Circadian Clocks in Neural Stem Cells & Their

CIRCADIAN CLOCKS IN NEURAL STEM CELLS & THEIR MODULATION OF ADULT NEUROGENESIS, FATE COMMITMENT & CELL DEATH

Astha Malik

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 2015

Committee:

Michael E. Geusz, Advisor

W. Robert Midden Graduate Faculty Representative

Roudabeh J. Jamasbi

Scott O. Rogers

Astha Malik

Michael E. Geusz, Advisor

Adult neurogenesis creates new neurons and glial cells from stem cells in the human brain throughout life, and it is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have been identified in the olfactory bulb and the hippocampus, but the role of any endogenous circadian oscillator cells in neurogenesis and their importance in learning and memory remains unclear. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from mPer1::luc mouse SVZ and

DG. Circadian bioluminescence rhythms were recorded in neurospheres maintained in culture medium that induces neurogenesis but not in one that maintains the stem cell state. Cell types were also characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Evidence was provided that neural stem progenitor cells (NSPCs) derived from the SVZ and DG of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Extremely rare percentages of mature neuronal cells were observed during ontogeny of rhythms. The bulk of the neurosphere cells were undifferentiated, indicating that they are the circadian clock cells producing timing signals. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To further test iv whether circadian clocks in NSPCs are necessary for growth, differentiation and cell survival, neurospheres were cultured from Bmal1-/- and Cry1-/-,2-/- knockout mice. Neurosheres from

Bmal1-/- knockout mice displayed unusually high differentiation into glia rather than neurons

according to GFAP and NeuN expression, respectively, and very few DCX and BetaIII tubulin- positive, immature neurons were observed. The knockout neurospheres also had areas visibly

devoid of cells and overall higher cell death. Neurospheres from mice lacking Cry1 and Cry2

showed significantly reduced growth. Altered NSPC proliferation and differentiation in these

mice may impede memory formation and could provide a way to identify circadian timing effects

in neurodegenerative disorders and impaired brain functions.

I dedicate my work to my family and my mentors, who have always supported me with words of

encouragement and perseverance. Without their love and support this would not have been

possible. vi ACKNOWLEDGMENTS

First of all I would like to thank my advisors, Dr. Michael E. Geusz and Dr. Roudabeh J.

Jamasbi for supporting me for the past four years. Dr. Geusz, is a very lively, enthusiastic and a pragmatic person. His enthusiasm, encouragement and resolute dedication to my work has kept me motivated and given me the strength to finish this project and provided me with a deeper understanding of the subject. I am also eternally thankful to Dr. Jamasbi. She is very kind, warm- hearted person who has always there when I needed her. She has nurtured me, given me advice and has helped me to become a better scientist. I am also indebted to my committee members,

Dr. Rogers, Dr. Phuntumart and Dr. Midden, whose passion for science has inspired me to take my research further to new heights. I was really lucky to find my soul-mate, Varun Singh, during my PhD training. I am really grateful to him and without his constant perseverance this would not have been possible. He has always been there to discuss frustrations- whether it be related to research or personal issues. I would also like to thank my family members (Rajni, Desh Bandhu and Arjun Malik) as well as my in-laws (Mrs. Manorma and Mr. Siddharth Singh). They have supported me with their blessings and love all throughout. They have been a great phone support too--It is funny because I used to call them at 2:00 am in the morning, on my way back home from work so that I don’t scared by the birds on the fourth floor in our building. I would also like to thank my friends in India (TNTC) and USA (Suheil Karkera, Mayank Gupta, Yury S.). I would also like to extend my regards to Deedee Wentland, Susan Schooner, and Chris Hess for their assistance with paper work. I would also like to thank Dr. Carol Heckman, Dr. Cayer for their guidance and assistance with the confocal and the inverted fluorescence microscope in the imaging facility, Dr. Paul Moore for helping me with statistics. Last but not the least; I would like to thank God for this opportunity and all my extended family members for their help. vii

TABLE OF CONTENTS

Page

PREFACE…………………………………………………………………………….…..... 1

CHAPTER I: INTRODUCTION ...... 3

Circadian rhythms...... 3

Peripheral oscillators ...... 6

Embryonic and adult neural stem cells ...... 8

Adult neurogenesis...... 9

Factors influencing adult neurogenesis ...... 11

Adult neurogenesis in the SVZ……………………………………….……………... 12

Adult neurogenesis in the DG ...... 14

Importance of neurogenesis ...... 17

Circadian rhythms and the cell cycle ...... 18

Circadian rhythms and adult neurogenesis ...... 22

CHAPTER II: DEVELOPMENT OF CIRCADIAN OSCILLATORS IN NEUROSPHERE

CULTURES DURING ADULT NEUROGENESIS ...... 24

Introduction …………………………………………………………………...... 24

Materials and methods…………………………………………………………….. . 27

Animals …………………………………………………………………….. 27

Neurosphere cultures…………………………………………………….. ... 27

Stem cell markers and confocal microscopy…………………………...... 28

Neurosphere bioluminescence imaging………………..…………………… 29

Data analysis………………...…………………………………………….. . 30 viii

Results………………...……………………...... 31

Circadian rhythms are rare in neurospheres maintained in SCM………….. 31

Circadian rhythms in mPer1 gene expression emerge in neurosphere during

differerentiation in SM or B27 medium …………………………………… 34

Stem cell state decline following transition to differentiation inducing

environments………………...…………………………………………….. . 38

Forskolin synchronizes circadian clocks within the neurospheres………. ... 43

Discussion…… ...... 45

Initiation of circadian rhythms during differentiation………………...… .... 45

Origins of neurosphere circadian rhythm ...... 47

Circadian rhythms in progenitor cell ...... 48

Possible importance of mPer1 in neurogenesis ...... 50

Conclusions…...……………………...... 52

CHAPTER III: CIRCADIAN CLOCKS ARE ESSENTIAL FOR NORMAL ADULT

NEUROGENESIS IN VITRO ………………………...... 55

Introduction …………………………………………………………………...... 55

Materials and methods ...... 58

Animals ...... 58

Neurosphere cultures ...... 58

Neurosphere bioluminescence imaging ...... 59

Immunohistochemistry ...... 60

Live/Dead stain ...... 60

Data analysis ...... 61 ix

Results………………...……………………...... 62

Circadian rhythms appear when neurospheres are allowed to

differentiate………………………………………………………………… 62

Circadian clock proteins are required for normal neurosphere

formation…………………………………………………………………… 63

Absence of circadian clock proteins result in greater cell death ...... 64

Circadian clocks proteins are required for normal neurosphere growth and

proliferation...... 65

Bmal1 is essential for neuronal fate commitment...... 66

Discussion………………...……………………...... 68

Circadian clocks in DG neurospheres ...... 68

Circadian clock proteins and neurosphere growth and formation ...... 72

Circadian clocks and neural differentiation ...... 74

Insights from DG neurospheres ...... 75

Clock, neurogenesis, and memory formation ...... 77

CHAPTER IV: CONCLUSIONS AND FUTURE DIRECTIONS ...... 86

Summary………………………………………...………………………………….. 86

Future directions……………………………………………………………………. 88

QNSCs the mysterious “kahuna”...... …... 88

Fate commitment in adult neurogenesis- “The road not taken” ...... 91

Circadian properties of NSPCs and clinical aspects...... 93

REFERENCES ………………………...... 97

APPENDIX A: LIST OF ABBREVIATIONS ...... 109 x

APPENDIX B: IACUC APPROVAL ...... 110 xi

LIST OF FIGURES

Figure Page

1 The molecular circadian oscillator ...... 5

2 The mammalian circadian timing system ...... 7

3 A diagram representing a sagittal section view of adult mouse brain ...... 10

4 Neurogenesis in the SVZ ...... 13

5 Neurogenesis in the subgranular zone of the hippocampus ...... 16

6 Circadian modulation of the cell clock ...... 21

7 Changes in mPer1 expression from ultradian to circadian during neurosphere cell

differentiation………………………………………………………………….…… 33

8 The rhythmic state of spheres during early and late exposure to three culture

conditions ………………………………………………………………….……….. 37

9 Emergence of circadian rhythms before fully differentiated neurons appear ...... 39

10 Synchronization of circadian neurospheres ...... 44

11 Images of spheres during differentiation ...... 53

12 Circadian rhythms during neurogenesis...... 54

13 Neurospheres display circadian rhythms in mPer1 gene expression during early

differentiation ……...……………………………………………………………… . 79

14 NSPCs are the dominant cell types during the first four days of DG neurosphere

differentiation ……………………………………………………………………… 80

15 Bmal1-/- neurospheres show altered growth patterns and increased cell death ...... 81

16 Cry1-/-, 2-/- neurospheres have reduced proliferation and growth in culture ...... 82

17 Neuronal commitment is diminished in Bmal1-/- DG neurospheres ...... 83 xii

LIST OF TABLES

Table Page

1 Summary of neurosphere circadian periods ...... 35

2 Cell types identified by markers for stem cells and differentiated cells in SM ...... 41

3 Cell types identified by markers for stem cells and differentiated cells after 1 or 4 days in

serum medium ……………………………………………………………………… 84

4 Percent positive mature neuronal and glial cells after 14 days of differentiation in B27

medium……………………………………………………………………………… 85 1

PREFACE

In this dissertation I will bring together concepts from developmental neuroscience, circadian rhythms, and learning and memory to understand the selective advantage provided by the circadian clock in the process of new neuron formation. Chapter One provides an overview of the circadian clock and the molecular players involved in its regulation. I will also discuss neural stem cells and new neuron formation in the adult brain after early embryonic development has ceased. Chapter Two describes how I identified and characterized circadian rhythms in neural stem cell populations derived from one stem cell-rich region of the mammalian brain—the subventricular zone. These unique circadian properties could enable targeting of neural stem cells with treatments at different times of the day to stimulate cell differentiation for repair and regeneration of the brain. Chapter Three presents my studies of another stem cell-rich brain region—the subgranular zone—and effects on neurogenesis from disruption of the circadian clock. Interestingly, I found a strong circadian regulation of gene expression in cells during neurogenesis. In vitro neurogenesis was also examined in two mutant mouse strains that lack a functional circadian clock and show deficits in cell survival and differentiation. Chapter Four brings together concepts and conclusions from this body of work and provides suggestions for future experiments on how biomedical approaches to healing the brain could benefit by exploiting clock control of neurogenesis.

There is a large body of literature on how circadian clocks might regulate the cell cycle in any tissue, but surprisingly not much is known about circadian regulation of adult neurogenesis which relies on cell proliferation. Reports have indicated the presence of a nonfunctional

2 circadian clock in embryonic stem cells, which to me was not unforeseen because a cell cycle synchronized to a circadian day might prove lethal for the fast-developing embryo that requires rapid cell proliferation. Nevertheless, I questioned whether a circadian clock, perhaps desynchronized from mitotic cycles, could perform important functions during the early stages of adult neurogenesis. Studies have also indicated that sensory stimuli acting as cues during learning can stimulate new memory formation more effectively when applied to mature mice at night. This possibility merited an exploration into whether neural stem cells and new neuron formation are regulated by the circadian clock.

CHAPTER I: INTRODUCTION

Circadian rhythms

The term “circadian” is derived from the Latin word “circa” and “dies” which mean

“about a day” and was first coined by Franz Halberg in 1959. Circadian rhythms are endogenous,

approximately 24-hr oscillations in behavior and physiology of an organism that function in

predicting changes in the environment in relation to the time of day [1]. Circadian rhythms

appear to have evolved to provide selective advantage to the animal by optimizing survival rate

and fitness. For example, it is most beneficial for rodents who are foraging for food or seeking

mates to do so in the dark, minimizing exposure to predators. At a much smaller level of

biological organization, the KaiC protein of cyanobacteria forms a hexamer during the day, most

likely to prevent DNA damage from daily UV radiation [2].

The master clock in mammals is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. The SCN contains approximately 20,000 neurons and has a “core” and

“shell” that serve different functions. The SCN core receives input from axons of the retinohypothalamic tract that carries signals regarding changes in ambient light cues detected by the eye. That information is then passed to cells in the shell that have autonomous circadian oscillations in neural firing rate and provide circadian timing information to the rest of the animal. Without this external light information that entrains the circadian clock to the 24-hr cycles of the environment, SCN cells exhibit a free-running rhythm with a period slightly different from 24 hours that is both genetically and developmentally determined [3, 4].

Circadian rhythms regulate various physiological functions such as the sleep-wake cycle and daily energy metabolism in mammals. Molecular oscillations of circadian clocks depend on

two interacting transcription/translation feedback loops (TTFL) that involve various clock and

clock-controlled genes. Levels of a heterodimer of the transcription factors CLOCK and

BMAL1 increase and bind to the E-box (CACGTG) or similar regulatory elements in the promoter regions of various genes. This step activates clock-controlled genes (CCGs) to carry out various rhythmic functions and also activates core clock genes such as Period (Per1, 2, and

3) and Cryptochrome (Cry1 and Cry2). Per and Cry mRNA in the cytoplasm are translated into

PER and CRY proteins. These proteins heterodimerize and translocate back into the nucleus [5] where they suppress their own transcription through negative feedback on the activation provided by CLOCK and BMAL1 activity, thereby decreasing their own production [6].

Adapted from: Kondratov et al., Nature Review Neuroscience [6].

Figure 1: The molecular circadian oscillator

Shown are the positive elements of the mechanism (red) and negative elements (yellow).

Transcription factors BMAL1 and CLOCK control activity of genes that activate an E-box or related element in promoter regions. BMAL1 and CLOCK heterodimerize and induce transcription of Period and Cryptochrome genes. PER and CRY proteins form complexes that inhibit their own transcription, representing the negative element of the TTFL. The transcription factor Rev-Erbα/β negatively regulates and RORα positively regulates BMAL1 expression.

Finally, the BMAL1: CLOCK complex regulates expression of circadian clock-controlled genes

(CCGs) that further control behavior and physiology of the organisms.

Peripheral oscillators

Autonomous rhythmic gene expression has been recorded in the peripheral tissues of

mammals, which includes the entire body and areas of the brain outside the SCN [7, 8]. The

TTFL can be observed in nearly every mammalian tissue [8, 9]. Without the input from the SCN,

peripheral circadian rhythms fade and tend to gradually disappear in these tissues. Hence, these

oscillators are termed “slave oscillators” [8, 10]. Studies have also shown that transplanting the

SCN region from one animal into SCN-lesioned animal results in restoration of behavioral rhythmicity with the period of the transplanted tissue, indicating that the SCN is not merely needed for rhythmicity but also provides the critical timing signal [7, 11].

The master circadian clock in the SCN uses several pathways to communicate with other

rhythmic tissues and orchestrate coherent rhythms at the organismal level. The SCN can communicate with other brain regions via chemical synapses [12, 13] and humoral signals [14].

Peripheral oscillators respond to SCN-driven timing cues through hormones and neural circuits

[15, 16]. To summarize, the SCN entrains to environmental light cycles as timing cues that synchronize its clock to the local time of day, and peripheral clocks entrain to complex combinations of SCN signals, thereby matching their intrinsic period to rhythmic entraining signals.

Adapted from: Gi Hoon Son et al., Frontiers in Neuroendocrinology [17].

Figure 2: The mammalian circadian timing system.

Signals from the SCN, the central circadian clock residing in the ventral hypothalamus, synchronize peripheral oscillators directly via rhythmic neural signals or hormones such as

glucocorticoids and indirectly via activity-dependent mechanisms such as body temperature,

metabolism, or food availability. The SCN thereby coordinates optimal functioning of clocks

throughout the body.

Embryonic and adult stem cells

During development, the zygote formed by fertilization of the egg is a totipotent, ultimate

stem cell that can produce an entire organism. The zygote divides further to form the morula and

then blastocyst stages. The inner cell mass of the blastocyst contains embryonic stem cells that

are pluripotent. Pluripotency is defined as the ability of cells to generate all body tissues except

the trophoblast (which gives rise to the placenta). During embryogenesis, germinal layers are

formed. Each germ layer (ectoderm, mesoderm, and endoderm) contains multipotent stem cells

that can give rise to mature cells of at least two or more lineages. Adult stem cells are therefore a

very specialized cell type that exhibits properties similar to cardinal features of embryonic stem

cells. One such property is the ability to proliferate and self-renew, through symmetrical cell division, and another is differentiation into mature cell types (to an organ or tissue-specific cell

type with a specific function) by dividing asymmetrically. These adult stem cells are rare and

usually found after development has ceased. Stem cells differentiate into progenitor (precursor)

cells, an intermediate cell type that further differentiates into a fully mature cell. Progenitor cells

are typically fate-committed to differentiate into a certain cell type and give rise to mature cells

during development [18, 19].

Neural stem cells (NSCs) by definition are multipotent stem cells and are found in the

embryonic neuroectodermal layer. In the adult they play a role in repair and regeneration and can

be subdivided into quiescent NSCs (qNSCs) and activated NSCs that are dividing symmetrically

or asymmetrically. NSCs can be isolated from the adult brain, cultured and maintained in vitro as

neurospheres, which are free floating three-dimensional spheroids formed from thousands of

cells. Neurospheres exhibit self-renewal and differentiation into new cell types in either the brain

or cultures and also provide a useful assay for studying various biological properties applicable to regenerative medicine [20].

Adult neurogenesis

Cell division and differentiation studies in the adult brain have greatly benefited from

autoradiographic detection of thymidine analogs, which incorporate into the DNA of dividing

cells. For decades it was believed that the brain composition is fixed and we die with the same

neurons that were present earlier in development [21]. This belief was challenged in the 1960’s

by the discovery of repair and regeneration in the adult brain by Altman, Das, Kaplan, and Hinds

using sophisticated electron microscopy data along with the technique mentioned above [22-24].

Adult neurogenesis is a process of new neuron formation from NSCs, which are the

somatic stem cells in the specialized niches of the brain. These regions have specialized

microenvironments that harbor and maintain NSCs for continuous production of new cells

throughout life. The SVZ (subventricular zone) and the SGZ (subgranular zone) have been

recognized as two major stem cell niches in the adult brain. NSCs are multipotent and were first

derived from the striatum region of the adult mouse brain in 1992 [25, 26].

Neurogenesis is a type of neural plasticity that produces new neurons such as granule

cells and interneurons in the hippocampus, olfactory bulb (OB), and other brain areas after

postnatal development has ceased [27]. Neurogenesis is a flexible process that is accelerated in

response to brain injury, exercise, dietary nutrients, and environmental cues such as odorants that

indicate challenges to survival or opportunities for feeding, mating, or defense of territory [28-

30].

Adapted from: Guo-li-Ming et al., Neuron [27]

Figure 3: A diagram representing a sagittal section view of adult mouse brain.

The two areas actively involved in adult neurogenesis are highlighted: the dentate gyrus (DG) in the hippocampal (HP) formation and the subventricular zone (SVZ) that lines the lateral ventricle

(LV). RMS: rostral migratory stream.

Factors influencing adult neurogenesis

New neuron formation is regulated by many factors including endogenous substances, environmental exposure to various compounds, and the physical, mental, and emotional state of the mind and body [29, 31-34]. Several studies have shown that wheel-running or treadmill

activity increases neurogenesis in male mice [35, 36]. Physical exercise during pregnancy has

also been shown to promote memory formation and elevate levels of neurotrophic factors in the

offspring [37]. The ability of exercise to induce adult neurogenesis results from multiple causes,

but several growth factors play important roles [38, 39]. Growth factors such as bone

morphogenic protein (BMP), brain derived neurotrophic factor (BDNF), fibroblast growth factor

(FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and ciliary

neurotrophic factor (CNTF) in the highly vascularized stem cell niche play a major role in

augmenting or curtailing the process of neurogenesis and survival of newly formed neurons

[40].

Environmental enrichment and exposure to novel objects can also enhance the survival of

newly generated cells [41]. New neuron formation in the brain is positively modulated by the

hormone melatonin, endogenous cannabinoids, flavonoids, vitamin E, resveratrol, folic acid,

curcumin, omega-3 fatty acids, and other factors. On the other hand, the rate of neurogenesis is

reduced by deficiencies in vitamin A, thiamine, folate, or zinc, or by high intake of fat, fructose,

ethanol, or capsaicin [42-45]. Inflammatory signals in the brain have also been shown to alter the

rate of neurogenesis. For example, TNF-alpha, an inflammatory mediator of NFkB, increases

NSC proliferation and neurosphere formation in vitro. NFkB is activated by LPS

(lipopolysaccharide) and epigenetically alters the NSC population and cell aggregation in vitro

[46-48].

Adult neurogenesis in the subventricular zone

Stem cells residing in the wall of the lateral ventricles proliferate and differentiate into neuronal cells which then migrate a relatively long distance to reach the OB. Neurogenesis is a complex process involving multiple molecular players to function synchronously and carry out the process effectively. During neurogenesis, the proliferating radial glia-like cells (NSCs) give rise to transit-amplifying cells (TAPs). TAPs further differentiate and give rise to neuroblasts that migrate as a chain to the OB by way of the rostral migratory stream (RMS), a tube lined with astrocyte-like cells. Upon reaching the OB, immature neurons disengage from the RMS and migrate radially and differentiate into OB interneurons [27, 49-52].

The OB relays olfactory sensory information to the olfactory cortex via mitral and tufted cells (MTCs). MTCs form dendrodendritic, glutamatergic, excitatory synapses on granule cells

(GCs), and GCs form GABAergic, inhibitory synapses on MTCs. MTCs are not replaced postnatally, but GCs and periglomerular cells (PGCs) are generated throughout life through neurogenesis. Survival of new GCs depends on the presence of appropriate olfactory signal experience during a critical time window 14-28 days after their generation [53-57].

In the OB, newly formed GCs form inhibitory synapses and outnumber MTCs, which

may enable olfactory circuits to refine odor discrimination signaling. Studies have also shown

that new neurons in the OB have enhanced synaptic plasticity, and a continuous supply of new neurons is essential for integration of odor memory with emotional significance into preexisting circuitry. Adult neurogenesis in the OB is also linked to reproductive behaviors and pheromone- associated behaviors, thereby aiding the animal’s survival [58-62].

Adapted from: Guo-li-Ming et al., Neuron [27]

Figure 4: Neurogenesis in the subventricular zone

Developmental stages and protein expression (stage-specific markers) during adult neurogenesis in the SVZ are highlighted. Activated radial glia-like cells in the SVZ differentiate into TAPs, which further differentiate into neuroblasts and other immature neuronal cells before forming interneurons (GCs and PGCs) in the OB.

Adult neurogenesis in the subgranular zone

The other major neurogenic region in the brain is the subgranular zone (SGZ) of the hippocampus. The neural stem cells lie in the narrow band of tissue between the granule cell layer and hilus, a highly vascularized microenvironment that maintains these cells. This niche is

located within the dentate gyrus (DG) of the SGZ [63].

Neurogenesis in the DG is governed by many factors including VEGF. During

neurogenesis in the DG, radial glia-like cells ultimately differentiate and give rise to granule

cells, the principal excitatory neurons. This process takes about 7 weeks in total. Granule cells

receive inputs from the entorhinal cortex and send projections to excite the pyramidal cells in the

CA3 region. The DG receives various other synaptic inputs from different brain areas such as

dopaminergic input from the ventral tegmental area, serotonergic input from the raphe nucleus,

and GABAergic input from local interneurons [19].

During neurogenesis, many factors are responsible for determining the fate and lineage

choice of the neural stem progenitor population. Tailless (Tlx) is one such transcription factor

that plays a key role in the transition from a glial to a neuronal cell fate [64, 65]. SIRT1 has also

been considered important for neuronal lineage commitment by inhibiting the Hes1 signaling

pathway. SIRT1 regulates proliferation and differentiation in the astrocytic lineage when the

redox status of cells is altered as under hypoxic conditions [66]. Some of these factors will be

discussed later in detail.

Radial glia-like cells differentiate into progenitor cells, which are the intermediate

precursor cells (Type 2a). These cells express glial markers but lack a glial morphology. At this

stage these cells commit to a particular fate and differentiate into a particular lineage. For

example, Type 2a cells differentiate into Type 2b cells (neuroblast-like cells, positive for

doublecortin X immunostaining) and then further into neuronal cells. Excitatory GABAergic synaptic input controls the newly formed cells, which then make contact with cells in the CA3 region after approximately 10 days. It takes several weeks before newly formed cells integrate into the existing circuitry. These nascent neurons exhibit a lower threshold for firing and long- term potentiation, which then helps to encode information and make fine distinctions between different spatial and temporal signals [61, 67, 68].

Adapted from: Guo-li-Ming et al., Neuron [27]

Figure 5: Neurogenesis in the subgranular zone of the hippocampus

Developmental stages and protein expression (stage-specific markers) during adult neurogenesis in the SGZ are highlighted. Quiescent radial glia-like cells are activated and undergo asymmetric cell division to give rise to progenitor cells [69], which further differentiate into neuroblast and immature neuronal cells before final mature neurons are formed.

Importance of neurogenesis

A causal relationship between increased hippocampal neurogenesis and improved

cognitive performance has been established. Many researchers have shown a correlation between

increased basal level neurogenesis in rodents and improved performance of memory-associated

tasks following treatment with neurogenesis-inducing factors (described above). Some studies,

however, report the contrary [70]. Improvements have been reported in acquisition and retention of memory in the Morris water maze (MWM) test, radial arm test, spatial memory consolidation, fear-conditioned memory, contextual fear memory, olfactory perceptual memory, and pattern separation following increased neurogenesis [71-74].

Selective ablation of neural stem cells in transgenic animals or depletion of the stem cell

pool by anti-mitotic treatments has been shown to impair place and object recognition memory

as well as contextual fear-conditioned memory. New neurons produced in the OB have been

shown to be critical for odor discrimination tasks, odor memory, and learning [34]. Overall,

neurogenesis in both the SVZ and DG has adaptive significance for animals and is likely

important for survival when, for example, avoiding predators, finding food, identifying mates,

nurturing progeny, and performing homing behavior

Recently it has been found that adult neurogenesis also persists in the human brain, and

impairment or alteration of neurogenesis has been widely associated with neuropsychiatric

disorders such as depression, schizophrenia, or epilepsy and neurodegenerative diseases such as

Parkinson’s disease [75, 76]. Pharmaceutical treatments of depression such as selective

serotonin reuptake inhibitors and other disorders may act in part by altering neurogenesis.

Despite our understanding of how newly formed neurons integrate into existing circuitry, exhibit

a low firing threshold, and thus show greater synaptic plasticity, we still lack an understanding of how hippocampal or olfactory bulb neurogenesis contributes to animal behavior.

Circadian rhythms and the cell cycle

It has been argued that evolutionary selective pressures produced a partitioning of cell

cycle phases so that they occur at a particular time of day, perhaps to synchronize DNA

replication, cell growth, and proliferation most efficiently with nutrient availability or behavioral

patterns. Several studies describe how cell cycles are modulated by the circadian clock. The clock modulates expression of various CCGs that can activate or repress other genes depending

on the tissue type. Studies have shown that up to 43% of protein coding genes oscillate cyclically

and more than half of the coding transcriptome in the body is circadian [77]. A complete

description of the cell cycle and its regulation is beyond the scope of this dissertation. However,

a brief overview of circadian modulation at different phases of the cell cycle is provided.

Control though gating by the circadian clock has been shown at many key checkpoints of

the cell cycle. For example, the DNA-replicating phase in the cycle is regulated by CCGs p20

and p21. Another CCG, Wee1, contains three E-box elements and regulates the G2/M transition

in the cell cycle by phosphorylating and inactivating the CDK1/Cyclin B complex. Circadian

clock proteins such as Period and Timeless are essential in the cell cycle for activating

checkpoint kinases Chk1 and Chk2 through ATM (ataxia telangiectasia mutated) and ATR

(ataxia telangiectasia and Rad3-related). These proteins cause cell cycle arrest during DNA

damage responses, thus providing time for repair.

Many cancer cells are shown to have a disrupted or altered circadian clock.

Overexpression of circadian genes causes cancer growth inhibition, and there are many possible

explanations for this result. For example, overexpression of Per2 leads to reduction in Wnt-β-

catenin activity, and overexpression of the clock protein CK1ε results in higher expression of

Cyclin A and Cyclin B. Cyclin D1 has also been shown to be under clock regulation. Period proteins inhibit transcription of c-myc and thereby promote Cyclin D1 expression, leading to increased cell proliferation [78-82].

Entry or exit from the cell cycle is tightly regulated in the organism by its circadian

timing mechanism. In mice, a peak in M phase entry is predicted to occur near the onset of

behavioral activity during the early night. When applied to humans the peak in mitotic events

coincides with our activity rhythms [83-85]. Modern lifestyles (environmental factors, altered

sleep, diet, etc.) are frequently associated with disruptions in the circadian timing mechanism

that can increase risk of developing various metabolic disorders, early onset of aging, and

neurodegenerative diseases such as Alzheimer’s disease and psychiatric disorders [86-90].

Circadian deregulation has been widely associated with susceptibility and progression of certain

cancers. For example, higher incidences of breast cancers and non-Hodgkins lymphoma are

reported in night-shift workers [91, 92].

Circadian clock genes can also have non-clock functions, and as mentioned above their

proteins serve a role in cell cycle regulation. Therefore clock and non-clock functions could

contribute to the observed associations between the clock and diseases. Nevertheless, the

accumulated evidence on disruption of circadian timing warrants the use of

chronopharmacotherapy for treatments in which the phase of the circadian cycle is taken into

consideration when designing patient therapy regimes. Specifically, efforts to exploit the

circadian clock should be made when developing and administering drugs to target cancer cells

20 at their most sensitive phase or to avoid phases of non-cancerous tissues when greatest cell damage can occur.

BMAL1/Clock RORa

WEE1 p21

CRY CDK2/ Cyclin E CDK1/ Cyclin B REV-ERB M p21

PER1 Cell G G1 X Cyclin D1 X c-myc BMAL1 2 Cycle PER2

G0 PI6ink 4a PER1 NONO S PER ATM Chk 2 P53

ATR Chk 1 CDK 4/6 Cyclin D P21 TIM

Modified from: Borgs L. et al., Cell Cycle [81]

Figure 6: Circadian modulation of the cell clock

Circadian regulation of the cell cycle is shown. G1/S, S/G2 and G2/M transitions in cell cycle are under tight regulation by the circadian clock proteins (Period, Rev-erbα, Rorα, BMAL1, and

Timeless) and clock-controlled genes (p20, p21, cyclinD1, Wee1), although the cell cycle can also disengage from the circadian clock. This figure was contributed by Shannon Turner, a work- study student. X: suppressed cyclin D1 activity by Bmal1 overexpression. Green: activation,

Red: inhibition.

Circadian rhythms and adult neurogenesis

The scientific and medical literature provides major insights into how circadian rhythms likely regulate neurogenesis. Three major signaling pathways have been deemed important for

stem cell maintenance and differentiation—the Wnt-β-catenin, Notch, and sonic hedgehog

pathways. Studies have shown that a balance between these pathways is essential for stem cell

maintenance and differentiation [93-95].

Signaling pathways consist of many effector genes that are members of the basic-helix-

loop-helix (bHLH) family of transcription factors. For example, the HES-1 bHLH protein

regulates early development of the brain by controlling the temporal pattern of cell division and

differentiation. The Hes-1 gene is required for self-renewal of adult and embryonic neural stem

cells by repressing their differentiation and allowing them to divide symmetrically. It is also

produced in other stem cells of the adult brain and assists with cell replacement [96, 97]. Hes-6 is

an inhibitor of Hes-1 that also serves a major function in neurogenesis [98]. During early

development of the nervous system, HES-1 levels oscillate in cells with a period of about 2-3

hours [99, 100]. Controlling HES-1 activity in the adult through regulation by the circadian

clock may be possible and also provides an opportunity for modulating effects of Hes-6. This

intervention might be further manipulated to elicit repair and regeneration of the damaged brain

more effectively at a particular time of day.

Another member of the bHLH gene family that may be under circadian control is

NeuroD1, which contains nine E-boxes in its regulatory region and is also essential for

development of the CNS, particularly for granule cell generation in the hippocampus and

cerebellum. NeuroD1 overexpression is sufficient to promote neuronal differentiation in adult

hippocampal neural progenitors, whereas its deletion results in decreased survival and maturation

of newborn neurons [101].

In summary, this dissertation is focused on circadian modulation of neurogenesis. Studies

have shown increased mitotic entry and neuron formation at night in rodents. DNA replication

mostly occurs during the daytime in mice housed in a 12 h light/12 h dark (LD) cycle [84]. The

most sensitive phase for NSC survival appears to be when DNA replication occurs, and this is synchronized to occur at the time when the animal is resting to avoid damage to replicating

DNA. In animals housed in LD cycles, cells constantly receive input from Zeitgebers (time- givers) originating in other clocks within the body. It is not clear, however, whether the circadian clock regulates neurogenesis through these factors or whether an endogenous stem cell clock plays a more direct role in regulation. It remains important to distinguish internal cell clocks from the various timing inputs that act on them. Only then can endogenous cellular timing signals be identified and distinguished from those generated elsewhere.

This project examined isolated neurospheres derived from the SVZ and DG to identify any endogenous circadian oscillations in neural stem progenitor cells (NSPCs) consisting of

NSCs and uncommitted progenitor cells (excluding neuroblasts). The hypothesis tested was that

NSPCs would not show circadian oscillations in mPer1 gene expression, as has been described for embryonic stem cells and other undifferentiated cells reported to lack a functional circadian clock [102, 103]. The results indicate that this hypothesis can be rejected because of distinct circadian oscillations in clock gene expression that were identified for the first time in adult neurospheres. Furthermore, results using neurospheres derived from knockout mice that fail to express circadian rhythms indicated that absence of core clock proteins significantly alters neurogenesis.

CHAPTER II: DEVELOPMENT OF CIRCADIAN OSCILLATORS IN NEUROSPHERE

CULTURES DURING ADULT NEUROGENESIS

Preface

This chapter was originally published as a research article in PLoS ONE March 2015

[104]. The original publication has been altered to meet BGSU’s specifications for formatting a dissertation. This publication identifies NSPCs in the SVZ and neuroblasts in the RMS to be circadian clock cells.

Introduction

Adult neurogenesis produces new neurons from neural stem progenitor cells (NSPCs).

This neural plasticity provides interneurons for the mammalian hippocampus, olfactory bulb

(OB), and other brain structures throughout life [27]. NSPCs follow a defined progression in cell differentiation that is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) near the lateral ventricles [105]. A daily rhythm in cell cycle entry of stem cells has been described in the adult mouse hippocampus [70], indicating that circadian pacemakers may regulate NSPC differentiation. Similarly, circadian gene expression rhythms have been identified in the hippocampus [106] and OB [107], possibly serving to optimize timing of neurogenesis [70] by providing more responsive cells when they are most needed for fine discrimination of sensory information [34]. Adult neurogenesis in many ways follows the behavior of embryonic stem cells, which undergo self-replication and also differentiate into progenitor cells that eventually give rise to various mature cell types [108]. Adult neural stem cells in the SVZ self-renew and produce neurons and glial cells sequentially through several

25 differentiation stages that appear transiently during neurogenesis and have identifiable cell markers [34].

Although in situ hybridization has shown that expression of the core circadian clock gene mPer2 oscillates in the mouse DG [109], what generates the circadian timing signal is unknown.

It remains unclear whether circadian rhythms occur in the heterogenous population of differentiating cells, mature neurons, or the mostly quiescent stem cells. The NSPCs of the DG may contain intrinsic circadian pacemaker capabilities. They may instead be driven by circadian pacemakers located in other cells within these brain regions or clocks elsewhere in the organism

[110, 111]. Bioluminescence imaging (BLI) of hippocampal explant cultures has revealed circadian rhythms in mPer2 expression indicating that autonomous circadian clocks are present

[106], but the source of the timing signal within this tissue has not been localized further. Daily rhythms in expression of a second clock gene Per1 in the intact DG are in phase with rhythms of the master circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) [112], suggesting that any NSPC circadian clocks within the DG, or possibly the SVZ, may also be coupled with the circadian timing system.

Circadian rhythms expressed in mouse or rat OB can function independently of the SCN

[111]. These oscillations appear to enhance olfactory responsiveness at night [111] and also interact with the SCN’s timing of daily behaviors [113]. Circadian rhythms in mPer1 and mPer2 gene expression are present in the mitral and tufted cells of the rat OB and the granule and mitral cells of the mouse OB [114]. Late embryonic neurons from the rat OB express circadian rhythms in action potential frequency [107]. Unlike the DG, progenitor cells of the SVZ produce immature neurons that migrate from the SVZ through the rostral migratory stream (RMS) to become interneurons of the OB [115]. Various sensory stimuli modulate OB neurogenesis. For

26 example, OB granule cells in mice undergo apoptosis at a higher rate following daily scheduled feeding [116], and olfactory cues must be available during a critical window for granule cell maturation between 2 and 4 weeks after neurogenesis in the SVZ [117]. Recently, it has been shown that suckling by pups synchronizes circadian rhythms in the OB of the dam [118].

Embryonic neural stem cells and differentiating stem cells of the adult testis lack detectable circadian rhythms [102, 119]. One possible explanation for this absence is the activity of stemness-maintaining genes producing factors that suppress differentiation. These gene regulators may not be compatible with functions of proteins such as mPer1, mPer2, or BMAL1 that serve in the circadian timing mechanism. As reviewed by Gimble et al., [120] studies suggest a close relationship between circadian and stem cell biology through hypoxia-induced transcriptional regulators [121, 122], chromatin remodeling enzymes [123], the cell cycle inhibitor p21WAF/CIP1 [124, 125], and Wnt signaling [126-128].

To determine when circadian rhythms first appear during adult neurogenesis, in relation to sequential differentiation events, we used a well-characterized paradigm of in vitro adult neurogenesis and applied BLI to monitor mPer1 gene expression continuously in mouse SVZ neurospheres. These non-adherent clusters of stem cells and progenitor cells in many ways resemble cells undergoing neurogenesis in vivo [129]. Neurospheres were induced to form in suspension cultures containing stem cell medium (SCM) that is devoid of serum but includes epidermal growth factor (EGF) and basic-fibroblast growth factor (FGF2) to suppress differentiation. An exchange with serum-containing medium (SM) or medium containing the serum supplement B27, without added EGF or FGF2, stimulates neurospheres to differentiate and attach as they transform into cell culture monolayers [130]. We describe a correlation between differentiation state of these neural stem cells and their circadian rhythm status.

Materials and Methods

Animals

Transgenic mPer1::luc mice expressing firefly luciferase under control by the mPer1 gene promoter [130] were bred and maintained in cycles of 12 h light and 12 h dark to entrain their circadian system. Animal procedures were approved by the BGSU Institutional Animal

Care and Use Committee and met the requirements of the NRC Guide for Care and Use of

Laboratory Animals.

Neurosphere cultures

Adult male or female C57BL/6 mice (3-5 months old) were euthanized using isoflurane.

Brains were removed quickly and coronal slices were made with a Brain Blocker (PA 001 Rat;

David Kopf Instruments, Tujunga, CA, USA) and the SVZ region was dissected. The tissue was washed 4-5 times in cold HBSS and then enzymatically digested with papain and DNAseI

(Worthington Biochemical, Lakewood, NJ, USA) for 25-30 min at 37°C, followed by 2-3 washes in DMEM with no added growth factors. The tissue was then mechanically triturated and passed through a 40 μm cell sieve (Falcon; BD Biosciences Discovery Labware, Bedford, MA,

USA). The cell suspension was washed and centrifuged for 5-6 min 4 times. The supernatant was discarded and the pellet was re-suspended in SCM, which consisted of DMEM with 10 ng/ml

FGF2, 20 ng/ml EGF (Life Technologies, Grand Island, NY, USA). Cells were plated at a density of 2.0-2.5 x 104 cells/ml in SCM. After 4-6 days, neurospheres were observed, as described in a previous study [131]. Between 7 and 10 days in culture, neurospheres of at least

50-µm diameter were collected along with the entire contents of the dish and centrifuged for 5

min at room temperature. The pellet was resuspended in 5-7 ml of SCM medium, triturated to form a cell suspension, and plated in fresh SCM, as described for neurosphere cultures [132,

133]. Each original dish was passaged into two dishes, and these secondary spheres were used for experiments.

Stem cell markers and confocal microscopy

Neurospheres were fixed in 100% methanol for 10 minutes and standard

immunocytochemistry was applied that was adapted from a previous study of enteric

neurospheres [134]. Immunofluorescence staining was used to identify neural stem progenitor

cells, neural progenitor cells, neurons and glia. Primary antibodies were used at the following

dilutions: chicken anti-Nestin (Aves Labs, Tigard, OR, USA) 1:1000; chicken anti-Dcx (Aves

Labs) 1:750; chicken anti-NeuN (Aves Labs) 1:1000; rabbit anti-BetaIII-tubulin (Cell Signaling

Technology, Danvers, MA, USA) 1:1000; mouse anti-GFAP (Cell Signaling Technology)

1:1000; rabbit anti-Musashi1 (Msi1, Cell Signaling Technology) 1:1000; rabbit anti-SOX2 (Life

Technologies) 1:500. Samples were rinsed after overnight incubation at 4˚C, and were incubated

for 2 hrs with appropriate Alexa488 and 458 secondary conjugated antibody (Life

Technologies). Confocal microscopy of spheres was performed with a DMI3000B inverted

microscope (Leica Microsystems, Buffalo Grove, IL, USA) equipped with a Spectra X LED light

engine (Lumencore, Beaverton, OR, USA), X-Light spinning-disk confocal unit (CrestOptics,

Rome, Italy) and a RoleraThunder cooled CCD camera with back-thinned, back-illuminated,

electron-multiplying sensor (Photometrics) with Metamorph software controlling image

acquisition and data analysis (Molecular Devices, Sunnyvale, CA, USA). Confocal images were

collected with 20X and 40X objectives using standard DAPI, fluorescein, and rhodamine filter

wavelengths.

Neurosphere bioluminescence imaging

Neurospheres maintained in culture dishes containing SCM were transferred manually

with 1 ml pipette tips to either SCM, DMEM containing 10% FBS (SM), or DMEM containing

the serum supplement B27 at the suggested dilution (Life Technologies). Approximately 10-15

spheres that were 100-200 µm in diameter were moved to a second 35-mm tissue culture dish containing 2 ml medium where they were imaged for up to 8 days to detect any circadian rhythms in bioluminescence. Media contained 100 U/ml penicillin and 100 µg/ml streptomycin.

All media used during BLI contained 10 mM Hepes with pH adjusted to 7.2 and bicarbonate levels adjusted for use in room air [135]. To provide synchronization of individual circadian oscillator cells to a common phase of the circadian cycle [136], some of the spheres in SM or

SCM were given 20 µM forskolin in 0.01% (v/v) DMSO for 2 hours, which was removed with two SCM exchanges immediately before 0.2 mM luciferin was added and BLI began.

During imaging, the culture dish was covered with a temperature-controlled optical window sealed with silicone grease and maintained at 37°C (Cell MicroControls, Norfolk, VA,

USA). Spheres were imaged with a back-thinned, back-illuminated CCD camera cooled to -90°C

(CH360; Photometrics, Tucson, AZ, USA) and a 50-mm Nikkor f/1.2 lens (Nikon, Melville, NY,

USA) combined with two close-up lenses (+10 and +4 diopter) that were used together. The field of view was 25% of the dish area, and the depth of field was greater than the height of the neurospheres. Neurospheres were illuminated with red LED light when focusing the camera to collect brightfield images and when handling cultures. Luminescence images were captured

with 2 x 2 binning and sequential 1-hr exposures over several days for a maximum of 8 days.

Images were analyzed using V++ (Photometrics) and ImageJ (NIH) software.

Data analysis

Bioluminescence images were processed to remove cosmic ray artifacts by keeping the

minimum value at each pixel when comparing every two frames in the time series. A single

region-of-interest (ROI) was drawn over each sphere at each frame in the time series. The ROI

was moved when needed to correct for any movement of the sphere, but it remained the same

size and shape. Spheres that produced a detectable signal for at least 5 days of imaging were

analyzed. The first 12 hours of imaging was excluded to eliminate the initial surge in

bioluminescence after luciferin was added. Detrending the BLI data was done by 24-point

running average subtraction as described previously [137]. A five-point running average was then applied, and the times when peaks occurred were measured using the Peak Analyzer routine in OriginLab 9.0 software (OriginLab, Wheeling, IL, USA). As described previously [135], we used a similar criterion to remove the effects from transient or damping signals to find the peak, which is the highest time point between a rising and a falling phase. Peaks, when identified by

Peak Analyzer, were accepted only if the amplitude was greater than or equal to 30% of the amplitude of the peak occurring before and the one during the next peak following the cycle.

Amplitude was calculated as the difference between the peak and the trough, which was the previous minimum after the last falling phase. Using the peak phase of each circadian cycle,

Rayleigh’s test for uniformity was performed using Oriana circular statistics (Kovach Computing

Services, Pentraeth, Wales, UK) to determine whether the phases of circadian rhythms were significantly clustered.

Confocal fluorescence images were collected in a Z-series, and frames that were approximately one third of the distance into the sphere were deconvolved with Autoquant 3D deconvolution. The percentage of neural stem cells was then measured using the Metamorph

Multi-Wavelength Cell Scoring routine after background intensity was subtracted based on the average intensity measurements from controls in which primary antibody was omitted.

Threshold for detection was 50% of the maximum pixel intensity. Other data set means were compared using Tukey’s multiple comparison test, Chi-square analysis, Mann-Whitney U test, and one-way analysis of variance (ANOVA) followed by Scheffe post hoc test (p< 0.05). Linear correlation was performed with OriginLab.

Results

Circadian rhythms are rare in neurospheres maintained in stem cell medium.

To identify the status of circadian rhythms in SCM, SVZ neurospheres were prepared from mPer1::luc mice [130] and imaged in SCM for 6-7 days. The first 3 and last 3-4 days

(early and late components) as well as the entire time series were analyzed. Measurements were made from spheres in four dishes. This procedure was repeated using spheres in SCM without the forskolin pulse (two dishes). Average bioluminescence intensity recorded over time from each sphere was characterized as either circadian (19-29 hrs, Fig. 7A), ultradian (<19 hrs, Fig.

7B), or non-rhythmic (>29 hrs or no significant oscillation) based on the strongest frequency component of a Lomb-Scargle spectral analysis after detrending the signal as described previously [136]. Only 2 of 9 were circadian in the forskolin-treated SCM group, and these oscillations lasted for only one cycle (Fig. 7A & C). One of 8 spheres in the non-forskolin group

was circadian (Fig. 7D). When imaged in SCM, irrespective of forskolin treatment, spheres

2 showed primarily ultradian mPer1 expression (chi-square test, χ 0.05,15 =24.996, p<0.05 followed by a Tukey multiple comparison post hoc test q∞ 0.05,15 =4.38, p<0.05). Many spheres had low- frequency oscillations that were beyond the circadian range and were not of further interest in this study.

Figure 7. Changes in mPer1 expression from ultradian to circadian during neurosphere cell

differentiation. Bioluminescence was recorded from individual spheres that were first treated

with forskolin and then maintained in SCM (A) or stimulated to differentiate in SM (E, F) or B27

medium (I, J). Bioluminescence was also recorded from spheres that were not treated with

forskolin before maintenance in SCM (B). Shown is the 5-point running-average of detrended

data as analog-to-digital units of the camera (ADUs). The proportion of spheres that were

ultradian (U), circadian (C) and non-rhythmic (N) after 4 days are shown with or without forskolin treatment for spheres in SCM (C, D), SM (G, H) and B27 medium (K). Arrows indicate when the 2-hr forskolin pulse ended.

Circadian rhythms in mPer1 gene expression emerge in neurospheres during

differentiation in serum medium or B27 medium.

Neurospheres were isolated from culture in SCM and moved to a second culture dish

containing SM to induce cell differentiation. Neurospheres were imaged with or without

forskolin synchronization. Analysis of the late component of the time series showed that 75% of

neurospheres were circadian (15 of 20) and 10% were ultradian (2 of 20) in SM after forskolin

synchronization (Fig. 1E-G), whereas 25% were circadian (2 of 8) and no ultradian rhythms were

detected (0 of 8) in the SM group not treated with forskolin (Fig. 7H). Significantly more

circadian rhythms were present in SM than in SCM, with or without forskolin synchronization

2 (Mann Whitney U test, p=0.02; χ 0.05, 5 =11.07, p<0.05, q∞ 0.05,5 =3.69, p<0.05). The proportion

of spheres expressing ultradian rhythms after forskolin treatment was not significantly different

in SM, SCM, or B27 medium (p>0.05).

Average periods of circadian spheres, based on peak-to-peak intervals, are shown in

Table 1. When the periods at the first and third cycles were compared to evaluate the stability of rhythms over time there was no significant difference between SM and B27 spheres (paired t- test, p>0.05). Both groups had been treated with forskolin. A linear regression was also used to identify any effect of time in culture on period for these two groups, and there was no significant change in either direction (SM: r=0.019, R2=0.011; B27: r=0.062, R2=0.005). Also, there was no significant correlation between amplitude and period when all spheres were analyzed (r=-0.124,

R2=-0.025, n=27) or when the SM and B27 groups were analyzed individually.

Average Mean Culture Spheres Spheres period amplitude ±SD condition tested circadian ±SD (ADUs) (hours) SCM with forskolin 9 2 23.5 ±6.3 26.21 ±13.6 treatment SCM without forskolin 8 1 22 38.13 ±26.2 treatment SM with forskolin 20 15 24 ±3.0 103.6 ±101.1 treatment SM without forskolin 8 2 21 ±2.8 61.5 ±42.0 treatment B27 medium with forskolin 8 7 21.71 ±3.3 84.52 ±27.5 treatment

Table 1. Summary of neurosphere circadian periods.

Spheres were imaged in SCM and SM with or without forskolin synchronization or in B27 medium. Mean amplitude of spheres that were circadian by Lomb-Scargle analysis was measured on the 2nd cycle during the last 3-4 days of imaging (late).

Periods were determined from peak-to-peak intervals of all cycles.

As a second way to induce differentiation, two dishes of SCM-grown spheres were given

forskolin treatment and then imaged in B27 medium. Analysis of the late component showed

that 87.5% of the neuropheres were circadian (Fig. 7I-K), and there was no significant difference

in the number of circadian spheres between the B27 and SM forskolin-treated groups (t-test,

p=0.315). No ultradian rhythms were detected in the B27 forskolin-treated group. Compared to

SCM, circadian rhythms were more frequently observed in forskolin-treated spheres imaged in

2 B27 medium (p=0.050; χ 0.05,5 =23.12, p< 0.05, q∞ 0.05,5 =3.76, p< 0.05).

To represent the fate that spheres followed in the three different media conditions, spheres were grouped by their initial state during the first 3 days of imaging (early) and their state during the final 3-4 days (late). These categories consist of nine paths that spheres could take during differentiation and are shown in Fig. 8A, in which “UU”, “CC”, and “NN” represent spheres that remained ultradian, circadian, or non-rhythmic throughout 5-7 days of imaging in SCM, SM, and

B27. The most common path taken by neurospheres in SM or B27 was to the circadian state during the late stage of imaging.

Figure 8. The rhythmic state of spheres during early and late exposure to three culture conditions. A: Spheres were maintained in either SCM, SM or B27 medium. Spheres were imaged immediately after a forskolin treatment to synchronize circadian clock cells or after no treatment. Shown is the percentage of spheres that began in a particular state (C: circadian, U: ultradian, N: nonrhythmic) during the first 3 days of imaging (early) and their state during the final 3-4 days (late) of imaging sessions. Under differentiating conditions (SM or B27) the three paths to the circadian state (UC, CC, and NC) were most commonly observed. B: The increase in the percentage of spheres showing circadian rhythms is correlated with an increase in the differentiation marker Nestin-/GFAP+ and negatively correlated with the decline in stem cell markers (Nestin+/GFAP+, Nestin+, and SOX2+) during 7 days in SM.

Stem cell state declines following transition into differentiation-inducing environments.

Following immunofluorescence staining for markers of stem cells and differentiated cells, it was clear that the population of identified NSPCs declined as differentiation progressed, but undifferentiated cells remained throughout the 7 days of BLI (Fig. 8B). The neurospheres did not fully differentiate into a complete monolayer cell culture during BLI. To characterize the extent of differentiation, partly differentiated cultures were fixed at different time intervals, after the 1st, 4th and 7th day of differentiation in SM or B27 medium, mimicking conditions during

BLI. NSPCs within neurospheres were identified by immunofluorescence using anti-SOX2

[138] (Fig. 9A-C), anti-Nestin and anti-GFAP (Nestin+/GFAP+, Fig. 9D-F), anti-Msi1 [139] (Fig.

9H), and anti-Nestin alone (Fig. 11A-C). Hoechst 3342 or propidium iodide (PI) were used to identify cell nuclei.

Figure 9. Emergence of circadian rhythms before fully differentiated neurons appear.

Spheres were synchronized by forskolin treatment and fixed after differentiation in SM or B27 medium, mimicking BLI conditions. Hoechst (blue) or propidium iodide (red) were used as nuclear stains. NSPCs were identified as SOX2+ (cyan; A-C: after 1, 4, 7 days in SM),

Nestin+/GFAP+ (yellow; D-F: after 1, 4, 7 days in SM; red: GFAP, green: Nestin) or Msi1+

(yellow; H: after 3 days in SM). Additional spheres were fixed after differentiation in medium with serum or B27 supplement to stain for progenitor cells as Dcx+ (Yellow; G, J: after 4 days in

SM or B27, respectively). Immature neuronal cells were identified as BetaIII-tubulin+ (Green; K: after 5 days in B27 medium), and mature neuronal cells as NeuN+ (Yellow; I: after 4 days in SM and Green; L: after 4 days in B27 medium). Scale bars=50 µm, and A-C, E, H, I, and K are at the same magnification.

During neurogenesis in the SVZ, neuroblast (type C) cells that are positive for doublecortin (Dcx), a marker for the neuroblast-like cells, migrate through the RMS to the OB

[140]. To determine whether neuroblast-like cells were present during BLI, neurospheres were immunostained for Dcx after 4 days in SM or B27 medium (Fig. 9G & J, respectively). Dcx+ cells were significantly more abundant in neurospheres maintained in B27 medium (57.71

±7.67%, 280, n=7) when compared to SM (Table 2; t =3.820, p<0.001). Mature neuronal cells were almost entirely absent when circadian rhythms were detected at the end of 4 days of differentiation in SM (Table 2, Fig. 9I) or B27 medium (1.30 ±1.1%, n=6, Fig. 9L), as determined by staining against the marker for terminally differentiated neurons NeuN [141].

After four days of differentiation in B27 medium, neurospheres were positive for BetaIII-tubulin

(41.40 ±7.1%, n=7, Fig. 9K), a marker for immature neurons [142]. No BetaIII-tubulin+ cells

41 were observed in neurospheres after 4 days of differentiation in SM (Fig. 11D). NeuN+ cells were present in neurospheres differentiating for 7-8 days in B27 (Fig. 11E.).

Cell type Day 1 Day 4 Day 7 47.11 ±4.6% (100, 27.38 ±8.9% (110, SOX2+/Hoechst* 90 ±6.9% (119, n=8) n=9) n=11) 83.90 ±6.4% (62, 48.12 ±7.8% (297, 25.59 ±8.1% (255, Nestin+/GFAP+/Hoechst n=7) n=9) n=11) 90.44 ±6.4% (102, 71.22 ±4.6% (182, 46.40 ±8.9% (202, Nestin+/PI n=10) n=9) n=7) 35.95 ±13.3% (128, DCX+/PI N.A. N.A. n=9) BetaIII-tubulin+/PI N.A. 1.28 ±1.1% (76, n=7) N.A. NeuN+/PI N.A. 1.96 ±2.6% (76, n=7) 2.58 ±4.0% (61, n=6) 16.59 ±6.5% (297, 25.04 ±5.1% (255, Nestin-/GFAP+/Hoechst 2.94 ±2.7% (62, n=7) n=9) n=11)

Table 2. Cell types identified by markers for stem cells and differentiated cells in SM.

Neurospheres were maintained in SM for the number of days indicated. Shown are the percentages of cells in optical sections that were positive for cell markers or combinations of markers followed by standard deviation. In parentheses are the total number of cells in the section, identified by nuclear stains (Hoechst 3342, propidium iodide), and the number of spheres analyzed (n). N.A. = not available. Cells were imaged with a 20X objective lens, except

40X was used where indicated (*).

We determined the relationship between the stem cell states of the SVZ cultures in differentiating medium to their rhythmicity. The BLI time-series data in Fig. 7 showing the percentage of rhythmic spheres was compared with the percentage of cells that were SOX2+,

Nestin+/GFAP+, or Nestin+ alone (without co-localization). Cells that were GFAP+/Nestin-

(mature astroglia) were also quantified to characterize the differentiation state of the culture.

Fig. 8B shows that a negative correlation exists between circadian rhythmicity and stem cell state of the sphere cultures (SOX2: slope= -0.8496 ±0.02085, R2= 0.9988, p=0.0156; Nestin+/GFAP+: slope= -0.7801 ±0.01338, R2= 0.9995, p=0.0099; GFAP+/Nestin-: slope= 0.2915 ±0.01603, R2=

0.9940, p=0.0336). As shown, the percentage of spheres that were rhythmic correlated with the decrease in stemness and increase in differentiation status.

Forskolin synchronizes circadian clocks within neurospheres.

Forskolin was used to synchronize clocks within spheres, but to verify that it was effective in these undifferentiated cultures we compared the phase at the first, second, and third peaks after the forskolin pulse for spheres expressing a circadian rhythm. In SM the 1st and 2nd peaks were clustered significantly near the predicted phase, approximately 24 hours after the treatment, according to the Rayleigh test (Fig. 10A). The mean vector occurred at 22:44 ±3.45 hrs SD (Z=3.98, p=0.014) and 22:55 ±4.03 (Z=2.96, p=0.047) for the 1st and 2nd peaks, respectively, where 0:00 indicates the end of the 2-hr forskolin pulse. The spheres were not significantly clustered by the third peak (Z=0.78, p=0.471, n=10 spheres for all peaks). The phases of spheres imaged in B27 medium were significantly clustered only during the first circadian cycle (Z=2.939, p=0.047, n=7), and the mean vector was at 10:42 ±3.56 hrs, about 12 hours out-of-phase with the SM group (Fig. 10B).

Figure 10. Synchronization of circadian neurospheres. A: Shown are the phases of individual neurospheres in SM expressing circadian rhythms plotted according to the first three peaks during imaging (left to right). Hour zero corresponds to the time when the forskolin treatment was removed and then projected over the next three days at 24-hour intervals. The mean vector of

phases for individual spheres (arrow) had a significant magnitude (Rayleigh test, p<0.05) only

during the first two cycles. B: Phases of spheres in B27 medium during the first circadian cycle

and mean vector showing significant clustering.

Although forskolin was used here to synchronize clock cells, it has been reported to have

differentiation-inducing properties as discussed previously [136]. In one study, 5 μM forskolin

in medium with 0.5% serum caused differentiation of mouse whole-brain neural stem cultures after

7 days of exposure [143]. To test whether the 2-hr forskolin pulse used here to provide

synchronization between spheres and within spheres caused differentiation, the percentage of cells

expressing the stem cell marker SOX2 was determined by immunofluorescence. There were no

significant differences in the prevalence of SOX2+ cells when comparing forskolin-treated and untreated spheres after 96 hrs in SM (t=-1.59, p>0.12). The percentage of SOX2+ cells in 4-day

SM spheres was 47.11% ±1.46 with forskolin treatment and 43.26% ±1.89 without treatmen

Discussion

Initiation of circadian rhythms during neurosphere differentiation

The circadian rhythms in mPer1 gene expression observed in individual SVZ neurospheres indicate that spheres contain a functional circadian clock while they differentiate in vitro. As predicted, stem cell markers were identified throughout the neurosphere, suggesting that circadian rhythms originated within NSPCs. Similarly, tumorspheres that form in vitro from cancer stem cells are also enriched with stem cell markers and express circadian rhythms in mPer2 activity [136]. Although some cells within the SVZ neurospheres may not contain a circadian clock, a substantial number of cells are rhythmic and are in an adequately close phase relationship with each other to provide a measurable ensemble circadian rhythm from entire spheres.

Neurospheres also displayed fast, ultradian oscillations of mPer1 gene expression, particularly when maintained in SCM. This is the first time that ultradian or circadian rhythms in

Per1 gene expression have been described in neural stem cell cultures, and it suggests that media conditions alter both the differentiation and rhythms of these cells. It agrees in principle with previous studies of mouse embryonic stem cells in which differentiation was correlated with circadian rhythmicity, and dedifferentiation suppressed circadian rhythms [102]. It is possible that the observed ultradian oscillations in mPer1 within SVZ neurospheres actually result from uncoupled cellular circadian oscillations that appear at the whole-neurosphere level as fast oscillations created by the multiple peaks of desynchronized rhythms. However, the adenylate cyclase activating agent forskolin was used to bring circadian oscillators into phase with each other. This treatment synchronizes circadian oscillators in rat-1 fibroblast cell cultures [144] and

tumorsphere cultures [136] with the first peak of the circadian oscillation in Per1 mRNA occurring about 20 hours after the treatment [144].

Switching neurospheres from SCM to either SM or B27 induced differentiation and increased the proportion of spheres expressing circadian rhythms. It is possible that this removal of EGF or FGF2 from the medium initiated emergence of circadian rhythms by allowing the cells to differentiate, suggesting that the more immature NSPCs are unable to generate circadian timing. There are two possible causes for this result: First, the necessary full set of core circadian clock genes are not yet expressed at this stage of differentiation. However, expression of the major core clock genes in mouse neurospheres has been reported [145]. Second, the clock genes are expressed, but the oscillator cannot operate because necessary non-rhythmic positive inputs are missing or an inhibitory factor is present in the spheres during early differentiation. It is also possible that the growth factors in SCM suppress functioning of the clock mechanism. It seems unlikely that either of the added growth factors can completely suppress circadian activity because circadian rhythms were detected in SCM, although these were rare during the late component of imaging sessions.

Stem cell state and circadian rhythmicity were negatively correlated, but the rhythmicity of spheres undergoing differentiation in vitro from the most stem-like state in SCM did take various paths, such as changing from a non-rhythmic or ultradian state to circadian. When

examining all of the possible paths, the neurospheres that were ultimately circadian during the

last 3-4 days of imaging were ones that had been given forskolin and then maintained in either

SM or B27 medium. Spheres under these medium conditions attached and began propagating

into neuroblast and glial-like cells that, by day 6 or 7, stained for Dcx and GFAP, respectively,

further indicating that more differentiated spheres are more likely to be circadian.

Origins of neurosphere circadian rhythms

It is likely that neurospheres are composed of many individual circadian oscillator cells as well as non-clock cells that are unable to sustain a circadian rhythm without input of timing information from other cells. Similarly, some brain areas when isolated as explant cultures produce circadian activity, whereas others do not. Several major brain structures have been grouped into three categories: endogenous circadian clocks, rapidly damping slave oscillators

(producing only a single cycle without timing input), and non-circadian (lacking observable circadian rhythms) [146].

One reason why circadian rhythms were not common in SCM spheres could be because individual circadian clock cells are present but they are not adequately synchronized to a common phase to be detected in the whole-sphere recordings. To test for this possibility, spheres in SCM were given a pulse of forskolin before imaging but the percentage of circadian spheres did not increase. It is possible, but seemingly unlikely, that the less differentiated cells present in

SCM are not responsive to forskolin but might contain a circadian clock. The circadian rhythms in spheres imaged in SM did respond to forskolin by showing a significantly clustered phase that was near the phase expected for this treatment, about 24 hours after the pulse. By the third cycle, the forskolin-treated SM spheres had drifted out of phase and were no longer clustered significantly, according to circular statistics. Spheres in B27 medium given a forskolin pulse were significantly clustered, but this occurred at a phase 12 hours away from the expected phase.

It is possible that the transition into B27 medium had its own phase-shifting effect that acted in combination with forskolin. B27 medium has been shown to elevate mPer1 expression in cortical astrocyte cultures [147], suggesting that it could cause a phase-shift by altering the level of this core clock component.

Although the forskolin-treated SM spheres were in SCM during the forskolin treatment, and so were mostly undifferentiated, some circadian clock cells must have been present for the forskolin to produce synchronization. Because the forskolin-treated SCM and SM spheres were initially in the same state of differentiation but SCM spheres showed few circadian rhythms for the next several days, it is likely that the clock cells present were too scarce to be detected within the larger cell population of non-circadian cells. In the SM spheres, on the other hand, these early synchronized clock cells likely proliferated in the presence of serum while other cells also differentiated and proliferated. It is possible that differentiating non-clock NSPCs began to function as circadian oscillators and was synchronized to the early clock cells through the close interactions present in neurospheres (gap junctions, NCAMs, integrin, etc.). Similarly, circadian clock cells may be present at very low numbers during early stages of embryonic development before circadian rhythms can be detected [148]. Therefore, the data are more reasonably explained if a small number of clock cells are present in neurospheres in SCM and these maintain a common phase and temporal order during the differentiation process in SM and B27 medium, while remaining NSPCs differentiate into clock cells.

Circadian rhythms in progenitor cells

As neurospheres differentiated in SM, the initial abundance of stem cell markers declined

(SOX2, Nestin, GFAP+/Nestin+), cells with differentiation markers increased (Dcx, BetaIII- tubulin, NeuN and GFAP), and the percentage of circadian spheres increased. The present study was not designed to determine whether individual, identified stem cells express circadian rhythms. Nevertheless, results did indicate that progenitor cells are functional circadian clocks because of the lack of mature neurons in spheres after 3-4 days in SM even though 50% of the

spheres were able to generate circadian rhythms. Bioluminescence images of spheres after 4

days in SM showed that the signal originated from cells throughout the spheres (Fig. 11F). The

astrocytes that were detected at this point in culture could have been responsible for generating

the rhythms because astroglial circadian clocks have been described in vitro [149]. Whether the

small minority of astrocytes present (16.5% according to GFAP+/Nestin- staining) were able to drive circadian rhythms in a much larger population of progenitor cells is not known. However, glial cell secretions can alter activity of neural circadian cells in drosophila [150] and mice [151].

In a previous study, circadian rhythms were described in neural progenitor-like cells, but these were in a glioblastoma-derived cell line rather than the non-transformed primary cultures used here [152]. The present results are not in agreement with a previous study of circadian gene expression in SVZ cell cultures in which a circadian clock appeared first in mature cells, and no circadian rhythms in differentiating neurospheres were reported [145]. Similarly, rapidly differentiating cells lack a detectable circadian rhythm during mouse spermatogenesis [153].

The circadian rhythms observed in neurospheres maintained in B27 medium for 4 days that were predominantly positive for Dcx indicates that circadian rhythms originate in neural progenitor cells, particularly neuroblasts (Fig. 12.), after their fate is determined to become interneurons

(granule or periglomerular cells) in the OB [154, 155]. It also suggests that circadian timing in neuroblasts may function during their migratory behavior in the RMS. We predict that neuroblasts become circadian granule cells upon final differentiation, although a previous study did not find circadian rhythms in the granule cell layer of the OB [107]. Nevertheless, circadian rhythms in mature olfactory granule cells may aid in discrimination between closely related odors, an important adaptive ability for which neural stem cells may be required [34], and may improve this sensation at times of day when that is most important [156]. Similarly, circadian

rhythms in SVZ progenitor cells might serve in establishing the time of day when final neuronal

differentiation occurs, optimizing availability of nascent cells with a lower threshold for the

excitation needed to perform odor discrimination [34].

Possible importance of mPer1 in neurogenesis

It is clear that mPer1 is expressed in spheres that are not showing circadian oscillations

through BLI. A question that remains is whether mPer1 in differentiating progenitor cells serves in the process of neurogenesis, similar to what has been observed for other clock genes. Studies described an increased expansion rate of neurospheres from mPer2-/- knockout mice that lack circadian rhythms [157]. Similarly, neural progenitor cell proliferation is increased in DG neurospheres from mPer2brdm1 mutant mice [158]. BMAL1 or CLOCK may also serve in

neurogenesis, with or without a functioning circadian clock, as shown by RNA inhibition that

decreased differentiation markers [145].

Along with a circadian function, mPer1 gene expression may also have an important role

when expressed in ultradian oscillations such as those observed in SCM neurospheres. These

rhythms may be working with stem cell-maintaining genes such as the hes family that are expressed in ultradian oscillations during neurogenesis and embryogenesis where they play an important role in repressing genes used in differentiation [159]. Neurogenesis and circadian oscillators both rely on a collection of basic-helix-loop-helix (bHLH) transcription factors, some

of which are shared between these two time-dependent processes. For example, one gene

promoter element used in circadian transcriptional control (an alternative E-box) includes the N-

box that binds the bHLH HES1 protein [160]. The circadian clock could also have a direct effect

51 on differentiation through its control of an E-box element of the Pax6 gene promoter [161].

Pax6 serves in determining the rate and direction of neurogenesis in the OB [162-164].

If circadian timing, rather than non-rhythmic clock gene expression, has a functional role in adult NSPCs during early stages of differentiation, circadian oscillations may modulate particular differentiation events [145]. In a similar way, daily oscillations in brain cortisol appear to gate cell proliferation in adult mouse hippocampus [112]. Again, coupling between circadian and stem cell-maintaining genes could serve in this control [120]. Alternatively, neurogenesis and circadian timing processes could act independently within the same cells despite predicted interactions between the bHLH transcription factors acting on N-box and E-box elements. The SVZ neurosphere cultures examined here provide a useful assay to investigate the role of circadian clocks and clock-controlled genes in adult neurogenesis. Understanding the relationship between circadian clock genes and neurogenesis could provide new targets for more effective treatments and prevention of neurological disorders such as Parkinson’s and

Alzheimer’s diseases that are suitable for stem cell therapies [6]. If circadian timing acts on differentiation, then circadian expression patterns may be manipulated to induce NSPCs to differentiate more readily into specific cell types needed to compensate for neural deficits.

Conclusions

This exploration of the circadian timing abilities of NSPCs identified autonomous circadian oscillators that are visible when growth conditions induce differentiation. Circadian rhythms appear in neurospheres before mature neurons are present, indicating that NSPCs, which are very prominent in neurospheres, also have functional circadian clocks. The results neither confirm nor deny existence of circadian clocks in the most undifferentiated neural stem cells, the radial glia-like cells. When NSPCs of the SVZ are allowed to differentiate into neuroblast-like cells of the RMS they appear to have circadian properties that could be adaptive for their unique transit to become OB interneurons.

Figure 11. Images of spheres during differentiation. Nestin+ cells (green) at days 1 (A), 4 (B),

and 7 (C) in SM with PI-stained nuclei (red). D: Lack of BetaIII-tubulin+ cells (green) with PI

(red) in a neurosphere at day 4 in SM. E: NeuN+ cells (green) with Hoechst-stained nuclei (blue)

at day 7 in B27 medium. Scale bars=50 µm. F: Three neurospheres in SM used for measuring

circadian rhythms in mPer1 expression. Top: Brightfield image at day 0. Bottom: Corresponding

bioluminescence image at day 4. Average maximum signal was 444 ADUs ±49.0 (SD). Each

pixel represents 61 x 61 µm.

Figure 12. Circadian rhythms during neurogenesis. A summary diagram predicting that neural stem cells (radial glia-like cells) residing in the SVZ lack a functioning circadian clock but can exhibit high-frequency oscillations in clock gene expression (green). They further differentiate into neuroblasts and enter the RMS where they exhibit circadian oscillations in clock gene expression (red). These cells migrate to the OB and differentiate into granule cells and may contribute to previously described OB circadian rhythms (red) [107].

CHAPTER III: CIRCADIAN CLOCKS ARE ESSENTIAL FOR NORMAL ADULT

NEUROGENESIS IN VITRO

Preface

This chapter will be sent for publication as a research article in PLoS ONE in 2015. The original manuscript has been altered to meet BGSU’s specifications for formatting a dissertation.

This publication identifies NSPCs in the DG to be circadian clock cells and highlights how the circadian clock regulates neurogenesis in vitro.

Introduction

Recent studies suggest that cellular circadian clocks may regulate adult neurogenesis and survival of newly formed neurons [70, 165], although circadian studies of neurogenesis in vitro are lacking. During adult neurogenesis, multipotent neural stem cells self-renew and differentiate to generate neurons. The dentate gyrus (DG) and the subventricular zone (SVZ) are two well-understood areas of the mammalian brain containing neural stem cells, which are maintained in a unique cellular environment. This niche for NSCs is emulated in vitro within neurospheres that are cultures derived from the DG and SVZ.

Circadian rhythms are endogenous, near-24-hour oscillations in gene expression, physiology, or behavior that are generated in animal cells by two interacting transcriptional- translational feedback loops in which core clock genes (e.g., Period, Cryptochrome, and Bmal1) are rhythmically activated [166]. The circadian clock can couple with the cell cycle [124] and modulate cell proliferation [167]. The circadian oscillator gates the G2/M checkpoint of the cell cycle via clock gene wee1 [168] and the G1/S transition via clock-controlled genes p20 and p21

[124, 169]. Cell cycle control over the circadian clock has also been shown, but is less well

understood than cell cycle regulation by the clock [170, 171].

Modulation of neurogenesis and NSC proliferation by an endogenous clock in the DG remains largely unexplored. Cortisol, melatonin, and various neurotransmitters under circadian clock control appear to regulate daily neurogenesis in the central nervous system [112, 172-

174]. Circadian rhythms in mPer2 expression have been reported in hippocampal explant cultures [106], although a separate study did not detect rhythms in the DG in vivo [158].

Hippocampal neural progenitor cells of mice divide more often at night [70, 84]. Disturbed sleep or alterations of circadian clock phase have also been shown to suppress neurogenesis as indicated by reduced expression of doublecortin (DCX), a marker of immature neurons [175].

Circadian rhythms influence learning, cognitive performance, and memory formation across different species [176-178]. Studies describe disruption of circadian rhythms altering learning and memory performance, spatial learning, intra and intersession habituation, place learning, long-term potentiation, and trace fear memory [106, 179-182]. Cryptochrome genes are also necessary for time-place learning [180]. These studies provide much evidence that a functional circadian clock is required for optimal memory formation and persistence [183].

During adult neurogenesis, newly made granule cells produced within the DG form functional hippocampal synapses that appear to provide improved performance of spatial memory tasks, enhanced mood, and neural repair [27, 184]. Because increased neurogenesis is associated with improved cognitive abilities in rodents, optimal circadian control of cell division that introduces new neurons into the hippocampal circuitry may also increase performance. For example, higher levels of cell proliferation in the DG of knockout mice lacking BMAL1 were shown in one study [70], whereas another described normal proliferation in the DG of Bmal1-/-

57 knockout mice [165]. Knockout of BMAL1 using lentivirus shRNA in primary mouse neuronal cultures caused increased cell death, and siRNA-mediated knockdown of Bmal1 showed similar effects [185]. Overexpression of Bmal1 in NIH3T3 cells produced an increase in cell proliferation [186]. In contrast, loss of mPER2 functioning increased DG NSPC proliferation

[158].

Circadian rhythms in clock gene expression are typically absent in embryonic or multipotent somatic stem cells but do appear in progenitor cells and more differentiated tissues [102,

152]. One important question is whether adult neural stem progenitor cells (NSPCs) are circadian clock cells that are capable of endogenous, sustained circadian rhythms. Our study identifies circadian rhythms in DG neurosphere cultures independent of rhythmic influences and timing cues from the animal or its environment. We also describe properties of neurosphere cultures from the DG of Bmal1-/- and Cry 1-/-, 2-/- double knockout mice that lack circadian rhythms. These results show that although the circadian clock is not required for neurosphere formation in vitro, its deficiency slows neurosphere growth, suppresses neuronal fate commitment, and increases apoptosis.

Materials and methods

Animals

Transgenic mPer1::luc mice [130] were bred and maintained in cycles of 12 h light and

12 h dark to entrain their circadian system. Animal procedures were approved by the BGSU

Institutional Animal Care and Use Committee and met National Institutes of Health guidelines.

All animal studies using Bmal1-/- [187] and Cry 1-/-, 2-/- mice [188] were conducted in

compliance with the CSU Committee of Animal Care and Use. Animals were 5-8 months old at

the time of tissue harvesting, except where noted. Bmal1+/- and wild-type C57BL/6 littermate

animals served as controls for the effects of Bmal1-/- knockout. Cry1-/- and wild-type C57BL/6 littermate mice served as controls to study the effects of cryptochrome gene double knockout on

sphere growth. When examining fate determination in NSPCs, control neurospheres were

prepared from age-matched (6-month-old) WT mice that were Bmal1-/- littermates.

Neurospheres were also made from 12-month-old mPer1::luc mice to serve as controls for

possible aging effects in Bmal1-/- knockouts.

Neurosphere cultures

Adult male C57BL/6 mice (5-8 months old), Bmal1-/- (6-8 months old), Cry 1-/-, 2-/- (6-8

-/- months old), and Cry1 (6-8 months old) animals were euthanized using isoflurane or in a CO2

chamber. The genetic background of all knockout mice was C57BL/6. Brains were removed

quickly, coronal slices were made with a Brain Blocker (PA 001 Rat; Kopf), and DG and SVZ

regions were dissected. The tissue was washed 4-5 times in HBSS and then enzymatically

digested with papain and DNAse I (Worthington) for 30 minutes at 37°C, followed by 2-3

59 washes in DMEM with no added growth factors. The tissue was then mechanically triturated and passed through a 40 μm cell sieve (Falcon; BD Biosciences Discovery Labware, Bedford, MA).

The cell suspension was washed and centrifuged 4 times for 5-6 min. The supernatant was discarded and the pellet was re-suspended in stem cell medium (SCM) which consisted of

DMEM with 10 ng/ml bFGF, 20 ng/ml EGF (Invitrogen) and 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were plated at a density of 2.5-3.0 x 104 cells/ml in SCM. Neurospheres were observed after 7-8 days in SVZ cultures and after 10-12 days in DG cultures. These neurospheres were then mechanically triturated and plated in 35-mm dishes. Secondary neurospheres that formed were used for all experiments [104].

Neurosphere bioluminescence imaging

Culture dishes containing neurospheres in DMEM and 10% FBS (SM) or stem cell medium (SCM) were covered with a temperature-controlled optical window sealed with silicone grease and maintained at 37°C (Cell MicroControls, Norfolk, VA). Spheres were treated for

2hrs with 20 µM forskolin immediately before adding luciferin and were imaged with a back- thinned, back-illuminated CCD camera cooled to -90°C (CH360, Photometrics, Tucson, AZ) and a 50-mm Nikkor f/1.2 lens (Nikon, Melville, NY). Cell dispersals were illuminated by red LEDs when focusing the camera and handling the cultures. Luminescence images were captured with

2 x 2 binning and sequential 1-hr exposures over several days for a maximum of 4 days. Images were analyzed using V++ (Photometrics) and ImageJ (NIH) software.

Immunocytochemistry

Neurospheres were placed into poly-D-lysine-coated glass-bottom dishes (Mattek) and

allowed to attach for 6 hours while in a thin film of SCM, DMEM with 10% fetal bovine serum

(FBS) and P/S (SM), or DMEM with B27M (Life Technologies, Grand Island, NY, USA) and

P/S (B27M). After the neurospheres attached, 2 ml of medium (SCM, B27M or SM) was added

to prevent loss of neurospheres. Neurospheres were fixed in 100% methanol for 10 minutes and

standard immunocytochemistry was performed. Immunofluorescence staining was used to

identify neural stem progenitor cells, neural progenitor cells, neurons, and astrocytes. Primary

antibodies were used at the following dilutions: chicken anti-NeuN (Aves Labs, Tigard, OR,

USA) 1:1000; chicken anti-Nestin (Aves Labs) 1:1000; rabbit anti-BetaIII-tubulin (Cell

Signaling Technology, Danvers, MA, USA) 1:1000; rabbit anti-Musashi1 (Msi1, Cell Signaling

Technology) 1:1000; rabbit anti-GFAP (Cell Signaling Technology) 1:1500; rabbit anti-SOX2

(Life Technologies) 1:500; cleaved caspase-3 (Cell Signaling Technology) 1:500. Samples were

rinsed after overnight incubation at 4˚C, and were incubated for 2 hours with appropriate

Alexa488 and 458-conjugated secondary antibody (Life Technologies). Confocal microscopy of

spheres was performed as mentioned in our previous study [104].

Live/Dead stain

Propidium iodide (PI) is only taken up by cells whose cell membrane integrity is

compromised [189]. Neurospheres from both wild-type and Bmal1-/- knockout animals were stained and incubated in PBS with PI (0.02mg/ml) for 5 minutes. PI was then washed out using

PBST (0.1% Triton in PBS), and neurospheres were fixed using 100% methanol for 10 minutes.

After fixation, neurospheres were washed with PBS to remove excess methanol, and cell nuclei

61 were stained using Hoechst3342 (5 ng/ml in PBS) for 5 minutes. This protocol was modified from a previously published study [190].

Data analysis

Bioluminescence images were processed and peaks were identified by a method similar to that described in our previous study [104]. Using the peak phase of each circadian cycle,

Rayleigh’s test for uniformity was performed using Oriana circular statistics (Kovach Computing

Services) to determine whether the phases of circadian rhythms were significantly clustered. The percentage of neural stem cells, neurons, astrocytes, and cleaved-caspase-3+ cells was measured using the Metamorph Multi-Wavelength Cell Scoring routine to create segmentation windows that show estimated areas occupied by positively-stained cells. Background intensity was subtracted based on the average intensity measurement from controls in which primary antibody was omitted. Threshold for detection was 30% of the maximum pixel intensity. Overall staining intensity for cleaved-caspase-3 was measured by drawing a region of interest (ROI) around the neurospheres and plotting a histogram to find the mean staining intensity. The means were then compared by using One-way analysis of variance (ANOVA) and T-test.

To evaluate neurosphere proliferation, primary neurospheres were triturated and plated at a density of 3.5-4.5 x 104 cells/ml in a 60-mm tissue culture dish. Neurosphere numbers were counted after secondary spheres were generated in the culture. Medium exchanges were done every 2-3 days and brightfield images were taken on day 14 and day 35 after plating. ImageJ software (NIH) was used to draw an ROI around neurospheres, and size measurements were made at day 14 and 35. ANOVA followed by Scheffe’s post-hoc test was used to compare average neurosphere area between Cry 1-/-, 2-/- , Cry1-/-, and wildtype.

Results

Circadian rhythms appear when neurospheres are allowed to differentiate.

To identify the status of circadian rhythms in NSPCs, DG neurospheres were prepared

from mPer1::luc mice and imaged in SCM or SM for 4 days after forskolin synchronization.

Total bioluminescence intensity recorded over time from each sphere was characterized as either

circadian (19 through 29-hr period) or non-rhythmic defined as less than 19-hr period (ultradian),

greater than 29-hr period or no significant oscillation [104]. All neurospheres that were imaged

while in SCM lacked circadian rhythms (n=8). One neurosphere in SCM exhibited an ultradian

oscillation. Neurospheres were mostly rhythmic in SM (7 of 8 imaged), but one had a low-

frequency (18.68 hr) oscillation in mPer1 gene expression (Fig. 13). Significantly more circadian

rhythms were recorded in SM than in SCM after forskolin synchronization (Tukey Multiple

2 Comparision post hoc test χ 0.05, 5 =11.07, p <0.05, q∞ 0.05,5 =2.472, p <0.05).

Average period of circadian spheres in the SM group, based on peak-to-peak intervals, was 22.12 hrs ±2.64. According to the Rayleigh test the 1st peaks of rhythms recorded in the SM

group were significantly clustered (Z=3.26, p=0.032). The mean vector occurred at 01:02 ±3.20

(SD) hrs, which was approximately 24 hours after the forskolin treatment. Time 0:00 indicates

the end of the 2-hr forskolin pulse.

NSPCs in the DG self-renew and produce neurons and glial cells through a sequence of

differentiation stages while identifiable cell markers appear transiently throughout neurogenesis

[27]. Circadian rhythms in DG neurosphere cultures were evident by BLI as early as day 1 of

differentiation in SM during up to 4 days of imaging. NSPCs were predominant in neurosphere

cultures during circadian rhythm ontogeny (Table 3) and were identified by confocal

immunofluorescence microscopy using anti-SOX2 (Fig. 14A & B), anti-Msi1 (Fig. 14C & D) and anti Nestin and GFAP (Fig. 14E) colocalization. BetaIII-tubulin+ (immature; Fig. 14F) and

NeuN+ (mature; Fig. 14G) neurons were nearly absent after 4 days of differentiation in SM.

Immunostaining also detected high mPER1 expression in the neurosphere core (Fig. 14H),

further indicating that the more stem-like cells populating the core are the source of the

bioluminescence.

Circadian clock proteins are required for normal neurosphere formation.

Critical genes such as Bmal1 (Arntl) serving in the timing mechanism of the circadian

clock are expressed in the SGZ, but studies of knockout mice lacking Bmal1 indicate that

circadian rhythms are not required for successful embryonic or adult neurogenesis [70]. Bmal1-/- knockout mice show arrhythmic locomotor activity under free-running conditions such as constant darkness (DD) [165, 187]. To determine whether a functioning circadian clock is necessary for neurosphere formation, we prepared spheres from both DG and SVZ of wild-type and Bmal1-/-mice.

Neurospheres could be cultured from both wild-type and Bmal1-/- knockout animals, but

distinct differences were observed in neurosphere morphology. An unusual feature was the

presence of large oval structures that appear in DG neurospheres from Bmal1-/- mice after they

are maintained in SCM for 15-20 days with medium exchanges every 2-3 days. These unusual

structures were dark when observed in brightfield at low magnification and were referred to as

“lacunae” because they were devoid of cells (Fig. 15A). The average percent area occupied by

lacunae was 5.15 ±4.89% (n=26) when entire spheres were examined. DG and SVZ

neurospheres from wildtype mice and SVZ neurospheres from Bmal1-/- mice lacked any

evidence of lacunae.

Live neurospheres were stained with PI to reveal dead or dying cells and were then fixed and stained with Hoechst3342 to identify all cell nuclei. Rather than distinct cells, only diffuse fluorescence was observed in the lacunae. We quantified the total number of live and dead cells using a multi-wavelength cell scoring routine (Fig. 15B, C, & G). The percentage of dead cells in

Bmal1-/- neurospheres increased significantly relative to wild-type (KO: 50.86 ±24.6%, WT: 18.2

±3.61%; t=2.62, p=0.03, n=4 spheres each). There was no obvious difference in neurosphere size

(as largest cross-sectional area) between Bmal1-/- and wild-type neurospheres when made from either DG or SVZ.

Absence of circadian clock proteins results in greater cell death.

Because of the aberrant morphological features (lacunae) observed in DG neurospheres,

we evaluated whether the circadian clock plays a role in regulation of cell death by measuring

cleaved caspase-3 staining intensity and the percentage of caspase-3+ cells in wild-type (Fig.

15D) and Bmal1-/- (Fig. 15E & F) neurospheres. Neurospheres were allowed to differentiate on poly-D-lysine-coated dishes in SM for 1 day before they were fixed for immunocytochemistry.

There was a significant increase in caspase-3 overall mean staining intensity in Bmal1-/- neurospheres (442.78 ±517.85 relative light units, n=9) as compared to wild-type neurospheres

(71 ±83.97, n=9, t=2.12, p=0.049). We also recorded a significant increase in the total percentage of cells positive for cleaved caspase-3 (Fig. 15G) in Bmal1-/- neurospheres (KO: 26.30 ±17.35%,

n=10; WT: 7.60 ±6.25%, n=8, t=-2.88, p=0.01).

Circadian clock proteins are required for normal neurosphere growth and proliferation.

In order to test whether observed defects in the neurosphere homeostasis were caused by

clock disruption or by some clock-independent function of BMAL1, we decided to test

properties of neurospheres generated from the DG and SVZ of another circadian mutant. We

used Cry 1-/-, 2-/- mice that are arrhythmic in DD but maintain daily activity patterns while in 24-

hr light/dark cycles (Vitaterna et al., 1999;Albus et al., 2002). To determine whether a

functioning circadian clock is necessary for overall growth and proliferation of neurospheres, we

counted and measured cross-sectional area of spheres from Cry 1-/-, 2-/-, Cry1-/-, and wild-type

mice. In the absence of both circadian clock CRY proteins we found significantly reduced cell

proliferation and lower numbers of secondary neurospheres (Fig. 16). Secondary DG spheres

from Cry 1-/-, 2-/- mice were on average smaller but not significantly different from the Cry1-/-

controls after 14 days in culture but were significantly different at the 35th day in culture

(F2,59=3.72, p<0.05). No significant difference was recorded in SVZ neurosphere cultures.

To determine whether the observed effects on proliferation were in response to absence

of one clock protein (Cry1-/-) or a non-functional circadian clock (Cry 1-/-, 2-/-) we also tested

neurosphere proliferation in wild-type age-matched littermate controls. Slower neurosphere

growth and proliferation were observed in Cry 1-/-, 2-/- mice as shown by significantly smaller

DG neurospheres from Cry 1-/-, 2-/- mice compared to wild-type littermates (Fig. 16) at Day 14

(F2,28=5.54, p<0.001) and at Day 35 (F2,59=3.72, p<0.05). On the other hand, no significant

difference was observed in SVZ neurosphere proliferation at day 14, but proliferation was

significantly reduced in both the Cry 1-/-, and Cry 1-/-, 2-/- knockout SVZ spheres at Day 35 in culture (F2,59=44.41, p<0.001). No lacunae were observed in neurospheres made from any of the

Cry knockout mice.

BMAL1 is essential for neuronal fate commitment.

To analyze whether Bmal1, an essential component of the circadian clock, is necessary

for neurogenesis in vitro, both secondary DG neurospheres from WT and KO dishes were

transferred to new 35mm poly-D-lysine-coated Mattek dishes with neural differentiation medium

(B27M). Neurospheres were allowed to differentiate for 4 or 6-7 days in B27M with no added stem cell-maintaining growth factors (bFGF and EGF). Immunocytochemistry was used to determine the percentage of neuroblasts (DCX+), immature neurons (BetaIII-tubulin+), and

astrocytes (GFAP+) in the culture. The percentage of DCX+ cells in Bmal1-/- DG spheres was

significantly lower relative to wild-type controls after 4 days of differentiation in B27M (KO:

5.42 ±7.91%, n=6; WT: 27.86 ±21.53%, n=7; t=3.42, p=0.01). When compared to wild-type

(Fig. 17A) the percentage of immature BetaIII+ neuronal cells, after differentiation in B27

medium at day 7, was significantly reduced in Bmal1-/- DG spheres (Fig. 17C) (KO: 5.88

±8.56%, n=9; WT: 55.79 ±7.38%, n=8; t= 4.93, p=0.001). In addition, these differentiated

neurospheres exhibited an increased astrocyte proliferation when compared to their wild-type

littermates (KO: 76.01 ±7.09%, n=9; WT: 5.22 ±4.19%, n=8; t= 32.13, p<0.001).

To rule out the possibility of delayed neuronal differentiation in the knockout cultures

that would not have been detected and to further determine whether BMAL1 regulates terminal

differentiation of neuronal cells, we allowed differentiation of the neurospheres in B27M for up

to 14 days. Confocal immunocytochemistry was performed using anti-NeuN antibody to

calculate the percentage of mature neuronal cells in the culture. We observed an obvious decline

in the number of fully mature neuronal cells in differentiated Bmal1-/- DG neurospheres (Fig.

17D). To test whether loss of circadian timing could explain these results we also examined Cry

1-/-, 2-/- neurospheres given B27M for 14 days. When comparing both of the arrhythmic

knockouts with 6-month-old (Fig. 17B, Table 4) and 12-month-old (Table 4) WT cultures there

+ -/- -/- -/- were significantly fewer NeuN cells in Bmal1 but not Cry 1 , 2 (F3,34=5.544, p=0.003)

neurospheres and both Bmal1-/- and Cry 1-/-, 2-/- had significantly more GFAP+ cells than WT

(F3,34=4.82 , p=0.006). These results (Fig. 17E) indicate that neuronal fate commitment depends

on non-clock functions of BMAL1, whereas glial proliferation is regulated by a circadian- dependent process, because it was observed in both knockouts.

To test for the possibility that the paucity of mature neurons in Bmal1-/- cultures was caused by previously described accelerated aging due to this mutation [191] neurospheres were prepared from 12-month-old WT mice. Both groups of neurospheres were maintained in B27M for 14 days and examined for NeuN+ and GFAP+ cells (Table 2). Only the Bmal1-/-neurospheres displayed significantly reduced evidence of neuron formation.

Discussion

Circadian clocks in dentate gyrus neurospheres

Bioluminescence imaging revealed circadian mPer1 activity in neurospheres maintained in serum medium. In contrast, spheres in SCM were arrhythmic. Furthermore, serum medium and B27M produced the temporal sequence of stem cell markers, neuron-specific proteins, and morphological changes predicted from previous studies of DG neurospheres undergoing differentiation and development in vitro. These results point to a strong dependency of circadian timing on the release of DG stem cells from molecular processes that maintain the stem cell state.

The lack of neurosphere rhythms in the undifferentiated state resembles the failure of embryonic stem cells to show circadian rhythms in gene expression until they are induced to differentiate [102]. Although it is possible that a small number of cells within SCM neurospheres were rhythmic but not detected, functioning of the circadian clock was suppressed overall perhaps by genes that maintain cell stemness, as suggested for SVZ neurospheres [104].

Alternatively, growth factors in SCM might inhibit the clock mechanism by excessively stimulating signal transduction pathways that are used in entraining the circadian clock to external 24-hr cycles, as proposed in a study of cancer stem cell tumorspheres [136]. These glioma tumorspheres nevertheless remained rhythmic in SCM, whereas circadian rhythms were absent in DG neurospheres indicating a greater suppression of timing processes.

Circadian rhythms were evident while DG neurospheres were differentiating in SM, as shown by immunocytochemistry identifying the stem cell markers SOX2, Msi1, Nestin, and

GFAP. For example, 42 ±6.40% of neurosphere cells were positive for SOX2 after Day 4 in

SM. However, no mature neuronal cells were present while circadian rhythms were detected,

suggesting that rhythms emerged in the NSPCs. This result was supported by mPER1

immunostaining indicating that bioluminescence originated in the stem cell-rich core of

neurospheres. These outcomes support a view that NSPC subpopulations in neurospheres are

circadian clock cells, in contrast to earlier studies asserting that circadian clocks first begin in

mature cells and are not operational in immature, differentiating cells [145, 153]. However,

additional support that differentiating stem cells are circadian is found in studies of

hematopoietic stem cells, embryonic cells of the hypothalamic suprachiasmatic nucleus (SCN),

and early embryonic stem cells that express circadian rhythms [148, 192, 193].

High-frequency oscillations in mPer1::luc expression were reported in SVZ neurospheres maintained in SCM [104] but were observed only once in the present study. Together, these two studies suggest that at least one of the core circadian clock genes can be modulated at higher frequencies much like the rhythmic expression of genes regulating early developmental events

[194, 195]. An alternative possibility is that the ultradian oscillations are formed from the output of two circadian cell populations that remain fixed in a phase relationship about 12 hours apart, similar to descriptions of other ultradian oscillations [196, 197].

Circadian rhythms were present as early as the second day after transfer to SM, permitting adequate time to evaluate their properties. The average period of DG neurospheres in

SM was shorter than 24 hrs, similar to that of the free-running circadian locomotor rhythm of the inbred C57BL/6 mouse line used in this study (23.84 hrs) [198]. On the other hand, hippocampal explant cultures from transgenic mice expressing a fusion protein of mPER2 and firefly luciferase display a circadian rhythm in bioluminescence of 25.08 hrs [106]. The phase of the neurosphere circadian rhythm was determined from the time of peak mPer1 expression. In SM,

70 this phase occurred as predicted following the forskolin treatment that was used to synchronize

NSPCs [144]. The average peak bioluminescence occurred at intervals about 24 hrs after the forskolin pulse ended, indicating an ensemble rhythm from multiple oscillating cells [136].

One model supported by the DG results here and previous work on SVZ neurospheres

[104] springs from NSPC heterogeneity and could explain the presence of rhythms during early differentiation events: At least two cell populations are considered, one that is non-circadian but substantial, and a second much smaller population that is circadian and entrains to the forskolin pulse. During differentiation, the minor population proliferates, producing a detectable ensemble circadian rhythm, whereas the non-circadian cells are diminished or depleted through asymmetric cell division. One possibility is that the original non-circadian cells are activated radial glial cells that are lost as they differentiate into NSPCs that entrain to the circadian cell population as proliferation proceeds. This two-cell-pool model describes emergence of circadian rhythms in mPer1 expression during neural differentiation and merits further testing. It predicts that circadian NSPCs can entrain to each other through cell contacts or paracrine factors, a premise supported by cell interactions reported in tumorspheres [136].

Although very few studies have examined circadian rhythms in the hippocampus maintained in vitro, evidence indicates that it contains a peripheral circadian oscillator distinct from that of the SCN [106]. However, a different in vitro study of clock genes in isolated hippocampal cultures did not detect circadian rhythms [137, 158]. Most studies of hippocampal circadian rhythms have analyzed rhythms in DG tissue harvested at intervals from animals housed in standard cycles of 12 hrs light and 12 hrs dark (LD) [158, 181, 199]. In one case, mice were in DD for at least 2 days before dissection, thereby avoiding immediate effects of entraining light signals on gene expression [70]. Nevertheless, indirect effects from circadian

locomotor activity or circadian oscillators elsewhere in the brain could have been responsible for

much of the rhythmic hippocampal activity observed in earlier studies. Circadian rhythms in

neural inputs to the hippocampus or rhythms in metabolic substrates and cortisol in the brain are

a few ways by which circadian rhythms may be driven [200]. By avoiding these external

influences, the DG neurosphere rhythms show that hippocampal cells are indeed capable of

endogenous circadian timing. Furthermore, cell phenotypes present in rhythmic neurospheres are

identifiable by immunofluorescence thereby suggesting which cells generate the BLI rhythm,

such as the mPER1-positive cells localized to the core.

Fluorescence imaging of fixed hippocampal sections of transgenic mice expressing a

DsRED and PER2 fusion protein are reported to show circadian rhythms in DG cells that appear

to be quiescent NSPCs (Type 1 cells, Sox2+/GFAP+) [70]. Evidence that the rhythmic cells were

Type 1 cells was indirect: High DsRED-PER2 fluorescence was inversely correlated with intensity of staining for Ki-67, a mitotic activity marker, suggesting that the circadian rhythm

originated in quiescent cells. However, DCX+ cells were also identified as Ki-67-negative,

providing an additional possible source of the rhythm [70]. Neuroblasts are DCX+ cells and undergo asymmetric cell division to become neurons but are distinct from Type 1 cells, which are multipotent and less differentiated [201]. An additional concern is that DsRED-PER2 rhythms that were recorded could have resulted from a rhythm in cell abundance in the DG, as purported, or from a circadian modulation of mPer2 promoter activity.

Circadian clock proteins and neurosphere growth and formation

Using Bmal1-/- and Cry 1-/-, 2-/- arrhythmic mice, we found that the circadian clock is

required for normal neurosphere growth and differentiation. Slower proliferation rates were

observed in secondary neurosphere cultures derived from DG of Cry 1-/-, 2-/- mice when

compared with age-matched Cry1-/- mice or wild-type littermates. The Cry1-/- control mice are

rhythmic but exhibit a circadian locomotor rhythm with a period 1 hr shorter than wild-type

[188]. Neurospheres generated from Cry 1-/-, 2-/- mice were significantly smaller and fewer

relative to controls, indicating that the lack of circadian timing production or absence of other

functions for cryptochrome proteins can suppress growth rates or induce increased apoptosis.

Deficits were also observed in Bmal1-/- DG neurospheres after culture in SCM for 15-20

days: Lacunae were abundant in Bmal1-/- DG neurospheres but were not present in DG neurospheres from wild-type littermates. Similarly, Bmal1-/- SVZ neurospheres lacked lacunae,

suggesting that DG NSPCs may be more sensitive than SVZ spheres to metabolic stress or other

challenges to survival in culture imposed by a loss of circadian timing. When compared with

WT controls, Bmal1-/- DG neurospheres had increased numbers of PI-positive cells, indicative of

damaged cells, particularly near the lacunae.

Increased levels of ROS have been reported in Bmal1-/- mice [202]. It is possible that

Bmal1-/- DG neurospheres also generate more ROS than WT spheres or are more sensitive to

ROS stress, leading to apoptosis. These possibilities are supported by the higher overall

immunofluorescence intensity for the apoptotic marker caspase-3 that we observed in Bmal1-/-

KO spheres. The percentage of cells positive for caspase-3 was also significantly elevated

relative to the control at the coverslip-level of confocal imaging sections, beneath attached

neurospheres. Access to culture medium would be low at this location, again suggesting that

loss of Bmal1 leads to greater sensitivity to stressors and increased cell death when cell survival

is challenged [191, 203].

A previous study of the circadian oscillator in mouse embryonic fibroblasts found that

loss of Cry1, 2 caused increased cell proliferation, but only under hypoxic conditions [204]. On

the other hand, there were no changes in cell proliferation of cryptochrome knockout cells in

normal culture conditions [205]. Surprisingly, we observed reduced cell proliferation in Cry 1-/-,

2-/- neurospheres, which might reflect differences between cell cultures and neurosphere culture

conditions or between fibroblasts and NSPCs. Nevertheless, studies should examine cell

proliferation specifically in the core of Cry 1-/-, 2-/- neurospheres because this area is typically more hypoxic.

There is a dearth of information about Bmal1-/- cells in culture. Recent studies using

Bmal1- modified cells suggest that the circadian clock alters the mitotic rate in different ways

depending on cell type. Hepatocytes cultured from Bmal1-/- animals exhibit a delay in the G1-S

phase of the cell cycle [124], whereas overexpression of this protein in NIH3T3 cells increases

the cell proliferation rate [186]. One study showed an increase in proliferation in the subgranular

zone of Bmal1-/- animals [70], whereas another reported normal proliferation and enhanced cell

survival in the SGZ in vivo [165]. We did not observe an increased proliferation rate in Bmal1-/-

DG or SVZ neurospheres. Sphere sizes were not significantly different from those of wild-type

animals.

Circadian clocks and neural differentiation

Recent studies found links between core circadian clock genes and factors which regulate adult neurogenesis. One report showed that Rev-erbα regulates neurogenesis through Fabp7 modulation [206]. NeuroD1, a neurogenic transcription factor, has been shown to be regulated by the BMAL1/Clock complex. The authors also reported a decline in the percentage of cells positive for the neuronal marker MAP2 after cell transfection with BMAL1 siRNA [145]. This interesting result agrees with the current study in which Bmal1-/- neurosphere cells failed to differentiate into neurons while in B27M. We confirmed our results using immature (BetaIII- tubulin+ and DCX+) and mature (NeuN+) neuronal markers and recorded very few cells of either phenotype.

Surprisingly, increased numbers of GFAP+ cells were observed when Bmal1-/- neurospheres were cultured in neuronal differentiation medium. Our results also agree with a recent study in which increased astrocyte numbers were observed in cerebral cortex and hippocampus of 6-month-old Bmal1-/- mice. Disruption of the circadian clock in the brain by deletion of Bmal1 was also shown to induce oxidative stress, astrogliosis, degeneration of axon terminals, and loss of neurons [185].

Some of the phenotypes observed specifically in Bmal1-/- neurospheres, including the lacunae, could be attributed to non-circadian functions of the protein. These clock independent processes would be expected to persist in Cry 1-/-, 2-/- but not Bmal1-/- spheres despite both types being arrhythmic. Also, the differences between the two knockout spheres could have been because the circadian clock was arrested at different phases of the cycle, causing the levels and activities of the many clock-controlled proteins to also differ.

Our in vitro results are similar to the aforementioned study. The low numbers of BetaIII- tubulin+, DCX+, and NeuN+ cells along with the increased numbers of GFAP+ cells in our cultures confirmed that Bmal1 is essential for normal neurogenesis. Our results do not agree with a study by Bouchard-Cannon et al. that reports an increase in Ki67+ cells and NeuN+ cells in 40- day-old Bmal1-/- animals [70]. The observed difference in our study might possibly be due to the age of the animal used. Another study reported no effect on proliferation in 60-day-old Bmal1-/- animals [165]. There are many possible pathways by which the clock might regulate neurogenesis. The circadian clock could directly affect differentiation through its control of an E- box element in the promoter region of neurogenic transcription factors such as NeruoD1, Pax6, etc. [145]. The promoter region of the NeuroD1 gene contains nine E-boxes [207, 208].

Circadian clocks may also regulate fate commitment by modulating miRNAs. For example, the

Clock/BMAL1 heterodimer regulates miRNA 219 [209] that promotes oligodendrocyte differentiation [210]. Our results indicate that loss of BMAL1 in NSPCs alters neuronal fate commitment capabilities and directs the NSPCs to an astroglial lineage.

Insights from DG neurospheres

The effects on growth of neurospheres we observed in response to both circadian clock knockouts indicate that the proteins serving in the oscillator’s timing mechanism are also important in neurogenesis. If, on the other hand, effects were only found in spheres from one knockout but not the other, it would be clear that the clock is not needed for normal neurogenesis to proceed. Nevertheless, the different effect on NSPCs observed in the two types of knockout neurospheres does suggest that they may be caused by deficits unique to the missing proteins. It seems equally likely that these differences are because of the different roles played by the

proteins in the timing mechanism. The proteins serve at different phases of the circadian cycle

many hours apart [211], and the protein expression patterns they induce are also distinct. The

phenotypes that the knockout spheres exhibit appear to be because of the missing circadian

timing and the unique state of clock proteins each knockout generates. For example, mPER2

protein levels are elevated and BMAL1 is at low levels in Cry1-/-, 2-/- mice [212]. How the many

downstream, clock-controlled genes that are under Bmal1 timing control [213] respond differently to suppressed rather than absent Bmal1 might explain why neurosphere features produced by the two knockouts differ: some of these effector genes may be induced under one condition but not the other.

Neurosphere cultures are also quite informative because they display endogenous capabilities and behaviors of NSPCs that can proceed independently of the neural regulation that controls stem cell proliferation and differentiation in the brain. Similar to the developing embryonic brain, adult neurosphere cultures reveal mechanisms by which neural cells arise solely from glial-like cell origins [214]. The sequence of progenitor cell types in sphere cultures may occur at a different rate than in situ, but eventually the immediate precursors of mature neurons and glia appear. Whether fully functional neurons are produced from the neurospheres used here remains to be determined. Nevertheless, it is clear that the decreased proliferation, increased apoptosis, and altered cell fate observed in the knockout spheres cannot be attributed to control or lack of control through neural signaling. The tendency to produce cells with astrocyte- like rather than neuronal characteristics is quite similar to the excessive gliosis observed in the

Bmal1-/- mouse brain [185]. This result indicates that a critical decision of progenitor cells in

setting the neurogenic yield depends on either a circadian timing event intrinsic to NSPCs or

merely to the presence of this key protein in these cells.

Clock, neurogenesis, and memory formation

Increased hippocampal neurogenesis is correlated with higher cognitive performance in animals. Neurogenesis-related improvements have been reported in acquisition and retention of memory in spatial memory consolidation (Morris water maze, radial arm test), fear-conditioned memory, contextual fear memory, olfactory perceptual memory, and pattern separation [215-

218]. Selective ablation of neural stem cells in transgenic animals or depletion of the stem cell pool by anti-mitotic treatments has been shown to alter place and object recognition memories

[71-73]. Neurogenesis in the olfactory bulb has been shown to be critical for odor discrimination tasks, odor memory, and learning [34, 74]. Overall, adult neurogenesis is important for its adaptive significance--for example, in predator avoidance, homing behavior, locating food, or identifying mates [34].

Similarly, the circadian timing system provides animals with an ability to anticipate predictable daily events that impact their survival or fitness. The core circadian clock proteins that serve in the timing mechanism are found throughout the hippocampus [181], and impairing their normal expression causes deficits in habituation, exploratory behavior, and learning [182].

Cry 1-/-, 2-/- mice exhibit impaired recognition memory, increased anxiety [219], and lack of time-place associations [180], although no deficits in working or long-term memory formation were reported. In contrast, Bmal1-/- mice show a diminished learning ability and have previously been reported to display phenotypes associated with accelerated aging [70, 191]. Per2-/- mice showed impaired trace-fear memory, suppressed long-term potentiation (LTP), and diminished

CREB phosphorylation [106]. Equivalent effects were observed in mPer1-/- mice in which spatial memory, CREB activation, and LTP declined [181, 220], further suggesting that Per

78 genes have additional effects on hippocampal functions, perhaps independent of their role in circadian timing.

The specialized ability of the hippocampus to replace its interneurons raises the possibility that a number of the described clock-related deficits are manifested through alterations in neurogenesis. The presence of circadian clock activity observed in this study during neurosphere differentiation encourages further examination of circadian protein influences on cell determination and proliferation. Circadian properties of the NSPCs could be exploited when modifying these cells to deliver treatments to the brain for correcting neurodegenerative diseases or brain trauma. For example, if differentiation into neurons and glia is gated by the clock, then the relative yield of the cell types might be manipulated through clock gene protein expression. Furthermore, by knowing the phase of NSPC rhythms in situ it may be possible to determine when the cells are least sensitive to deleterious effects of medications.

Furthermore, delivery of cancer chemotherapies could be timed to a specific phase of the NSPC rhythm to minimize stem cell toxicity and impaired adult neurogenesis.

Figure. 13. Neurospheres display circadian rhythms in mPer1 gene expression during

early differentiation. DG neurospheres grown in stem cell medium were transferred to serum

medium (SM) immediately after a synchronizing forskolin treatment ending at time zero. Shown

are 5-point running averages of intensity measurements from images collected hourly. Note that

rhythms can be observed as early as the second day in SM. ADUs: analog-to-digital units of the camera.

Figure 14. NSPCs are the dominant cell types during the first four days of DG neurosphere differentiation. SOX2+ (green) cells after A: 1 day in SM, B: 4 days in SM. Msi1+ (green) cells after C: 1 day in SM and D: 4 days in SM. E: Nestin+/GFAP+(yellow) cells at day 4 in SM indicating that radial glial-like cells persist after 4 days of differentiation in SM. Neurospheres lack immature and mature neuronal cells after differentiating 4 days in SM as shown by F:

BetaIII+ (green) and G: NeuN+ (green). Nuclei were stained with propidium iodide (red). H:

The source of the bioluminescence signal indicated by mPER1+ (green) cells in the neurosphere core after 3 days in SM. All nuclei were stained with Hoechst3342 (blue) unless specified. Scale bar=50 µm.

Figure 15. Bmal1-/- neurospheres show altered growth patterns and increased cell death.

A: brightfield image of Bmal1-/- DG neurosphere (arrows indicate large and small lacunae).

Live/dead stain using propidium iodide (red) and Hoechst3342 (blue) shows higher cell death near the lacunae in B: Bmal1-/- DG neurospheres when compared to C: wild-type controls.

After 2 days of differentiation in SM, caspase-3+ cells were detected in D: wild-type neurospheres and Bmal1-/- neurospheres at E: 20x and at F: 40x magnification. Nuclei were stained with propidium iodide in (D-F). G: Percentage of caspase3+ and propidium iodide- positive cell staining in DG neurospheres from Bmal1-/- and wild-type littermates. Scale bar=50

µm.

Figure 16. Cry1-/-, 2-/- neurospheres have reduced proliferation and growth in culture.

The average size of DG and SVZ neurospheres from Cry1-/-, 2-/-, wild-type, and Cry1-/- mice were compared at days 14 and 35 in vitro while in SCM. Shown is the total area of all spheres of each dish (as µm2). Asterisk indicates significant difference from controls (p<0.05).

Figure 17. Neuronal commitment is diminished in Bmal1-/- DG neurospheres. In wild-type

DG neurospheres the sequence of cell types during differentiation in B27 medium parallels events during in situ neurogenesis. A: Immature neurons, shown by BetaIII-tubulin (green) at day 7 and lacking GFAP co-localization (red). B: Mature neurons, shown by NeuN (green) at day 14 and lacking GFAP (red). In Bmal1-/- neurospheres an increased number of astrocyte

proliferation and reduced neuronal differentiation is shown by C: BetaIII-tubulin (green) along

with GFAP (red) at day 7, and D: NeuN (green) along with GFAP (red) at day 14. All nuclei

were stained with Hoechst (blue). Scale bar=50 µm. E: Percentage of positive cells for DCX

(neuroblasts), BetaIII-tubulin and NeuN at days 4, 7, and 14 after differentiation in B27 medium,

respectively.

Cell type Day 1 Day4

SOX2+ 74.83 ±14.93% (n=6) 35.00 ±6.40% (n=7)

Msi1+ 81.21 ±14.90% (n=5) 50.11 ±12.77% (n=7)

Nestin+/ GFAP+ N.A. 47.35 ±10.09% (n=6)

Beta-III+ N.A. 0.62 ±1.32% (n=7)

NeuN+ N.A. 0.16 ±0.27 (n=9)

Table 3. Cell types identified by markers for stem cells and differentiated cells after 1 or 4 days in serum medium. DG neurospheres were allowed to differentiate in SM for 1 or 4 days as indicated. Shown are the average percentages of cells positive for cell markers followed by standard deviation. Total numbers of spheres analyzed are indicated in parentheses. N.A.: not available.

Genotype Percent NeuN+ cells Percent GFAP+ cells

Bmal1-/- 1.39 ±3.44% (n=10) 53.58 ±15.85% (n=10)

Cry1-/-, 2-/- 41.04 ±9.07% (n=12) 23.41 ±8.98% (n=12)

C57 BL6 (6-month-old WT) 53.56 ±15.84% (n=8) 3.55 ±4.00% (n=8)

mPer1::luc (12-month-old 49.75 ±7.13% (n=9) 9.79 ±10.18% (n=9)

WT)

Table 4. Percent positive mature neuronal and glial cells after 14 days of differentiation in B27 medium. Neurospheres were made from the DG tissue harvested from mice and were allowed to differentiate in B27 medium for up to 14 days. Shown are the percentages of positive mature neuronal (NeuN+) and astrocytes (GFAP+) cells followed by standard deviation. n: the total number of spheres analyzed.

CHAPTER IV: CONCLUSIONS AND FUTURE DIRECTIONS

Summary

1. NSPCs harvested from the SVZ exhibit circadian rhythms in mPer1 gene expression.

Neurospheres in SCM are enriched in NSPCs and are abundantly present during

ontogeny of the circadian rhythm in SM or B27 medium.

2. Type-A cells (the neuroblasts) which migrate to the OB via the RMS are also a subset of

circadian clock cells that seed the OB with new interneurons. These clock cells may

undergo differentiation at a particular time of the day to allow controlled timely repair

and regeneration in the brain.

3. NSPCs harvested from the DG also exhibit circadian rhythms in mPer1 gene expression.

4. DG neurospheres cultured from Bmal1-/- knockout mice, that lack circadian rhythms,

have abnormal growth and development relative to wild-type controls. Lacunae were

observed only in Bmal1-/- DG neurospheres maintained in SCM and could be a result of

increased ROS levels previously reported in Bmal1-/- mouse brain.

5. Increased cell death was also reported in Bmal1-/- DG neurospheres and was quantified by

a live/dead assay and confocal immunofluorescence microscopy to localize cleaved-

caspase-3.

6. Increased astrogliosis was recorded, relative to their wild-type control, when Bmal1-/- DG

neurospheres were allowed to differentiate in neural differentiation medium.

7. Lack of immature and mature neuronal immunostaining in Bmal1-/- DG neurospheres was

confirmed by stage-specific immunofluorescence markers and confocal

immunocytochemistry. Very sparse neuroblast (DCX+), immature neurons (BetaIII-

tubulin+) and mature neuronal (NeuN+) cells were observed after 4, 7 or 14 days in

neuronal differentiation medium in vitro.

8. Significant reduction was recorded in proliferation and growth of DG neurospheres

cultured from Cry1-/- , 2-/- knockout mice relative to Cry1-/- knockout and wild-type

controls.

9. Circadian timing or clock proteins are essential for normal neurosphere formation,

proliferation, growth, fate commitment, neuronal differentiation, and cell survival in

vitro.

10. The circadian clock gates the process of neurogenesis. Peak clock gene expression

coincides with a peak in cell cycle phases in different cell types. The phase and peak of

clock gene expression identified in NSPCs and Type-A cells in this study should be used

to target drugs at different times of the day.

Future directions

Quiescent neural stem cells (qNSCs) - the mysterious “kahuna”

QNSCs are a most primitive type of cells that are mostly in G0 phase of the cell cycle

[221]. The present study was not designed to identify circadian properties of qNSCs and we

cannot rule out the possibility that qNSCs could be functional circadian clock cells. They are

present in the cultures while BLI was performed. It is possible that these cells could be seeding

the rhythmic progeny (NSPCs and the neuroblasts) identified in our system. Nevertheless,

examining circadian properties of qNSCs is important to develop chrono-pharmacotherapeutics

for neurological disorders.

The first question needing to be addressed is whether qNSCs oscillate with a circadian

rhythm. If they do, then treatments could be designed to stimulate qNSCs to enter the cell cycle,

and that should be possible by understanding how mitosis is regulated by the circadian clock.

For example, it would be useful to know the phase of qNSC rhythms when they can be most

easily induced to divide. A neurogenic stimulating agent could be administered at a particular

phase of the clock gene expression rhythm to stimulate differentiation of qNSCs to an activated radial glia-like state (NSPCs).

If the qNSCs do not have a functional circadian clock, then a second question is whether circadian rhythms can be induced, perhaps by manipulating levels of core clock proteins, and then using the molecular actions of the clock on the cell cycle to stimulate qNSC cell division.

We may find that initiation of the circadian clock in these cells also induces differentiation because of the important regulators that may be under clock control, as mentioned in Chapter I.

Although examining any stem cells is a challenging task because of their scarcity in vivo

and in vitro, cell sorting can be used to purify these cells in vitro. It is however difficult to

maintain qNSCs in a quiescent state in vitro without additional growth factors [222], unlike

tumorospheres which secrete their own growth factors [223]. Growth factors such as bFGF and

EGF activate the qNSCs in culture medium which then undergo symmetric or asymmetric cell

division to give rise to activated NSCs. Not much is known about the interactions of such

growth factors with the circadian clock. It is possible that the additional growth factors would

interfere with the timing mechanism by constantly activating MAPK pathways that shift the

phase of the circadian clock [136]. Additional challenges could result from not knowing the

effects of cell sorting on the phase and timing of core clock genes; the laser light and cell

handling by the sorter could depolarize cells and alter calcium levels though voltage-sensitive

Ca2+ channels, causing shifts in phase or other effects on the clock. A Ca2+/cAMP-response element in the mPer1 gene promoter is known to produce phase shifts and probably serves in the entrainment of the clock to light cycles [224]. Despite these limitations, isolation and culture of pure stem cell populations would be quite valuable for studies of any intrinsic circadian properties they may have.

Many studies do not take into consideration the heterogeneity of the tissue or cell type when analyzing circadian properties. An apparent lack of rhythms in the cells as a whole can be caused by a failure to synchronize cells within the culture or tissue as discussed in the previous chapters in which forskolin was used for this purpose. For example, one study by Kimiwada et al., did not identify circadian rhythms in NSC’s from the SVZ possibly because of cell heterogeneity and lack of synchronization [145]. I used confocal immunofluorescence microscopy exhaustively to characterize the cell types present in the neurosphere cultures, but it

90 was not possible to identify circadian rhythms within individual cell types. The neurosphere cultures used in this study are a useful assay to analyze circadian properties of the NSPCs in vitro, but additional imaging techniques applied to monitoring circadian rhythms more selectively within the spheres according to cell type should be explored. This approach would also help in describing how many qNSCs are present and their circadian properties.

Fate commitment in adult neurogenesis- “The road not taken”

Increased astrocyte proliferation, cell death, and a significant decrease in neuronal cells

were observed in Bmal1-/- mice under neuronal differentiation conditions. Similar reduced

proliferation was also recorded in cryptochrome knockout mice. A question remaining

unanswered by this study is whether the observed effects on the knockout neurospheres could be

manifested by a non-clock function of these clock proteins outside the circadian timing

mechanism. The oscillations in the level of the clock proteins or, instead, just the protein

expression itself could be needed to prevent the switch to the abnormal phenotypes observed in

knockout neurospheres. To rule out the possibility of a non-clock role of clock proteins, it would

be interesting to rescue the clock in the NSPCs. For example, the circadian clock could be

rescued in BMAL1 knockout neurospheres by using BMAL2, a paralog of BMAL1 as shown in

a previous study [225].

It is also possible that the abnormalities observed in the neurospheres harvested from the

circadian arrhythmic mice could have manifested early in the developmental process. It would be

interesting to use tissue-specific conditional clock gene knockout mice in which specific genes

could be targeted at different stages of development, thereby ruling out the possibility of clock

knockout effects during development.

Phenotypic alterations observed in both Bmal1-/- and Cry1-/- , 2-/- mice could have also been due to different amounts of some effector genes controlling the neurogenesis process downstream of the circadian core clock genes. To better understand how these CCGs could explain the effects seen in the mutants they might be induced conditionally and in combinations to rescue the mutations. Although there may be many candidate CCGs to screen, ones cited in

Chapter I should be examined. They may be found to serve as important links between the clock and neurogenesis. As mentioned earlier, disruption of circadian rhythms has also been widely associated with altered memory formation, learning capabilities and animal survival [226].

Therefore, modulation of fate determination in these neural stem cells by the circadian clock or clock proteins warrants much further examination.

Circadian properties of NSPCs and clinical aspects

“And further still at an unearthly height,

One luminary clock against the sky

Proclaimed the time was neither wrong nor right.

I have been one acquainted with the night.”

– Robert Frost

Neural stem cells are believed to give rise to cancer stem cells (CSCs) in the brain that have many of the properties of embryonic and adult neural stem cells including self-renewal and multipotency [227]. CSCs are thought to differentiate and produce the diverse proliferating cancer cells of recurrent solid tumors and blood cancers after unsuccessful therapeutic treatments. NSPCs were shown to exhibit circadian rhythms in mPer1 gene expression in this study, and CSCs from a rat glioma cell line were also shown to exhibit circadian rhythms in clock gene expression in a previous study [136]. The expression of core clock genes changes throughout the day and coincides with timing of cell cycle entry and exit in non-cancer cells.

Some CCGs have been shown to peak in early afternoon and others later in the night [228]. The phase relationships between CCGs in cancers cells and CSCs are far less understood but could be used to time drug delivery.

Chemotherapeutic agents like gamma-secretase inhibitors that suppress the Notch pathway of stem cells are beginning to be used to specifically target and eliminate the CSC population within tumors [229]. To target CSCs more effectively, it is plausible to administer high-dosage treatments at the phase of clock gene rhythms when these cells are most vulnerable.

Ideally, the phase differences observed in the two studies, of CSCs and NSPCs, could be used to

design a drug treatment regimen which would coincide with the circadian phase of NSPCs

during which damage to these cells and the non-cancer cell populations of the body would be

minimal. This approach could thereby help in improving the prospects of long-term patient

survival without the damaging effects of chemotherapy on cognition, also known as the

“chemobrain” [230]. Whether this disorder is caused by loss of neural stem cells is not yet

known, but cells designed to target CSCs are more likely to have this effect. How much of a loss

of qNSCs or NSPCs is acceptable remains unclear, but such deficits early in life are likely to be

particularly concerning because adult neurogenesis is needed for learning and memory. In the

elderly, however, NSCs are less abundant, suggesting that any lost to chemotherapy or radiation

therapy might have a greater impact on cognition.

The circadian timing mechanism and the core clock proteins that were shown to be important for NSPC differention in this study could also be exploited to design treatment regimens to help patients suffering from Alzheimer’s and Parkinson’s diseases. By exploiting links between the circadian clock proteins and differentiation events, as they become better established, NSPCs could be directed to needed neuronal cell types in specific brain areas to compensate for cell loss. Loss of circadian timing or clock proteins as shown in this study was a crucial factor in determining the astroglial versus non-neuronal cell fate.

It may also be possible to design drugs which can serve as circadian clock protein agonists so as to alleviate the effects from loss of neurons in certain neurodegenerative diseases.

The bHLH transcription factors have been suggested previously to determine the fate of multipotent progenitors [231]. Therefore, it is likely that the level of the circadian clock proteins can be manipulated to control both the timing of differentiation and cell fate.

Lastly, this study did not explore the circadian properties of oligo-progenitor cells or the oliogodendrocytes. It has been shown previously that the oligo-progenitor cell numbers peak during the night in animals housed in an LD cycle [232]. Although these rhythms were in an LD cycle, in vitro studies are warranted to identify endogenous circadian clock control.

Consequently, cells needed to repair demyelinated brain areas in, for example, multiple sclerosis patients may be produced more effectively after understanding the role of clock proteins or clock timing in differentiation and NSPC proliferation [233].

The lab is lovely, dark and deep,

But I have deadlines to keep,

And lines to write before I sleep,

And lines to write before I sleep.

(Inspired by Robert Frost)

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APPENDIX A: LIST OF ABBREVIATION

SCN Suprachiasmatic nucleus bHLH Basic helix loop helix BMAL1 Brain muscle and ARNT like 1 PER Period CRY Cryptochrome CCG Clock controlled gene OB Olfactory bulb SVZ Subventricular zone DG Dentate gyrus RMS Rostral migratory stream NSC Neural stem cell SCM Stem cell medium SM Serum medium EGF Epidermal growth factor FGF Fibroblast growth factor BLI Bioluminescence imaging Hes-1 Hairy and enhancer of split-1 Msi-1 Musashi-1 SOX 2 Sex determining region Y-box 2 GFAP Glial fibrillary acidic protein DCX Doublecortin PI Propidium iodide 110

APPENDIX B: IACUC APPROVAL

DATE: February 16, 2015

TO: Michael Geusz FROM: Bowling Green State University Institutional Animal Care and Use Committee

PROJECT TITLE: [695209-4] Molecular Biology and Biomedical Applications of Circadian Clock Cells IACUC REFERENCE #: 14-012 SUBMISSION TYPE: Revision

ACTION: APPROVED with comment APPROVAL DATE: February 16, 2015 EXPIRATION DATE: February 15, 2018 REVIEW TYPE: Administrative Review

Thank you for your submission of Revision materials for the above referenced research project. The Bowling Green State University Institutional Animal Care and Use Committee has APPROVED your submission with comment. All research must be conducted in accordance with this approved submission. Please make sure that all members of your research team read the approved version of the protocol.

The comment was:

Minor wording clarifications were made to the protocol application (Item II.E). Please see the final approved protocol application.

Report all NON-COMPLIANCE issues regarding this project to this committee.

Please note that any revision to previously approved materials must be approved by this committee prior to initiation. Please use the Addendum Request form for this procedure.

This project requires Continuing Review by via Progress Report on an annual basis. Please use the Annual Renewal form for this procedure.

If you have any questions, please contact the Office of Research Compliance at 419-372-7716 or [email protected]. Please include your project title and reference number in all correspondence with this committee.

This letter has been electronically signed in accordance with all applicable regulations, and a copy is retained within Bowling Green State University Institutional Animal Care and Use Committee's records.

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