 150. Rodents were

housed under standard conditions in 12-h light:12-h dark (L:D) cycles or constant darkness

(D:D) and given food and water ad libitum. All animals were maintained and cared for in full

compliance with NIH guidelines for the humane treatment and care of animals under protocols

approved by IACUC of the University of Illinois.

Tissue collection and brain slicing

Mice were sacrificed in the day, and reduced slices placed into a brain slice chamber.

Slices were perfused continuously with Earle’s balanced salt solution (EBSS) supplemented with

24.6 mM glucose, 26.2 mM sodium bicarbonate and 5 mg/L gentamicin and saturated with 95%

O2 / 5% CO2. At the appropriate CT (CT 2, 6, 10, 14, 18, or 22), tissue was collected from the

chamber and frozen on dry ice. Tissue from three mice was pooled for each time point. For

phalloidin and DNase histochemistry, mice were transcardially perfused with 50 ml cold 0.9%

saline, followed by 50 ml 4% paraformaldehyde, at the appropriate time of day (ZT 2, 6, 10, 14,

18, or 22).

Phalloidin and DNase staining

Whole brains from perfused mice were post-fixed in 4% paraformaldehyde overnight and

then sliced into 40-µm sections using a vibratome. These were collected into Watson’s

cryoprotectant for storage at -20°C. Upon thorough rinsing to remove cryoprotectant, sections

were blocked in bovine serum albumin (BSA, Sigma, St. Louis, MO) for 90 min and then

incubated with rhodamine-phalloidin and Alexa-fluor 488-DNase (phalloidin: 2 units/mL,

29

DNase: 10 µg/mL, in 1% BSA/0.03% Triton X-100-PBS; Invitrogen, Carlsbad, CA) for 2 h at

room temperature. Slices were then incubated with DAPI (0.2 µM, Invitrogen) for 5 min at

room temperature. Slices were mounted to gel-coated glass slides and coverslipped using

Prolong Gold Anti-Fade solution (Invitrogen). Staining was evaluated using a Zeiss Meta 510

LSM confocal microscope or a Nikon fluorescent microscope.

F-:G-actin separation assay and western blotting

For each sample, SCN-containing reduced slices from 3 mice were pooled. One hundred

µl of cell lysis buffer (20 mM HEPES pH 7.2, 100 mM NaCl, 1 mM sodium orthovanadate, 1%

Triton X-100) containing protease inhibitor (Complete Protease Inhibitor Cocktail, Roche,

Indianapolis, IN) was added to each sample and then samples were lysed by trituration. Thirty-

five µl of this whole cell lysate was collected, spun briefly to remove membrane debris, and the

supernatant saved as the “total actin” sample. The remaining cell lysate was centrifuged at

10,000 x g for 20 min. The supernatant containing G-actin was collected. The pellet, containing

F-actin, was then redissolved in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 1%

NP40, 0.5% sodium deoxycholate) containing protease inhibitor (Complete Protease Inhibitor

Cocktail, Roche) for 1 h, and then centrifuged again at 12,000 x g for 30 min. This supernatant

was collected as the F-actin fraction and the pellet was discarded. Total protein content from

each sample was determined using the Micro BCA Protein Assay (ThermoFisher Scientific,

Waltham, MA).

Separated samples were resolved by western blotting. Twenty-five µg of total protein

from each sample was mixed with Laemmli buffer and denatured at 95°C for 5 min. Samples

were then separated by 8% SDS-PAGE and transferred to nitrocellulose (Biorad, Hercules, CA).

Following blocking in 5% milk, membranes were probed with anti-β-actin polyclonal antibody

30

(Sigma) overnight at 4°C. As a loading control, blots were probed with anti-α-tubulin (Sigma).

Membranes were developed using Supersignal chemiluminescent substrate (Thermofisher

Scientific) and visualized using a Biochemi Imaging System (UVP, Upland, CA). Levels of

total, G-, and F-actin were determined using densitometry and compared to tubulin as a loading

control.

Results

Actin polymerization state oscillates in the SCN around the circadian cycle

To examine the endogenous state of the actin cytoskeleton across the day-night cycle,

tissue from mice was collected at various points around the day and night and actin state was

evaluated using two methods: 1) F- and G-actin separation and 2) phalloidin and DNase

histochemistry. Differential centrifugation of SCN tissue lysate followed by western blot

analysis allows for the ratio of F-actin:G-actin to be measured. Using this technique in wild-type

C57 mice, the ratio of F-:G-actin peaks in early night, at CT 14 (Fig. 2.2). At this time of night,

F-:G-actin is >25% higher than at all other time points examined (p < 0.05, Student’s t-test (CT 6

vs CT 14), p < 0.001, Student’s t-test (CT 10 vs. CT 14), n = 4).

Phalloidin, which binds specifically to F-actin 151, 152, and DNase, which binds

specifically to G-actin 153, 154, can be used to evaluate actin state. By measuring the level of each,

a ratio of phalloidin:DNase can also be used to determine F-:G-actin 153. Staining the tissue of

mice collected around the day and night with these two compounds also yielded a significant

oscillation in actin state, as a peak in the F/G-actin ratio is demonstrated at ZT 14. However, an

additional peak at ZT 2 is observed (Fig. 2.3, p < 0.01, 1-way ANOVA, n = 4).

31

Actin polymerization is dependent on a functional molecular clock

In order to evaluate whether the changes in actin state in the SCN are driven by an

oscillating molecular clock, F- and G-actin state was measured by phalloidin and DNase staining

in tissue from mice that lack the gene encoding a core molecular clock protein, BMAL1. These

BMAL1 - /- mice are arrhythmic under constant conditions. F-:G-actin ratios measured from

these mice do not show any significant changes around the circadian day (Fig. 2.4, p = 0.9313, 1-

way ANOVA, n = 3), suggesting that a functional clock must be present to drive the oscillation

in actin polymerization state.

Discussion

The findings presented above demonstrate an oscillation in F-actin state in the cells of the

SCN in mice across the day-night cycle. This oscillation is similar to that seen in rat tissue

(Kandalepas and Gillette, unpublished), which was evaluated using F- and G-actin separation

and western-based techniques. In rats, the F-:G-actin ratio peaked between CT 10 and CT 14,

whereas in mice I report a clear peak at CT 14, using the same technique in mice to measure

actin state. This slight difference between the two rodent species is not unexpected, as the

endogenous period of mice is shorter than in rats, and consequently the phase response curve is

not identical 23, 155.

This study also utilized a second method to measure actin state, phalloidin and DNase

histochemistry. Rhodamine-conjugated phalloidin is commonly used in laboratories to visualize

cell shape. In this study, however, the presence of actin in every cell resulted in difficulties

measuring overall changes in staining in a specific brain region. As such, the differences

measured between various times of day are much smaller, though significant. Additionally,

32

because the F- and G-actin separation technique requires tissue from 3 mice to be pooled while

staining does not, individual differences between animals may result in small differences and a

higher error in staining from one set of mice to another. Conversely, the phalloidin and DNase

staining technique allows for a more specific documentation of actin remodeling in the SCN

only, whereas the western-based F- and G-actin separation will always contain some non-SCN

hypothalamic tissue in the reduced slices. These differences in technique may also account for

inconsistencies between the two.

Regardless of these potential discrepancies, both techniques suggest an oscillation of

actin state in the SCN which peaks in the early night in mice. In the early night, the SCN is

sensitive to light from the visual system, acting through a glutamate-based signaling pathway to

produce a delay of clock state. As preliminary findings from our lab and data in chapter 3 show,

actin depolymerizes as part of this signaling pathway. F-actin content is high at this time of the

night, which may result in the more robust depolymerization that is seen compared to mid-day

(Tyan and Gillette, unpublished observations).

Oscillation of cytoskeletal elements is not restricted to the SCN. A recent study

measured both actin and tubulin polymerization state in the hippocampus of rats, and found that

actin state changed in this brain region in light vs. dark conditions, while tubulin state did not

oscillate significantly 156. In the hippocampus, this actin oscillation was shown to be regulated

by melatonin, which is secreted in the absence of light 157, because pinealectomy abolished this

effect 156.

The oscillation of actin state measured in the SCN of wild-type mice was abolished in

BMAL1 -/- mice. These mice are able to entrain to light-dark cycles, but once placed in constant

conditions, such as total darkness (D:D), they become arrhythmic rapidly 150. They have been

33

used to evaluate the effect of molecular arrythmicity on various aspects of physiology, including

effects on metabolism and insulin secretion 158-161, the cardiovascular system 162-166, oscillations

in the oxidation and reduction state of the SCN 167, along with several others 168-175. In this

study, staining with phalloidin and DNase yielded measurable F-:G-actin ratios, but they did not

vary significantly across the day-night cycle. This suggests that the oscillation of the core clock

protein BMAL1 is involved in the regulation of actin polymerization state. It remains to be seen

whether the abolished oscillation in actin state is specific to BMAL null mice. BMAL1 has been

shown to be an important regulator of skeletal muscle tissue, as BMAL1 -/- mice show

abnormalities in microfilament organization in muscle cells 169. Therefore, examining actin state

in other models of arrythmicity, such as long-term exposure to total light (L:L) or the double

knockout of two period genes, the Per1 -/- / Per2 -/- mouse, may provide further details about

the regulation of actin by the clock.

34

Figures

Fig. 2.1 BMAL1 -/- mice display arrythmicity in constant conditions. A. DNA gel electrophoresis is used to identify the genotype of BMAL1 -/- mice. The wild-type BMAL1 band runs at 399 bp, and the mutant band runs at 600 bp. Mice displaying only the mutant band are used for experiments. B. Actograms showing running-wheel behavior for a wild-type (top) and BMAL1 -/- (bottom) mouse. In L:D conditions, both show entrainment, but when placed into constant D:D conditions, the BMAL1 -/- rapidly becomes arrhythmic. Image from Bunger et al. 2000.

KO WT

A

B

35

Fig. 2.2 Actin polymerization state oscillates around the day-night cycle. A. F- and G- actin fractions separated by centrifugation and quantified by western blotting technique are normalized to total, unseparated actin levels. B. F/G-actin ratio peaks in the early night, around CT 14. C. Total actin levels do not show a peak of actin at CT 14. n = 4, * p < 0.05, *** p < 0.001, 1-way ANOVA with Tukey’s post-test.

2 6 10 14 18 22

F-actin

actin

tubulin

Circadian Time (CT)

G-actin

actin

tubulin

total actin

actin

tubulin

A

B C

36

Fig. 2.3 Actin polymerization state measured by staining oscillates around the day-night cycle. Rhodamine-phalloidin (red), and Alexa-fluor 488-DNase (green) staining of wild-type mouse SCN tissue collected around the day-night cycle shows a peak in F-:G-actin at ZT 2 and ZT 14. DAPI staining (blue) was used to define the borders of the SCN. n = 4, p < 0.01, 1-way ANOVA with Tukey post-test, scale bar = 100 um.

ZT22

ZT18

ZT14

ZT10

ZT6

ZT2

37

Fig. 2.4 Actin polymerization state measured by staining in arrhythmic mice does not oscillate around the day-night cycle. Rhodamine-phalloidin (red), and Alexa-fluor 488-DNase (green) staining of BMAL1 -/- mouse SCN tissue collected around the day-night cycle does not show any significant differences in F-:G-actin. DAPI staining (blue) was used to define the borders of the SCN. n = 3, p = 0.9313, 1-way ANOVA, scale bar = 100 um.

CT22

CT18

CT14

CT10

CT6

CT2

38

CHAPTER THREE

LIGHT EXPOSURE ALTERS THE CYTOSKELETON IN THE SCN

39

Abstract

Activation by light of a glutamate-mediated signaling pathway results in a delay of the circadian

clock in the early subjective night. While many components of this pathway are known,

including a requirement for calcium (Ca2+) influxes, nitric oxide production, and activation of

ryanodine receptors and subsequent Ca2+ release from internal stores, downstream components

are less clear. The findings presented here support the hypothesis that the actin cytoskeleton is a

required component of this light signaling pathway. Brief light pulses in the early night induce

transient decreases of polymeric actin. Additionally, exogenous application of actin-disrupting

chemicals induce light-like phase delays, while blocking actin depolymerization blocks light-

induced delays of the clock. These findings provide evidence for the necessity of actin

rearrangement in the SCN during light-induced signaling.

Introduction

Aligning an organism to its ambient environmental condition is essential for carrying out

appropriate daily physiological processes. The central circadian clock in mammals plays a

critical role in maintaining this homeostasis, and is located in mammals in the homeostatic center

of the brain, the hypothalamus, in the suprachiasmatic nucleus (SCN) 14, 15. To maintain this

homeostasis, the clock receives time cues from its surroundings and adjusts itself accordingly,

sending timing information to various tissues.

One important time cue is environmental light. The SCN receives light through a

retinohypothalamic projection. Light exposure during an organism’s subjective night signals that

it is out of phase with its environment. The result is a shift in timing of the circadian clock to

40

realign it with the environment. In the early subjective night, light exposure signals that sunset is

later than anticipated, and results in a delay of the clock 51.

A series of signaling events occur in the cells of the SCN that facilitate this change of

clock phase. Light signals from the eye are transmitted through the retinohypothalamic tracts

(RHT) to the SCN, releasing the excitatory neurotransmitter glutamate (Glu) onto the cells 60.

This Glu activates a variety of receptors, including the NMDA receptor, and the resultant Ca2+

influx is responsible for further downstream events 23, 65, 67. Nitric oxide synthase is activated by

this Ca2+ influx 23, 68, 69. In the early night, the nitric oxide produced results in activation of

ryanodine receptors on internal cell membranes, which, when activated, release Ca2+ from

internal stores into the cytoplasm 71. The processes occurring downstream of this second wave

of Ca2+ influx are the subject of the current study.

Intracellular Ca2+ changes have been associated with a wide variety of cell signaling

events 138. A common effect of increasing cytosolic Ca2+ in neurons is cytoskeletal

reorganization 121. In the hippocampus, neurons receive input signals through Glu acting through

NMDA and AMPA receptors on dendritic spines 92, 93, which results in a local increase in Ca2+ 94-

96. One effect of this Ca2+ increase is rearrangement of actin, allowing the dendritic spines to

change to a shape most effective for new memory formation 93, 176, a primary function of the

hippocampus. In fact, actin rearrangements have been found to be required for memory

formation. In vitro, chemicals that prevent actin from polymerizing, latrunculin B (Lat B) and

cytochalasin D (Cyto D), were incubated with hippocampal brain slices before electrical or

chemical stimulation. This pre-incubation was able to block long-term potentiation from

occurring, thereby preventing the electrical state that the hippocampal cells must achieve for

learning to take place 115, 116. Actin changes have also been shown to be required in vivo, as the

41

same actin-stabilizing chemicals injected into the amygdala of mice or rats before fear

conditioning block the learned response of freezing after a sound pulse 118, 119.

Preliminary findings suggest actin dynamics are also involved in light signaling in the

SCN. Stimulation of the light signaling pathway by Glu in SCN brain slices results in a transient

decrease in polymerized, or F-actin, although total actin levels do not change. This decrease in

F-actin seems to be required for a delay in the clock, as stabilization of actin by the chemical

jasplakinolide (Jasp) before application of Glu blocks the Glu-induced delay in vitro.

Conversely, breaking down F-actin using the chemicals Lat A or Cyto D is sufficient to induce a

delay in the clock similar to that seen with Glu application 130.

The importance of actin dynamics in the hippocampus and the preliminary findings

presented above led me to examine actin state as a result of light in the early night in the SCN.

Using a centrifugation and western blot-based assay, I found that a brief light pulse can induce a

decrease in F-actin in SCN tissue. This was verified by a second method using histochemistry to

measure actin state with phalloidin and DNase staining. Additionally, actin state changes can

adjust the clock. I found that a delay can be induced in onset of wheel-running behavior through

exposure to the actin depolymerizer, Lat A. Conversely, stabilizing F-actin by injection of the

compound Jasp can attenuate light-induced delays in early night, thereby demonstrating the

necessity of actin depolymerization in the SCN after exposure to light.




ME) were used for this study. These rodents were housed under standard conditions in 12-h

42

light:12-h dark (L:D) cycles or constant darkness (D:D) and given food and water ad libitum.

All animals were housed and cared for in full compliance with NIH guidelines for the humane

treatment and care of animals and animal protocols have been approved by IACUC at the

University of Illinois.

In order to determine circadian time for animals living in constant conditions, mice were

housed singly in cages equipped with a running wheel. Running-wheel activity was measured by

rotation of the wheel. This rotation causes a magnet on the wheel to pass a hermetically-sealed

switch, which completes a circuit. This information was processed on a computer containing

LabView software and then plotted into actogram form using Clocklab Analysis running in

Matlab (Actimetric Inc., Wilmette, IL). Onset of activity, which by convention occurs at

circadian time (CT) 12, was used to determine the treatment time for each individual mouse.

Intra-SCN cannulation surgeries

Surgeries were performed at approximately 6 weeks of age (18-22 g). At least 1 h prior

to the procedure, mice received buprenorphine (0.03 mg/kg i.p.) or carprofen (5 mg/kg i.p.) as an

analgesic. Under deep anesthesia (60 mg/kg sodium pentobarbital i.p. or 1-2% isofluorane),

mice were secured into a stereotaxic device in order to minimize movement and for correct

placement of the cannula. After exposing the skull, a small machine screw (0-80 x 1/16, Plastics

One, Roanoke, VA) was drilled into the skull to help secure the cannula. Either a guide cannula

(26 ga, 11.2 mm total length, Plastics One) or a microdialysis guide (CMA7, CMA

Microdialysis, Chelmsford, MA) was aligned with bregma and then adjusted to coordinates

experimentally determined to correspond to the SCN in mice (guide cannula: anterior-posterior -

0.1 mm, medial-lateral -0.1 mm, dorsal-ventral -2.0 mm; microdialysis guide: anterior-posterior -

0.1 mm, medial-lateral -0.1 mm, dorsal-ventral -3.4 mm). A small hole was then drilled into the

43

skull at this location, and the cannula was inserted into the brain. For microdialysis guides, a

small peg was glued to the skull to be used for tethering (Harvard Apparatus, Holliston, MA).

Superglue and cranioplastic cement were used to secure the entire apparatus. A 33-ga stylet was

placed into the cannula to maintain its patency. Mice received buprenorphine (0.05 mg/kg i.p.)

or carprofen (5 mg/kg i.p.) as a post-operative analgesic 12-24 h following surgery and

additionally as needed. Mice were then allowed to fully recover for at least 10 days before

undergoing any further treatments.

Wheel-running activity monitoring

Upon recovery, mice were individually housed in a cage containing a running wheel,

with two cages residing in a circadian activity monitoring system (CAMS). These are light-tight,

ventilated, and the lighting is specifically controlled for each CAMS by computer. After

adjusting to the wheel, mice were placed into these CAMS in constant darkness (D:D). Running

wheel activity was measured by rotation of the wheel. This rotation causes a magnet on the

wheel to pass a hermetically-sealed switch, which completes a circuit. Onset of activity, which

by convention is designated as CT 12, was used to evaluate changes in the clock state. This

information was sent to a computer with LabView software. The information was then plotted

into an actogram using Clocklab Analysis running in Matlab (Actimetric Inc.). The magnitude

of phase shift was determined by measuring the distance between the computer-generated

regression lines plotted against onset of activity before and after treatments.

Behavioral treatments

After mice recovered fully and wheel-running activity was established, mice were

removed from their cages at the appropriate CT in dim red light (<1 lux). For acute injections,

mice were gently restrained and a 10-μl Hamilton syringe fitted with a 33-ga infusion cannula

44

extending 3.4 mm below the guide cannula (Plastics One) was placed into the guide cannula. A

volume of 300 nl of the chemical of interest was then injected into the SCN over a period of 30

sec. The stylet was then replaced to maintain patency throughout the experiment.

For microdialysis perfusions, the mouse was removed from the cage prior to CT 12 and a

probe (CMA Microdialysis) extending 2 mm below the guide cannula was inserted through the

guide and attached to the animal using a head-block tether system (Harvard Apparatus).

Animals were placed in a circadian activity monitoring system (CAMS) designated for these

collections, in a cage without a wheel, and attached to fluid swivels (Harvard Apparatus) to

allow free movement throughout the perfusion. The probe was perfused continuously at a rate of

1 µl/min with artificial cerebrospinal fluid (aCSF: 0.13 M NaCl, 4 mM KCl, 0.75 mM NaH2PO4

[anhydrous], 2 mM Na2HPO4, 1 mM dextrose, 2 mM MgCl2, 1.7 mM CaCl2), until the

appropriate CT, when the chemical of interest was prepared in aCSF and perfused through the

probe. Upon completion of the experiment, the mouse was unhooked from the probe and

returned to the home cage and wheel for behavioral monitoring.

Unless otherwise stated, acute light pulses were administered for 5 min at 20 lux in a

designated CAMS, while the microdialysis light pulses were administered for 10 min at 20 lux.

The chemicals that were injected or perfused are as follows: jasplakinolide (20 µM, Enzo Life

Sciences, Plymouth Meeting, PA) and latrunculin A (100 µM, Calbiochem, Gibbstown, NJ).

Both of these were dissolved in aCSF containing ≤0.05% DMSO for acute injections and ≤0.5%

DMSO for microdialysis infusions. Vehicle injections were composed of aCSF containing

DMSO at the same concentration as that used in chemical treatments.

45

Histology

Upon completion of the experiment, the injection site was verified by injection of 300 nl

of 0.1% thionin into the guide cannula or infusion of 0.1% methylene blue dye through the

microdialysis probe at 1 µl/min for 20 min. Mice were sacrificed, brains were sliced coronally

and Nissl-stained with cresyl violet using standard procedures 177, and cannula placement was

verified using light microscopy (Fig 3.1).

Tissue collection and brain slicing

Light pulse experiments used mice in the early night (ZT 14 or CT 15). Mice were

removed from their home cage at ZT 14 under dim red light (~1 lux). After receiving a light

pulse, mice were sacrificed by cervical dislocation and brains quickly removed. Whole brains

were placed into powdered dry ice to immediately freeze the tissue, which was then stored at

-80°C until being sliced. For F-:G-actin separation experiments, SCN tissue was removed from

these brains by sectioning whole brains into 160-µm sections using a cryostat. The SCN were

selectively removed from 4 of these slices using a 2-mm tissue punch and kept frozen until

processing for actin content. For phalloidin staining experiments, whole brains were sliced on a

cryostat into 30-µm sections, mounted directly to slides, and kept frozen until being fixed in 4%

paraformaldehyde for 30 min and allowed to dry for further staining.

F-:G-actin separation assay and western blotting

For each sample, SCN-containing reduced slices from 3 mice were pooled. One hundred

µl of cell lysis buffer (20 mM HEPES pH 7.2, 100 mM NaCl, 1 mM sodium orthovanadate, 1%

Triton X-100) containing protease inhibitor (Complete Protease Inhibitor Cocktail, Roche,

Indianapolis, IN) was added to each sample and then samples were lysed by trituration. Thirty-

five µl of this whole cell lysate was collected, spun briefly to remove cellular debris, and the

46

supernatant saved as the “total actin” sample. The remaining cell lysate was centrifuged at

10,000 x g for 20 min. The supernatant, containing G-actin, was collected. The pellet,

containing F-actin, was then redissolved in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1%

SDS, 1% NP40, 0.5% sodium deoxycholate) containing protease inhibitor (Complete Protease

Inhibitor Cocktail, Roche) for 1 h, and then centrifuged again at 12,000 x g for 30 min. This

supernatant was collected as the F-actin fraction and the pellet was discarded. Total protein

content from each sample was determined using the Micro BCA Protein Assay (ThermoFisher

Scientific, Waltham, MA).

Separated samples were resolved by western blotting. Twenty-five µg of total protein

from each sample was mixed with Laemmli buffer and denatured at 95°C for 5 min. These

samples were then separated by 8% SDS-PAGE and transferred to nitrocellulose (Biorad,

Hercules, CA). Following blocking in 5% milk, membranes were probed with anti-β-actin

polyclonal antibody (Sigma, St. Louis, MO) overnight at 4°C. As a loading control, blots were

probed with anti-β-tubulin (Sigma). Membranes were developed using Supersignal

chemiluminescent substrate (ThermoFisher Scientific) and visualized using a Biochemi Imaging

System (UVP, Upland, CA). Levels of total, G-, and F-actin were determined using

densitometry and compared to tubulin as a loading control.

Phalloidin and DNase staining

Slides containing 30-µm sections of whole mouse brains were rinsed thoroughly to

remove excess fixative. After blocking in bovine serum albumin (BSA, Sigma) for 90 min,

slides were incubated with rhodamine-phalloidin and Alexa-fluor 488-DNase (phalloidin: 2

units/mL, DNase: 10 µg/mL, in 1% BSA/0.03% Triton X-100-PBS; Invitrogen, Carlsbad, CA)

for 2 h at room temperature, and then slides were incubated with DAPI (0.2 µM, Invitrogen) for

47

5 min at room temperature. Slides were coverslipped using Prolong Gold Anti-Fade solution

(Invitrogen) and allowed to dry. Staining was evaluated using a Zeiss Meta 510 LSM confocal

microscope or a Nikon fluorescent microscope.

Results

Brief light exposure causes rearrangement of actin

In order to determine how light exposure affects actin state, mice received pulses of light

for 1-2, 4, or 6 min at ZT 14, at which point the mice were sacrificed and whole brains collected

by snap-freezing. These were then kept frozen during sectioning and removal of the SCN, which

was then analyzed by F- and G-actin separation in order to determine the F/G-actin ratio. As

controls, mice were collected at ZT 14 under dim red light, previously shown to cause no delay

in rhythms (Arnold and Gillette, unpublished observations). Using this technique, light exposure

for 1-2 min induces significant decreases in the F-:G-actin ratio by 15% ± 6% compared to

control animals (p < 0.05, Student’s t-test). However, by 4 min, F-actin levels are not

statistically different from controls (Fig. 3.2).

Changes in actin state were also evaluated using phalloidin and DNase staining. By

analyzing fluorescence intensity of these two compounds and comparing phalloidin:DNase, the

F/G-actin ratio can be determined 153. For this experiment, mice were placed in total darkness

(D:D) for 3 days, upon which they received a 1-min light pulse at CT 15 and mice were

sacrificed and whole brains snap-frozen. As controls, mice were placed in D:D for 3 days and

tissue was collected at ~CT 15 under dim red light. Tissue was sectioned frozen and post-fixed

before staining with rhodamine-conjugated phalloidin and Alexa Fluor-488-conjugated DNase to

analyse actin state. Utilizing this method, animals receiving light show a significant decrease in

48

F/G-actin of 3.52 ± 0.81% compared to control animals (Fig. 3.3, p < 0.05, Student’s t-test).

These experiments indicate that a brief light pulse induces actin depolymerization in the SCN.

Infusion of the actin depolymerizing compound latrunculin A induces phase delays in behavior

To examine the effects of direct F-actin depolymerization on running-wheel behavior in

mice, the actin depolymerizing agent latrunculin A was infused through a microdialysis probe at

a rate of 1 µl/min from ZT 13-14, after which mice were placed into constant darkness and onset

of running-wheel behavior was analyzed for changes in phase. Infusion of 100 µM Lat A

resulted in a delay of 0.28 ± 0.10 hr, while infusion of vehicle (0.5% DMSO in aCSF) induced a

delay of 0.004 ± 0.09 hr (Fig. 3.4, p < 0.05, Student’s t-test). The delay induced by Lat A is not

significantly different from the delay of 0.40 ± 0.15 hr induced by a 10 min, 20-lux light pulse at

ZT14 (p = 0.84).

Actin depolymerization is necessary for light-induced phase delays

In order to examine the necessity of F-actin depolymerization in the phase response to

light, Jasp was administered directly to the SCN of freely behaving mice immediately before

exposing the mice to light at CT 14. Following treatment, mice were allowed to run for 10 days,

and the onset in wheel-running was compared before and after treatment to assess changes in

clock state (Fig. 3.5). In these mice, a 5-min, 20-lux pulse of light at CT 14 elicited a 0.88 ± 0.08

hr delay in wheel-running onset, while injection of vehicle (artificial CSF/0.05% DMSO)

immediately before the light pulse resulted in a delay of 0.90 ± 0.14 hr. In contrast, injection of

vehicle alone, under dim red light with no light pulse following the injection, elicited a 0.14 ±

0.07 hr delay. Injection of 20 µM Jasp, in a vehicle of aCSF containing 0.05% DMSO,

immediately before a light pulse resulted in a significantly attenuated delay of 0.55 ± 0.09 hr

compared to light-pulsed controls (p < 0.05, Student’s t-test). Twenty µM Jasp in vehicle

49

injected at CT 14 without a light pulse elicited a delay of 0.10 ± 0.12 hr. These results suggest

that stabilizing the actin cytoskeleton using Jasp can block the light-induced delay in the clock in

vivo.

Discussion

These findings further our evidence that actin dynamics are an important component of

the light signaling pathway. I have shown that a brief light pulse can induce significant changes

in actin polymerization in the SCN, using two methods to measure actin state. This supports the

finding from our lab that glutamate exposure to SCN in a tissue chamber at CT 14 induces F-

actin breakdown. However, the in vivo changes in actin are less robust than the in vitro changes

previously generated in the laboratory using Glu. One reason for this smaller change in actin

state, a subset of animals that do not respond to short pulses of light, is examined in the

appendix.

F/G-actin separation experiments with mice show that a very short pulse of light in the

early night is enough to elicit a change in actin state, but that this change is very brief. Indeed,

after 4 and 6 min, no measurable difference in actin state can be measured, implying that the

actin change is rapid and transient, and returns to basal levels before 4 min. This is in contrast to

in vitro data, where the decrease in F-actin is significant at 5 min. This difference can possibly

be accounted for by the differences in the mode of stimulation. Glu is applied as a microdrop to

the surface of the slice and must diffuse into SCN tissue in a chamber in vitro. During light

exposure of a whole, intact mouse in vivo, the signal is transmitted through the eye and along the

optic nerve. While Glu diffusion into SCN tissue is based on Fick’s Law of Diffusion, intact

synaptic transmission is faster by a factor of 109 178, so activating neurons in the eyes of the

50

intact animal and then measuring changes in a transient state in the SCN cells will occur much

more quickly. Additionally, the tissue used for the original in vitro experiments was a reduced

SCN “punch”, whereas in vivo experiments used whole animals, containing all neuronal

connections intact. It is possible that changes are smaller because of some form of inhibition in

the whole brain system, whereas the SCN punches would contain only SCN cells and little

extraneous tissue, and therefore allow larger changes to occur. This phenomenon has been

demonstrated previously in our lab, as Glu-induced changes in firing rate in recording

experiments induces larger phase shifts than light-induced changes in wheel-running activity at

the same time of day 23. Nevertheless, light pulses for very brief periods seem to be able to

induce statistically significant changes in F-actin state.

Rhodamine-phalloidin and Alexa-488-DNase staining of SCN tissue also yielded

significant differences in actin state after brief light pulses. As with the western-based method,

the changes are much smaller than those seen using staining in vitro of a brain slice after Glu

treatment. Similar to the results presented in chapter 2, this staining technique also seems to

yield smaller changes in F/G-actin than western-based methods. In an attempt to increase the

differences between controls and light-pulsed mice, the method of tissue collection for this study

was different than the one used for the western-based F/G-actin experiment. In this study, mice

were placed into constant conditions of total darkness (D:D) for three days prior to tissue

collection, and then a light pulse was administered at approximately CT 15, in order to increase

the probability that each mouse responded to the brief (1-2 min) pulse of light. The appendix

section of this dissertation discusses the importance of this technique further. Using this

collection method, staining results show a small but significant decrease in the ratio of

phalloidin:DNase staining, indicating a decrease in the F/G-actin ratio.

51

A second result from this study also matches findings in vitro. When actin is

depolymerized by direct infusion of Lat A into or near the SCN of a mouse, the onset of wheel-

running is significantly delayed compared to a vehicle infusion. This follows previous work

from the lab using rat brain tissue, in which bath application of Lat A from CT 12-14 induced a

delay in peak firing rate that was of the same magnitude as the delay induced by Glu application

130. In vitro work also included actin depolymerization using a second compound, Cyto D. Bath

application of this compound to rat SCN slices also induced Glu-like delays in peak firing rate.

Intra-SCN injections with this chemical were not able to induce changes in the clock.

This discrepancy may be due to the different ways in which the two chemicals act to

break down F-actin. Cytochalasins, compounds isolated from fungi, act by binding to the

growing end of the actin filament (the barbed end), preventing further polymerization, and

therefore eventually resulting in a decreased level of F-actin 179, 180. On the other hand,

latrunculins, which are produced by sea sponges, bind to G-actin monomers in a 1:1 ratio,

preventing the formation of further polymers 181. It is possible that in order to sufficiently

depolymerize actin, a higher concentration of Cyto D is need in vivo than for Lat A. It is also

possible that longer exposure to the chemical is needed, due to differences in cell permeability of

the two compounds. Regardless, using Lat A to break down F-actin in the SCN results in a

phase delay, indicating that directly depolymerizing the actin cytoskeleton is sufficient to induce

a delay in clock phase.

The final result from this study suggests that actin depolymerization is required for the

phase delay induced by light in early night. Jasplakinolide is a peptide isolated from a sea

sponge that binds strongly and selectively to F-actin and prevents its depolymerization 182.

When Jasp is injected directly into mice at CT 14, either into or near the SCN, the delay in

52

wheel-running onset induced by light is significantly attenuated. This follows in vitro results, in

which pre-incubation of SCN slices with Jasp blocked the Glu-induced delay in peak time of

extracellular activity recordings. Although delays in running-wheel onset were not completely

blocked like those in recordings, the results are still significant.

The differences can be accounted for by several factors. First, the method of delivery is

different. For electrophysiology, Jasp is administered to the SCN tissue by bath application.

Jasp is dissolved in tissue culture media and this is placed into the tissue chamber, completely

surrounding the small slice of brain, for 20-30 min before adding glutamate. For in vivo

delivery, Jasp is injected through a cannula into a living brain. This limits the volume of liquid

that can be delivered, and therefore the amount of Jasp that the mouse receives. Also, bath

application of Jasp allows for continuous delivery of the chemical to the brain slice during the

treatment, while an injection delivers a bolus of chemical all at once. In the living brain,

extracellular fluid or cerebrospinal fluid is constantly circulating. The liquid injection delivered

to the animal is likely quickly diffused in the tissue, so that Jasp is not able to get into cells and

bind to actin as well. Finally, as discussed above, the mode of transmission of light and

glutamate are different, and this likely affects how Jasp works to block the phase delay. A light

pulse is given to the mouse immediately following the acute injection of Jasp, while Glu is

delivered to a brain slice while it is still exposed to the bath application of Jasp. Because of this,

it is possible that the effects of stabilizing actin are not present throughout the entire light pulse,

which could account for the incomplete blockade of the delay in vivo.

A second factor that may account for the smaller attenuation by Jasp in vivo is the

injection technique. The implanted cannula is aimed at one SCN in the mouse brain, so that

during an injection, Jasp should only be administered to the cells on one side of the brain. This

53

likely accounts for the partial inhibition of the light pulse’s effects. However, for this study, the

data from mice in which the cannula was implanted into the 3rd ventricle, at the level of the optic

chiasm, was also included. Therefore, bilateral exposure is possible. Again, though, injection

into the ventricle, which contains a large volume of CSF, dilutes the compound further, and so

exposure of Jasp to the SCN is still limited.

These results, combined with the in vitro studies previously undertaken in the laboratory,

demonstrate the involvement of transient actin dynamics in mediating light-induced delays of the

clock in early night.

54

Figures

Fig. 3.1 Validation of cannula placement in intra-SCN surgeries in mouse. The cresyl-violet dye track is clearly visible in these Nissl-stained hypothalamic slices (50 µm). A-B: acute cannula, C-D: microdialysis probe. Scale bar = 500 µm.

dye track

A

D

B

C

55

Fig. 3.2 Light induces a transient decrease in F-actin. Light pulses of 1-2 min induce a decrease in the F-:G-actin ratio of 15 ± 6% relative to non-light pulsed controls (0 min). Tubulin levels do not change and are used as a loading control. n = 14 for 0, n = 12 for 1-2 min, n = 6 for 4 and 6 min, * p < 0.05, Student’s T-test (0 vs 1-2 min).

Actin

Tubulin

0 min

G F G F G F G F

2 min 4 min 6 min

6'4’2’0’

total actin

Actin

Tubulin

*

56

Fig. 3.3 Light induces a decrease in F/G-actin ratio by staining. Light pulses of 1-2 min induce a decrease in the ratio of F-actin (rhodamine-phalloidin, red) to G-actin (Alexa-488 DNase, green) of 3.52 ± 0.81% compared to control animals (DD). DAPI staining (blue) was used to define the borders of the SCN. n = 7, p < 0.05, Student’s t-test, scale bar = 100 um.

DD LP

57

Fig. 3.4 Direct actin depolymerization in vivo induces phase delays. A 10 min, 20-lux LP at ZT 14, after which the mouse was placed into total darkness, resulted in a delay in wheel-running of 0.40 ± 0.15 hr. Infusion at a rate of 1 µl/min into or near the SCN of 100 µM latrunculin A (Lat A) induced a delay of 0.28 ± 0.10 hr, while infusion of vehicle (0.5% DMSO in aCSF) resulted in a delay of 0.004 ± 0.09 hr. Yellow arrow indicates time of light pulse, green box indicates infusion times, n = 7, * p < 0.05, Student’s t-test (vehicle vs Lat A).

LatAVehicleLP

58

Fig. 3.5 Stabilizing the cytoskeleton in vivo attenuates the light-induced delay of the clock. Injection of vehicle (0.05% DMSO/aCSF) at CT 14 elicited a delay in onset of wheel-running of 0.14 ± 0.07 hr, while injection of 20 µM jasplakinolide (Jasp) induced a delay of 0.10 ± 0.12 hr. Injection of vehicle followed by a 5 min, 20-lux light pulse (LP) elicited a delay of 0.90 ± 0.14 hr. Injection of Jasp immediately followed by a 5 min, 20-lux LP caused a delay of 0.55 ± 0.09 hr. LP alone induced a delay of 0.88 ± 0.08 hr. Yellow arrows indicate time of treatment, n = 8, * p < 0.05, Student’s t-test (Jasp + LP vs vehicle + LP, vs LP, and vs Jasp), ** p < 0.01, Student’s t-test (Jasp + LP vs vehicle).

LPVehicle Jasp Vehicle + LP Jasp + LP

59

CHAPTER FOUR

ACTIN CHANGES IN THE SCN ENGAGE DOWNSTREAM EFFECTORS OF CLOCK STATE

60

Abstract In the circadian system, light exposure in the night activates a signaling pathway through

glutamate resulting in upregulation of the clock genes, Period 1 and Period 2. This upregulation

is mediated in part through cAMP response elements (CREs), through activation of the

transcription factor, cAMP response element binding protein (CREB), which itself is activated

by MAP kinase activity. In Chapter 3, I provided evidence that transient actin depolymerization

is required for early night light signaling, but a connection between these cytoskeletal changes

and known downstream transcriptional events has not been found. In this study, I found that

treatments depolymerizing F-actin significantly increase the level of activated phospho-ERK, a

component of the MAP kinase pathway. Depolymerizing F-actin also resulted in a trend of

increased phospho-CREB. When CRE-mediated transcription was blocked in SCN slices,

latrunculin A-induced delays in the clock were blocked, indicating a role for actin-mediated

regulation of CREB. Finally, treatments that depolymerize F-actin significantly induce PERIOD

2 (PER2), as measured by the clock-gene reporter, PERIOD2::LUCIFERASE. Stabilizing actin

with Jasplakinolide (Jasp) blocks the Glu-induced rise in PER 2. This study supports a role for

actin dynamics in mediating transcriptional changes in the central circadian clock.

Introduction

The suprachiasmatic nucleus (SCN) in the ventral hypothalamus houses the central

circadian clock in mammals. The cells of this pair of nuclei keep time endogenously, and the

phasing of the time base can be reset in order to align the internal clock of the animal with the

external environment. The most important environmental signal to reset the clock is light. Light

from the eyes signals the cells of the SCN through glutamate (Glu), and the signaling pathway

61

that is activated results in a change of clock phase that is time-of-day dependent. In the early

subjective night, light induces a delay of the clock. At this time, activation of the glutamate-

mediated signaling pathway has been shown to increase expression of the clock genes, Period 1

(Per1) and Period 2 (Per2) 36, 183.

Connecting glutamate release with these transcriptional changes is a series of signaling

components, many of which have been characterized, though some remain unidentified. In the

early night, NMDA receptor activation by Glu results in a series of signaling events that include

nitric oxide production and consequent activation of ryanodine receptors within the cell 23, 71.

The calcium (Ca2+)-induced Ca2+ release that accompanies the activation of these receptors

induces a transient rearrangement of the actin cytoskeleton and activation of a MAP kinase

pathway. Results from chapter 3 indicate that the actin breakdown is required in order for a

light-induced phase delay to occur. It is not currently known, however, how changes in actin

could mediate downstream transcriptional effects that underlie the phase delay. In this study I

will address this issue.

In mammals, two period genes, Per1 and Per2, have been found to be involved in light

signaling. Light pulses in the night induce rapid expression of Per1 mRNA, peaking around 30

min after light exposure. This induction of Per1 is seen in both early and late subjective night,

but not in subjective day. Expression of Per2, on the other hand, is much slower, with increases

seen 3 h after light exposure 36, 183. Also, induction of Per2 is restricted to light exposure in the

early subjective night 35. Expression of Per1 has been found to be required for light-induced

phase advance, as knockdown of Per1 using antisense oligodeoxynucleotides (ODN) blocks the

light and glutamate-induced changes in the clock 184. Also, mice containing a mutant Per1 do

62

not advance after a light pulse in late night. Conversely, Per2 mutant mice do not delay in early

night after a light pulse, indicating a requirement for Per2 in early night phase delay 185.

Both period genes contain at least one CRE sequence within their promoters 91. This

sequence binds the activated transcription factor PCREB, mediating gene expression for a wide

range of physiological processes, including long-term potentiation and memory formation,

spermatogenesis, and pituitary function 186. In the SCN, PCREB induction is seen within 5 min

of a light pulse at night 87, as well as due to application of several of the known components of

the light signaling pathway, including Glu 88, 187, and NO donors 88. Additionally, PCREB is

induced in JEG-3 cells transfected with mPer1 or mPer2 promoters containing a luciferase

transcriptional reporter upon addition of 12-o-tetradecanoylphorbol 13-acetate (TPA), which

activates MAPK and its downstream targets, including PKA 91. PCREB has been shown to bind

to the promoters of both Per1 and Per2 91, supplying additional support that PCREB mediates, at

least in part, the transcription of these genes as a response to light exposure in the night.

One activator of CRE-mediated transcription is the mitogen activated kinase (MAPK)

pathway 84, 188. This pathway has been found to be induced by light or Glu in early night 82.

Extracellular-signal regulated kinases (ERKs), kinases of the MAPK cascade, are phosphorylated

within 5 minutes of light exposure at CT 15 82. Inhibition of MAPK/ERK Kinase (MEK) by

intracerebroventricular application of the inhibitors U0126 or PD98059 attenuates the light-

induced delay in early night in behavior, indicating a role for ERK in mediating phase delays 78,

83. MAP Kinases have been found to lie downstream of both CAMKII and Glu-induced Ca2+ 83,

84, but their specific position within the light signaling pathway has yet to be established.

In this chapter, I present evidence that actin depolymerization can induce activation of

ERK, and a trend toward induction of the active form of CREB. Additionally, I demonstrated

63

the requirement of actin remodeling for CRE-mediated transcription. Finally, I show that actin

depolymerization can increase PERIOD 2 levels. These experiments utilize a transgenic mouse

that contains a modified Period 2 gene encoding the PERIOD 2 protein fused to luciferase,

resulting in a PER2::LUC reporter 189. By measuring luciferase activity, PER 2 abundance can

be tracked and quantified. These experiments will provide the first evidence that actin

rearrangements in the SCN mediate the transcriptional changes required for a light-induced

phase delay in the early night.




ME) were used for this study. Mice containing a transgene that encodes PER 2 fused to

luciferase (PER2::LUC mice) were obtained from Jackson Laboratories and bred in-house. For

recording experiments, 6-12 week old Long Evans/BluGill rats were used (University of

Illinois). These rodents are housed under standard conditions in 12-h light:12-h dark (L:D)

cycles or constant darkness (D:D) and given food and water ad libitum. All animals are housed

and cared for in full compliance with NIH guidelines for the humane treatment and care of

animals and animal protocols have been approved by IACUC at the University of Illinois.

Tissue collection and treatments

Mice are sacrificed before ZT 10 by cervical dislocation and brains quickly removed. A

block of tissue containing the hypothalamus was made and then a 500-µm slice containing the

SCN was isolated using a tissue chopper. For biochemical analysis, the SCN was then removed

from these slices using a 2 mm tissue punch and this was placed into a brain slice chamber.

64

Slices were perifused continuously with Earle’s balanced salt solution (EBSS) supplemented

with 24.6 mM glucose, 26.2 mM sodium bicarbonate and 5 mg/L gentamicin and saturated with

95% O2 / 5% CO2. At the designated CT, treatments were applied one of two ways: 1) Glu (10

mM) was applied directly to the SCN as a 2-µl microdrop; 2) actin-altering chemicals and CRE-

decoy were applied as a bath application: latrunculin A (1 µM; Calbiochem, Gibbstown, NJ),

cytochalasin D (0.1 µM; Calbiochem), jasplakinolide (2 µM; Enzo Life Sciences, Plymouth

Meeting, PA), CRE-decoy (1 µM, Operon, Huntsville, AL). Reduced slices were collected by

snap-freezing on dry ice, and kept at -80°C until further processing.

Western blotting

For each sample, 2 SCN-containing reduced slices were used for each N. Seventy-five µl

of RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium

deoxycholate) containing protease (Complete Protease Inhibitor Cocktail, Roche, Indianapolis,

IN) and phosphatase inhibitors (EMD Chemicals, Gibbstown, NJ) was added to each sample and

then samples were lysed by trituration. Samples were spun at 10,000 rpm for 5 min and the

supernatant was saved in a fresh tube. Total protein content from each sample was determined

using the Micro BCA Protein Assay (ThermoFisher Scientific, Waltham, MA).

Samples were resolved by western blotting. Twenty-five to thirty µg of total protein

from each sample was mixed with Laemmli buffer and denatured at 95°C for 5 min. These

samples were then separated by 8% SDS-PAGE and transferred to nitrocellulose (Biorad,

Hercules, CA). Following blocking in 5% milk, membranes were probed with anti-phospho-

CREB (PCREB, Ser133) antibody (Upstate, Billerica, MA) or anti-phospho-p42/p44 (PERK)

antibody (Cell Signaling Technology, Danvers, MA) overnight at 4°C. As controls, blots were

probed with anti-CREB (made in-house) or anti-p42/p44 (ERK, Cell Signaling Technology).

65

Membranes were developed using Supersignal chemiluminescent substrate (ThermoFisher

Scientific) and visualized using a Biochemi Imaging System (UVP, Upland, CA). Levels of

PCREB or PERK were determined using densitometry and compared to CREB or ERK, which

show no changes between treatments.

Luciferase assays

Hypothalamic slices or reduced slices containing the SCN were collected following

treatment and immediately frozen in dry ice. Each slice was lysed by trituration in 100 µl of cell

culture lysis reagent (Promega, Madison, WI). Lysates were centrifuged for 5 min at 10,000 rpm

and the supernatant was saved in a fresh tube. Sixty µl of this lysate was then placed in one well

of a white 96-well microlite 2+ plate (ThermoFisher Scientific) and PER2::LUC levels were

measured in a luminometer (Luminoskan, MTX Lab Systems, Vienna, VA). Total protein levels

in each sample were evaluated using a BCA Protein Assay Kit (ThermoFisher Scientific) and

luminescence in relative light units was normalized to total protein for each sample.

Electrophysiology

Spontaneous firing rhythms were assessed in SCN brain slices using a single unit activity

recording technique 28. Mean firing rate for an individual cell was measured over a period of 4

min, and these were then grouped into 2-h running averages with a 15-min sliding window.

Average firing rate was plotted against the circadian time of recording, and time-of-peak for each

experiment was determined from this plot for the highest firing rate. Phase shifts were assessed

by comparing time-of-peak for each treatment experiment with that of the mean of vehicle-

treated control experiments.

66

Results

Actin depolymerization induces activation of the MAP Kinase pathway

To evaluate the effect of actin depolymerization on activation of downstream signaling

elements, SCN tissue was treated with a continuous bath application of 1 µM latrunculin A (Lat

A) at CT 15 and collected 5, 15, 30, and 60 min after the start of treatment on dry ice. As a

control, tissue was collected at CT 15 immediately prior to Lat A treatment. Tissue was

analyzed using western procedures probing for anti-phospho-p42/p44 (PERK). In this study,

actin depolymerization induced PERK formation within 5 min, with a 33 ± 5 % increase that

peaked at 15 min after the start of exposure to Lat A with a 46 ± 7 % increase. Thirty minutes

after beginning Lat A exposure, PERK continued to be induced by 45 ± 11 %, returning to basal

levels by 60 min (Fig. 4.1, p < 0.001, ANOVA with Dunnett’s post-test). These results indicate

that the MAP kinase pathway is activated by F-actin breakdown in the SCN.

Actin depolymerization may alter PCREB

To evaluate the effect of actin dynamics on CRE-mediated transcription, SCN tissue was

treated with a continuous bath application of 1 µM Lat A at CT 15 and collected on dry ice 5, 15,

30, and 60 min after the start of treatment. As a control, tissue was collected at CT 15 before Lat

A treatment began. Tissue was then analyzed for induction of PCREB using western procedures,

probing for anti-Ser-133-phospho-CREB antibody. Using this technique, depolymerization of

the actin cytoskeleton with Lat A trended toward a 19.2 ± 5.8 % increase in phospho-CREB at

30 min (Fig. 4.2, p = 0.0822, Student’s t-test).

67

CRE-mediated transcription is mediated through actin dynamics

In order to physiologically connect actin dynamics to transcriptional changes in the SCN,

I used a CRE oligodeoxynucleotide (CRE-decoy) to examine whether CRE-mediated

transcription plays a role in actin-based signaling in the SCN. CRE-decoy was preincubated (1

h, CT 14-15) with rat SCN slices in early subjective night, and then these slices were co-

incubated with the actin destabilizer, Lat A (1 h, CT 15-16). As previously observed, Lat A

alone induced a 2.42-hr delay in peak firing rhythm compared to control (peak time = CT 9.08 ±

0.22 vs. CT 6.67 ± 0.08, respectively) when administered to slices from CT 15-16. CRE-decoy,

applied from CT 14-16, effectively blocked this Lat A-induced delay in firing rhythms in rat

(peak time = CT 6.75 ± 0.25). CRE-decoy alone did not affect timing (peak time = CT 6.92 ±

0.08). (Fig 4.3, p < 0.0001, 1-way ANOVA with Dunnet’s post-test, n = 3.)

Actin depolymerization induces an increase in PER 2

In order to examine how the actin cytoskeleton affects Per2 expression, SCN slices from

mice containing the gene encoding the PER2::LUC fusion protein were treated with actin-

rearranging chemicals and analyzed for luciferase activity. When 10 mM Glu or 0.1 µM

cytochalasin D (Cyto D), an actin depolymerizing agent, were added to slices, luciferase activity

increased 1.32 ± 0.20-fold and 1.64 ± 0.18-fold after 6 h, respectively, compared to EBSS-

treated controls. This indicates that PER 2 levels are increased by these treatments. Conversely,

when these slices were pre-incubated with Jasp, which stabilizes F-actin, Glu treatment did not

induce luciferase activity (0.85 ± 0.13-fold vs. control), while a preliminary control experiment

with Jasp treatment alone also did not induce luciferase activity (0.52-fold). (Fig 4.4, p < 0.05, 1-

way ANOVA with Dunnett’s post-test)

68

Discussion

The results of this study provide further evidence that the actin dynamics shown in

chapter 3 are an important component of the light signaling pathway. Direct actin

depolymerization induces ERK activation, an important step in the MAP kinase signaling

pathway. This suggests that changes in actin state occur upstream of MAPK pathway activation.

Prior to this study, MAP kinase elements were found to lie downstream of Ca2+ influx 84. The

findings in this chapter continue to support this position for MAPK in the signaling pathway.

Additionally, Lat A-induced F-actin breakdown resulted in an increase in the active form

of CREB, PCREB. Though not statistically significant, this result is suggestive of actin’s

regulation of CRE-mediated transcription. As with PERK, the neurons of the hippocampus that

undergo actin changes after activation also show increased PCREB, which is linked to increased

expression of several genes important for the consolidation phase of long-term potentiation 106,

107. A second result that supports the role of actin in the regulation of CRE-mediated

transcription was found using electrophysiological recordings of SCN slices. When the CRE-

decoy is incubated in SCN slices, activated CREB and other CRE-binding proteins will bind to

the decoy oligodeoxynucleotide molecules, essentially serving as a blocker of transcription that

is mediated through CRE 89. When this decoy was preincubated with the SCN slice before and

during Lat A application, the delay in peak firing rate that is caused by Lat A was blocked. This

implies that Lat A works to induce a delay in the clock through CRE-mediated transcription.

These two experiments together also suggest that the changes in actin state are upstream of

activation of CREB. Because ERK activation, which is upstream of CREB activation, is induced

by actin depolymerization, and the MAPK pathway is one regulator of CREB 84, 188, these

conclusions are plausible.

69

A final study examined PERIOD 2, a protein that is upregulated following light exposure

in early night 35, 36. This study found that direct actin depolymerization results in an

upregulation of PER 2, an important event in the early night for a light-induced phase delay.

This supports previous findings from our lab, in which per2 mRNA was found to increase 2 hr

after treatment of rat SCN with Cyto D, measured by qPCR (Kandalepas and Gillette,

unpublished). Additionally, pre-incubation of rat SCN tissue with Jasp, followed by treatment

with Glu, blocked the induction of per2 seen with Glu exposure (Mitchell and Gillette,

unpublished), and pre-incubation of SCN tissue with Jasp blocked the Glu-induced rise of PER 2

(Fig. 4.4). The findings from this study, which evaluated PER 2 protein, corroborate these

earlier findings examining mRNA. Taken together, this data indicates that actin dynamics play

an important role in transcriptional regulation of the clock.

70

Figures

Fig. 4.1 F-actin depolymerization induces MAPK signaling. Treatment of SCN reduced slices from mice from CT 15-16 with latrunculin A (Lat A) significantly increased PERK at 5 (1.33 ± 0.05-fold), 15 (1.46 ± 0.07), and 30 (1.45 ± 0.11) min following start of treatment. PERK levels returned to basal levels by 60 min. n = 3, p < 0.001, 1-way ANOVA with Dunnet’s post-test.

PERK

ERK

0’ 5’ 15’ 30’ 60’

71

Fig. 4.2 F-actin depolymerization may alter PCREB levels. Treatment of SCN reduced slices from mice from CT 15-16 with latrunculin A (Lat A) resulted in a trend toward an increase in PCREB by 1.19 ± 0.06 – fold at 30 min following start of treatment. n = 3, p = 0.0822, Student’s t-test, 0 vs. 30 min.

PCREB

CREB

0’ 5’ 15’ 30’ 60’

72

Fig. 4.3 Actin dynamics regulate CRE-mediated transcription. Control, untreated SCN slices from rat show a peak in spontaneous neural activity at circadian time (CT) 6.67 ± 0.08). 1-h bath application of the actin-destabilizer latrunculin A (Lat A) from CT 15-16 induces a 2.42-h delay (peak time = 9.08 ± 0.22). Incubation with CRE-decoy (from CT 14-16) blocks the phase-delaying effects of Lat A incubation (CT 15-16, peak time = 6.75 ± 0.25), while CRE-decoy alone does not shift the time of peak in this preliminary experiment (peak time = 6.92 ± 0.08). n = 3, p < 0.0001, 1-way ANOVA with Dunnet’s post-test.

control

LatA

CRE-decoy + LatA

CRE-decoy

A

Circadian Time (Hrs)

0 6 12 18 0 6 1202468

10Aver

age

Firin

g R

ate

(Hz)

02468

1002468

10120

2468

1012

control

LatA

CRE-decoy + LatA

CRE-decoy

A

73

Fig. 4.4 Actin dynamics influence clock gene expression in the SCN. Treatment of an SCN brain slice from mouse at CT 14 with Glu increases PER 2 expression by 1.32 ± 0.20-fold at 6 hr from the start of treatment, while cytochalasin D (Cyto D) induces a 1.64 ± 0.18-fold increase in PER 2 at 6 hr. Pre-incubation with the actin stabilizer jasplakinolide (Jasp) blocks the Glu-induced increase of PER 2, with a 0.85 ± 0.13-fold induction 6 hr later. A preliminary experiment found that Jasp alone does not increase PER 2 levels. n = 5 control, n = 4 Glu, n = 2 Cyto D and Jasp + Glu, n = 1 Jasp. * p < 0.05, ANOVA with Dunnett’s post-test.

74

CHAPTER FIVE

SUMMARY AND CONCLUSIONS

75

Actin has been implicated in an ever-growing list of physiological roles. As the main

component of contractile cells, actin has long been known to be critical for muscle function. It is

also ubiquitous in the cytoplasm of all eukaryotic cells, forming microfilaments of the

cytoskeleton, a function that allows for the overall shape and integrity of cells to be maintained.

Until recently this scaffolding was thought to do little more than support. More recently,

however, the actin cytoskeleton has been shown to be highly dynamic, allowing for a wide

variety of cell types to respond to various environmental and intracellular responses, several of

which are reviewed below.

In many motile types of cells, such as the protozoa species of Amoeba and Euglena, as

well as immune cells such as macrophages, actin rearrangements are responsible for movement.

F-actin polymerization occurs at the leading edge and depolymerization occurs at the lagging

edge, propelling the cell forward. This allows the cell to move toward chemoattractant

substances it encounters in the environment 122, 137.

Shape changes due to actin rearrangements are also important in the cells of the

hippocampus. When the dendrites of these neurons are activated by glutamate, a series of

signaling events leads to increased polymerization of actin in the dendritic spines, which

increases their size to allow for more contact with the stimulating cell. This is one step in the

formation of new memories 115, 116.

The microfilament scaffold is also involved in signaling. In rat hepatoma cells, a signal

of oxidative stress is mediated through actin. When the stressor activates surface receptors and

subsequent downstream signaling molecules, actin is depolymerized, which releases a bound

protein, Keap 1. Upon release from actin, Keap 1 relinquishes its hold on the transcription factor

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Nrf2. Nrf2 then translocates to the nucleus where it mediates transcription of important factors

that aid in the response to oxidative stress 123, 124.

Yet another function of the actin cytoskeleton has been demonstrated in secretory cells,

such as chromaffin cells in the adrenal medulla, β cells in the pancreas, and magnocellular

neurons of the supraoptic nucleus. These cells have been shown to release their vesicles of

peptide hormone upon rapid and transient actin depolymerization occurring just beneath the cell

membrane at the edge of the cell. This actin “net” is thought to block vesicles from reaching the

plasma membrane until a releasing stimulus, regulating release of hormone from the cell 125, 128,

129. In these experiments, stabilization of actin by jasplakinolide (Jasp) can block vesicular

release 125, while disruption of F-actin by latrunculin A (Lat A) or cytochalasin B (Cyto B) can

induce hormonal secretion 125-128, suggesting actin’s importance in secretion.

The data presented in this dissertation demonstrate a role for actin dynamics in the

circadian clock within the hypothalamic suprachiasmatic nucleus (SCN). In chapter 2 I show

that actin state oscillates in mouse SCN in a circadian fashion, which is dependent on a

functioning molecular clock. A variety of signaling components oscillate, suggesting a role in

signaling at specific times of the day. In the SCN, light in early night induces a specific set of

signaling components, ultimately resulting in a delay of the clock. F-actin levels are highest in

the SCN during early night, allowing for a robust depolymerization of the cytoskeleton at this

time of the day-night cycle. Loss of this oscillation in actin state in BMAL1 null mice may

implicate the molecular clock’s regulatory role in actin state. However, further studies may be

needed to fully characterize this regulatory mechanism. The use of another arrhythmic model

would strengthen the finding. Additionally, identification of the signaling elements that directly

mediate the change of actin polymerization state have yet to be elucidated. The number of

77

proteins that are known to bind to or indirectly regulate actin polymerization state is very large,

and likely one or more of these compounds is directed by the clock to mediate actin state. Until

these are identified, the main conclusion that can be made about the regulation of actin state is

that functional BMAL1 is necessary for the circadian oscillation.

In chapter 3, I show that light exposure induces a rapid and transient decrease of F-actin,

strengthening previous in vitro results from the lab. When this change in actin state is prevented

by direct exposure of the SCN to Jasp, the delay induced by light in the onset of wheel-running is

significantly attenuated. A delay can be induced by application in vivo of the actin

depolymerizing agent Lat A. These results also complement in vitro findings from the lab on the

effects of glutamate in the SCN brain slice. The requirement for a rapid and transient change of

actin state is reminiscent of the secretory cells of the SON. Both the SCN and the SON are

highly peptidergic, releasing peptides after stimulation in order to affect a downstream

physiological target 190, 191. While actin changes in the SON have been shown to occur at the

cortex of the cell, the intracellular location of actin changes in the SCN have not yet been

identified. These neurons are some of the smallest in the brain, with an average soma size of 5-

15 µm, 10,000 of which are packed into each dense nucleus. Together with the extensive glial

network in the SCN 16, 192, this prevents easy visualization of individual cells in the SCN,

compared to the large cells of the SON. Thus, identifying the specific intracellular location of

actin change in the SCN is difficult.

An alternative possibility for actin changes in the SCN is that the breakdown of actin

releases a bound protein, which, upon release, mediates further signaling events. Precedence for

this is seen in rat hepatoma cells, in which actin depolymerization as a result of oxidative stress

results in untethering of a transcription factor, Nrf2, which directly mediates expression of

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effector genes 123, 124. In chapter 4, I showed that light-induced actin rearrangements in the SCN

induce activation of components of the MAP Kinase pathway and CRE-mediated transcription.

Other possible components of this pathway downstream of actin have not been identified, but it

is possible that one or more molecules could bind actin or an actin-binding protein. Upon the

transient breakdown of the cytoskeleton, these would be released from actin and act on further

signaling machinery to mediate the activation of MAPK. Work is being undertaken in the lab to

identify F-actin-bound proteins that decrease their binding to actin upon Glu stimulus.

A third explanation for the changes in actin state is provided by the intense study of the

neurons of the hippocampus. In these cells, actin rearrangements result in a change of shape of

the dendrites, which, in these cells, is important for increasing synaptic strength during formation

of memory. My results with light stimulation, as well as those previously established using Glu,

indicate that activation of the light signaling pathway decreases F-actin polymerization in the

SCN. These changes were measured in whole SCN, including all cells in the nuclei. These

results do not provide any information about the cell type or location of the actin change. As

mentioned before when discussing the SON cells, examining individual cells in the SCN is

difficult due to their small size and high density. Consequently, it is not known where in the

cells actin changes are occurring, or if light or Glu stimulation results in any change of shape in

the SCN cells. Clearly, localizing the changes in actin state within cells will provide valuable

information.

In chapter 4, I began to explore the effects that actin rearrangements have on downstream

events in the SCN cells. By inducing actin filament breakdown using Lat A, I showed an

increase in ERK phosphorylation, as well as a trend toward an increaes in PCREB. I also

blocked the Lat A-induced delay in the clock using CRE-decoy. These experiments demonstrate

79

that actin changes in the cells are activating signaling events with known effects on transcription

82, 84, 90, 91. The final experiment in this chapter shows an increase in PER 2 upon cytoskeletal

breakdown using Cyto D, while stabilization of F-actin using Jasp prevented the Glu-induced

rise in PER 2. This work supports previous findings from the lab that direct actin

depolymerization by Cyto D induces per2 mRNA, while stabilization by Jasp blocks the Glu-

induced rise in per2 mRNA. These findings are the first demonstration that actin remodeling

directly affects clock gene expression. These experiments examined the clock gene Per2, which

is involved only in early-night light signaling 35, 36. The specificity of Per2 to the early night

makes it an attractive target for actin dynamics, but the effects of actin dynamics on other clock

genes cannot be ruled out.

This study has contributed new information about the role of actin in light signaling, but

further work remains in order to understand this process. As mentioned previously, isolating the

intracellular location of actin change in the SCN cells is a critical future experiment. Once the

location of actin change within the cells is known, predictions about the regulation of the actin

state as well as possible downstream effectors can be made.

Another potential study is to examine other cytoskeletal changes in the SCN. The

findings in this work measured actin state, but many other types of cytoskeletal proteins also play

important roles in various cell functions. Microtubules, composed of tubulin, are one example.

These structures play a role in the cell cycle, during spindle formation in mitosis, as well as in

axonal transport and as a structural component of cell structures such as cilia. Melatonin,

another important modulator of clock phase, has been shown to induce changes in microtubule

structure, both inducing polymerization and depolymerization depending on the presence or

absence of calcium (Ca2+), respectively 193. Conversely, microtubule breakdown regulates

80

melatonin receptors by enhancing their function and preventing desensitization 194-196.

Interestingly, another study from the Gillette lab found changes in actin state mediated by

melatonin, which are required for the melatonin-induced advances in the clock 197. If melatonin

induces changes in both tubulin and actin, it is possible that Glu does, as well. This is just one

example of a potential future study of the cytoskeleton. Neurofilament and glial fibrillary acidic

protein (GFAP) are two types of intermediate filaments in the brain 198, and how these

cytoskeletal elements participate in signaling or in clock function needs to be examined.

Figure 5.1 summarizes the light signaling pathway in early night. While many advances

have been made to identify the factors involved in this pathway, clearly, much work remains.

81

Figure

Fig. 5.1 Early night light signaling induces phase delays of the clock. RHT = retinohypothalamic tract, Glu = glutamate, NMDA-R = N-methyl-D-aspartate receptor, Ca2+ = calcium (i denotes intracellular), NOS = nitric oxide synthase, NO = nitric oxide, Ry-R = ryanodine receptor, PERK1/2 = phosphorylated extracellular-signal regulated kinases 1 & 2, PCREB = phosphorylated cAMP response-element binding protein, CRE = cAMP response element, Per1/2 = Period 1 and Period 2.

82

APPENDIX

LIGHT PULSE LENGTH DETERMINES ROBUSTNESS OF THE DELAY OF CLOCK PHASE

83

Introduction

In chapter 3 of this dissertation, I present evidence that light exposure in the early night

results in a rapid and transient depolymerization of actin state. The changes measured in actin

were seen with light pulses of 1-2 min, but by 4 min of light, no changes were seen in actin state.

However, the changes seen in actin in mice receiving 1-2 min of light did not become

significantly different until 12 experiments were completed. This is due to a subset of

experiments in which F/G-actin did not decrease upon light exposure. The 1-2 min light pulse

data presented in fig. 3.2 has been re-plotted in fig. A.1, showing the data from all 12

experiments plotted individually. These results led me to hypothesize that some mice lack a

response to such short pulses of light. These mice, termed “non-responders,” do not respond to

short light pulses as robustly as they would to standard light pulses of 5 min or longer. I have

examined wheel-running behavior and induction of a marker of activation of the light signaling

pathway, PERK, in mice receiving brief light pulses compared to longer exposure to light.

Results show variable responses of behavioral changes to short light exposure, as well as little to

no PERK induction with brief pulses of light.




ME) were used for this study. These rodents were housed under standard conditions in 12-h

light:12-h dark (L:D) cycles or constant darkness (D:D) and given food and water ad libitum.

All animals were housed and cared for in full compliance with NIH guidelines for the humane

84

treatment and care of animals and animal protocols have been approved by IACUC at the

University of Illinois.

Mice were individually housed in a cage containing a running wheel, with two cages

residing in a circadian activity monitoring system (CAMS). These are light-tight, ventilated, and

the lighting is specifically controlled for each CAMS by computer. After adjusting to the wheel,

mice were placed into these CAMS in constant darkness (D:D). Running wheel activity was

measured by rotation of the wheel. This rotation causes a magnet on the wheel to pass a

hermetically-sealed switch, which completes a circuit. Onset of activity, which typically is

designated as CT 12, was used to evaluate changes in the clock state. This information was sent

to a computer containing LabView software. The information was then plotted into an actogram

using Clocklab Analysis running in Matlab (Actimetric Inc., Wilmette, IL). The magnitude of

phase shift was determined by measuring the distance between the computer-generated

regression lines plotted against onset of activity before and after treatments.

Tissue collection and preparation

Following light pulse, mice were transcardially perfused with 50 ml cold 0.9% saline,

followed by 50 ml 4% paraformaldehyde. Whole brains from perfused mice were post-fixed in

4% paraformaldehyde overnight and then sliced into 40-µm sections using a vibratome. These

were collected into Watson’s cryoprotectant for storage at -20°C.

Immunohistochemistry

Upon thorough rinsing to remove cryoprotectant, slices were quenched in 1% H2O2/0.3%

Triton X-100-PBS for 30 min, blocked in 5% normal goat serum/0.3% Triton X-100-PBS for 1

h, and then incubated in anti-phospho-p42/p44 (PERK) antibody (Cell Signaling Technology,

Danvers, MA) in PBS containing 2% normal goat serum/0.3% Triton X-100 for 36-48 h at 4°C.

85

Slices were then incubated in biotin-conjugated anti-rabbit antibody (1.5 µg/ml) for 1 h at room

temperature before ABC conjugation and DAB staining according to directions provided by the

manufacturer (Vectastain Elite ABC kit, Vector Labs, Burlingame, CA). Staining was evaluated

using a Nikon fluorescent microscope.

Results

Brief light pulses in early night produce variable delays in behavior

In order to examine whether light pulses of 1-2 min affect individual mice differently, 4

mice received 7 light pulse treatments: 2 initial 1-min pulses, followed by 2, 4, 6, and 15 min of

bright (>500 lux) light at ZT 14. After treatment, mice were placed into constant dark conditions

(D:D), and running wheel activity was monitored for 10 days between each light administration.

Each mouse received all treatments, plus an additional 1-min light pulse as the final treatment to

test whether mice respond more robustly after running for a longer duration. The magnitude of

their responses for each dose of light is presented in figure A.2. Individual data is plotted, as

well as the averaged results. Of the 4 mice, 3 delayed in response to 1 min of light, though

variably, but the fourth advanced. A second 1-min pulse of light yielded delays of a different

magnitude in 2 of the 4 mice. Two min of light yielded the same result. By 4 min, all 4 mice

showed a delay in wheel-running onset that was approximately 0.5-hr or larger in magnitude.

Examples of each response are shown in the actograms in figure A.3. This small study indicates

that individual mice respond to short light pulses variably, but all mice respond with a delay of

equal magnitude to light pulses of 4-min or longer.

86

Brief light pulses in early night do not induce a robust induction of signaling components

A second experiment was undertaken to examine PERK induction with short light

pulses. Light pulses of 15-min in early night robustly increase phosphorylation of ERK in the

SCN (Fig. A.5 A) 82. Prior to histochemical examination of PERK, mice were evaluated for

running-wheel behavior after 2 separate, 1-min light pulses to identify potential responders and

non-responders. The results of this and representative actograms are given in figure A.4. Of the

6 mice, 2 responded to one of the 1-min LPs with a large advance in running-wheel onset, and in

all cases, there was variability between mice, and, unexpectedly, from treatment to treatment

within the same mouse.

After completion of the behavioral assays, the same mice received a 1-min light pulse of

bright (>500 lux) light at ZT 14, after which they were returned to dim red light. As controls, 6

mice were perfused in dim red light, shown to cause no changes in the clock (Arnold and

Gillette, unpublished observations), and collected as for the light-pulsed mice. Brain slices from

these mice were probed for PERK to determine if the signaling pathway in the SCN was

activated. Results from 1 representative set of mice are shown in figure A.5 B. For comparison,

results from a mouse that received a 15-min light pulse are also shown. Mice receiving 1 min of

light show very little difference in PERK staining from the dark controls. This second

experiment demonstrates that brief light pulses, while causing behavioral delays in some mice,

do not induce the signaling components as robustly as longer light pulses.

Discussion

These two small studies demonstrate the variability in response of mice to very brief light

pulses in early night. For both sets of mice, 1-min light pulses induce a range of responses from

87

advances of >1 hr to delays of >1 hr. As the length of light exposure increases, the number of

mice responding with a delay of over 30 min increases, so that with 4 or more min of light, all

mice are delaying. When activation of a signaling element, PERK, was evaluated by

immunohistochemistry, all 6 mice that were probed for PERK show low levels of PERK

induction following a 1-min LP. This suggests that the light signaling pathway in these mice is

not activated in the majority of cells in the SCN, as PERK induction after 5 min or more of light

exposure typically is robust and distributed through most of the middle of the SCN 82.

These studies were carried out after examining the individual replicates of a light pulse

experiment measuring actin change. The 12 repeats of this experiment resulted in the F/G-actin

ratio ranging from 53% of control to 112% of control levels. Even though tissue from 3 mice

were pooled for each treatment in each replicate, if one or more of the mice is a non-responder,

this could account for the variability in this experiment. Unfortunately, because actin changes

occur so quickly following activation of the signaling pathway, it is not possible to expose mice

to longer durations of light in order to guarantee a response. The study presented in this

appendix, however, helps explain a possible source of the high variability in these experiments.

88

Figures

Fig. A.1 Light induces a transient decrease in F-actin. Light pulses of 1-2 min induce a decrease in the F-:G-actin ratio of 15 ± 6% relative to non-light pulsed controls. Individual points are plotted over the average value for each time (solid line). * p < 0.05, Student’s T-test, n = 12.

89

A

B

Fig. A.2 Varying lengths of light pulse affects magnitude of delay. A. Mice #1-4 received bright (>500 lux) pulses of light of the given lengths, and then running-wheel onset was assessed for changes in clock state. B. Summary of responses for all mice. All 1-min LPs were pooled from (A) for the result shown in (B). LP = light pulse.

Mouse

1 2 4 6 15-1.5

-1.0

-0.5

0.0

min of LP

Phas

e C

hang

e (h

rs)

90

Fig. A.3 Varying lengths of light pulse affects magnitude of delay. Representative actograms from 1 mouse receiving 1-15 mins of bright light. Yellow arrow indicates light pulse treatment time, and the resulting phase change (in hrs) is given above the actogram along with the treatment. LP = light pulse.

1min LP

delay = 0.27

2min LP

delay = 1.01

4min LP

delay = 0.87

1min LP

delay = 0.77

6min LP

delay = 1.43

15min LP

delay = 1.28

1min LP after

delay = 0.58

91

B

Fig. A.4 Variable responses to short light pulses. A. Each representative actogram shows the response of 1 mouse to a 1-min pulse of bright light at ZT 14. The response is shown above each actogram in hrs. B. Summary of 6 mice for each 1-min light pulse (LP).

92

Fig. A.5 Induction of PERK by light depends on light pulse length. A. Bright light exposure for 15 min at ZT 14 induces a robust increase in PERK staining compared to a non light-pulsed control. B. Bright light exposure for 1 min at ZT 14 does not result in a robust increase in PERK induction. 1-min images are representative of 6 replicates. Scale bar = 100 µm.

Control Light pulse

15-min LP

1-min LP

A

B

93

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