EXPLORING THE ROLE OF CALCIUM-BINDING IN THE MOUSE SUPRACHIASMATIC NUCLEUS

Tyler M. Birkholz

A Thesis

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

MASTER OF SCIENCE

August 2019

Committee:

Michael Geusz, Advisor

Verner Bingman

Raymond Larsen

ii ABSTRACT

Michael Geusz, Advisor

The master circadian clock in the hypothalamic suprachiasmatic nucleus of vertebrates receives information from the retina that entrains the clock to cycles of light and darkness in the animal’s environment. This timing signal is important for maintaining the appropriate organization of circadian rhythms generated throughout the body, which have many effects on health, development, and aging. One potential influence on entrainment is from retinal light signals acting on SCN cells that have protein expression patterns typical of stem cells. These stem-like cells may have plasticity in their interactions allowing circadian rhythms generated in the SCN to be modified when needed to adapt to changing environmental or internal conditions.

Calcium-binding in SCN cells have important functions in the entrainment process, but the role of one of these proteins, calreticulin (CALR), has not been examined in the SCN. This study characterized the spatial pattern of CALR-expressing SCN cells and their distribution among (MAP2-positive), glial cells (GFAP-positive), and stem-like cells (SOX2- positive) by using immunocytochemistry and confocal fluorescence microscopy. Cells expressing significant levels of CALR were found throughout most of the SCN, with fewer in the core region. In the SCN, 86.9% of CALR cells were classified as neurons, 19.1% as astrocytic glial cells, and 60.33% as stem-like cells according to immunofluorescence imaging. To determine whether CALR expression can be induced by a stimulus that can entrain the SCN circadian clock a group of mice were exposed to two hours of light in the early portion of the night and were compared with mice remaining in darkness. Although SCN neurons did not show iii a significant response to the stimulus, non-neuronal cells including glial cells did, showing a two-fold increase in percentage of CALR-positive cells. We conclude that a substantial number of the CALR cells, 98.6%, express stem-marker SOX2 elevated, suggesting CALR could have an important role in this enigmatic cell type. We also conclude that SCN glial cells respond to the excitation from an entraining light signal with an increase in number that express elevated

CALR, suggesting a role for this ubiquitous protein in Ca2+-regulated events during entrainment. v

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 11

1. Animals ...... 11

2. Brain sections and ICC ...... 11

3. Metamorph multi-wavelength cell scoring routine ...... 12

4. Statistical analysis ...... 13

RESULTS ...... 14

1. CALR and SOX2 at ZT8 ...... 14

1.1. CALR and SOX2 ICC ...... 14

2. CALR and MAP2 at ZT4 and ZT11 ...... 18

2.1. CALR and MAP2 ICC (ZT4) ...... 18

2.2. CALR and MAP2 ICC results (ZT11) ...... 18

3. CALR and GFAP at ZT5 and ZT7...... 22

3.1. CALR and GFAP ICC (ZT5) ...... 22

3.2. CALR and GFAP ICC (ZT7) ...... 22

4. Test of CALR induction by light at ZT17 ...... 25

4.1 CALR and MAP2 ICC induction control ...... 25

4.2. CALR and MAP2 ICC induction experimental ...... 26

4.3. Statistical analysis of ICC results ...... 26

DISCUSSION ...... 32

1. CALR and SOX2 ...... 32 vi

2. CALR and MAP2...... 33

3. CALR and GFAP ...... 35

4. CALR induction by a light pulse and possible role in entrainment to the

light cycle ...... 36

REFERENCES ...... 43

APPENDIX A. METAMORPH CELL COUNTING DATA ...... 52 vii

LIST OF FIGURES

Figure Page

1 Proposed mechanism for light induction of circadian regulation and

entrainment ...... 3

2 Daily expression of calreticulin (Calr), (Calm1), (Calb2), and

(Calb1) RNA in SCN of mice maintained in a light/dark cycle ...... 7

3 Calr in SCN of mice maintained in darkness ...... 8

4 SCN cells expressing CALR and SOX2 ...... 15

5 Summary of SOX2 and CALR expressing cells within the SCN ...... 17

6 Summary of MAP2 and CALR expressing cells within the SCN ...... 20

7 SCN cells expressing MAP2, GFAP, and CALR ...... 21

8 Summary of GFAP and CALR expressing cells within the SCN ...... 24

9 SCN cells expressing MAP2 and CALR...... 28

10 Summary of MAP2 and CALR expressing cells within the SCN ...... 31 viii

LIST OF TABLES

Table Page

1 RNA of major Ca2+ -binding proteins expressed in mouse SCN ...... 6

2 CALR and SOX2 metamorph cell counting data ...... 16

3 CALR and MAP2 cells in SCN ...... 19

4 CALR and GFAP cells in SCN ...... 23

5 Proportions of cells expressing CALR and MAP2. Control ...... 29

6 Proportions of cells expressing CALR and MAP2. Experimental ...... 30

7 T-TEST analysis of CALR light induction ...... 31 1

INTRODUCTION

The circadian system regulates various near-24-hour behavioral and physiological rhythms through a series of central and peripheral oscillators (1-3). The principal circadian pacemaker is a spindle shaped brain area known as the suprachiasmatic nucleus (SCN), which is located just above the optic chiasm. External cycles of light and dark entrain the circadian clock to the period and phase of the day through retinal light signals that pass through the retinohypothalamic tract (RHT) to the SCN. The mechanism behind entrainment involves the transcription-translation feedback loop of core clock BMAL1, CLOCK, PER, and CRY; along with the oscillations of a regulatory loop involving REV-ERBα and RORα that positively and negatively regulate BMAL1 oscillations (4-11). These autonomous oscillations in the TTFL of the core circadian genes, which have been shown to occur endogenously within individual isolated cells (12), are altered by intracellular Ca2+ signals (13) which also mediate much of the light response of the circadian system (14).

Because light induces intracellular calcium release within SCN neurons (15) and intracellular calcium affects the TTFL of core clock genes, there have been numerous investigations of several calcium-binding proteins in the SCN (16-19). Calreticulin (CALR), also known as calregulin, is a major calcium-binding protein within the lumen of the (ER). SCN cells show circadian rhythms in cytoplasmic Ca2+ levels (20), but it is not known whether this is also true in the ER. Ca2+ release from the ER appears to be an important component of SCN entrainment (Figure 1), but any specific role of CALR in this process has yet to be examined.

The roles of other calcium-binding proteins such as calretinin, calbindin, calmodulin and calmodulin II (CaMKII) have been examined extensively within the SCN to try to

2 elucidate what roles they may have in altering cytosolic calcium levels to influence the circadian clock. Calretinin has been shown to be present within rat SCN neurons where it is most likely modulating the regulation of calcium during synaptic transmission (17). Calbindin has been shown to buffer intracellular free calcium (21) and it is required in order to generate behavioral circadian rhythms based on SCN lesion studies (22). Calmodulin and CaMKII have also been shown to be involved in the enhancement of circadian rhythm phase shifts through the activation of NMDA receptors by photic stimulation, which causes an influx of extracellular calcium that will then bind to calmodulin and activate CaMKII (18). These studies have focused mainly on cytosolic calcium signals, yet calcium signals from the ER and other intracellular compartments have not been thoroughly examined in relation to circadian rhythms.

The mechanism of light entrainment of SCN neurons via glutamatergic signaling from the RHT has been examined previously (15). Light input is received by SCN neurons through the

RHT which releases glutamate. Glutamate activates NMDA-type glutamate receptors allowing

Ca2+ influx. The intracellular calcium binds to ryanodine receptors (RyR) of the ER causing more Ca2+ release. This large efflux of calcium from the ER activates CaMKII pathways that activate gene transcription. However, any role or influence of CALR on calcium within this mechanism is unknown.

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Figure 1. Proposed mechanism for light induction of circadian gene regulation and entrainment. Image modified from Ikeda, 2004 (15).

4

CALR performs a large variety of functions such as protein folding, Ca2+ homeostasis, sarcoplasmic/ER Ca2+ATPase (SERCA) regulation, integrin protein control, and cell adhesiveness modifications (23, 24). It is composed of three structural and functional domains:

The N-domain, P-domain, and C-domain, all of which play a distinct role in the protein’s function. The N and P-domains are involved in functions and protein interactions, whereas the C-domain is responsible for binding calcium, buffering ER calcium levels, and acting as a sensor for chaperone/protein interactions (23). It has also been shown that the P- domain of CALR binds to the C-terminal tail of SERCA2b and alters its function based on ER calcium levels, and that high concentrations of intraluminal ER CALR block SERCA calcium influx, even after ER stores have been depleted (25, 26). This inhibition prevents the influx of cytosolic calcium into the ER, which may allow CALR to perform its role in protein folding without being inhibited by binding and buffering intraluminal ER calcium. Essentially, CALR monitors ER Ca2+ levels and can respond to Ca2+ release from the ER to the cytosol and serves in controlling store-operated Ca2+ entry (SOCE) from outside the cell (23, 25).

Calcium mobilization from internal stores is an important modulator of gene transcription

(27) and this is the most likely way that CALR interacts with the circadian clock molecular mechanism. Circadian rhythms, generated in the SCN through feedback loops acting on gene expression, synchronize the network of circadian oscillators in each major organ system based on photic information the SCN receives through the RHT (13). Excitatory, glutamatergic, light signals via the RHT produce SCN cytoplasmic Ca2+ responses (28) that entrain the SCN clock to the environmental light cycle by altering gene transcription within SCN neurons (20). Note that this external signal to the SCN is not required for the circadian clock to function. Circadian

5 rhythms persist under conditions where there is no entraining signal, such as when rodents are maintained in conditions of continuous darkness and constant air temperature.

The entraining light signal causes Ca2+ influx from outside the SCN cell causing Ca2+ to be released from the ER, but the effects of this additional ER Ca2+ on the clock are not yet clear.

It has been reported, however, that a stress response induced by sustained depletion of ER Ca2+ in mouse NIH/3T3 fibroblasts (26) or HeLa cells (29) increases CALR gene expression.

However, this response occurs 8-16 hours after stimulating Ca2+ store depletion and is generally too slow to transfer an entraining light signal to the molecular clock mechanism, which is phase shifted quickly during entrainment (26, 29, 30).

Nevertheless, the CALR gene and upstream regulatory sequences contain elements that suggest CALR could be induced quickly by Ca2+ signaling pathways acting through the transcription factors AP-1 (Fos and Jun) or NFAT according to a bioinformatics analysis and published results (26, 31). More rapid CALR gene induction (1-hour) has been reported following a heat shock treatment that might act through c-Jun activation (30). This study will test for rapid CALR induction by a light exposure known to phase shift the clock during entrainment.

Another process that controls CALR expression is the rhythmic output from the circadian clock, as shown by the CircaDB database, raising the possibility that evolutionary pressures have selected for more of the protein to be available at a particular time of day. Exploring CALR protein activity at this phase of the cycle could help in understanding the role of CALR in SCN functions. The CALR promoter contains the E-box regulatory element, suggesting the circadian clock regulates the CALR gene directly. CALR RNA levels also fluctuate daily in the SCN of mice maintained in a typical 12 hours of light:12 hours of dark (LD) cycle according to the

SCNseq database (http://www.wgpembroke.com/shiny/SCNseq (32). Interestingly, calbindin and

6 calretinin, do not show this rhythm, again suggesting that CALR has an important function near the peak or trough of its rhythm (Table 1, Figure 2, Figure 3). Although CALR mRNA is rhythmic, we will need to measure the protein levels at different phases of the cycle to verify that

CALR is also expressed rhythmically.

Table 1. RNA of major Ca2+-binding proteins expressed in mouse SCN.

Peak Gene Protein Fluctuating SCN enriched Phase

CALR Calreticulin Yes ZT14 No

Calm1 Calmodulin Yes ZT12 No

Calb2 Calretinin No ZT17 No

Calb1 Calbindin No ZT6 No

Data indicate fluctuation, peak phase, and SCN enrichment relative to other brain expression of four major calcium-binding proteins found within the SCN. Only CALR and Calm1 fluctuate significantly, suggesting their rhythmic gene expression is driven by the light/dark (LD) cycle or the circadian clock or both. These calcium-binding proteins are not significantly enriched in the SCN suggesting they are not expressed primarily in this brain area. Data obtained from the SCNseq database (32). ZT is the time of the LD cycle where dusk = ZT 12.

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Figure 2. Daily expression of calreticulin (Calr), calmodulin (Calm1), calretinin (Calb2), and calbindin (Calb1) RNA in SCN of mice maintained in a light/dark cycle. Calr, Calm1, and Calb2 show peak expression during the night, however only Calr and Calm1 have significantly oscillating expression. Figures obtained from SCNseq database (19).

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Figure 3. Calr gene expression in SCN of mice maintained in darkness. Period estimate = 25.74 hours by Lomb-Scargle analysis (CircaDB database).

Because of the role of intracellular Ca2+ in development we will also test whether CALR co-localizes with SOX2, which plays an important role in neurogenesis. Cell proteins associated with neurogenesis, including SOX2, are expressed in the SCN (31, 33, 34), suggesting that the apparent immature state of the SCN is important for clock functions such as entrainment. CALR is expressed on the surface of cells where it interacts with α-integrins, which is an important protein of cell-cell interactions with the that are modified during development and stem cell differentiation (35, 36). We will use immunocytochemistry (ICC) to characterize the cell types and cell compartments where CALR is present to determine whether it is associated with SCN stem-like properties.

The ER is essential in eukaryotic cells for its role in protein folding (37, 38). However, various factors can cause an accumulation of misfolded proteins within the ER which is known as endoplasmic reticulum stress; this will cause a reaction known as the unfolded protein response (UPR) in eukaryotic cells (39). The UPR activates intracellular pathways that regulate expression of various genes in order to maintain proper protein folding

9 within the ER under stress; However, if proper protein folding within the ER does not occur, apoptosis will be induced (40). Alterations to the expression levels of CALR have been shown to induce UPR within cells, suggesting that alterations to CALR may also activate gene expression through UPR signaling pathways (41, 42, 43). There are interactions between the UPR signaling pathways and clock genes which also suggests that UPR within the SCN may activate clock genes (44), so CALR induction due to intracellular in the SCN may be able to also induce circadian genes by activating the UPR pathway; however, this interaction has yet to be examined.

In light of the various roles of CALR and its effects on intracellular calcium levels, we examined the protein through two specific aims. Specific Aim 1 was to characterize the protein expression pattern of calreticulin (CALR) across the mouse SCN and identify SCN cells types expressing CALR. To accomplish this aim we used ICC to determine the co-localization of

CALR expression with various neuronal, glial, and stem cell markers. These results identified the types of SCN cells that express CALR and also cells co-expressing CALR with stem cell markers. We believe stem-like properties in the SCN allow for plasticity of SCN neurons enabling entrainment to different rhythmic internal and external signals. It has yet to be shown that CALR co-localizes with stem cell markers, but this is likely because of various CALR roles in metastasis, cell-cell communication, and integrin-mediated calcium signaling (13, 24). How these CALR functions might also serve in the SCN is not yet known. Brain sections were imaged as described below in Methods Details. Cell counting software was used to determine the distribution of CALR across cell categories.

Specific Aim 2 was to determine whether CALR expression oscillates in the SCN throughout the day and is inducible in the SCN by stimuli that entrain the circadian clock. Mice

10 were prepared for CALR ICC during the early day and early night and possibly two other phases.

Two measures of daily induction were examined: intensity of ICC signal in individual cells and total number of CALR-positive cells. First, we tested whether CALR expression is inducible in the SCN by nighttime light exposure that shifts the phase of the circadian clock in mice. Levels and distribution of CALR protein expression were examined in the SCN by ICC immediately after a 2-hour light exposure at different phases of the light cycle using a protocol that has been shown to generate phase shifts and induce the immediate-early gene Fos in the SCN by light

(45).

Our analysis of the CALR gene promoter suggests it is inducible by Ca2+ through the c-

AMP response element binding protein (CREB), a transcription factor that activates Fos and the core circadian clock gene mPer1 (46). We have also identified putative NFAT-binding elements within the CALR promoter and enhancer, suggesting that activation of NFAT through the calcium-binding protein calcineurin could activate CALR through the light entrainment pathway

(47). NEUROD and DREAM, Downstream Regulatory Element (DRE) Antagonist Modulator, are additional transcription factors that could activate CALR transcription in response to Ca2+ signals (48). The continuous increase in CALR mRNA throughout the day shown in SCNseq data (Fig. 1) could result from early induction of CALR by CREB, NFAT1, NEUROD or

DREAM, followed by more activation through AP1, and then additional induction from sustained ER Ca2+ store depletion. The intensity of CALR expression, number of CALR-positive cells, and phenotypes of induced cells was determined using Metamorph cell counting software and comparisons between treated and untreated SCN.

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MATERIALS AND METHODS

1. Animals

Inbred C3HBL/6 (B6) and outbred C3H mice were bred and maintained in cycles of 12 h light and 12 h dark (LD) to entrain their circadian system. Mice were fed ad libitum. Mice of both sexes, 1-8 months old, were used. For the induction experiment, B6 mice were given light, using the standard colony fluorescent lighting, at Zeitgeber time (ZT) 15 (three hours after light offset) for two hours. They were then rapidly euthanized by isoflurane anesthesia and decapitated. Brains were removed surgically, and brain slices were prepared for immunocytochemistry (ICC). Brains of control mice for light-exposure experiments were removed at ZT17 under red LED light. The protocols were approved by the BGSU Institutional

Animal Care and Use Committee and they agree with the National Institutes of Health guidelines.

2. Brain sections and ICC

Coronal brain sections (150 µm-thick) were examined by ICC to identify neural stem cells, neurons, , and CALR-positive cells. Tissue was harvested during the middle of the light portion of the LD cycle, to avoid evoking a phase shift of the clock and then trimmed to an area enclosing SCN and surrounding tissue. Sections containing the SCN were fixed in 4% paraformaldehyde for 60 minutes and standard ICC was then performed. Primary were used at these dilutions: rabbit anti-SOX2 (Life Technologies) 1:500; mouse anti-CALR (sc-

373863, Santa Cruz Biotechnology) 1:500; rabbit anti-CALR (SK252573, Invitrogen) 1:200; mouse anti-GFAP (sc-33637, Santa Cruz Biotechnology) 1:500; chicken anti-MAP2 (MAP,

AVES) 1:1000. After overnight incubation in primary at 4oC, sections were incubated for 2 hours with appropriate Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 648-conjugated

12 secondary antibodies (Life Technologies). Confocal microscopy was performed with a

DM13000B 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 Rolera Thunder cooled-CCD camera with back- thinned, back-illuminated, electron-multiplying sensor (Photometrics). Metamorph software controlling image acquisition (Molecular Devices, Sunnyvale, CA, USA) was used for data analysis along with ImageJ software (NIH). Confocal images were collected in a Z-axis series with 10X or 20X objective lenses and standard DAPI, fluorescein, rhodamine and Cy5 filter wavelengths. Background intensity was subtracted based on intensity measurements from an area outside the SCN lacking distinguishable cells. Neurons and glia were not detected in the SCN of control sections in which only primary antibody was omitted. The distribution of cell types within the SCN was determined with the Metamorph Multi-Wavelength Cell Scoring routine.

3. Metamorph multi-wavelength cell scoring routine

The Metamorph Offline program was used for the cell scoring routine. Each wavelength

(DAPI, SOX2 or MAP2 or GFAP, CALR) image stack (10x) was opened, and the Hoechst+ frame that best represents SCN cells was chosen for all of the images. A background reduction was performed using the background and shading correction process, which allows the removal of background signal based off of an area that has no defined cells. This process is repeated for all of the wavelengths. The borders of the SCN were traced based on Hoechst+ staining, and copied onto a new image, which allows the SCN to be removed from the background image to prevent cells from outside of the SCN being counted. This process was repeated for all wavelengths. The multi-wavelength cell scoring app was then chosen, and the intensity above background was found for each image. This was done by measuring the signal of each image,

13 and taking out the bottom ¼ of the signal, which allows the cell scoring routine to only count cells that have an intensity that’s above ¼ of the background value, which are also co-localized with Hoechst staining. Once the intensity above local background was found, the cell scoring routine was performed, and the data was sent to an excel file showing the co-localization patterns and a segmented image. All of this was done by an undergrad student in the lab who was blind to which wavelength represented which protein. For the induction experiment, the undergrad student was blinded to which group was the control and which group was the experimental.

Errors in some of the drawing of the SCN boundary made by the undergrad student were fixed and redone in every experiment, however.

4. Statistical analysis

Statistics were performed using OriginLab software (Microcal). We performed T-Tests to compare CALR+/MAP2+, CALR+/MAP2-, and total CALR+ cells in the light induction experiments. We also used one-way ANOVA and Tukey post-hoc tests to test for differences at a significance level of p<0.05. Groups compared were the percentages of total CALR+,

CALR+/MAP2+, and CALR+/MAP2- cells across different Zeitgeber time points for 4 groups:

ZT4 and 5; ZT7, ZT8, and ZT11; ZT17-control; and ZT17-experimental.

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RESULTS

1. CALR and SOX2 at ZT8

Our lab has previously looked at stem markers within SCN cells and found that there were 20.31% of cells that expressed SOX2 (49). The idea of stem-like cells in the SCN is important because it may help to explain how SCN cells are able to adapt and have plasticity in order to entrain to different signals such as light and temperature. Since CALR has been shown to interact with integrin, and integrin is important in cell-cell communication and stem cells (50-

56), we hypothesized that calreticulin would co-localize with stem cell marker SOX2 in the

SCN.

1.1. CALR and SOX2 ICC

We found that there was a high degree of co-localization between SOX2 and CALR in the SCN. SOX2 localization was found mostly within the shell region of the SCN with a distinct lack of staining in most of the core region; CALR localization was found mostly in the ventral portion of the SCN, however there were CALR+ cells in both the shell and core regions (Figure

4). CALR+ refers here to elevated CALR expression that is clearly above basal levels likely to be expressed in all non-muscle cells. For this reason we only considered as positive any intensity measurements that were above 25% of the maximum intensity of the staining observed at each wavelength.

Almost all of the CALR+ cells were also SOX2+ (98.6%), while less than half of the

SOX2+ cells were also CALR+ (42.6%) (Figure 5). On average there were 60.34% total SOX2+ cells, and 26.09% total CALR+ cells (Table 2). Using statistical software (OriginLab) we found that of the total cells, 34.60 ±7.366 were SOX2+, 0.360 ±0.139 were CALR+, and 25.73

15

±6.834% were SOX2+/CALR+ (Figure 5). Tissue was harvested from these animals at ZT8 in their light cycle.

Figure 4. SCN cells expressing CALR and SOX2. Co-localization of SOX2 with CALR in the left and right SCN of Mus musculus. The SCN boundary was determined from the pattern of Hoechst-stained cell nuclei (blue). Shown is high co-localization (yellow) of SOX2 (green) and CALR (red), with most co-localization within the shell region of the SCN. There was a distinct lack of staining within some of the core region for both SOX2 and CALR. Scale bar = 35 µm.

16 Table 2. CALR and SOX2 metamorph cell counting data.

Age SOX2- / SOX2+ / CALR+ / SOX2+ / CALR+ Total Total (Days) CALR- CALR- SOX2- SOX2+ CALR+ 43 36.39 45.92 0.6802 17.01 62.93 17.69 41.83 13.15 0.3984 44.62 57.77 45.02 43 43.48 42.03 0 14.49 56.52 14.49 35.51 37.32 0.3623 26.81 64.13 27.17 Avg. 39.30 34.61 0.3602 25.73 60.34 26.09 Metamorph cell counting routine. Shown are percentages of total cells as determined by Hoechst+ staining. N=2 mice.

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Figure 5. Summary of SOX2 and CALR expressing cells within the SCN. Percentages are relative to the total number of cells as identified by Hoechst+ nuclei. Of all CALR+ cells, 98.6% co-localized with SOX2, and less than half of all the SOX2+ cells (42.6%) co-localized with CALR.

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2. CALR and MAP2 at ZT4 and ZT11

There have been extensive studies on neurons within the SCN, and it has been shown that

SCN neurons express MAP2 (57-59). It has been shown that neurons of the ventral SCN are entrained by light signals passing through the RHT, which stimulates glutamate release which then causes calcium-induced calcium-release, altering the TTFL of circadian clock genes (19).

Since calreticulin is the major calcium-binding protein within the ER, and the calcium induced calcium release from the ER is important in the entrainment pathway, we hypothesized that there would be high degrees of co-localization within SCN neurons involved in the entrainment pathway.

2.1. CALR and MAP2 ICC (ZT4)

We found that there was a high degree of co-localization between MAP2 and CALR in the SCN. Almost all of the MAP2+ cells were also CALR+, and almost all of the CALR+ cells were also MAP2+ (Table 3). We found that of the total cells in the SCN region, as identified by

Hoechst+ nuclear staining, 6.484 ±1.918% were MAP2+/CALR-, 6.771 ±2.475% were

CALR+/MAP2-, and 54.83 ±5.884%were MAP2+/CALR+ (Figure 6). MAP2 and CALR localization was found within both the shell region and core regions of the SCN (Figure 7).

Tissue was harvested at ZT4.

2.2. CALR and MAP2 ICC results (ZT11)

There was a high degree of co-localization between MAP2 and CALR in the SCN.

Almost all of the MAP2+ cells were also CALR+, and almost all of the CALR+ cells were also

MAP2+ (Table 3). We found that 8.970 ±5.520% were MAP2+.CALR-, 15.28 ±9.850% were

CALR+/CALR-, and 34.78 ±19.02% were MAP2+/CALR+ (Figure 6). CALR was found within both the shell region and core regions of the SCN (Figure 7). Tissue was harvested ZT11. There

19 appeared to be more co-localization at ZT 4 (54.83%) compared to ZT11 (34.78%), however there were less CALR+/MAP2- non-neuronal cells at ZT4 (6.771%) compared to ZT11

(15.28%). Of the total CALR+ cells, 82.3% co-localized with MAP2, and of the total MAP2+ cells 82.6% co-localized with CALR (Figure 6). On average, there were 58.72% total MAP2+ cells and 58.88% total CALR+ cells between both experiments (Table 3).

Table 3. CALR and MAP2 cells in SCN.

Age MAP2- / MAP2+ / MAP2- / MAP2+ / Total Total

(Days) CALR- CALR- CALR+ CALR+ (%) MAP2+ CALR+ (%) (%) (%) (%) (%) 54 21.18 7.882 5.419 65.52 73.34 70.94 103 36.70 19.03 5.437 38.83 57.85 57.86 52.91 15.89 7.364 23.84 39.73 31.20 25.81 4.301 3.226 66.67 70.97 69.94 22.86 7.143 1.429 68.57 75.71 69.99 183 23.94 16.43 25.35 34.27 50.70 59.62 21.74 15.46 3.865 58.94 74.4 62.81 50.16 3.560 17.48 28.80 32.36 46.28 36.20 2.509 10.39 50.90 53.41 61.29 Avg. 32.39 10.25 8.884 48.48 58.72 58.88 Shown are percentages of total cells as determined by Hoechst+ staining. N=5 mice.

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Figure 6. Summary of MAP2 and CALR expressing cells within the SCN. Percentages are relative to the total number of cells as identified by Hoechst+ nuclei. Of all CALR+ cells 82.3% co-localized with MAP2, and of all MAP2+ cells 82.6% co-localized with CALR.

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Figure 7. SCN cells expressing MAP2, GFAP, and CALR. (Top Left, Top Right): Co- localization of MAP2 with CALR in the left and right SCN. The SCN boundary was drawn to enclose the SCM region identified by the Hoechst staining pattern (blue). There was a high degree of co-localization (yellow) between MAP2 (green) and CALR (red) in both the shell and core SCN regions. There appeared to be higher CALR staining ventrally and near the ependymal cells of the 3rd ventricle. Top left represents ZT4, while the top right represents ZT11. (Bottom left, Bottom Right): Co-localization of GFAP with CALR in the left and right SCN. The SCN boundary was drawn to enclose the SCM region identified by the Hoechst staining pattern (blue). There was a small degree of co-localization (yellow) between GFAP (green) and CALR (red) in both the shell and core SCN regions. There appeared to be higher co-localization in the core region however. Bottom left represents ZT5, and bottom right represents ZT7. Scale bar = 35 µm.

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3. CALR and GFAP at ZT5 and ZT7

Glial cells play an important role in the entrainment of the SCN (60,61), and studies have shown that GFAP is a marker for glia within the SCN (60-63). However, GFAP has also been described as a stem marker (64) suggesting that its presence may also be in immature cells of the

SCN. Our lab has previously shown GFAP+ cells within the SCN where we saw 9.31% ±3.35,

(n = 4 slices, p = 0.035, t-test) of total GFAP+ cells in SCN slices (49). Calreticulin is the major calcium-binding protein of all non-muscle cells, and due to its various functions we hypothesized that CALR would co-localize with GFAP in SCN glia/stem-like cells.

3.1. CALR and GFAP ICC (ZT5)

We found that there was a low degree of co-localization between GFAP and CALR in the

SCN, almost all of the CALR+ cells were GFAP- (Table 4). GFAP localization was found within both the shell region and core regions of the SCN with high density staining in the core; CALR localization was found evenly in both the shell and core regions (Figure 7). We found that of the total cells, 2.726 ±0.967% were GFAP+, 25.20 ±3.113% were CALR+, and 5.511 ±1.229% were

GFAP+/CALR+ (Figure 8). Tissue was harvested at ZT5.

3.2. CALR and GFAP ICC (ZT7)

In the second experiment we also found that there was a low degree of co-localization between GFAP and CALR in the SCN; almost all of the CALR+ cells were GFAP- (Table 4).

The average of the total GFAP+ cells was 10.98%, and the average of the total CALR+ cells was

32.25% (Table 4). GFAP localization was found within both the shell region and core regions of the SCN; CALR localization appeared to be found evenly in both the shell and core regions

(Figure 7).

We found that 6.459 ±3.825% were GFAP+, 26.30 ±9.692% were CALR+,

23 and 6.331 ±2.916% were GFAP+/CALR+ (Figure 8). Of the total CALR+ cells, only 19% co- localized with GFAP, and of the total GFAP+ cells, 56.1% co-localized with CALR (Figure 8).

Tissue was harvested at ZT7.

Table 4. CALR and GFAP cells in SCN. Age GFAP- / GFAP+ / CALR+ / GFAP+ / Total Total (Days) CALR- CALR- GFAP- CALR+ (%) GFAP+ CALR+ (%) (%) (%) (%) (%) 72.41 5.172 17.24 5.172 10.34 22.41 77 72.73 1.212 23.64 2.424 6.636 26.06 62.16 3.378 28.38 6.081 9.459 34.46 58.94 1.141 31.56 8.365 9.506 39.93 77 80.12 17.39 2.484 0 17.39 2.485 58.62 5.836 21.49 14.06 19.89 35.54 56.59 1.286 35.37 6.752 8.036 42.12 44.80 0.1890 48.58 6.427 6.616 55.01 Avg. 63.30 4.451 26.09 6.160 10.98 32.25 Shown are percentages of total cells as determined by Hoechst+ staining. N=4 mice.

24

Figure 8. Summary of GFAP and CALR expressing cells within the SCN. Percentages are relative to the total number of cells as identified by Hoechst+ nuclei. Of all CALR+ cells, only 19% co-localized with GFAP, and of all GFAP+ cells, 56.1% co-localized with CALR.

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4. Test of CALR induction by light at ZT17

It has been shown that SCN neurons are involved in the light entrainment pathway, and that calcium induced calcium release from the ER plays an important role in modifying the

TTFL of circadian clock genes (15, 20). Calreticulin is the major-calcium binding protein of the

ER which buffers the amount of free calcium available in the lumen of the ER (25). CALR gene activation has been shown to be activated by ER depletion of calcium, but this was seen after a long-sustained calcium depletion which is not fast enough for light entrainment. However, the

CALR gene and upstream regulatory sequences contain elements that suggest CALR could be induced quickly by Ca2+ signaling pathways acting through the transcription factors AP-1 (Fos and Jun) or NFAT according to a bioinformatics analysis and published results (26, 31). More rapid CALR gene induction (1-hour) has also been reported following a heat shock treatment that might act through c-Jun activation (30).

In light of this, we hypothesized that CALR may be involved in the light entrainment pathway by altering the amount of free calcium that is available for calcium induced calcium release from the ER in order to alter the TTFL of circadian core clock genes, and that CALR gene induction will occur after receiving light. In order to test for this, we had a control group that was euthanized under normal dark conditions at ZT17, and an experimental group that was euthanized after receiving a light pulse from ZT15-ZT17. We then performed ICC to look at

CALR and MAP2 SCN cells to look for differences in neuronal and non-neuronal cells between groups.

4.1. CALR and MAP2 ICC induction control

In the control group we found that MAP2 and CALR localization was found within both the shell region and core regions of the SCN (Figure 9). There was a high degree of co-

26 localization between MAP2 and CALR in the SCN; almost all of the CALR+ cells were also

MAP2+ (Table 5). The average of the total MAP2+ cells was 71.15% and the average total

CALR+ cells was 62.89% (Table 5). We found that of the total cells, 16.84 ±5.343% were

MAP2+, 6.520 ±1.591% were CALR+, and 54.31 ±4.891% were MAP2+/CALR+ (Figure 10).

Of the total CALR+ cells, 86.3% co-localized with MAP2, and of the total MAP2+ cells, 76.3% co-localized with CALR (Figure 10). Tissue was harvested at ZT17 without animals receiving the 2-hour light pulse.

4.2. CALR and MAP2 ICC induction experimental

In the experimental group we again saw that MAP2 and CALR localization was found within both the shell region and core regions of the SCN (Figure 9). There was a high degree of co-localization between MAP2 and CALR in the SCN; almost all of the CALR+ cells were also

MAP2+ (Table 6). The average total MAP2+ cells was 54.61%, and the average total CALR+ cells was 65.60% (Table 6). We found that 4.104 ±0.945% of the cells were MAP2+, 15.60

±3.534% were CALR+, and 50.51 ±5.595% were MAP2+/CALR+ (Figure 10). Of the total

CALR+ cells, 76.9% co-localized with MAP2, and of the total MAP2+ cells, 92.5% co-localized with CALR (Figure 10). Tissue was harvested at ZT17 immediately after mice received two hours of light.

4.3. Statistical analysis of ICC results

Using student’s T-test there did not appear to be any significant difference in the number of CALR+/MAP2+ or the total CALR+ cells between the control and experimental groups, however we did see almost double the amount of CALR+/MAP2- cells in the experimental group compared to the control. In order to verify that there was a statistical difference we performed three T-tests to look at CALR+ cells, CALR+/MAP2+ cells, and total CALR+ cells (Table 7).

27

We verified that there was a statistical significant difference between the CALR+/MAP2- cells in the control vs. experimental group, and that there was not any statistical difference in the

CALR+/MAP2+ or the total CALR+ cells between groups.

We also used a One-way ANOVA and Tukey test to determine whether the percentage of total CALR+, CALR+/MAP2+ or CALR+/MAP2- cells differs according to the ZT when the tissue was harvested. We found that there was a significant difference (p<0.05) between the total

CALR+ cells during the day (ZT 7, 8, and 11 grouped) and night using the control group at ZT

17 and the experimental group, which received two additional hours of light during their early night (ZT 15-17). The total CALR+ group was lower during the day (ZT 7, 8, & 11) than at night

(ZT17). We did not see a significant difference between the CALR+/MAP2+ cells in any group.

Although there was also no significant difference between the CALR+/MAP2- cells in any group, when we only compared the CALR+/MAP2- (non-neuronal) cells there was a significant difference between control and experimental (light induction) groups. This ANOVA test agreed with the T-tests described above.

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Figure 9. SCN cells expressing MAP2 and CALR. Co-localization of MAP2 with CALR in the left and right SCN of mice euthanized under dark conditions with no light signal from ZT15-17 (Top left, Top right). The SCN boundary was drawn to enclose the SCM region identified by the Hoechst staining pattern (blue). There was a high degree of co-localization (yellow) between MAP2 (green) and CALR (red) in both the shell and core SCN regions. Co-localization of MAP2 with CALR in the left and right SCN of mice euthanized under after receiving a light signal from ZT15-17 (Bottom left, Bottom right). The SCN boundary was drawn to enclose the SCM region identified by the Hoechst staining pattern (blue). There was a high degree of co-localization (yellow) between MAP2 (green) and CALR (red) in both the shell and core SCN regions. There did not appear to be any difference in the staining pattern after light treatment. Scale bar = 35 µm.

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Table 5. Proportions of cells expressing CALR and MAP2. Control.

Age MAP2- / MAP2+ / CALR+ / MAP2+ / Total Total (Days) CALR- CALR- MAP2- CALR+ MAP2+ CALR+ (%) (%) (%) (%) (%) (%) 50 28.74 8.38 7.784 55.10 63.48 62.88 29.46 8.915 14.73 46.90 55.82 61.63 22.75 7.059 3.529 66.67 73.73 70.20 30.51 6.618 10.29 52.57 59.19 62.87 75 17.28 3.704 4.938 74.07 77.77 79.01 17.83 17.05 1.550 63.57 80.62 65.12 95 20.83 55 0 24.17 79.17 24.17 17.84 26.29 10.80 45.07 71.36 55.87 95 15.73 18.54 5.056 60.67 79.21 84.27 Avg. 22.33 16.84 6.520 54.31 71.15 62.89 Shown are percentages of total cells as determined by Hoechst+ staining. N=5 mice.

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Table 6. Proportions of cells expressing CALR and MAP2. Experimental.

Age MAP2- / MAP2+ / CALR MAP2+ / CALR+ Total Total (Days) CALR- CALR- + / (%) MAP2+ CALR+ (%) (%) MAP2- (%) (%) (%) 76 24.26 5.882 0 69.85 75.73 69.85 32.71 5.607 21.96 39.72 45.32 61.68 27.97 2.098 16.78 53.15 55.25 69.93 88 26.94 2.591 21.76 48.70 51.29 70.46 30 0.7143 15.71 53.57 54.28 69.28 36.91 1.928 27.55 33.61 35.54 61.16 18.33 2.5 4.167 75 77.50 79.17 33.62 6.034 1.724 58.62 64.65 60.34 95 41.85 9.585 26.20 22.36 31.95 48.56 Avg. 30.29 4.104 15.09 50.51 54.61 65.60 Shown are percentages of total cells as determined by Hoechst+ staining. N=6 Mice.

31

Figure 10. Summary of MAP2 and CALR expressing cells within the SCN. Percentages are relative to the total number of cells as identified by Hoechst+ nuclei. Of all CALR+ cells in the control, 86.3% co-localized with MAP2, and of all MAP2+ cells 76.3% co-localized with CALR (Left). Of all CALR+ cells in the experimental group, 82.3% co-localized with MAP2, and of all MAP2+ cells, 92.5% co-localized with CALR (right).

Table 7. T-TEST analysis of CALR light induction. CALR+ / MAP2- CALR+ / MAP2+ TOTAL CALR+ Control vs. 0.0488* 0.6162 0.6426 Experimental Control vs. ZT4 0.9338 0.9473 0.7868 Control vs. ZT11 0.4686 0.4140 0.5114 Control vs. ZT4/ZT11 0.4448 0.5013 0.5382 * = p<0.05

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DISCUSSION

1. CALR and SOX2

Unlike cortical brain areas, the dentate gyrus and subventricular zone of the lateral ventricles (65-68), the adult SCN has not been extensively examined for cells with stem like markers. Several labs have, however, identified cells in the adult SCN that express proteins characteristic of stem cells and neurogenesis, although neurogenesis has not been demonstrated conclusively. Also, our lab has provided evidence of cells expressing additional stem cell markers in the SCN (49, 33), but any confirmation for the role of these proteins in the circadian clock has yet to be provided. This study is the first to examine co-localization patterns of CALR with SOX2, a stem cell marker that has been shown to be expressed in the SCN of mice (49).

CALR interacts with integrins in a variety of roles (24, 50-53), and various integrins are known to be important in stem cells primarily by connecting the to proteins of the extracellular matrix (54-56). These connections require modification when differentiating stem cells or progenitor cells migrate. Cell migration in the adult SCN has not been shown, but astrocytes display circadian rhythms in morphology that could regulate circadian rhythms (69).

Network interactions such as these have been suggested as one way the population of circadian clock cells in the SCN alters its ensemble rhythmic output, most likely providing adaptive physiological changes elsewhere in the body (50). Due to the possible connection between

CALR and the stem-like cells of the SCN we hypothesized that there would be co-localization between SOX2 and CALR, particularly because of their mutual relationships with integrin.

We found that 25.73 ±6.834% of cells were SOX2 and CALR positive, and that almost all of the CALR+ cells were also SOX2+ (98.6%). While a majority of CALR cells were

SOX2+, almost half of the SOX2+ cells were CALR+ (42.6%). There were 34.60 ±7.465%

33

SOX2+/CALR- cells which may represent a subpopulation of stem-like cells within the SCN that lack elevated CALR expression. This feature may indicate these cells are not involved in cell- cell communication mediated through CALR-integrin interactions. In contrast, SOX2+/CALR+ cells may be involved in increased cell-cell communication through integrin interactions, which may provide tighter synchronization of SCN cells to daily rhythms that affect the core circadian clock genes. It could allow increased plasticity and adaptability to different timing signals that can shift or alter our circadian rhythms.

Co-localization of calreticulin with SOX2 is an important finding that has not been shown before in the SCN. These findings provide further evidence for the presence of stem cell markers within the SCN, which may be playing a role in the plasticity and adaptability of SCN cells. The protein interactions between CALR and SOX2 need to be examined, and studies should examine their co-localization patterns together with integrins to determine whether there is increased cell-cell communication between CALR, SOX2, and a specific type of integrin in

SCN cells.

2. CALR and MAP2

Several studies have shown MAP2+ neurons within the SCN (57-59) as expected for this general neuronal marker. Studies have also shown that excitatory light signals via the RHT produce cytoplasmic Ca2+ responses (28) that entrain the SCN clock to the environmental light cycle within SCN neurons (20). CALR is the major calcium binding protein of the ER and buffers the amount of free calcium available for calcium release (23,25), and calcium mobilization from internal stores is an important modulator of gene transcription (27).

In light of this role in neurons, we hypothesized that there would be high degrees of co- localization between MAP2 and CALR.

34

In the first CALR/MAP2 experiment we found that 54.83 ±5.884% of cells were CALR and MAP2 positive, and that most of the CALR+ cells were also MAP2+ and vis versa.

However, there was a small subpopulation of neuronal cells (MAP2+/CALR-) within the SCN that do not appear to have elevated CALR expression (6.484 ±1.918%). There were also 6.771

±2.475% of CALR+ cells that did not co-localize with MAP2.

In the second CALR/MAP2 experiment we found that 34.78 ±19.02% of cells were

CALR and MAP2 positive, and that again most of the CALR+ cells were also MAP2+ and vis versa. We saw that there was a small subpopulation of neuronal cells (MAP2+/CALR-) within the SCN that do not appear to have elevated CALR expression (8.970 ±5.520%). There were also

15.28 ±9.850% of CALR+ cells that did not co-localize with MAP2.

CALR is the major calcium-binding protein of all non-muscle cells, so we would expect to see ubiquitous CALR expression throughout all of the SCN cells. In both experiments we found small populations of cells where we do not show substantial CALR expression; This result suggests that the cells expressing CALR are doing so at elevated levels. We speculate that those neurons with elevated CALR expression are involved in the synchronization of light signals, and the cells lacking elevated CALR expression may not be involved in the entrainment pathway. It is also possible that the small populations of cells that we observed did not undergo significant

ER stress to induce calreticulin gene expression through the activation of the UPR. Further studies are needed to find out why some SCN cells lack elevated CALR expression.

The CALR+/MAP2- cells represent SCN glial cells, which could be reactive or non- reactive astrocytes, however, we do not know the exact type of glial cell. It could also be possible that the CALR+/MAP2- cells represent non-neuronal stem-like cells that are developing

35 due to GFAP also being a potential neural stem cell marker (70,71), but further studies are needed to elucidate the cell types of non-neuronal CALR+ SCN cells.

Another important finding of the study is the quantification of CALR and MAP2 co- localization in the SCN. It showed that significant, detectable CALR expression, what we counted here, is mainly in neurons. We do not know the mechanism or distinct role that CALR may have within these neurons, but with the current understanding of CALR functions we can speculate that it may be acting as a calcium buffer or playing a role in the UPR response to alter circadian gene expression. Further studies are needed to characterize any specialized role of

CALR in the SCN, other than typical maintenance of protein folding and ER Ca2+ regulation.

Studies should address whether it has a unique role in the entrainment pathway distinct from other Ca2+-binding proteins, particularly in SCN glial cells where it may serve as more than just a protein chaperone of the ER.

3. CALR and GFAP

There have been extensive studies showing GFAP+ glial cells within the SCN (60-63) and they have also shown that glial cells play in important role in the entrainment pathway

(60,61). We also know that CALR is the major calcium-binding protein of the ER, and buffers the amount of free calcium available for calcium release (23,25), and that calcium mobilization from internal stores is an important modulator of gene transcription (27). So we hypothesized that CALR would co-localize with GFAP, however we anticipated lower levels of co- localization due to SCN neurons playing a much larger role in the light entrainment pathway.

In the first experiment we found that 5.511±1.229% of cells were CALR+ and GFAP+, and that most of the CALR+ cells did not co-localize with GFAP (25.20 ±3.113%). There were

2.726 ±0.967% of GFAP+/CALR- cells as well. In the second experiment we found that 6.331

36

±2.916% of cells were CALR and GFAP positive. Again we saw that most of the CALR+ cells did not co-localize with GFAP (26.30 ±9.692%), while 6.459 ±3.825% of cells were

GFAP+/CALR- cells.

Because CALR is the major calcium-binding protein of all non-muscle cells we expected to see expression within GFAP+ SCN cells. However, we observed a small population of

GFAP+/CALR- cells. This again suggests that not every SCN cell expresses elevated calreticulin levels, which means that the glial cells expressing elevated CALR may be playing a role in the entrainment pathway. The non-expressing GFAP+ cells could possibly be non-reactive astrocytes or stem-like cells within the SCN that are not involved in entrainment pathways.

This is the first study to look at the co-localization between GFAP and CALR within the

SCN of mus musculus. We found that most of the CALR+ cells did not express GFAP, but there were small populations that had co-localization. We expect these to be SCN glial cells that are involved in the entrainment pathway, however further studies are needed in order to elucidate the role of these glial cells. Further experiments may involve looking at the calcium dynamics of the

SCN in conjunction with ICC to look for elevated or altered CALR expression within SCN glial cells.

4. CALR induction by a light pulse and possible role in entrainment to the light cycle

We showed previously that CALR co-localizes with MAP2 to a high degree, and due to the calcium buffering roles of CALR we wanted to test whether or not it responds to light in a way that would indicate it is involved in the light entrainment pathway described by Ikeda

(Figure 1). This is the first study to try to elucidate the mechanism behind CALR induction within the SCN of mice. In order to test for light induction of CALR we first examined bioinformatics data, shown above Figures 2 and 3, to identify when CALR mRNA expression

37 would be at its peak. We found that CALR mRNA expression was at its peak around ZT15 for animals in LD, which is three hours after 12 hours of light exposure (Figure 2). For animals in

DD, CALR mRNA expression showed a ~25hr rhythm and peaked when the animals would normally be in darkness under LD conditions (Figure 3). This data suggests that CALR mRNA is rhythmic within the SCN, and that sustained light exposure may activate CALR expression, although its expression is still rhythmic under DD conditions. Due to these suggestive results acquired from databases we hypothesized that CALR will be induced by light, and that it may be playing a role in the light induction pathway by buffering the calcium response of the light entrainment pathway within SCN neurons.

To test for induction of CALR expression by a light treatment that is known to cause phase shifts of the SCN circadian clock, and to test for ceiling effects in expression levels as well, we decided to test two groups of mice under differing light conditions. The control group was under a standard 12:12 LD schedule, and were brought into the lab in darkness at ZT17, and then tissue was immediately harvested under red light to test for CALR expression shortly after its mRNA expression should be at its peak. The experimental group was also under a standard

12:12 LD schedule, however they were given light from ZT15-ZT17 followed by tissue harvesting in room light to test for effects of light on CALR expression. There were three sets of experiments in total performed months apart, providing us N=5 for the control group, and an

N=6 for the experimental group. For the Metamorph cell-scoring routine the undergrad observer was blind to which groups were the control or the experimental. There was an uneven amount of males and females in both groups; There were 2 males, 3 females in the control and 5 males, 1 female in the experimental group. Due to this difference there is a possibility that there were sex

38 differences between the groups, however there are not any studies that suggest that there are sex differences in the expression of calreticulin.

For the control group, we found that 54.31 ±4.891% of cells were CALR and

MAP2 positive, and that most of the CALR+ cells were also MAP2+ and vis versa. As we saw in the previous CALR/MAP2 experiments, there was a small subpopulation of neuronal cells

(MAP2+/CALR-) within the SCN that do not appear to have elevated CALR expression (16.84

±5.343%). There were also 6.520 ±1.591% of CALR+ cells that did not co-localize with MAP2.

For the experimental group, we found that 50.51 ±5.595% of cells were CALR and MAP2 positive, and that again most of the CALR+ cells were also MAP2+ and vis versa. As we saw in the previous experiments, there was a small subpopulation of neuronal cells

(MAP2+/CALR-) within the SCN that do not appear to have elevated CALR expression (4.104

±0.945%). There were also 15.60 ±3.534% of CALR+ cells that did not co-localize with MAP2.

Once we had the results from the cell-scoring routine we ran a students T-Test to look for significant differences between the cell populations of the control and experimental groups. The results of these tests can be seen in Table 7 above. We found no significant difference between the control and experimental groups among the MAP2+ cells, including the

MAP2+/CALR+ cells. This was a surprising result because we hypothesized that light would induce CALR expression based on the pathway described by Ikeda (15). However, we tested expression levels when they should have been at their peak, so if there is a ceiling effect in the expression levels of calreticulin, then the light pulse we gave would have no effect.

It is also possible that two hours of light is simply not enough of a stimulus to deplete the

ER of enough calcium to activate CALR induction, and instead a long sustained light pulse may be needed to simulate the long light exposure an animal normally receives during the day.

39

Further studies may involve increasing the length of the light stimulus to see if a sustained exposure will increase expression. Further studies may also try to get around the potential ceiling effect by testing at times of the day when CALR expression will be at its lowest. One of the best studies would be to look at animals kept under DD conditions; a light pulse could be given to a group once they have entrained to the DD light schedule to look at whether CALR is inducible by light in animals kept under constant darkness. However, since CALR expression is also induced by the UPR which is activated under ER stress, a light pulse may simply act as a significant enough stressor to trigger UPR; therefore, it might not be light itself but stress which activates CALR induction.

One of the exciting finds from this experiment was that there was indeed a significant difference between the control/experimental groups with their CALR+ cells. These are SCN cells that did not express MAP2, suggesting that they are non-neuronal cells. It has been shown that

SCN glial cells play an important role in entrainment (64, 65). We found that giving a light pulse during the subjective night of the mice caused almost a two-fold increase in the glial cells expressing CALR, suggesting that it is light inducible within SCN glial cells. These cells may be involved in the entrainment pathway and CALR may be acting as a calcium buffer to alter the gene transcription within them, however further studies are needed to verify that these cells are involved in light entrainment and that CALR is actually playing a role in altering calcium dynamics of these cells. However, since CALR is also induced by UPR, it may be that light is acting as a stressor which is then activating CALR through the UPR pathway. It is not clear as to why this stress would only affect the glial cells and not the neurons of the SCN. Further studies are needed in order to resolve this conundrum, possibly examining calcium dynamics within

SCN glia followed by ICC to look for altered CALR expression.

40

Following this discovery, we ran a One-way ANOVA to test for differences between total

CALR+ cells, CALR+/MAP2+ cells, and CALR+/MAP2- cells across different ZTs. We had four groups (ZT4 and 5; ZT7, 8, and 11; ZT17-control; and ZT17-experimental). These results indicated the CALR+/MAP2- cells responded to light with an increase in percentage of cells expressing CALR.

The results also indicated that there were less total CALR+ cells during the day compared to night, which coincides with SCNseq and CircaDB mRNA data that shows CALR fluctuating rhythmically where it is low during the day and high during the night. We did not find a significant difference in the CALR+/MAP2+ cells between any of the groups, suggesting that the number of CALR+ neurons does not change through these different ZTs. We then looked at CALR+/MAP2- cells across different ZTs and again did not detect any significant difference between any of the groups. However, when considering only the CALR+/MAP2- control and experimental groups at ZT17 there was a significant difference (p<0.05) suggesting that light did have an effect on the number of CALR+/MAP2- non-neuronal cells within the SCN. This coincides with what we saw using the T-Test analysis. We conclude that the glial cells may be using CALR in entrainment to light, yet further studies are needed to test whether these non- neuronal cells are reacting to light through the RHT or reacting to stress induced in the mice from receiving light during the night.

The identity of the cells responding to light can be further clarified by considering what is currently known about SCN SOX2 expression and results from the current study. SOX2 has been shown to be expressed in 20.31% of mouse SCN (49) and 70% of rat SCN cells previously

(72), and ~67% in the current study. We found 19.1% of CALR+ cells are GFAP+; the

41 remaining CALR+ cells included neurons. In agreement, 86.9% of CALR+ cells were MAP2+, and therefore neurons.

Considering only the GFAP+ cells, 58.1% of these were CALR+. Previously, we determined that 91.2% of GFAP+ SCN cells are SOX2 (49). By treating these percentages as probabilities and combining them, we can then estimate that 52.99% of GFAP+ SCN cells are both SOX2+ and CALR+, indicating CALR is expressed at significant levels in the stem-like cells. This result encourages further studies of how CALR could serve in SCN stem-like cells.

Further, because the significant response to light at ZT17 was in the non-neuronal cells (MAP2-) the stem-like cells are likely to participate in this response to an entraining light signal.

Although we found a substantial number of SCN cells positive for SOX2, far fewer were detected in our previous study (49). This difference could be explained by the decline in Sox2 gene expression in SCN of mice entrained to a light cycle, as shown in the SCNseq database. In this case, Sox2 expression declines about 4-fold between ZT6 and about ZT14, when our previous ICC tissue was collected for SOX2 analysis. In the current study SCN tissue for this experiment was harvested at ZT8. Note that SCNseq did not show a significant fluctuation in

Sox2 expression.

Once we found a significant difference between the control (darkness) and experimental

(2-hour light) groups in the percentage of CALR+ cells, we decided to run another t-test for any significant differences between the control group and the MAP2 experiments performed earlier during the day (ZT4 and ZT11). This test allowed us to compare CALR protein expression levels between the day and night. We found that there was again no significant difference among the

MAP2+ cells, along with the MAP2+/CALR+ cells (Table 7). Further studies can look at the populations of SCN glia expressing CALR during the day and during the night to look for

42 significant differences, and to see what role these cells may be playing in the light entrainment pathway.

These studies are the first to examine CALR protein expression within the SCN of mice.

Since CALR is the major calcium-binding protein of all non-muscle cells we hypothesized that it would be ubiquitously expressed throughout all of the cells, yet we found distinct populations of cells lacking elevated CALR expression. This is important because it allows us to begin to understand why we see elevated CALR expression in certain cells but not others. We hypothesized that we would find CALR expression with neurons, glia, and stem-like cells and we found its expression within all of these cell types. This finding is important because it is the first to distinguish SCN cell types expressing CALR. We also hypothesized that CALR expression would be inducible by light through the entrainment pathway in SCN neurons.

Although we did not find that light induces CALR expression in SCN neurons during the early night, it should also be examined in the late night, around ZT22, when SCNseq indicates CALR gene expression is lowest in SCN of mice in a light cycle. We tested at ZT15 because the 2-hour light stimulus does induce phase shifts at this phase.

Interestingly, we did see CALR induction by light within SCN glial cells. This is a very important finding because it allows us to begin understanding the mechanistic role that CALR may play in the entrainment pathway. Accumulating evidence indicates a role for SCN astrocytes in entrainment and in generating circadian rhythms. Considering the extensive list of known

CALR functions and its differing roles according to cell type or organ location it will be exciting to confirm and characterize a unique SCN role for CALR in future studies.

43

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APPENDIX A. METAMORPH CELL COUNTING DATA

Table 1: Mean, standard deviation, and standard error of metamorph cell counting data. CALR/SOX2 ZT 8 MEAN SD SEM DAPI 39.30 3.945 1.973 SOX2 34.60 14.73 7.365 CALR 0.360 0.279 0.139 CALR/SOX2 25.73 13.67 6.834 CALR/MAP2 ZT 4 MEAN SD SEM DAPI 31.92 10.79 4.405 MAP2 6.484 4.699 1.918 CALR 6.771 6.061 2.475 CALR/MAP2 54.83 14.41 5.884 CALR/MAP2 ZT 11 MEAN SD SEM DAPI 40.97 22.23 12.83 MAP2 8.970 9.561 5.520 CALR 15.28 17.06 9.850 CALR/MAP2 34.78 32.95 19.02 CALR/GFAP ZT 5 MEAN SD SEM DAPI 66.56 7.066 3.533 GFAP 2.726 1.934 0.967 CALR 25.20 6.227 3.113 CALR/GFAP 5.511 2.457 1.229 CALR/GFAP ZT 7 MEAN SD SEM DAPI 60.91 14.54 7.272 GFAP 6.459 7.649 3.825 CALR 26.30 19.39 9.692 CALR/GFAP 6.331 5.832 2.916 CALR/MAP2 Cont. MEAN SD SEM DAPI 22.33 5.818 1.439 MAP2 16.84 16.03 5.343 CALR 6.520 4.774 1.591 CALR/MAP2 54.31 14.67 4.891 CALR/MAP2 Exp. MEAN SD SEM DAPI 30.29 6.986 2.329 MAP2 4.104 2.835 0.945 CALR 15.60 10.60 3.534 CALR/MAP2 50.51 16.78 5.595 Shown are the mean, standard deviation, and standard error for all of the experiments using OriginLab software.