THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE

DEPARTMENT OF KINESIOLOGY

THE EFFECT OF STRAIN AND EXERCISE ON HYPOTHALMIC GENE EXPRESSION IN C57BL/6J AND DBA/2J MICE

JAMES METKUS SPRING 2013

A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biology with honors in Kinesiology

Reviewed and approved* by the following:

Dena Lang, Ph. D. Research Associate in Kinesiology Thesis Supervisor

Jinger Gottschall Assistant Professor of Kinesiology Honors Adviser

* Signatures are on file in the Schreyer Honors College.

i

ABSTRACT

Osteoporosis is a degenerative bone disease that affects a majority of Americans, specifically those of older age. Within the last few decades, researchers have begun to consider neurological pathways that contribute to osteoporosis and other bone disorders, including hormonal effects on osteoporosis or other bone disorders. There have been decades of studies

focusing on exercise and it has been shown that exercise has positive effects on bone mass by

increasing bone density and gene expression within bone. However, there is a lack of research on

how exercise affects gene expression within the brain or specific parts of the brain, such as the

hypothalamus.

In this study, 77 480 day old C57BL/6 (B6) and DBA/2J(D2) female mice were divided

into three treatment groups: a treadmill running group (14 B6 and 14 D2 mice), a tower climbing

group (13 B6 and 12 D2) and finally a sedentary control group (14 B6 and 10 D2). After 5 weeks

of exercise treatment, RNA was extracted from the hypothalamus of each mouse, homogenized

and underwent Nanodrop and Bioanalyzer assessment for quality control. Two samples were

pooled onto three gene chips for each treatment group in order to measure gene expression within

the hypothalamus. Gene expression was then compared across to identify differential gene

expression as a function of mouse strain and/or exercise. While the exercise treatment did not

produce significant differences in gene expression, important differences that were suggested

from this study include the expression of Insulin Growth factor-1(IGF-1) in B6 tower climbing as

compared to B6 sedentary controls. In terms of strain differences, around 497 genes were

differentially expressed between B6 and D2 mice. The identification of genes such as IGF-1

within the hypothalamuses of B6 and D2 mice may help provide greater insight into how differences in activity levels and genetics may lead to the elucidation of new pathways for the prevention as well as treatment of osteoporosis and other bone disorders. ii

TABLE OF CONTENTS

List of Figures ...... iv

List of Tables ...... v

Acknowledgements ...... vi

Chapter 1 Introduction ...... 1

1.1 Study aims...... 2 1.2 Study limitations ...... 2

Chapter 2 Literature Review ...... 3

2.1 Neurological Control of Bone Remodeling ...... 3 2.1a Exercise Induced Neurogenesis...... 3 2.2 Hypothalmic-Pituitary-Gonadal-Skeletal Axis ...... 4 2.2a Estrogen and Follicle Stimulating Hormone ...... 4 2.3 Hypothalmic-Pituitary-Adrenal Axis ...... 5 2.4 Leptin is correlated to high turnover in osteoporosis ...... 6 2.4a Cocaine and Amphetamine-Regulated Transcript ...... 7 2.4b Sympathetic Nervous System Pathway ...... 7 2.4c Neuropeptide Y ...... 8 2.4d Insulin like Growth Factor ...... 8 2.4e Insulin ...... 9 2.5 Strain Differences between B6 and D2 mice ...... 9

Chapter 3 Materials and Methods ...... 12

3.1 Animals and Experimental Design ...... 12 3.2 Tissue Harvesting ...... 14 3.3 RNA Extraction ...... 14 3.3a. Preparation ...... 14 3.3b. RNA Extraction ...... 15 3.3c. RNA quality analysis ...... 15 3.4 RNA Pooling and Labeling ...... 17 3.5 Microarray Processing and Data Analysis ...... 18

Chapter 4 Results ...... 19

4.1 Differential Expression as a Function of Genetic Strain ...... 19 4.2 Differential Expression as a Function of Exercise Treatment ...... 37 iii

Chapter 5 Discussion ...... 39

5.2 Analysis of Treatment Effect ...... 42 5.3 Recommendations for Future Investigation ...... 44 Appendix A Treadmill Operator Protocol ...... 45 Appendix B Tower Design...... 47 References ...... 48

iv

LIST OF FIGURES

Figure 2-1 Sun et al HPG axis [7] ...... 4

Figure 2-2 Estrogen’s effects on bone resorption ...... 5

Figure 2-3 Leptin Osteoblast Pathway [14] ...... 7

Figure 2-4 Insulin’s effects on bone resorption [24] ...... 9

Figure 3-1 Schematic of experimental design...... 12

Figure 5-1 Design of Tower for Exercise Intervention ...... 47

v

LIST OF TABLES

Table 3-1Protocol of settings used during treadmill running intervention ...... 13

Table 3-2 Summary of Chip Data used for Microarray Analysis ...... 16

Table 4-1 Genes expressed in B6 mice over D2 with a FC of greater than 5 ...... 19

Table 4-2 Genes expressed higher in B6 over D2 mice with a FC of 3 or 4 Part I ...... 21

Table 4-3 Genes expressed higher in B6 over D2 mice with a FC of 3 or 4 Part II ...... 22

Table 4-4 Genes expressed higher in B6 over D2 mice with a FC of 2 Part I ...... 24

Table 4-5Genes expressed higher in B6 over D2 mice with a FC of 2 Part II ...... 25

Table 4-6Genes expressed higher in B6 over D2 mice with a FC of 2 Part III ...... 26

Table 4-7Genes expressed higher in D2 over B6 mice with a FC of 5 or greater ...... 28

Table 4-8Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part I ...... 29

Table 4-9Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part II ...... 30

Table 4-10Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part III ...... 31

Table 4-11Genes expressed higher in D2 over B6 mice with a FC of 2 Part I ...... 33

Table 4-12Genes expressed higher in D2 over B6 mice with a FC of 2 Part II ...... 35

Table 4-13Genes expressed higher in D2 over B6 mice with a FC of 2 Part III ...... 36

Table 4-14 Genes Involved in Hypothalmic Neurogenesis ...... 38

Table 4-15 Genes Involved in HPG Axis ...... 38

Table 4-16 Genes involved in the Insulin Pathway ...... 38

Table 5-1 Basal gene expression for the PVN in Tsolakidou et al. supports data from author’s study [35] ...... 41

vi

ACKNOWLEDGEMENTS

I first must thank both of my parents for their love and constant support during my college career, giving me everything I need to obtain an honors education at Penn State.

I would also like to thank my honors advisers, Dr. Stephen Schaeffer who helped me navigate my way through a Biology degree while being able to also prepare myself for a medical education, as well as Dr. Jinger Gotshall who was one of the many welcoming faces within the

Kinesiology department.

I also would like to thank the Pennsylvania Space Grant Freshman Undergraduate

Research Program, which put me in contact with the Biomechanics lab and allowed me to begin my thesis work and undergraduate research career as a freshman student. I am extremely thankful for Dr. Dena Lang for allowing me to take part in skeletal research and teach me proper research and lab etiquette as a freshman and throughout my college career. I would also like to thank Dr.

Holly Preston and Nicole Irizarry who as students in Dr. Lang’s lab helped pave the way for my research as well as serve as a constant support system and role model throughout my time within the lab. I am so grateful for Dena working with me throughout these four years providing me the opportunity to complete this thesis and gain a great understanding for the research process, always being there for me for guidance and support. I would also like to thank Dr. David Blizzard for allowing me to assist with dissections as well as Craig Paul and Candace Price from the Penn

State Microarray Facility for their role and hard work in generating the microarray data used in this study.

This work was funded by NIH/NIA grant K25 AG0251

Chapter 1 Introduction

Osteoporosis is a disease of bone that weakens bones to the point that they become fragile and break easily. The disease is characterized by thinning cortices and trabeculae. Because osteoporosis occurs with no symptoms, people afflicted with it may not know they have osteoporosis making it a “silent” disease [1]. In the United States, more than 40 million people either already have osteoporosis or at a high risk of developing osteoporosis due to decreasing bone mass [1]. While it can strike at any age, osteoporosis is more common among older adults, especially older women. Older women are more susceptible because of the hormone changes that occur after menopause. It has been shown that estrogen has a positive effect on increasing bone mass. Menopause in effect drastically decreases a woman’s production of estrogen, leading to a decrease in bone mass. In summary, women are more likely to develop osteoporosis as they age

[1]. Many components contribute to a bone’s ability to resist fracture including bone density, architecture, and composition, and are all important aspects of skeletal quality.

Over the past few decades, there has been a greater emphasis on researching the neuroskeletal basis of skeletal quality. Because hormones have such a huge effect on bone formation and resorption through the action of osteoblasts and osteoclasts, research has focused on how treatments normally prescribed for increasing bone mass and/or strength, namely exercise, which has been shown to have a positive effect on bone mass, affect the hypothalamus and other structures within the brain [2]. The hypothalamus is the central hub for release of hormones to the rest of the body. Therefore, the aim of this study is to investigate whether or not exercise affects neuro-skeletal pathways and if there are genetic differences both in the response 2 to exercise as well as between the two inbred strains of mice used in this study. Through this

process, it can be hoped that further research could lead to tailored exercise programs based on

genetic predisposition ultimately improving skeletal health for our aging populations [1].

1.1 Study aims.

The hypothesis for study is based off of neuroendocrine pathways between the brain and bone tissue that may help in increasing bone mass and quality:

1. The treatment groups undergoing exercise will experience a down regulation in

osteoclast pathways in comparison to control groups.

2. The treatment groups undergoing exercise will experience an upregulation of

osteoblast activity in comparison to control groups.

The overall goal of this study is to determine how gene expression within the hypothalamus changes as a function of exercise and genetic mouse strain as well as the differences in the response to exercise of two genetically different inbred mouse strains.

1.2 Study limitations

1. Because some of the animals were more willing to participate in the exercise

intervention programs compared to others, the data might be skewed in terms of the

amount of physical activity experienced by each animal. This is more of a concern in

the tower climbing exercise group.

2. Because some of the RNA during extraction was found to be contaminated, it

couldn’t be used in the study and therefore limited the breadth of data from which to

select from to be used on the chips for the microarray.

Chapter 2

Literature Review

2.1 Neurological Control of Bone Remodeling

Bone remodeling deals with two distinct levels of regulation: paracrine and systemic regulation. Paracrine regulation deals with factors released by either osteoclasts or osteoblasts, the two cells associated most directly with bone remodeling. Some of these released factors include osteoprotegerin, RANKL, and IGF. The systemic regulation of bone remodeling is exerted by hormones, such as estrogens, androgens, PTH, and leptin, as well as other mechanical and neural signals [3][4].

2.1a Exercise Induced Neurogenesis

Motor activity has been shown to increase adult hippocampal neurogenesis, as well as facilitate recovery from brain injury such as stroke, and enhance cognitive function in B6 and D2 mice. For instance, exercise affects the level of protein kinase C (PKC). The PKC second messenger system has been shown to be involved with acetylcholine, serotonin, glutamate and norepinephrine release in the hippocampus [5]. Overall, a relationship has been found between

PKC within the hippocampus and spatial learning performance in rats. Exercise has also been found to increase brain-derived neutrophic factor (BDNF), a molecule that has importance in learning and memory [6].

4 2.2 Hypothalmic-Pituitary-Gonadal-Skeletal Axis

Since estrogens have a large influence on bone remodeling, they are an important point of regulation for bone mass. The Hypothalmic-Pituitary-Gonadal-Skeletal Axis is defined as the hypothalamus releasing Gonadotropin-releasing hormone (GnRH) which directly stimulates

Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) leading to the production of hormones such as inhibins, progesterone, and estrogens. All three of these hormones have been demonstrated to directly or indirectly impact bone remodeling [8]. Specifically, Sun et al [2006] helped further develop the pathway that shows the effect of estrogen on bone remodeling, an important discovery since menopause effectively halts estrogen production in women, making them more susceptible to osteoporosis [7].

Figure 2-1 Sun et al HPG axis [7]

2.2a Estrogen and Follicle Stimulating Hormone

Estrogen prevents osteoporotic bone loss by lessening bone resorption by osteoclasts.

Estrogen support helps regulate the life span of mature osteoclasts, which are the major cell type 5 involved in bone resorption by the activation of the Fas/FasL system. The Fas/FasL system is thought to be activated by estrogen and leads to osteoclast apoptosis [8]. Estrogen effectively protects bone by inducing a signal that is thought to originate in osteoblasts and leads to the death of premature osteoclasts [9]. Imai et al [2007] reports on a pathway for this process as detailed below:

Figure 2-2 Estrogen’s effects on bone resorption

2.3 Hypothalmic-Pituitary-Adrenal Axis

Corticosteroids are widely used in the consumer world because of their effectiveness against many inflammatory diseases Their long term overuse however causes corticosteroid osteoporosis [10]. Corticosteroids increase bone loss by inhibiting osteoblast activity. This 6 inhibition of activity has been marked by a decrease in the biomarker osteocalcin, which is used

to measure the activity of osteoblasts [11].

2.4 Leptin is correlated to high turnover in osteoporosis

Leptin is a 16 kDa protein that plays a role in regulating energy expenditure, including hunger, and metabolism. Leptin is mainly produced by adipocytes in proportion to fat size stores.

In terms of bone remodeling, leptin can affect bone metabolism by direct signaling from the brain through the hypothalamus, decreasing cortical bone mass [11]. There are a number of controlling agents of leptin’s effects, including serotonin [12]. Leptin also modulates bone mass through hypothalamic receptors within the brainstem, which stimulate neurons who utilize serotonin as a neurotransmitter. Serotonin binding to the Beta 2 Adrenergic receptors on osteoblasts down regulates its activity, as well as down regulating the activity in adipocytes, regulating the amount of leptin produced by adipocytes [13]. A summary of this pathway can be seen below, taken from

Driessler et al. [14]. 7

Figure 2-3 Leptin Osteoblast Pathway [14]

2.4a Cocaine and Amphetamine-Regulated Transcript

Under the control of leptin, CART has been shown to regulate bone resorption [15].

CART is a molecule synthesized by neurons of the arcuate hypothalamic nuclei, acting on osteoclasts by modulating Receptor activator of nuclear factor kappa-B ligand (Rankl) expression, which is in charge of triggering osteoclastic activity [16]. CART effectively decreases the amount of Rankl, causing osteoclastic activity to be inhibited [15].

2.4b Sympathetic Nervous System Pathway

Sympathetic signaling occurs via beta2-adrenergic receptors present on osteoblasts that control bone formation downstream of leptin. Sympathetic nervous system favors bone resorption 8 by increasing expression in osteoblast progenitor cells of the osteoclast differentiation factor

RankL [17]. This process occurs by phosphorylation by Protein Kinase A of ATF4, a cell specific transcription factor essential for osteoblast differentiation and function [18].

2.4c Neuropeptide Y

It has been suggested that Neuropeptide Y (Y2) is a critical modulator of leptin's regulation of cortical bone. Through using Y2 knockout mice (Y2−/−), it was revealed these mice had a two-fold greater bone volume in both male and female Y2−/-- mice compared to wild type mice due to elevated osteoblastic activity and no decrease in osteoclastic activity. Y2 acts downstream of leptin and is negatively regulated by serum leptin levels such that reduced leptin levels within the body results in an increase in Y2 levels [19]. Y2 and leptin receptors are localized on NPY-ergic neurons in the arcuate nucleus of the hypothalamus, showing Y2 also has a role in the regulation of energy homeostasis [20].

2.4d Insulin like Growth Factor

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. Mice that have IGF-1 knocked out exhibited a decrease in bone mineral density. It has been shown that IGF and other insulin growth factor complexes upregulate osteoblastic differentiation, leading to an increase in osteoblastic activity [21].

9 2.4e Insulin

Insulin and bone each play an important role in each other’s metabolisms [22]. Insulin

binding to insulin binding receptors on osteoblasts causes a decrease in Osteoprotegerin (Opg),

which causes increase bone resorption by osteoclasts. The acidic pH in the resorption lacuna

activates, via decarboxylation, osteocalcin which then goes on to stimulate insulin secretion in the

beta cells of the pancreas as well as promote insulin sensitivity in peripheral organs [23]. Overall,

insulin helps link bone remodeling with glucose homeostasis [24].

Figure 2-4 Insulin’s effects on bone resorption [24]

2.5 Strain Differences between B6 and D2 mice

More than 70% of the variability in human bone density can be attributed to genetic factors as shown in studies using twins, osteoporotic families and individuals with rare heritable 10 bone disorders [25]. The inbred strains of mice, C57BL/6J (B6) and DBA/2J (D2), have different

peak bone densities in such bones as their femurs and therefore serve as models for studying

genetic regulation of bone mass [25]. Long bones in D2 mice were smaller compared with those

in B6 mice, although they were found to be stronger per unit area of bone [43]. One study

revealed that total femur density comparisons between B6 and D2 mice show strain differences

arise as early as 2 months of age in the mice. It was shown D2 mice have significantly higher

femur densities (0.70 + 0.01 mg/mm3) compared to B6 mice (0.61 + 0.01 mg/mm3) at 12 months of age [48]. In terms of marrow, D2 marrow was found to have an 11 fold higher frequency of hematopoietic stem cells in comparison with B6 mice, furthering the point that D2 has a higher peak bone masses [49].

In terms of muscle, four hindlimb muscles, two fast twitch, one slow twitch and one mixed were 11-34 % heavier in the B6 strain as compared to the D2 strain [47]. For longevity, D2 mice have a significantly shorter lifespan, 21 months, as compared to B6 mice, 29 months [50].

Looking at lung surface, D2 mice have a larger alveolar air production as well as a smaller fixed lung volume per lung weight compared to B6 mice [51].

In terms of exercise, both strains of mice have been heavily tested and show great heterogeneity in terms of how each strain deals with different workloads of exercise. In terms of endurance exercise, the response of animals to a given exercise can be quite variable [26]. In volunteer wheel running for example, D2 mice outperformed B6 mice. D2 mice also have been shown to have a higher relative cardiac mass and function as well as high levels of ANF and B-

MHC mRNA, both of which show increased cardiac output [26]. Other strain differences include

B6 mice developing obesity at a faster rate when on a high fat diet. B6 mice are also more likely to develop seizures when exposed to loud noises as a neonate [27]. The hippocampus of

C57BL/6J is 5 mg (20%) heavier than that of DBA/2J as well [28]. While all these differences should be taken∼ into consideration, it should be noted that exercise involves a variety of complex 11 factors and therefore performance in exercise related tasks varies not just between strains but within them as well, making the results of any study interesting and unique when dealing with exercise trials.

Based on the above information, it can therefore be hypothesized that there will be a significant difference in gene expression between the two strains such that D2 mice will have more genes upregulated that function to increase bone density and skeletal quality as compared to

B6 mice. This reasoning can be attributed to previous findings that D2 mice have a higher bone density, specifically in their femur as well as a higher peak bone mass in comparison to B6 mice

[43][49].

Chapter 3

Materials and Methods

3.1 Animals and Experimental Design

Seventy seven adult (480 day old) female mice divided between C57BL/6 (B6) and DBA/2J (D2) inbred mouse strains were exposed to treadmill running(14 B6 mice and 14 D2 mice), tower climbing (13 B6 and 12 D2) or served as non-exercised controls

(14 B6 and 10 D2) (Figure 3-1). The five week exercise intervention was conducted with

five cohorts staggered over a span of nine months with approximately 3 mice from each

strain by treatment group in each cohort.

77 Female Mice

B6 Strain D2 Strain n = 41 n = 36

Treadmill Tower Control Treadmill Tower Control n = 14 n = 13 n =14 n = 14 n = 12 n =10

Figure 3-1 Schematic of experimental design.

Mice in the treadmill group were run on a rodent treadmill 5 days per week for 5

weeks. The speed, incline and duration were gradually increased until a target speed of 15

m/min at a 25 degree incline for 30 minutes was attained. The mice were run for one

week at the maximum incline, speed and duration (Table 1). See Appendix A for

instructions to treadmill operators on conditions for refusal to exercise. The tower 13 climbers were housed in a standard mouse cage attached to a 120 cm tall mesh wire tower with a diameter of 17 cm, with water bottles placed at the top of the tower (See Appendix

B). Tower climbers remained in the towers 24 hours per day 7 days/week for a 5 week period. To train the mice to climb during the first week, the water bottles were put at the bottom of the tower and gradually raised to the top. The mice then climbed to the top of the towers to drink during the remaining weeks of the intervention.

Table 3-1Protocol of settings used during treadmill running intervention

Incline Speed Duration (min) Week Day (degrees) (m/min) 1 1 5 10 10 1 2 5 10 20 1 3 5 10 20 1 4 5 10 30 1 5 10 10 20 2 8 10 10 30 2 9 10 12 30 2 10 15 12 20 2 11 15 12 30 2 12 15 13 30 3 15 20 13 20 3 16 20 13 30 3 17 20 14 30 3 18 25 14 20 3 19 25 14 30 4 22 25 14 30 4 23 25 15 30 4 24 25 15 30 4 25 25 15 30 4 26 25 15 30 5 29 25 15 30 5 30 25 15 30 5 31 25 15 30 5 32 25 15 30 5 33 25 15 30

14 All of the mice were housed in individual cages in the same room with monitored temperature and humidity, and with food and water available ad libitum. The lights were set on a reverse 12 hour light/dark cycle and treadmill mice were exercised during the dark cycle when mice are known to be more active [28]. All procedures complied with and were approved by the Pennsylvania State University Institutional Care and Use

Committee (IACUC#: 22463).

3.2 Tissue Harvesting

Approximately two hours after the last exercise exposure, either after treadmill running or removal from the cages attached to towers, mice were euthanized by cervical dislocation. After euthanasia the head was separated from the body of the mouse. The brain was recovered from the skull by cutting along the midline of the dorsal skull.

Immediately after recovery, brains were inserted into a microtube, which was in turn immersed in liquid nitrogen. Brain tissue was stored at -80 °C until preparation for hypothalamic RNA extraction.

3.3 RNA Extraction

3.3a. Preparation

At a minimum of 16-24 hours prior to extraction, individual brains were removed from the microtube where they had been stored at -80 °C and moved into a second tube containing 15 Ambion RNAlater-ICE (cat# AM7030) at a ratio of 10 volumes relative to the total brain mass

(assumed to be 400 mg).

3.3b. RNA Extraction

Total RNA from the hypothalamus of all mice (n=77) was extracted using Qiagen

RNeasy Fibrous Tissue Mini Kit (cat # 74704). There were two deviations to the protocol provided with the kit. In order to avoid overflow of the Qiazol lysis reagent in the micro tube, the amount of reagent was reduced from 1000µL to 800µL. Homogenization was accomplished using a rotor-stator homogenizer. The sample was then transferred to a new tube leaving any remaining foam to be discarded as is specified by the Department of

Environmental Health and Safety. Deviation from the Qiagen kit protocol additionally occurred during aqueous and organic separation of the homogenate and chloroform solution where the sample was centrifuged for 20 min at room temperature rather than

4ºC.

3.3c. RNA quality analysis

The concentration and quality of the total RNA samples were assessed by the

Penn State Genomics Core Facility using a Thermo Scientific Nanodrop and Agilent

Bioanalyzer. The Nanodrop was used to obtain concentrations and 260/280 ratios. Ratios between 1.8 and 2.1 were considered as an indication of good quality, while ratios under

1.8 were considered unusable because such a value indicates sample degradation or contamination. The Bioanalyzer calculated RIN (RNA Integrity Number) values which 16 were used to evaluate the quality of the RNA as well. The RIN scale ranges from 1 to 10 with a score of 10 indicating intact or high quality RNA where a score of 1 indicates sample degradation. Samples with RIN values below 8 were not used. The Bioanalyzer also provided a pseudo-gel image and an electropherogram. These additional tools were employed to evaluate the quality of the RNA. For a detailed chart of the samples used, see Table 3-2.

Table 3-2 Summary of Chip Data used for Microarray Analysis

Group Chip Sample ID ng/ul 260/280 RIN 28s/18s B6 Control 373 758.49 2.06 9.0 1.1 1 B6 Control 355 455.16 2.01 9.1 1.6 B6 Control 374 388.97 2 9.1 1.5 2 B6 Control 319 683.75 2.01 9.2 1.3 B6 Control 375 438.96 1.98 9.1 1.6 3 B6 Control 339 777.98 2.07 9.1 1.2 D2 Control 322 1807.39 2.13 8.1 1.3 4 D2 Control 377 351.95 2.05 8.2 1.4 D2 Control 306 1190.88 2.03 8.5 1.4 5 D2 Control 341 903.21 2.1 9.0 1.1 D2 Control 304 263.06 2.08 8.6 1.4 6 D2 Control 324 790.01 2.14 9.7 1.8 B6 Tower 315 362.23 2.05 8.4 1.0 7 B6 Tower 387 359.94 1.99 8.9 1.6 B6 Tower 369 509.3 2.03 9.2 1.3 8 B6 Tower 333 797.57 2.07 9.3 1.3 B6 Tower 314 507.62 2.08 9.4 1.8 9 B6 Tower 367 530.49 2.06 9.5 1.4 D2 Tower 335 662.44 2.04 9.4 1.3 10 D2 Tower 389 426.43 2.06 8.7 1.5 D2 Tower 336 653.64 2.1 9.4 1.3 11 D2 Tower 352 662.63 2.13 8.9 1.4 D2 Tower 372 621.51 2.07 8.9 1.4 12 D2 Tower 317 198.36 1.96 9.1 1.5 B6 Treadmill 326 369.27 2.07 8.6 1.3 13 B6 Treadmill 307 272.08 1.98 8.8 1.5 B6 Treadmill 14 327 508.32 2.04 8.8 1.4 17

B6 Treadmill 343 660.47 2.11 8.8 1.3 B6 Treadmill 362 470.48 2.03 8.9 1.2 15 B6 Treadmill 344 560.52 2.08 9.3 1.3 D2 Treadmill 312 700.38 2.06 9.4 1.2 16 D2 Treadmill 364 1262.92 2.13 9.6 1.6 D2 Treadmill 311 1188.74 2.08 8.5 1.0 17 D2 Treadmill 347 348.14 2.09 8.2 1.1 D2 Treadmill 310 759.92 2.06 8.9 1.3 18 D2 Treadmill 384 410.07 2.04 8.9 1.2

3.4 RNA Pooling and Labeling

RNA samples from two biological replicates were pooled for each microarray,

reducing the sample variance. For each mouse strain by exercise group being evaluated (6 groups total; B6 and D2 strains split into 3 treatment groups: non-exercised controls, treadmill runners and tower climbers), the RNA samples were ranked according to quality (14 B6 Treadmill, 13 B6 Tower, 14 B6 Control, 14 D2 Treadmill, 12 D2 Tower,

10 D2 Control).

The six best RNA samples in terms of quality were chosen in each group. These six samples were then pooled in pairs of two for a total of three pooled samples per group. The resulting 18 samples were then labeled by the Penn State Genomics Core

Facility staff using Ambion MessageAmp II-Biotin Enhanced Kit (cat # AM1791) and protocol. From each pooled sample, 300 ng of RNA was labeled (kit range = 50-

5000ng). The amplified and labeled RNA samples were then quantified and evaluated at the Penn State Genomics Core Facility once again using the Agilent Technologies

Nanodrop and Bioanalyzer. The Nanodrop results provided the concentration of 18 amplified RNA and the Bioanalyzer results were used as a visual indication that the

samples amplified properly (using the pseudo-gel and electropherogram images).

Amplified RNA (15μg) was then fragmented using the buffer and protocol provided with

the Ambion kit. The samples were evaluated once again using the Bioanalyzer which

provided visual assurance that the samples were completely fragmented using the

pseudo-gel and electropherogram results.

3.5 Microarray Processing and Data Analysis

Gene expression was examined using the Affymetrix GeneChip Mouse Gene 1.0

ST Array and 18 experiments (chips) were conducted for the brain tissue. Three chips

containing similar samples from different mice were used as biological replicates for each

strain (B6 or D2) and treatment (control, treadmill running or tower climbing) group.

Labeled and fragmented samples were processed (hybridized and scanned) at the Penn

State Genomics Core Facility using the Affymetrix platform.

The resulting microarray data was analyzed using statistical software, R, with

Bioconductor and Linear Models for Microarray Data (LIMMA) packages. The data were

normalized using Robust Multichip Averaging (RMA) and differential expression was deemed significant at p ≤ 0.05 after appropriate adjustment for multiple comparisons using False Discovery Rate (FDR). Differential expression was compared relative to mouse strain and exercise treatment within brain tissue [52].

Chapter 4

Results

4.1 Differential Expression as a Function of Genetic Strain

Comparison between the two mice genetic strains used in the study (B6 vs. D2) showed the differential expression in over genes when evaluated for significance over a variety of different comparisons. Genes were considered significant with an adjusted p-value < 0.05 as well as a minimum fold change of 2 or higher.

A total of 343 genes were expressed significantly higher in B6 compared to D2 mice. 51 genes were expressed higher in B6 mice with a fold change of 5 or greater (Table 4-1). 150 genes were expressed higher in B6 mice with a fold change of 3 or 4 (Table 4-2 and Table 4-3). 142 genes were expressed higher in B6 mice with a fold change of 2 (Table 4-4, Table 4-5 and Table

4-6).

A total of 339 genes were expressed significantly higher in D2 compared to B6 mice. 37 genes were expressed higher in D2 mice with a fold change of 5 or greater (Table 4-7). 144 genes were expressed higher in D2 mice with a fold change of 3 or 4 (Table 4-8 and Table 4-9 and

Table 4-10). 158 genes were expressed higher in D2 mice with a fold change of 2 (Table 4-11 and

Table 4-12 and Table 4-13).

Table 4-1 Genes expressed in B6 mice over D2 with a FC of greater than 5

Gene Symbol Gene Title adj.P.Val FC Acp1 acid phosphatase 1, soluble 0.000 13 Agxt2l1 alanine-glyoxylate aminotransferase 2-like 1 0.000 12 Armcx1 armadillo repeat containing, X-linked 1 0.000 5 20

Actr6 ARP6 actin-related protein 6 homolog (yeast) 0.000 13 Cd38 CD38 antigen 0.002 5 Cdc5l cell division cycle 5-like (S. pombe) 0.001 12 Creg1 cellular repressor of E1A-stimulated genes 1 0.000 5 Ccl21c chemokine (C-C motif) ligand 21C (leucine) 0.000 19 Chrna3 cholinergic receptor, nicotinic, alpha polypeptide 3 0.015 5 Cbx7 chromobox homolog 7 0.000 5 Commd7 COMM domain containing 7 0.000 5 Cyb5r3 cytochrome b5 reductase 3 0.000 14 Defa20 /// Defa3 defensin, alpha, 20 /// defensin, alpha, 3 0.000 491 Eapp E2F-associated phosphoprotein 0.000 15 Entpd4 ectonucleoside triphosphate diphosphohydrolase 4 0.000 5 Fcrls Fc receptor-like S, scavenger receptor 0.000 15 Fggy FGGY carbohydrate kinase domain containing 0.000 23 Hspa8 /// LOC624853 heat shock protein 8 /// hypothetical LOC624853 0.000 10 Hspb6 heat shock protein, alpha-crystallin-related, B6 0.000 6 Hbb-b1 hemoglobin, beta adult major chain 0.025 8 Hmgb1 high mobility group box 1 0.003 9 Klk6 kallikrein related-peptidase 6 0.012 7 Klra3 killer cell lectin-like receptor, subfamily A, member 3 0.000 10 Mrpl35 mitochondrial ribosomal protein L35 0.000 6 Mkl2 MKL/myocardin-like 2 0.000 14 Myoc Myocilin 0.000 13 Myo7a myosin VIIA 0.000 5 Nfu1 NFU1 iron-sulfur cluster scaffold homolog (S. cerevisiae) 0.000 5 Olfr113 olfactory receptor 113 0.000 6 Pon2 paraoxonase 2 0.000 5 Pttg1 pituitary tumor-transforming gene 1 0.000 55 Plekhh2 pleckstrin homology domain containing 0.000 10 Pdxdc1 pyridoxal-dependent decarboxylase domain containing 1 0.000 8 Rnasel ribonuclease L (2', 5'-oligoisoadenylate synthetase) 0.000 6 ribosomal protein L21 0.000 9

Rpl3 ribosomal protein L3 0.000 387 Rps15a ribosomal protein S15A 0.000 5 Rps2 ribosomal protein S2 0.000 5 Rplp0 ribosomal protein, large, P0 0.000 6 Sec24d Sec24 related gene family, member D (S. cerevisiae) 0.000 5 Srp54c signal recognition particle 54C 0.000 6 Sirpb1a signal-regulatory protein beta 1A 0.004 5 Sat2 spermidine/spermine N1-acetyl transferase 2 0.000 10 Tas2r131 taste receptor, type 2, member 131 0.000 7 Tcrg-V3 /// Tcrg-V2 T-cell receptor gamma, variable 3 /// T-cell receptor gamma, variable 2 0.004 6 Trim12a tripartite motif-containing 12A 0.000 11 21

Trim30d tripartite motif-containing 30D 0.000 5 Tnfaip6 tumor necrosis factor alpha induced protein 6 0.000 6 Ublcp1 ubiquitin-like domain containing CTD phosphatase 1 0.000 5018 Usmg5 upregulated during skeletal muscle growth 5 0.002 5 Zfp125 zinc finger protein 125 0.000 333

Table 4-2 Genes expressed higher in B6 over D2 mice with a FC of 3 or 4 Part I

Gene Symbol Gene Title adj.P.Val FC Oxsm 3-oxoacyl-ACP synthase, mitochondrial 0.002 4 Phgdh 3-phosphoglycerate dehydrogenase 0.001 3 Abhd10 abhydrolase domain containing 10 0.001 3 Abhd14a abhydrolase domain containing 14A 0.000 3 Acot11 acyl-CoA thioesterase 11 0.000 3 Adipor2 adiponectin receptor 2 0.006 3 Arl3 ADP-ribosylation factor-like 3 0.000 3 Aldh7a1 /// Phax aldehyde dehydrogenase family 7, member A1 /// phosphorylated adaptor 0.000 4 A2m alpha-2-macroglobulin 0.002 4 Ang6 angiogenin, ribonuclease A family, member 6 0.046 3 Arnt2 aryl hydrocarbon receptor nuclear translocator 2 0.000 3 Arid5b AT rich interactive domain 5B (MRF1-like) 0.001 3 B3gat2 beta-1,3-glucuronyltransferase 2 (glucuronosyltransferase S) 0.001 3 B2m beta-2 microglobulin 0.002 3 Bex1 brain expressed gene 1 0.011 3 Cml3 /// Cml5 camello-like 3 /// camello-like 5 0.001 3 Cmbl carboxymethylenebutenolidase-like (Pseudomonas) 0.033 3 Cartpt CART prepropeptide 0.039 3 Cd33 CD33 antigen 0.000 3 Cks2 CDC28 protein kinase regulatory subunit 2 0.033 3 Cetn4 centrin 4 0.005 3 Ccl28 chemokine (C-C motif) ligand 28 0.001 3 Cldn10 claudin 10 0.001 3 F3 coagulation factor III 0.011 3 Ccdc124 coiled-coil domain containing 124 0.003 3 Ccdc56 coiled-coil domain containing 56 0.000 3 Chchd5 coiled-coil-helix-coiled-coil-helix domain containing 5 0.003 3 Cda cytidine deaminase 0.003 3 Cyb5r4 cytochrome b5 reductase 4 0.004 3 Defa21 /// Defa22 defensin, alpha, 21 /// defensin, alpha, 22 0.035 3 Dhrs11 dehydrogenase/reductase (SDR family) member 11 0.005 3 Dlg4 discs, large homolog 4 (Drosophila) 0.000 3 Darc Duffy blood group, chemokine receptor 0.000 3 22

Dnm3os dynamin 3, opposite strand 0.008 3 Dnahc7a /// Dnahc7b dynein, axonemal, heavy chain 7A /// dynein, axonemal, heavy chain 7B 0.001 3 Dnahc7b dynein, axonemal, heavy chain 7B 0.015 3 Dbndd2 dysbindin (dystrobrevin binding protein 1) domain containing 2 0.003 3 Entpd2 ectonucleoside triphosphate diphosphohydrolase 2 0.000 3 Entpd3 ectonucleoside triphosphate diphosphohydrolase 3 0.001 3 Efhd1 EF hand domain containing 1 0.014 3 Epas1 endothelial PAS domain protein 1 0.010 3 Efnb3 ephrin B3 0.011 3 Eps8l1 EPS8-like 1 0.002 3 Exosc9 exosome component 9 0.006 3 Fan1 FANCD2/FANCI-associated nuclease 1 0.000 3 Fancl Fanconi anemia, complementation group L 0.000 4 Fancl /// Vrk2 Fanconi anemia, complementation group L /// vaccinia related kinase 2 0.001 3 Fggy FGGY carbohydrate kinase domain containing 0.040 3 Fmn2 formin 2 0.013 3 Fut10 fucosyltransferase 10 0.000 3 Gabrb2 gamma-aminobutyric acid (GABA) A receptor, subunit beta 2 0.001 3 Gabrr2 gamma-aminobutyric acid (GABA) C receptor, subunit rho 2 0.023 3 Gaa glucosidase, alpha, acid 0.038 3 Glul glutamate-ammonia ligase (glutamine synthetase) 0.000 3 Gapdhs glyceraldehyde-3-phosphate dehydrogenase, spermatogenic 0.000 4 Gatm glycine amidinotransferase (L-arginine:glycine amidinotransferase) 0.008 3 Glra2 glycine receptor, alpha 2 subunit 0.038 3 Gpnmb glycoprotein (transmembrane) nmb 0.001 3 Hebp2 heme binding protein 2 0.000 3

Table 4-3 Genes expressed higher in B6 over D2 mice with a FC of 3 or 4 Part II

Gene Symbol Gene Title adj.P.Val FC Hpcal1 hippocalcin-like 1 0.004 3 Hist1h4c histone cluster 1, H4c 0.001 3 Hdac1 histone deacetylase 1 0.000 4 Hapln2 hyaluronan and proteoglycan link protein 2 0.013 3 Idua iduronidase, alpha-L- 0.001 3 Ighg Immunoglobulin heavy chain (gamma polypeptide) 0.031 3 Il33 interleukin 33 0.021 4 Ica1 islet cell autoantigen 1 0.000 3 Ivd isovaleryl coenzyme A dehydrogenase 0.000 4 Kpna2 karyopherin (importin) alpha 2 0.003 4 Klhl1 kelch-like 1 (Drosophila) 0.009 3 Klra6 killer cell lectin-like receptor, subfamily A, member 6 0.002 3 23

Kif1c kinesin family member 1C 0.002 3 Lcmt2 leucine carboxyl methyltransferase 2 0.002 3 Mro Maestro 0.001 3 Man2b1 mannosidase 2, alpha B1 0.000 3 Miip migration and invasion inhibitory protein 0.000 3 Mcm6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) 0.000 3 Mis12 MIS12 homolog (yeast) 0.004 3 Mrpl41 mitochondrial ribosomal protein L41 0.000 3 Mal myelin and lymphocyte protein, T-cell differentiation protein 0.002 3 Mobp myelin-associated oligodendrocytic basic protein 0.011 3 Naaa N-acylethanolamine acid amidase 0.000 4 Asah1 N-acylsphingosine amidohydrolase 1 0.005 3 Ntsr2 neurotensin receptor 2 0.000 4 Ninj2 ninjurin 2 0.010 3 Nlrp1c-ps NLR family, pyrin domain containing 1C, pseudogene 0.001 4 Mpg N-methylpurine-DNA glycosylase 0.001 3 Nsun7 NOL1/NOP2/Sun domain family, member 7 0.002 4 Nudt19 nudix (nucleoside diphosphate linked moiety X)-type motif 19 0.001 4 Ociad1 OCIA domain containing 1 0.000 3 Olfr231 olfactory receptor 231 0.031 3 Olfr611 olfactory receptor 611 0.004 3 Odc1 ornithine decarboxylase, structural 1 0.007 4 Oscar osteoclast associated receptor 0.003 4 Pank2 pantothenate kinase 2 0.000 3 Plac9 placenta specific 9 0.002 3 Polr1a polymerase (RNA) I polypeptide A 0.000 4 Pfdn2 prefoldin 2 0.001 3 Pcolce2 procollagen C-endopeptidase enhancer 2 0.007 3 Psmb6 proteasome (prosome, macropain) subunit, beta type 6 0.002 3 Rabl2 RAB, member of RAS oncogene family-like 2 0.001 3 Rassf8 Ras association (RalGDS/AF-6) domain family (N-terminal) member 8 0.037 3 Rassf2 Ras association (RalGDS/AF-6) domain family member 2 0.004 4 Rec8 REC8 homolog (yeast) 0.000 3 Rbp1 retinol binding protein 1, cellular 0.000 3 Rnase6 ribonuclease, RNase A family, 6 0.002 3 Rnps1 ribonucleic acid binding protein S1 0.001 3 Rpl3 ribosomal protein L3 0.001 3 Rpl31 ribosomal protein L31 0.001 3 Rps4y2 ribosomal protein S4, Y-linked 2 0.000 4 Rplp0 ribosomal protein, large, P0 0.000 3 Rnf122 ring finger protein 122 0.000 3 Rnf208 ring finger protein 208 0.002 3 Rnf213 ring finger protein 213 0.000 3 24

Sspn Sarcospan 0.000 3 Sgk1 serum/glucocorticoid regulated kinase 1 0.002 3 Sft2d2 SFT2 domain containing 2 0.000 4 Sprr2a2 /// Sprr2a3 small proline-rich protein 2A2 /// small proline-rich protein 2A3 0.040 3 Sprr2a3 /// Sprr2a1 small proline-rich protein 2A3 /// small proline-rich protein 2A1 0.040 3 Slc39a2 solute carrier family 39 (zinc transporter), member 2 0.009 3 Srd5a1 steroid 5 alpha-reductase 1 0.000 3 Tcrg-V1 /// Tcrg-V2 T-cell receptor gamma, variable 1 /// T-cell receptor gamma, variable 2 0.048 3 Tada1 transcriptional adaptor 1 0.000 3 Tmem159 transmembrane protein 159 0.000 4 Tmem63a transmembrane protein 63a 0.048 3 Trim30e-ps1 tripartite motif-containing 30E, pseudogene 1 0.000 3 Tnnt2 troponin T2, cardiac 0.001 3 Ttll3 tubulin tyrosine ligase-like family, member 3 0.000 3 Ubb /// Gm1821 ubiquitin B /// ubiquitin pseudogene 0.005 4 Usp53 ubiquitin specific peptidase 53 0.004 3 Uba2 ubiquitin-like modifier activating enzyme 2 0.001 3 Ulk4 unc-51-like kinase 4 (C. elegans) 0.000 3 Vamp8 vesicle-associated membrane protein 8 0.001 3 Vit Vitrin 0.000 3 Vmn2r96 vomeronasal 2, receptor 96 0.012 4 Zfp277 zinc finger protein 277 0.006 3 Zfp458 zinc finger protein 458 0.001 3 Zfp738 zinc finger protein 738 0.003 3 Zfp87 zinc finger protein 87 0.016 3 Zfyve21 zinc finger, FYVE domain containing 21 0.000 3

Table 4-4 Genes expressed higher in B6 over D2 mice with a FC of 2 Part I

Gene Symbol Gene Title adj.P.Val FC Bdh1 3-hydroxybutyrate dehydrogenase, type 1 0.006 2 Nt5c 5',3'-nucleotidase, cytosolic 0.001 2 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 Adamts2 motif, 2 0.046 2 Abhd14b abhydrolase domain containing 14b 0.003 2 Acaa1a acetyl-Coenzyme A acyltransferase 1A 0.000 2 Atf7ip activating transcription factor 7 interacting protein 0.020 2 Aldh9a1 aldehyde dehydrogenase 9, subfamily A1 0.020 2 Anxa3 annexin A3 0.021 2 Dars2 aspartyl-tRNA synthetase 2 (mitochondrial) 0.008 2 biregional cell adhesion molecule-related/down-regulated by oncogenes (Cdon) Boc binding protein 0.042 2 Bex1 /// Bex4 brain expressed gene 1 /// brain expressed gene 4 0.029 2 Btbd11 BTB (POZ) domain containing 11 0.037 2 25

Capns1 calpain, small subunit 1 0.005 2 Clic4 chloride intracellular channel 4 (mitochondrial) 0.017 2 Chmp4b chromatin modifying protein 4B 0.028 2 F2rl2 coagulation factor II (thrombin) receptor-like 2 0.048 2 Coq2 coenzyme Q2 homolog, prenyltransferase (yeast) 0.000 2 C1qbp complement component 1, q subcomponent binding protein 0.023 2 C1qb complement component 1, q subcomponent, beta polypeptide 0.022 2 Crim1 cysteine rich transmembrane BMP regulator 1 (chordin like) 0.003 2 Cyb5r4 cytochrome b5 reductase 4 0.004 2 Cox7a2l cytochrome c oxidase subunit VIIa polypeptide 2-like 0.001 2 Cyp2r1 cytochrome P450, family 2, subfamily r, polypeptide 1 0.015 2 Dcaf8 DDB1 and CUL4 associated factor 8 0.021 2 Defb8 defensin beta 8 0.006 2 Dhdds dehydrodolichyl diphosphate synthase 0.039 2 Dennd1b DENN/MADD domain containing 1B 0.014 2 Dync2li1 dynein cytoplasmic 2 light intermediate chain 1 0.002 2 Dynll1 dynein light chain LC8-type 1 0.028 2 Eltd1 EGF, latrophilin seven transmembrane domain containing 1 0.015 2 Esr1 estrogen receptor 1 (alpha) 0.032 2 Etohi1 ethanol induced 1 0.027 2 Fbxo44 F-box protein 44 0.038 2 Fcgr3 Fc receptor, IgG, low affinity III 0.008 2

Table 4-5Genes expressed higher in B6 over D2 mice with a FC of 2 Part II

Gene Symbol Gene Title adj.P.Val FC Fgf2 fibroblast growth factor 2 0.001 2 Fxyd6 FXYD domain-containing ion transport regulator 6 0.006 2 Gpr37l1 G protein-coupled receptor 37-like 1 0.003 2 Gtf3a /// Mtif3 general transcription factor III A /// mitochondrial translational initiation factor 3 0.012 2 Gapt Grb2-binding adaptor, transmembrane 0.025 2 Gadd45gip1 growth arrest and DNA-damage-inducible, gamma interacting protein 1 0.004 2 H2-Ke2 H2-K region expressed gene 2 0.016 2 Hfm1 HFM1, ATP-dependent DNA helicase homolog (S. cerevisiae) 0.001 2 Hmgb1 /// Hmgb1l high mobility group box 1 /// high mobility group box 1-like 0.026 2 Hmgb2 high mobility group box 2 0.010 2 H2-Eb1 histocompatibility 2, class II antigen E beta 0.027 2 H2-K2 histocompatibility 2, K region locus 2 0.004 2 Hist1h1c histone cluster 1, H1c 0.004 2 Hpgd hydroxyprostaglandin dehydrogenase 15 (NAD) 0.002 2 Inpp5a /// Nkx6-2 inositol polyphosphate-5-phosphatase A /// NK6 homeobox 2 0.023 2 Igtp /// Irgm2 interferon gamma induced GTPase /// immunity-related GTPase family M member 2 0.003 2 26

Iltifb interleukin 10-related T cell-derived inducible factor beta 0.006 2 Iffo2 intermediate filament family orphan 2 0.023 2 Ina internexin neuronal intermediate filament protein, alpha 0.005 2 Ift27 intraflagellar transport 27 homolog (Chlamydomonas) 0.025 2 Josd2 Josephin domain containing 2 0.007 2 Katnal2 katanin p60 subunit A-like 2 0.005 2 Klrc2 killer cell lectin-like receptor subfamily C, member 2 0.010 2 Klra8 killer cell lectin-like receptor, subfamily A, member 8 0.015 2 Klc1 kinesin light chain 1 0.000 2 Kank4 KN motif and ankyrin repeat domains 4 0.020 2 Lpin1 lipin 1 0.028 2 Litaf LPS-induced TN factor 0.023 2 Lsm4 LSM4 homolog, U6 small nuclear RNA associated (S. cerevisiae) 0.013 2 Mr1 major histocompatibility complex, class I-related 0.004 2 Mlec Malectin 0.015 2 Manba mannosidase, beta A, lysosomal 0.022 2 Mxra7 matrix-remodelling associated 7 0.045 2 Med21 mediator complex subunit 21 0.032 2 Med8 mediator of RNA polymerase II transcription, subunit 8 homolog (yeast) 0.001 2 Mta3 metastasis associated 3 0.017 2 Mri1 methylthioribose-1-phosphate isomerase homolog (S. cerevisiae) 0.006 2 Mrpl48 mitochondrial ribosomal protein L48 0.003 2 Mzt1 mitotic spindle organizing protein 1 0.017 2 Mmd2 monocyte to macrophage differentiation-associated 2 0.004 2 Myo1d myosin ID 0.024 2

Table 4-6Genes expressed higher in B6 over D2 mice with a FC of 2 Part III

Gene Symbol Gene Title adj.P.Val FC Ndufa9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 0.011 2 Nav1 neuron navigator 1 0.011 2 Nmnat2 nicotinamide nucleotide adenylyltransferase 2 0.001 2 Nudcd1 NudC domain containing 1 0.038 2 Olfr1040 olfactory receptor 1040 0.041 2 Palmd palmdelphin 0.010 2 Park2 Parkinson disease (autosomal recessive, juvenile) 2, parkin 0.001 2 Ppib /// Snx22 peptidylprolyl isomerase B /// sorting nexin 22 0.002 2 Prdx2 peroxiredoxin 2 0.002 2 Pip5k1b phosphatidylinositol-4-phosphate 5-kinase, type 1 beta 0.042 2 Plscr4 phospholipid scramblase 4 0.009 2 Pllp plasma membrane proteolipid 0.049 2 Pkd2l1 polycystic kidney disease 2-like 1 0.033 2 27

Pgap2 post-GPI attachment to proteins 2 0.003 2 Kctd5 potassium channel tetramerisation domain containing 5 0.002 2 Paqr8 progestin and adipoQ receptor family member VIII 0.000 2 Prrg4 proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) 0.018 2 Pcsk6 proprotein convertase subtilisin/kexin type 6 0.003 2 Pofut1 protein O-fucosyltransferase 1 0.000 2 Pink1 PTEN induced putative kinase 1 0.021 2 Pgpep1 pyroglutamyl-peptidase I 0.000 2 Rab5a RAB5A, member RAS oncogene family 0.005 2 radial spoke 3A homolog (Chlamydomonas) /// radial spoke 3B homolog Rsph3a /// Rsph3b (Chlamydomonas) 0.001 2 Rgl2 ral guanine nucleotide dissociation stimulator-like 2 0.000 2 Reep5 receptor accessory protein 5 0.002 2 Rsl1 regulator of sex limited protein 1 0.001 2 regulatory factor X-associated ankyrin-containing protein /// nuclear receptor 2C2- Rfxank /// Nr2c2ap associated protein 0.003 2 Rpl34 ribosomal protein L34 0.003 2 Rnf213 ring finger protein 213 0.007 2 Selplg selectin, platelet (p-selectin) ligand 0.002 2 Serpina1d serine (or cysteine) peptidase inhibitor, clade A, member 1D 0.043 2 Spink10 serine peptidase inhibitor, Kazal type 10 0.024 2 Spint1 serine protease inhibitor, Kunitz type 1 0.009 2 Sirpb1a /// LOC100038947 /// signal-regulatory protein beta 1A /// signal-regulatory protein beta 1-like /// signal- Sirpb1b regulatory protein beta 1B 0.002 2 Scnm1 sodium channel modifier 1 0.004 2 Sstr1 somatostatin receptor 1 0.025 2 S1pr1 sphingosine-1-phosphate receptor 1 0.010 2 Stard9 START domain containing 9 0.000 2 Stard9 START domain containing 9 0.011 2 Soat1 sterol O-acyltransferase 1 0.001 2 Sdhaf1 succinate dehydrogenase complex assembly factor 1 0.000 2 Sult2b1 sulfotransferase family, cytosolic, 2B, member 1 0.002 2 Stx6 syntaxin 6 0.002 2 Sys1 SYS1 Golgi-localized integral membrane protein homolog (S. cerevisiae) 0.001 2 Tac2 tachykinin 2 0.034 2 Tasp1 taspase, threonine aspartase 1 0.016 2 Tas2r120 taste receptor, type 2, member 120 0.016 2 Txnip thioredoxin interacting protein 0.036 2 Tcea1 transcription elongation factor A (SII) 1 0.002 2 Tmc4 transmembrane channel-like gene family 4 0.006 2 Tmem56 transmembrane protein 56 0.021 2 Tmem59l transmembrane protein 59-like 0.009 2 Trim3 tripartite motif-containing 3 0.000 2 Tnni1 troponin I, skeletal, slow 1 0.007 2 28

Tsc22d3 TSC22 domain family, member 3 0.039 2 Tubg2 tubulin, gamma 2 0.014 2 Tssc1 tumor suppressing subtransferable candidate 1 0.002 2 Ttyh2 tweety homolog 2 (Drosophila) 0.010 2 Ubiad1 UbiA prenyltransferase domain containing 1 0.001 2 Vps13d vacuolar protein sorting 13 D (yeast) 0.006 2 Vmn1r180 vomeronasal 1 receptor 180 0.002 2 V1rd19 /// V1rd18 vomeronasal 1 receptor, D19 /// vomeronasal 1 receptor, D18 0.002 2 Wnk1 WNK lysine deficient protein kinase 1 0.040 2 Xrcc2 X-ray repair complementing defective repair in Chinese hamster cells 2 0.016 2 Zfp459 zinc finger protein 459 0.024 2 Zfp874a zinc finger protein 874a 0.001 2 Zfp9 zinc finger protein 9 0.036 2

Table 4-7Genes expressed higher in D2 over B6 mice with a FC of 5 or greater

Gene Symbol Gene Title adj.P.Val FC a disintegrin-like and metallopeptidase (reprolysin type) with Adamts3 thrombospondin type 1 motif, 3 0.001 5 Abi3bp ABI gene family, member 3 (NESH) binding protein 0.028 11 Adi1 acireductone dioxygenase 1 0.000 12 Adora2a adenosine A2a receptor 0.034 5 Adat2 adenosine deaminase, tRNA-specific 2, TAD2 homolog (S. cerevisiae) 0.000 5 Alad aminolevulinate, delta-, dehydratase 0.000 11 Ano3 anoctamin 3 0.001 11 Ano3 /// Muc15 anoctamin 3 /// mucin 15 0.013 5 Abcb8 ATP-binding cassette, sub-family B (MDR/TAP), member 8 0.000 5 Bex4 brain expressed gene 4 0.021 6 Arpp19 cAMP-regulated phosphoprotein 19 0.000 5 Ctse cathepsin E 0.000 8 Cnot8 CCR4-NOT transcription complex, subunit 8 0.002 5 Cd59a /// Cd59b CD59a antigen /// CD59b antigen 0.009 5 Fabp7 fatty acid binding protein 7, brain 0.017 5 Gabra2 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 2 0.000 9 29

Gsr glutathione reductase 0.000 6 Glo1 glyoxalase 1 0.000 7 Gpr88 G-protein coupled receptor 88 0.046 5 Hdhd3 haloacid dehalogenase-like hydrolase domain containing 3 0.000 10 LOC641050 hypothetical protein LOC641050 0.033 7 Mlycd malonyl-CoA decarboxylase 0.000 10 Mela melanoma antigen 0.000 49,671 Npl N-acetylneuraminate pyruvate lyase 0.000 5 Nmbr neuromedin B receptor 0.016 5 Ocel1 occludin/ELL domain containing 1 0.000 8 Olfr208 olfactory receptor 208 0.000 8 Pla2g4e phospholipase A2, group IVE 0.000 13 Kcnv1 potassium channel, subfamily V, member 1 0.039 5 Prss41 protease, serine, 41 0.000 5 Pcdhb3 protocadherin beta 3 0.000 9 Pcdhb7 protocadherin beta 7 0.000 6 Rasgrp1 RAS guanyl releasing protein 1 0.013 5 Rpa3 replication protein A3 0.003 6 Rfk riboflavin kinase 0.000 14 Rpl29 ribosomal protein L29 0.000 6 Serpina1a serine (or cysteine) peptidase inhibitor, clade A, member 1A 0.000 23 Sh3rf2 SH3 domain containing ring finger 2 0.009 5 Snrpa1 small nuclear ribonucleoprotein polypeptide A' 0.000 6 Snord53 small nucleolar RNA, C/D box 53 0.000 608 Slc15a2 solute carrier family 15 (H+/peptide transporter), member 2 0.000 8 Stab2 stabilin 2 0.000 136 Tdpoz3 TD and POZ domain containing 3 0.016 12 Tmem132b transmembrane protein 132B 0.000 6 Wdfy1 WD repeat and FYVE domain containing 1 0.000 6 Zfp640 zinc finger protein 640 0.001 6 Zfand1 zinc finger, AN1-type domain 1 0.000 443

Table 4-8Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part I

Gene Symbol Gene Title adj.P.Val FC Hmgcs2 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 0.002 4 Mtrr 5-methyltetrahydrofolate-homocysteine methyltransferase reductase 0.001 4 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 Adamts3 motif, 3 0.001 3 Actn2 actinin alpha 2 0.014 4 Adal adenosine deaminase-like 0.010 3 Arl15 ADP-ribosylation factor-like 15 0.002 4 Acer2 alkaline ceramidase 2 0.026 4 30

Ang4 angiogenin, ribonuclease A family, member 4 0.015 3 Anxa4 annexin A4 0.037 3 Ano2 anoctamin 2 0.036 4 Acap3 ArfGAP with coiled-coil, ankyrin repeat and PH domains 3 0.002 4 Agap2 ArfGAP with GTPase domain, ankyrin repeat and PH domain 2 0.031 4 Atp2b1 ATPase, Ca++ transporting, plasma membrane 1 0.010 4 Atp6ap1l ATPase, H+ transporting, lysosomal accessory protein 1-like 0.001 3 Atg16l2 autophagy related 16 like 2 (S. cerevisiae) 0.001 4 Bcl11a B-cell CLL/lymphoma 11A (zinc finger protein) 0.005 3 Bcl11b B-cell leukemia/lymphoma 11B 0.016 3 Baiap2 brain-specific angiogenesis inhibitor 1-associated protein 2 0.022 4 Btbd9 BTB (POZ) domain containing 9 0.000 3 Cacng3 calcium channel, voltage-dependent, gamma subunit 3 0.015 4 Camk4 calcium/calmodulin-dependent protein kinase IV 0.003 4 Clip4 CAP-GLY domain containing linker protein family, member 4 0.017 3 Car11 carbonic anhydrase 11 0.000 4 Car7 carbonic anhydrase 7 0.038 3 Catsperg1 /// cation channel, sperm-associated, gamma 1 /// cation channel, sperm-associated, Catsperg2 gamma 2 0.001 4 Cd59b CD59b antigen 0.029 3 AK129341 cDNA sequence AK129341 0.005 4 Cdc40 cell division cycle 40 homolog (yeast) 0.005 3 Cenpb centromere protein B 0.027 4 Ccl17 chemokine (C-C motif) ligand 17 0.000 3 Cx3cl1 chemokine (C-X3-C motif) ligand 1 0.002 4 Chn1 chimerin (chimaerin) 1 0.034 3 Chi3l1 chitinase 3-like 1 0.047 3 Clic1 chloride intracellular channel 1 0.008 3 Cckbr cholecystokinin B receptor 0.046 3 Coq9 coenzyme Q9 homolog (yeast) 0.001 4 Ccdc72 coiled-coil domain containing 72 0.002 4

Table 4-9Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part II

Gene Symbol Gene Title adj.P.Val FC Cnksr2 connector enhancer of kinase suppressor of Ras 2 0.023 4 Cpne5 copine V 0.022 3 Cpne8 copine VIII 0.005 3 Cttnbp2 cortactin binding protein 2 0.047 4 Ctxn1 cortexin 1 0.033 3 Cox18 COX18 cytochrome c oxidase assembly homolog (S. cerevisiae) 0.000 3 Cuedc1 CUE domain containing 1 0.034 4 Celf2 CUGBP, Elav-like family member 2 0.008 4 31

Celf2 CUGBP, Elav-like family member 2 0.010 4 Arpp21 cyclic AMP-regulated phosphoprotein, 21 0.002 4 Ccna2 cyclin A2 0.000 4 Cdk17 /// Elk3 cyclin-dependent kinase 17 /// ELK3, member of ETS oncogene family 0.001 4 Cdkl5 cyclin-dependent kinase-like 5 0.005 4 Cyp4f15 cytochrome P450, family 4, subfamily f, polypeptide 15 0.002 4 Dkkl1 dickkopf-like 1 0.021 4 Dpp7 dipeptidylpeptidase 7 0.015 3 Dlx5 distal-less homeobox 5 0.029 4 D3Bwg0562e DNA segment, Chr 3, Brigham & Women's Genetics 0562 expressed 0.009 4 Dnajc24 DnaJ (Hsp40) homolog, subfamily C, member 24 0.002 3 Drd1a dopamine receptor D1A 0.008 4 Dusp1 dual specificity phosphatase 1 0.003 3 Dnahc6 dynein, axonemal, heavy chain 6 0.001 4 Dnahc8 dynein, axonemal, heavy chain 8 0.007 4 Eml5 echinoderm microtubule associated protein like 5 0.028 4 Eml5 echinoderm microtubule associated protein like 5 0.035 4 Erc1 ELKS/RAB6-interacting/CAST family member 1 0.011 4 Epha3 Eph receptor A3 0.022 3 Epha4 Eph receptor A4 0.027 3 Epha7 Eph receptor A7 0.017 4 Ephx4 epoxide hydrolase 4 0.043 4 Exoc5 exocyst complex component 5 0.014 4 Far2 fatty acyl CoA reductase 2 0.041 3 Fosb FBJ osteosarcoma oncogene B 0.038 3 Fcho1 FCH domain only 1 0.004 3 Flrt2 fibronectin leucine rich transmembrane protein 2 0.021 3 Fndc1 /// fibronectin type III domain containing 1 /// fibronectin type III domain-containing protein LOC100039091 1-like 0.000 3 Fmo2 flavin containing monooxygenase 2 0.032 3 Foxg1 /// 3110039M20Rik forkhead box G1 /// RIKEN cDNA 3110039M20 gene 0.043 3 Foxp1 forkhead box P1 0.027 3 Fras1 Fraser syndrome 1 homolog (human) 0.027 3 Fv1 Friend virus susceptibility 1 0.002 3 Gpr126 G protein-coupled receptor 126 0.009 3

Table 4-10Genes expressed higher in D2 over B6 mice with a FC of 3 or 4 Part III

Gene Symbol Gene Title adj.P.Val FC Gabra4 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 4 0.016 3 Gabrd gamma-aminobutyric acid (GABA) A receptor, subunit delta 0.026 3 Glo1 glyoxalase 1 0.000 3 Gnrh1 gonadotropin releasing hormone 1 0.013 3 32

Gzmk granzyme K 0.000 3 Grasp GRP1 (general receptor for phosphoinositides 1)-associated scaffold protein 0.026 3 Gng7 guanine nucleotide binding protein (G protein), gamma 7 0.017 3 Gmpr guanosine monophosphate reductase 0.028 3 Gucy1b3 guanylate cyclase 1, soluble, beta 3 0.004 3 H2-Ke6 H2-K region expressed gene 6 0.011 3 Hes7 hairy and enhancer of split 7 (Drosophila) 0.017 3 Homer1 homer homolog 1 (Drosophila) 0.002 3 Inhba inhibin beta-A 0.001 3 Itpr1 inositol 1,4,5-triphosphate receptor 1 0.004 3 Il17d interleukin 17D 0.013 3 Iqgap1 IQ motif containing GTPase activating protein 1 0.005 3 Isoc2b isochorismatase domain containing 2b 0.001 3 Kcnip2 Kv channel-interacting protein 2 0.017 3 Lmo7 LIM domain only 7 0.018 3 Mael maelstrom homolog (Drosophila) 0.000 3 Me3 malic enzyme 3, NADP(+)-dependent, mitochondrial 0.000 3 Man1a mannosidase 1, alpha 0.001 3 Mir128-1 microRNA 128-1 0.024 3 Mir9-2 microRNA 9-2 0.001 3 Mgll monoglyceride lipase 0.004 3 Mudeng MU-2/AP1M2 domain containing, death-inducing 0.019 3 Mctp1 multiple C2 domains, transmembrane 1 0.037 3 Nxph1 neurexophilin 1 0.007 3 Npas2 neuronal PAS domain protein 2 0.007 3 Npy neuropeptide Y 0.021 3 Olfr193 olfactory receptor 193 0.011 3 Pde10a phosphodiesterase 10A 0.016 3 Pih1d1 /// Aldh16a1 PIH1 domain containing 1 /// aldehyde dehydrogenase 16 family, member A1 0.000 3 Plk2 polo-like kinase 2 (Drosophila) 0.004 3 Kcnh3 potassium voltage-gated channel, subfamily H (eag-related), member 3 0.034 3 Kcnq5 potassium voltage-gated channel, subfamily Q, member 5 0.008 3 Ppp1r1b protein phosphatase 1, regulatory (inhibitor) subunit 1B 0.044 3 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 0.048 3 Pcdhb10 protocadherin beta 10 0.010 3 Pcdhb8 protocadherin beta 8 0.000 3 Rarres1 retinoic acid receptor responder (tazarotene induced) 1 0.000 3 Rarb retinoic acid receptor, beta 0.022 3 Sfrp1 secreted frizzled-related protein 1 0.006 3 Scg5 secretogranin V 0.000 3 Szt2 seizure threshold 2 0.000 3 Szt2 seizure threshold 2 0.004 3 33

Serpina1a /// serine (or cysteine) peptidase inhibitor, clade A, member 1A 0.032 3 Serpina3n serine (or cysteine) peptidase inhibitor, clade A, member 3N 0.000 3 Slitrk6 SLIT and NTRK-like family, member 6 0.026 3 Snrpe small nuclear ribonucleoprotein E 0.000 3 Slc22a2 solute carrier family 22 (organic cation transporter), member 2 0.007 3 Syngap1 synaptic Ras GTPase activating protein 1 homolog (rat) 0.038 3 Ugcg UDP-glucose ceramide glucosyltransferase 0.002 3 Zfp58 zinc finger protein 58 0.004 3 Zfp760 zinc finger protein 760 0.005 3

Table 4-11Genes expressed higher in D2 over B6 mice with a FC of 2 Part I

Gene Symbol Gene Title adj.P.Val FC Jhdm1d jumonji C domain-containing histone demethylase 1 homolog D (S. cerevisiae) 0.024 2 Kalrn kalirin, RhoGEF kinase 0.008 2 Kptn Kaptin 0.009 2 Kitl kit ligand 0.014 2 Leprotl1 leptin receptor overlapping transcript-like 1 0.002 2 Lrrc51 leucine rich repeat containing 51 0.001 2 Lrrc7 leucine rich repeat containing 7 0.017 2 Lcorl ligand dependent nuclear receptor corepressor-like 0.027 2 Lix1 limb expression 1 homolog (chicken) 0.022 2 Lpl lipoprotein lipase 0.013 2 Lsm5 LSM5 homolog, U6 small nuclear RNA associated (S. cerevisiae) 0.045 2 Mal2 mal, T-cell differentiation protein 2 0.016 2 Me1 malic enzyme 1, NADP(+)-dependent, cytosolic 0.020 2 Mfap3l microfibrillar-associated protein 3-like 0.015 2 Mir9-3 microRNA 9-3 0.003 2 34

Mgst3 microsomal glutathione S-transferase 3 0.014 2 Mrpl20 mitochondrial ribosomal protein L20 0.006 2 Ndfip2 Nedd4 family interacting protein 2 0.002 2 Nlk nemo like kinase 0.014 2 Ntn4 netrin 4 0.002 2 Neurl1B neuralized homolog 1B (Drosophila) 0.036 2 Neto1 neuropilin (NRP) and tolloid (TLL)-like 1 0.019 2 Nin Ninein 0.000 2 Necab1 N-terminal EF-hand calcium binding protein 1 0.001 2 Nudc nuclear distribution gene C homolog (Aspergillus) 0.041 2 Nr4a1 nuclear receptor subfamily 4, group A, member 1 0.039 2 Nup35 nucleoporin 35 0.014 2 Olfr1507 olfactory receptor 1507 0.019 2 Olfr176 /// Olfr177 olfactory receptor 176 /// olfactory receptor 177 0.027 2 Osbpl8 oxysterol binding protein-like 8 0.021 2 Parvg parvin, gamma 0.006 2 Pdzd2 PDZ domain containing 2 0.029 2 Ppil4 peptidylprolyl isomerase (cyclophilin)-like 4 0.017 2 Fars2 phenylalanine-tRNA synthetase 2 (mitochondrial) 0.002 2 Phactr1 phosphatase and actin regulator 1 0.010 2 Pten phosphatase and tensin homolog 0.049 2 Pigz phosphatidylinositol glycan anchor biosynthesis, class Z 0.001 2 Pitpnm2 phosphatidylinositol transfer protein, membrane-associated 2 0.036 2 Pde1b phosphodiesterase 1B, Ca2+-calmodulin dependent 0.017 2 Pde5a phosphodiesterase 5A, cGMP-specific 0.009 2 Sh3bp1 SH3-domain binding protein 1 0.001 2 Snhg11 small nucleolar RNA host gene 11 0.002 2 Slc24a4 solute carrier family 24 (sodium/potassium/calcium exchanger), member 4 0.030 2 solute carrier family 25 (mitochondrial carrier; adenine nucleotide Slc25a31 translocator), member 31 0.000 2 Spata2L spermatogenesis associated 2-like 0.035 2 Spred1 sprouty protein with EVH-1 domain 1, related sequence 0.037 2 Stard4 StAR-related lipid transfer (START) domain containing 4 0.016 2 Stard10 START domain containing 10 0.019 2 Senp3 SUMO/sentrin specific peptidase 3 0.001 2 Sycp1 synaptonemal complex protein 1 0.000 2 Synpr synaptoporin 0.046 2 Syt14 synaptotagmin XIV 0.016 2 Stxbp2 syntaxin binding protein 2 0.018 2 Tbc1d23 TBC1 domain family, member 23 0.031 2 Tctex1d2 Tctex1 domain containing 2 0.002 2 Tns1 tensin 1 0.025 2 Txndc9 thioredoxin domain containing 9 0.025 2 35

Thoc7 THO complex 7 homolog (Drosophila) 0.041 2 Tor3a torsin family 3, member A 0.003 2 Sp9 trans-acting transcription factor 9 0.043 2 Tmem132b transmembrane protein 132B 0.001 2 Tmem149 /// U2af1l4 transmembrane protein 149 /// U2 small nuclear RNA auxiliary factor 1-like 4 0.000 2 Tmem167 transmembrane protein 167 0.026 2 Trps1 trichorhinophalangeal syndrome I (human) 0.016 2 Trim9 tripartite motif-containing 9 0.004 2 Tmod4 tropomodulin 4 0.004 2 Tnfrsf19 tumor necrosis factor receptor superfamily, member 19 0.003 2 Tyro3 TYRO3 protein tyrosine kinase 3 0.001 2 B3galt2 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2 0.011 2 Ugdh UDP-glucose dehydrogenase 0.033 2 Uck2 uridine-cytidine kinase 2 0.010 2 Zfp385b zinc finger protein 385B 0.021 2 Zfp658 zinc finger protein 658 0.008 2 Zfp69 zinc finger protein 69 0.004 2 Zfp74 zinc finger protein 74 0.039 2 Zfp81 zinc finger protein 81 0.022 2 Zmynd17 zinc finger, MYND domain containing 17 0.028 2 Zswim6 zinc finger, SWIM domain containing 6 0.015 2

Table 4-12Genes expressed higher in D2 over B6 mice with a FC of 2 Part II

Gene Symbol Gene Title adj.P.Val FC Pion pigeon homolog (Drosophila) 0.001 2 Plekha5 pleckstrin homology domain containing, family A member 5 0.033 2 Kctd1 potassium channel tetramerisation domain containing 1 0.008 2 Kctd16 potassium channel tetramerisation domain containing 16 0.015 2 Kcnj10 potassium inwardly-rectifying channel, subfamily J, member 10 0.039 2 Kcnab1 potassium voltage-gated channel, shaker-related subfamily, beta member 1 0.006 2 Kcna4 potassium voltage-gated channel, shaker-related subfamily, member 4 0.034 2 Kcnh1 potassium voltage-gated channel, subfamily H (eag-related), member 1 0.018 2 Psme4 proteasome (prosome, macropain) activator subunit 4 0.038 2 Prmt8 protein arginine N-methyltransferase 8 0.037 2 Prkch protein kinase C, eta 0.048 2 Ppp1r12b protein phosphatase 1, regulatory (inhibitor) subunit 12B 0.001 2 Ppp4r2 protein phosphatase 4, regulatory subunit 2 0.048 2 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 0.027 2 36

Pcdha4-g protocadherin alpha 4-gamma 0.001 2 Pcdhb11 protocadherin beta 11 0.024 2 Pcdhb2 protocadherin beta 2 0.013 2 Pcdhb4 protocadherin beta 4 0.001 2 Pcdhb6 protocadherin beta 6 0.018 2 Prune2 prune homolog 2 (Drosophila) 0.004 2 Pdp1 pyruvate dehyrogenase phosphatase catalytic subunit 1 0.019 2 Rapgef5 Rap guanine nucleotide exchange factor (GEF) 5 0.002 2 Rapgefl1 Rap guanine nucleotide exchange factor (GEF)-like 1 0.012 2 Rasd2 RASD family, member 2 0.026 2 Rragd Ras-related GTP binding D 0.031 2 Rgs4 regulator of G-protein signaling 4 0.041 2 Rxfp1 relaxin/insulin-like family peptide receptor 1 0.005 2 Arhgap10 Rho GTPase activating protein 10 0.048 2 Arhgap33 Rho GTPase activating protein 33 0.043 2 Arhgap4 Rho GTPase activating protein 4 0.000 2 Rpl13 ribosomal protein L13 0.006 2 Rps18 ribosomal protein S18 0.020 2 Rps20 ribosomal protein S20 0.001 2 Rps6ka3 ribosomal protein S6 kinase polypeptide 3 0.044 2 Rps9 ribosomal protein S9 0.000 2 Rwdd3 RWD domain containing 3 0.002 2 Ryr1 ryanodine receptor 1, skeletal muscle 0.009 2 Slfn8 schlafen 8 0.001 2 Scrg1 scrapie responsive gene 1 0.002 2 Sec14l1 SEC14-like 1 (S. cerevisiae) 0.011 2 Szt2 seizure threshold 2 0.006 2 Szt2 seizure threshold 2 0.000 2

Table 4-13Genes expressed higher in D2 over B6 mice with a FC of 2 Part III

Gene Symbol Gene Title adj.P.Val FC Sh3bp1 SH3-domain binding protein 1 0.001 2 Snhg11 small nucleolar RNA host gene 11 0.002 2 Slc24a4 solute carrier family 24 (sodium/potassium/calcium exchanger), member 4 0.030 2 solute carrier family 25 (mitochondrial carrier; adenine nucleotide Slc25a31 translocator), member 31 0.000 2 Spata2L spermatogenesis associated 2-like 0.035 2 Spred1 sprouty protein with EVH-1 domain 1, related sequence 0.037 2 Stard4 StAR-related lipid transfer (START) domain containing 4 0.016 2 Stard10 START domain containing 10 0.019 2 Senp3 SUMO/sentrin specific peptidase 3 0.001 2 Sycp1 synaptonemal complex protein 1 0.000 2 37

Synpr synaptoporin 0.046 2 Syt14 synaptotagmin XIV 0.016 2 Stxbp2 syntaxin binding protein 2 0.018 2 Tbc1d23 TBC1 domain family, member 23 0.031 2 Tctex1d2 Tctex1 domain containing 2 0.002 2 Tns1 tensin 1 0.025 2 Txndc9 thioredoxin domain containing 9 0.025 2 Thoc7 THO complex 7 homolog (Drosophila) 0.041 2 Tor3a torsin family 3, member A 0.003 2 Sp9 trans-acting transcription factor 9 0.043 2 Tmem132b transmembrane protein 132B 0.001 2 Tmem149 /// U2af1l4 transmembrane protein 149 /// U2 small nuclear RNA auxiliary factor 1-like 4 0.000 2 Tmem167 transmembrane protein 167 0.026 2 Trps1 trichorhinophalangeal syndrome I (human) 0.016 2 Trim9 tripartite motif-containing 9 0.004 2 Tmod4 tropomodulin 4 0.004 2 Tnfrsf19 tumor necrosis factor receptor superfamily, member 19 0.003 2 Tyro3 TYRO3 protein tyrosine kinase 3 0.001 2 B3galt2 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2 0.011 2 Ugdh UDP-glucose dehydrogenase 0.033 2 Uck2 uridine-cytidine kinase 2 0.010 2 Zfp385b zinc finger protein 385B 0.021 2 Zfp658 zinc finger protein 658 0.008 2 Zfp69 zinc finger protein 69 0.004 2 Zfp74 zinc finger protein 74 0.039 2 Zfp81 zinc finger protein 81 0.022 2 Zmynd17 zinc finger, MYND domain containing 17 0.028 2 Zswim6 zinc finger, SWIM domain containing 6 0.015 2

4.2 Differential Expression as a Function of Exercise Treatment

One of the aims of the study was to investigate the genes within the hypothalamus that were differentially expressed as a result of exercise. While many genes showed differential 38 expression, once adjustments were made, all of the genes were found to not be significant with an

adjusted p value greater than 0.05.

One of the pathways elucidated and described earlier in the study was neurogenesis due

to exercise treatments. For a list of genes involved in neurogenesis, see Table 4-14.

Table 4-14 Genes Involved in Hypothalmic Neurogenesis

Results Gene Title P-value Adj P-value Fold Change B6TO>C Nlgn3 neuroligin 3 0.039 0.99 1.33 B6TO

Alkaline Phosphatase has been found to be involved in bone reformation in conjunction with the

Hypothalamic-Pituitary-Gonadal Axis previously described. For genes that interact with the HPG axis, see Table 4-15.

Table 4-15 Genes Involved in HPG Axis

Results Gene Title P-value Adj P-value Fold Change B6TO>C Alpl alkaline phosphatase, liver/bone/kidney 0.005 0.99 1.76

Finally, Insulin has been found to have a dual role in both Bone reformation and

Glucose Homeostasis along with a variety of genes. For a list of genes involved in the

Insulin-Bone Pathway, see Table 4-16.

Table 4-16 Genes involved in the Insulin Pathway

Results Gene Title P-value Adj P-value Fold Change B6TO>C Igf2 insulin-like growth factor 2 0.049 0.99 2.42 B6TR>C Igfbp6 insulin-like growth factor binding protein 6 0.015 0.99 2.29 B6TR>C KL klotho 0.015 0.99 4.48

Chapter 5

Discussion

The genetic factors contributing to skeletal quality has been a topic of study that has made great strides over many decades. One of the discoveries from the research is the fact that the expression of many genes plays a role in the structure and quality of the skeletal system.

The ability to undergo endurance activities depends on the relationship of many different systems within the body including cardiovascular, nervous and musculoskeletal systems to name a few. Animals exhibit variation in voluntary physical activity with a significant component of this variation being attributed to genetic factors. Within this study there was both voluntary activity, tower climbing, in which the mice could modulate how many times they climbed, as well as involuntary activity in treadmill running. Studies have shown that there are differing genetic contributions to involuntary and voluntary activities. For instance, studies have shown a positive correlation between body mass and voluntary wheel performance in a variety of inbred mouse strains.

5.1 Analysis of strain effect

As seen above, 497 genes were found to be differentially expressed between B6 and D2

mice, helping confirm the original hypothesis of strain differences having an effect on

hypothalamic gene expression.

Despite genetic similarities, inbred strains of mice all are unique when it comes to

various physiological variables. For instance, two potassium channel genes, Kcnj9 and Kcnj10 40 are candidates for the seizure susceptibility Quantitative Trait Loci (QTL) in Qrr1. Qrr1 is a regulatory region that plays a role in the expression of a group of genes and subsequent phenotypes. Kcnj9 has over a 2-fold higher expression in D2 mice as compared to B6 mice. This is in agreement with previous findings that indicate that D2 is the more seizure prone strain of the two. [29]

In terms of bone metabolism, genes that were expressed to a greater extent in D2 mice compared to B6 mice include GnRH1, which encodes for Gonadotropin-releasing hormone, or GnRH.

GnRH, as discussed above, plays a key role in the HPG axis, released from the hypothalamus, causing the upregulation of the release of FSH and LH from the pituitary gland which causes the release of sex hormones, such as estrogen, from the gonads [30]. Research has shown that D2 mice have a greater baseline amount of estrogen present within their blood as compared to B6 mice, agreeing with previous findings [53].Estrogen has already been shown to have a positive impact on bone, increasing BMD. Since it has already been shown that D2 mice have a greater

BMD compared to B6 mice, the results are in agreement with the previous findings [7].

Similarly, Fatty Acid Binding Protein 7, or fabp7, within the brain was upregulated in D2 mice as compared to B6 mice. Increased expression of fabp7 allows for more fatty acids to be transported into a cell. The now bound fatty acids make their way to storage in adipocytes [31].

Recent research has shown that short chain fatty acids, through a G protein coupled cascade, stimulate leptin production in adipocytes. As shown above, leptin has been shown to increase osteoblasteogenesis, increasing BMD in D2 mice [32]. In addition, D2 mice have been found to have elevated leptin levels as compared to B6 mice when fed diets with varying fat content [54]

This is in agreement with previous research that D2 mice have a greater BMD as compared to B6 mice.

In terms of B6 mice, Interleukin-33, or Il33, was upregulated in B6 mice compared to D2 mice. Increased expression of IL-33 has been shown to increase bone resorption in IL-33 41 deficient mice, decreasing the trabecular bone mass. Studies have shown that IL-33 increases the

expression of Tnfsf11, the gene encoding RankL, which has previously been shown to upregulate

osteoclast differentiation and lower trabecular bone mass [33]. Overall, previous research has

shown that D2 mice possess a greater hardness in trabecular bone as well as a greater hardness in

tibial hardness in cortical bone as compared to B6 mice [55].

Similarly, B6 mice were also found to have increased expression of osteoclast associated

receptor, or OSCAR. OSCAR has been found to be associated with RANKL-simulating

osteoclastogenesis, effectively decreasing bone mass in B6 mice as compared to D2 mice [34].

Both the greater expression of OSCAR and IL-33 are in agreement with previous research of B6

having a lower BMD as compared to D2 mice.

For further support of some of the genes differentially expressed in the hypothalamus

between these two strains, Tsolakidou et al’s study shows certain genes expressed within the

Paraventricular nucleus (PVN) of the hypothalamus, known as the stress control brain region,

supports many of the genes found to be differentially expressed within this study, as seen in Table

5-1. One of the differences between the author’s study is that gene extraction occurred for the

entire hypothalamus in the author’s study whereas in Tsolakidou et al’s study focused on gene

expression within a specific portion of the hypothalamus [35].

Table 5-1 Basal gene expression for the PVN in Tsolakidou et al. supports data from author’s study [35]

Fold Change Fold Change reported by in author's Result Gene Title Tsolakidou study D2 > B6 Baiap2 brain-specific angiogenesis inhibitor 1-associated protein 2 2.34 4 D2 > B6 Rsp6 ribosomal protein S6 1.52 2 D2 > B6 Pde1b phosphodiesterase 1B, Ca2+-calmodulin dependent 1.5 2 B6 > D2 Kif1c kinesin family member 1C 1.45 3 D2 > B6 Rgs4 regulator of G-protein signaling 4 1.61 2 D2 > B6 Prkcz protein kinase C, zeta 1.71 2

42

5.2 Analysis of Treatment Effect

Although no genes were found to be differentially expressed because of exercise treatment after adjustment for multiple comparisons, many genes of interest were identified at an unadjusted p < 0.05 that are supported in the literature.

Such genes include Insulin binding growth factor, which was upregulated in both B6

treadmill runners and B6 tower climbers compared to B6 controls. Research has shown a

complex interaction between exercise, serum insulin like growth factor-1 (IGF) levels and

neurogenesis. Within the brain there are areas that are both IGF dependent and IGF1 independent

when it comes to the brain’s response to exercise in the form of neurogenesis. The main areas of

the brain where IGF1 plays a prominent role is the hippocampus, where IGF1 influences neural

plasticity as well as neurogenesis [36]. Both liver and bone derived IGF-1 can stimulate

longitudinal bone growth. Though both are produced by different organs, liver and bone derived

IGF have the potential to replace each other due to their overlapping activation sites. IGF’s main

target is immature proliferating chondrocytes within the resting zone of the epiphyseal growth

plate within long bones. Low IGF levels have been shown to result in lower growth rates as well

as decreased bone density, indicating that normal IGF levels are required for normal bone growth.

[37].

In the last decade, a new hormone, klotho, has been discovered as an important factor in

calcium homeostasis. Klotho is expressed to a large degree within the mouse kidneys. It

hydrolyzes extracellular sugar residues on the Transient Potential ion channel TRPV5 which

activates the protein allowing for normal calcium resorption in the body leading to regular bone

thickness. Inactivation of klotho in mice leads to bone aberrations such as osteoporosis like 43 symptoms and hypercalcemia [39]. Other studies have shown that knocking out klotho in mice causes impairment in both osteoblastgenosis and osteoclastgenesis, the latter through the upregulation of osteoprotegerin, an osteoclastogenesis inhibitor [40]. Because exercise has been found to be beneficial for increasing BMD, the increased expression of klotho within the B6 treadmill group as compared to the B6 control group is in agreement with previous findings.

Alkaline phosphatase was also found to be differentially expressed in the B6 tower climbing group as compared to the B6 control group. Lowered levels of alkaline phosphatase have been associated with osteoporosis and decreased cortical and trabecular bone density, specifically the hypomineralization of growth plates and bones. Therefore, tissue non-specific alkaline phosphatase, or TNAP, has catalytic activity in promoting bone mineralization. TNAP accomplishes bone mineralization by hydrolyzing inorganic pyrophosphate, resulting in increased calcium deposition on bones [41]. Estrogen promotes alkaline phosphatase activity, further proving estrogen’s positive impact on bone growth and density [42].

As mentioned before, exercise promotes neurogenesis not just in the hypothalamus, but in all regions of the brain [5]. Several genes of note were differentially expressed in the exercise group as compared to the control group: neutoligin, neuroexophilin and protocadherin beta 5.

Neuroligin is a transmembrane molecule that binds neurexins which help create synaptic connections within the brain, specifically the hypothalamus. Neuroligins are thought to help form the initial connection at the synapse [43]. Neuroexophilin is a ligand of alpha neurexins that plays a role in efficient neurotransmitter release from the synapses of many regions of the brain, including the hypothalamus. They affect calcium channels, which play an important role in neurotransmitter release [44]. Protocadherin beta 5 is part of a superfamily of cadherin that is comprised of cell surface receptors that maintains specific connections between neurons, seen by how some of them are localized in the synapses [45]. All three of these genes helped in the 44 process of neurogenesis within the brain which is promoted through exercise and physical

activity.

5.3 Recommendations for Future Investigation

Because this study looked at mRNA expression within the hypothalamus, future work focused on the tissues related to the hormones regulated by the hypothalamus, such as adipose and uterine tissues, would further elucidate downstream pathways affected by gene expression

within the hypothalamus. Also, since adipose and uterine tissues factor into the bone

resorption/deposition pathways, studying these tissues would provide more insight into these

indirect pathways related to central nervous system control on bone.

Another area for future investigation could be focused on how age affects gene

expression within the hypothalamus. The results presented here are part of a larger study that

included both young adult and aged mice of the same inbred mouse strains. This provides the

opportunity to measure how exercise affects gene expression in both young and old mice and to

identify age dependent and independent gene expression within the brain itself.

Studies could also be done on different portions of the brain. Areas such as the

hippocampus have been the focus of research over the past decade and continued research on the

effects of exercise on gene expression in other regions of the brain would add to this line of

research.

Further areas of investigation include looking at specific nuclei or portions of the

hypothalamus. Additional insight could be gained through the investigation of gene expression

within certain regions of the hypothalamus, such as the paroventricular nuclei, and how exercise

affects gene expression within specific portions of the organ and the subsequent effects on bone

Appendix A

Treadmill Operator Protocol

Protocol used to monitor the mice during running on the treadmill and actual setting used

throughout duration of treatment (speed, incline, duration).

Technician/Treadmill Operator:

1) Fill out log-in sheet with animal ID, group, treadmill inclination, exercise intensity, and duration.

2) Verify the following treadmill settings: 0.76 mA (equivalent to 4 on the intensity dial) and a repetition rate of 2 pulses per second (equivalent to 5 on the repetition rate dial).

Acclimation:

3) Prior to each exercise session each mouse should be placed on the treadmill in its respective lane with the belt unmoving and shock grids off but with the belt motor on. The mice should be left undisturbed for 5 minutes.

Warm-up:

4) Turn on the shock grids and start the belt. The animals should be warmed up at the beginning of each session. The belt speed should start at 10 m/min and slowly be ramped up with an acceleration of 1 m/min2.

Exercise Training Regimen:

5) The timer should be started as soon as the belt speed reaches 10 m/min.

Treadmill Monitoring:

The red lamp next to the Repetition Rate knob indicates each time there is a shock pulse present at the shocker grid.6) The technician will observe the animals during the entire exercise period.

Record the following: a) Using a stop watch: record the time of each shock. b) Time between each consecutive shock. c) Number of times each animal is willing to receive 2 seconds or more of shocking rather than return to the treadmill (this is equivalent to 4 consecutive shocks). d) If the animal remains on the bar for a period longer than 5 seconds (this is equivalent to 10 consecutive shocks). 46 7) Once the animal has received 1 interval of 2 seconds of shock (equivalent to 1 interval of 4 consecutive shocks) the technician should gently nudge the mouse on its rump to encourage the mouse to run.

8) If at any time the animal becomes exhausted during the exercise session the shock grid must be deactivated for that lane. If the electric stimulus is removed the technician will note the time and whether the animal resumes running during the remainder of the session.

9) If the electric stimulus is removed, the technician should continue to nudge the mouse with their hand to encourage the mouse to run. The technician should stop nudging the animal after 5 attempts to encourage the animal to run.

The stimulus consists of 200 millisecond 0.74 mA pulses that will be delivered at a rate of 2 pulses per second. Two (200 millisecond) pulses are equivalent to 0.4 seconds of shock in a 1 second period. This is equivalent to 0.8 seconds of shock in a 2 second period and 2 seconds of shock in a 5 second period.

The criteria for discontinuing shock exposure are as follows:

A) If the animal spends more than 5 consecutive seconds on the shock grid without attempting to reengage the treadmill.

(10 consecutive shocks)

(equivalent to 2 seconds of shock in a 5 second consecutive period)

B) The third time a mouse is willing to sustain 2 seconds or more of shocking rather than return to the treadmill.

(3 X 4 consecutive shocks = 12 shocks)

(equivalent to 3 periods of 0.8 seconds of shock in a 2 second consecutive period)

(equivalent to 2.4 seconds of shock)

C) When the animal sustains a total of 20 shocks within any 5 minute period regardless of the spacing of the shocks during the 30 minute exercise period. (equivalent to 4 seconds of shock in a 5 minute period)

D) When the animal sustains a total of 50 shocks regardless of the spacing of the shocks during the 30 minute exercise period.

(equivalent to 10 seconds of shock)

10) Animals that repeatedly refuse to run after five attempts will be removed from the exercise intervention group. Records on each animal’s performance will be maintained and the number of shocks each animal receives during each session will be recorded by the observer. 47 Appendix B

Tower Design

Figure 5-1 Design of Tower for Exercise Intervention 48

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ACADEMIC VITA James Metkus [email protected] ______Education

B.S., Biology, 2013, The Pennsylvania State University, State College, Pennsylvania

Honors and Awards

• Dean’s List Pennsylvania State University Fall 2009, Spring 2010, Fall 2010, Spring 2011, Fall 2011, Spring 2012, Fall 2012

• Pennsylvania SpaceGrant Consortium Freshman Undergraduate Research Program Research Assistantship: awarded December 2009

• Academic Excellence Scholarship Schreyer Honors College Pennsylvania State University 2009-2013

• Edward C. Hammond Jr. Memorial Scholarship, Eberly College of Science, Pennsylvania State University Fall 2012

Association Memberships/Activities

• Atlas THON Benefitting the Penn State Dance Marathon • Presidential Leadership Academy • Emergency Room Volunteer at Mount Nittany Medical Center • Clinic Intern at University Health Services at the Pennsylvania State University

Research Experience

• Dr. Dena Lang, Genetic Determinants of Skeletal Strength and Architecture, Biomechanics Lab, Pennsylvania State University

o Perform mouse brain RNA extractions and analysis to determine the effects of various types of exercise on the mouse hypothalamus.

o Knowledge and experience using Materials Testing System to assess skeletal strength

o Knowledge and experience using a Micro CT scanner to assess skeletal architecture and density