Medicine, Faculty of
DSpace
UBCV
Baehring, Stella
Master of Science - MSc
University of British Columbia
Insulin resistance in the brain is associated with Alzheimer’s disease, the most common form of dementia with women being more affected than men. Diabetes is a metabolic disorder signified by dysfunctional insulin release and action, and it is a risk factor for Alzheimer’s Disease. Interestingly, administration of insulin to the brain is an effective experimental treatment to improve learning and memory impairments in Alzheimer’s Disease patients. This suggests an important role of insulin action local to the brain. Increasingly more research reports that insulin can be synthesized in the hippocampus, the brain region responsible for learning and memory, and one of the only areas in which new neurons can form throughout adulthood. However, not much is known about how brain-derived insulin effects adult neurogenesis and what factors modulate its expression. This study aimed to confirm insulin production in the hippocampus, assess its modulation by diet and exercise, and identify how brain-derived insulin loss affects adult neurogenesis in female and male mice. I hypothesized that insulin expression is most abundant in the hippocampus, that a high carbohydrate diet decreases insulin expression while exercise increases its expression, and that brain-derived insulin loss decreases adult neurogenesis. To examine these aims I utilized a genetically engineered mouse model with deletion of the Ins2 gene (expressed in the brain) while leaving the Ins1 gene (expressed only in the pancreas), and thus normal glucose homeostasis, intact. In line with my prediction, Ins2 mRNA was high in the hippocampus compared to other regions. Ins2 mRNA was expressed at higher levels in hippocampi of females than males. Contrary to my prediction, Ins2 expression was higher in females eating a high carbohydrate diet and exercise blunted this effect. Surprisingly, the deletion of Ins2 alleles led to increased cell proliferation and this effect was sexually dimorphic. Overall, these results suggest brain-derived insulin differs between sexes and influences adult neurogenesis in mice, highlighting the potential of brain-derived insulin for therapeutic research of Alzheimer’s Disease.
THE EFFECTS OF BRAIN-DERIVED INSULIN LOSS ON ADULT NEUROGENSIS IN ADGED MALE AND FEMALE Ins2 KNOCKOUT MICE by Stella Baehring B.A., The University of British Columbia, 2018 Cert. Ed., Academy of Social Pedagogy, 2012 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Cell and Developmental Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2022 © Stella Baehring, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: THE EFFECTS OF BRAIN-DERIVED INSULIN LOSS ON ADULT NEUROGENSIS IN ADGED MALE AND FEMALE Ins2 KNOCKOUT MICE submitted by Stella Baehring in partial fulfillment of the requirements for the degree of Master of Science in Cell and Developmental Biology Examining Committee: James D Johnson, PhD, Professor, Cellular and Physiological Sciences, UBC Supervisor Jason S Snyder, PhD, Associate Professor, Department of Psychology, UBC Supervisory Committee Member Shernaz X Bamji, PhD, Professor, Cellular Physiological Sciences, UBC Supervisory Committee Member Liisa AM Galea, PhD, Professor, Department of Psychology, UBC Additional Examiner Additional Supervisory Committee Members: Kiran K Soma, PhD, Professor, Department of Psychology, UBC Supervisory Committee Member iii Abstract Insulin resistance in the brain is associated with Alzheimer’s disease, the most common form of dementia with women being more affected than men. Diabetes is a metabolic disorder signified by dysfunctional insulin release and action, and it is a risk factor for Alzheimer’s Disease. Interestingly, administration of insulin to the brain is an effective experimental treatment to improve learning and memory impairments in Alzheimer’s Disease patients. This suggests an important role of insulin action local to the brain. Increasingly more research reports that insulin can be synthesized in the hippocampus, the brain region responsible for learning and memory, and one of the only areas in which new neurons can form throughout adulthood. However, not much is known about how brain-derived insulin effects adult neurogenesis and what factors modulate its expression. This study aimed to confirm insulin production in the hippocampus, assess its modulation by diet and exercise, and identify how brain-derived insulin loss affects adult neurogenesis in female and male mice. I hypothesized that insulin expression is most abundant in the hippocampus, that a high carbohydrate diet decreases insulin expression while exercise increases its expression, and that brain-derived insulin loss decreases adult neurogenesis. To examine these aims I utilized a genetically engineered mouse model with deletion of the Ins2 gene (expressed in the brain) while leaving the Ins1 gene (expressed only in the pancreas), and thus normal glucose homeostasis, intact. In line with my prediction, Ins2 mRNA was high in the hippocampus compared to other regions. Ins2 mRNA was expressed at higher levels in hippocampi of females than males. Contrary to my prediction, Ins2 expression was higher in females eating a high carbohydrate diet and exercise blunted this effect. Surprisingly, the deletion of Ins2 alleles led to increased cell proliferation and this effect was sexually iv dimorphic. Overall, these results suggest brain-derived insulin differs between sexes and influences adult neurogenesis in mice, highlighting the potential of brain-derived insulin for therapeutic research of Alzheimer’s Disease. v Lay Summary Alzheimer’s disease and diabetes are two chronic health conditions that are affecting more people as our population is aging. Both conditions share an underlying dysfunction of insulin, the main metabolic hormone. Clinical trials have shown that memory impairments can be improved when insulin is given directly to the brain. Previously, researchers discovered that insulin can be produced in the brain but how exactly it contributes to brain function and the development of new brain cells during adulthood is not clear. This study directly tested how brain-produced insulin can affect the generation of new neurons in both sexes. To do this, I used a specific mouse model that can delete the production of insulin in the brain. The results of this study will contribute to our understanding of brain-produced insulin and its potential for future therapeutic applications. vi Preface Chapter 2 is based on preliminary work conducted in UBC by Dr. Timothy O’Leary and Grace Li, and others in the lab, supervised by Dr. James D. Johnson. Diet and exercise experiments were done in collaboration with Dr. Krystal’s Laboratory at the BC Cancer Research Centre where Dr. Ingrid Elisia conducted the animal work and provided me with the isolated brain tissue for analysis. All qPCR experiments were done by Stella Baehring with support from undergraduate student Sophia Provenzano. All experiments on adult neurogenesis were designed, executed, and analyzed by Stella Baehring under the supervision of Dr. James D. Johnson. All projects were approved by the University of British Columbia’s Research Ethics Board and procedures were followed with strict compliance in accordance with the guidelines provided by the University of British Columbia Animal Care (A21-0135 and A14-075). vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 Insulin production and action on the brain ..................................................................... 1 1.1.1 Pancreas-derived insulin production ........................................................................... 1 1.1.2 Insulin transport to the brain ....................................................................................... 2 1.1.3 Insulin action and production within the brain ........................................................... 3 1.1.4 Brain-insulin action in disease .................................................................................... 4 1.2 Learning and memory ..................................................................................................... 6 1.2.1 The hippocampal formation ........................................................................................ 7 1.2.2 Dentate gyrus .............................................................................................................. 8 1.3 Adult hippocampal neurogenesis .................................................................................... 9 1.3.1 Modulating factors of adult neurogenesis ................................................................. 12 1.3.2 Links to insulin synthesis .......................................................................................... 13 viii 1.4 Preliminary data ............................................................................................................ 14 1.5 Study aims and hypothesis ............................................................................................ 18 Chapter 2: Materials and methods .............................................................................................20 2.1 Experimental animals and mouse model ...................................................................... 20 2.1.1 Overview of experimental mice ................................................................................ 20 2.1.2 Transgenic knockout of Ins2 and genotyping ........................................................... 20 2.2 Ins2 expression experiments ......................................................................................... 21 2.2.1 Tissue preparation ..................................................................................................... 21 2.2.2 Diet and exercise conditions ..................................................................................... 22 2.2.3 Analysis of Ins2 gene expression .............................................................................. 24 2.3 Effects of Ins2 gene knockout on adult neurogenesis ................................................... 24 2.3.1 Experimental design.................................................................................................. 24 2.3.2 Measuring neurogenesis with Click-iT chemistry and immunostaining .................. 25 2.3.3 Analysis..................................................................................................................... 26 Chapter 3: Results........................................................................................................................27 3.1 Ins2 mRNA levels across brain regions ........................................................................ 27 3.2 Ins2 mRNA Levels in exercise and diet conditions ...................................................... 28 3.3 EdU and DCX staining in Ins2 knockout mice ............................................................. 29 3.3.1 EdU+ cells are differentially expressed within sexes in Ins2 genotypes .................. 32 3.3.2 No difference in DCX+ cells between sexes and Ins2 genotypes ............................ 33 Chapter 4: Conclusion .................................................................................................................34 4.1 Brain-derived insulin characterization .......................................................................... 34 4.2 Diet and exercise effect Ins2 expression ....................................................................... 35 ix 4.3 Considerations of sex differences in data interpretation ............................................... 37 4.4 Brain-derived Insulin loss affects cell proliferation differently in females and males . 38 4.5 Brain-insulin loss does not affect the number of immature neurons ............................ 41 4.6 Strengths and limitations............................................................................................... 42 4.7 Overall conclusions ....................................................................................................... 43 Bibliography .................................................................................................................................44 x List of Tables Table 1. Primer sequences for Ins2 knockout mice. ..................................................................... 21 xi List of Figures Figure 1: The dentate gyrus of the hippocampus and stages of adult neurogenesis ..................... 11 Figure 2: Ins2 knockout mice have the same peripheral metabolic profile as Ins2 controls. ....... 15 Figure 3. Assessment of anxiety-like behaviour in male and female Ins2-/- mice. ....................... 16 Figure 4. Female Ins2 knockouts show impaired spatial memory and downregulation of Cyclin D1. ................................................................................................................................................. 17 Figure 5. Diet and exercise conditions for measuring Ins2 expression.. ...................................... 23 Figure 6. Schematic of EdU Click-iT method for assessing cell proliferation. ............................ 25 Figure 7. Increased levels of Ins2 mRNA in the hippocampus were sexually dimorphic. .......... 27 Figure 8. Ins2 mRNA is significantly higher in mice that ate a High Carb Western (HC-Western) diet and did not exercise.. ............................................................................................................. 28 Figure 9. Staining controls for cellular proliferation (EdU) and immature neurons (DCX). ....... 29 Figure 10. Staining of EdU and DCX in Ins2 genotypes of male and female mice. .................... 30 Figure 11. Magnification of EdU+ and DCX+ cells. .................................................................... 31 Figure 12. Cell proliferation in female and male mice responded differently to Ins2 loss........... 32 Figure 13. Immature neuron numbers are not affected by Ins2 loss in male and female mice.. .. 33 xii List of Abbreviations BBB Blood brain barrier BrdU 5-bromo-2’deoxyuridine Ccnd1 Cyclin D1 CNS Central nervous system DCX Doublecortin EdU 5-ethynyl-2’-deoxyridine GCL Granule cell layer GLUT1 Glucose transporter 1 GLUT3 Glucose transporter 3 GLUT4 Glucose transporter 4 IGF1 Insulin-like growth factor 1 IGF2 Insulin-like growth factor 2 Ins1 Insulin 1 Ins2 Insulin 2 MCL Molecular cell layer NeuN Neuronal nuclei NPC Neuronal progenitor cell PDI Pancreas-derived insulin PI3K Posphoinositide-3-kinase PML Polymorphic layer xiii Acknowledgements First and foremost, I would like to thank my supervisor Jim for giving me the opportunity to reach beyond my comfort zone, for his patience, optimism, and gentle guidance. I am so fortunate that I was able to explore this scientific endeavor with resources beyond financial support. I am truly appreciative of the mentorship Jim has offered me. Next, I would like to thank my committee members Shernaz, Jason, and Kiran for their expertise, patience, and support. Thanks to Cal and Cyndi who have provided me so much structural support during my studies. Your kindness is immeasurable and was always there when I needed it. The biggest thanks to Dr. Tim O’Leary who has not only set the stage for my project but was always there to support me with this wide range of knowledge in theory and experimental design. I am truly grateful for all the scientific exchanges we have had, especially during the Covid pandemic where having casual discussion with fellow scientist was a rare occasion. Leanne, without your continuous encouragement, support, and managerial skills I would not know how I would have gotten through the most challenging times. Sophia, my lovely student, thank you so much for standing with me and helping me with the experiments. I know you will accomplish great things going forward. I also want to thank all my lab members who have provided me with the environment to grow and be surrounded by much brilliance. Papa, Michelle, Vance, Christin, Maria, Anna, Fabienne, and Theresa – my German family. Without your love none of this would have been possible. Thank you for lifting me up when I could not do it for myself. xiv Dedication To my mom – the bravest woman I have ever known. The reason I have dared to step into scientific discovery. The reason I found back to my heart. Thank you for gifting me with this beautiful life. If it wasn’t for your courage, unbreakable spirit, and incredible strengths, I would not be here today. Always loved and never forgotten. From here to eternity.1 Chapter 1: Introduction 1.1 Insulin production and action on the brain 1.1.1 Pancreas-derived insulin production Insulin is a 51-amino-acid long peptide hormone with its key roles in the control of glucose homeostasis, metabolism, and cell growth (Weiss et al., 2000). Since the discovery of insulin by Banting and Best (Banting & Best, 1922) the pancreas has been recognized as the central organ for insulin production and therapeutic target for diseases of insulin dysfunction (Busnardo et al., 1983; Lewis & Brubaker, 2021). Elevated levels of blood glucose stimulate insulin release from pancreatic β-cells into the blood stream. Once insulin reaches target cells, such as those in muscle, liver, and adipose tissue, it binds to the insulin receptor and signals the uptake of glucose into the cell resulting in returning blood glucose to homeostatic levels (Kahn & White, 1988). In peripheral organs insulin stimulates the translocation of glucose transporter 4 (GLUT4), a glucose transporter in insulin sensitive tissue (Shepherd & Kahn, 1999). In contrast, the brain’s glucose uptake relies on glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3) which are inulin-independent (Mueckler, 1994). Despite not playing a major, direct role in glucose uptake, insulin receptors are abundant in the central nervous system (CNS) with highest expression in olfactory bulbs, cerebral cortex, hippocampus, hypothalamus, choroid plexus, and cerebellum (Mazucanti et al., 2019; Wozniak et al., 1993), pointing to a more complex role of insulin action on the brain. 2 1.1.2 Insulin transport to the brain Although there is clear evidence for insulin action on the brain due to insulin receptor abundance, how circulating insulin reaches the brain remains to be fully understood. The most studied pathway of insulin entry into the brain is via the blood brain barrier (BBB) transport. The BBB is comprised of a highly specialized endothelial cell layer separating the blood and brain interstitial fluid (Serlin et al., 2015). Together with pericytes, immune cells, basement membrane, and astrocytes, endothelial cells form the neurovascular unit which regulates the transport and function of the BBB. The brain endothelial cells are the cellular units dominantly responsible for the restrictive nature of the of BBB by preventing solute movement between blood and brain interstitial fluid due to tight junctions (Virgintino et al., 2004). Interestingly, early studies showed that insulin binds to the endothelium with high specificity (van Houten & Posner, 1979). Later studies discovered insulin receptors as potential receptor-mediated transporters by endothelial cell-mediated internalization of insulin to cross the BBB (King & Johnson, 1985). One study showed that IR blockers inhibit insulin uptake by brain tissue and later studies, investigating potential mechanisms, reported that insulin receptors, not insulin-like growth factor 1 (IGF-1) receptors, initiated brain endothelial cell insulin uptake requiring lipid raft-mediated endocytosis. It did not, however, require posphoinositide-3-kinase or mitogen-activated protein kinases activation, commonly known in downstream insulin signaling. Thus, IRs of the brain endothelial cells show to be critical for insulin transport from the periphery to the brain (Gray et al., 2014; Meijer et al., 2016). A recent historical review of insulin transport on the brain reported that studies examining regional differences in insulin BBB binding found high intensity binding activity in the neocortex, hippocampus, and hypothalamus whereas studies investigating insulin transport found 3 low activity in midbrain regions but high activity in peripheral brain regions such as olfactory bulbs, medulla, and hypothalamus (Rhea & Banks, 2021). In addition, most studies investigating insulin action on the brain use direct insulin injections or nasal administration of insulin allowing to examine local effects of the CNS bypassing insulin action from the circulation (Havrankova et al., 1981; Lv et al., 2020; Stockhorst et al., 2004). This highlights that insulin has significant effects on the brain independent of circulatory transport. Together, these observations create new questions leading to a more recently discovered and debatable topic: Insulin production by the brain. 1.1.3 Insulin action and production within the brain Insulin-like peptides, which include insulin, IGF1 and IGF2, serve as important growth factors and neuromodulators for normal brain development by affecting cell proliferation, survival, differentiation, and refinement of neural circuitries (Fernandez & Torres-Alemán, 2012). Brain insulin signalling plays a key role in metabolism and regulation of mood, behavior, and cognition (Beddows & Dodd, 2021; Soto et al., 2019). Early studies of insulin action on the brain employed direct brain administration of insulin, either via injection or nasal spray (Gray & Barrett, 2018). Most of these studies developed their methods under the assumption that insulin in the brain is of pancreatic origin. Interestingly, with increasing reports of insulin being produced within the brain itself, such studies received new relevance because direct injection bypasses the insulin transport and is thus an ideal proxy for studying insulin action independent of circulatory insulin (Lv et al., 2020). Although still controversial, evidence to support brain-derived insulin synthesis is mounting (Csajbok & Tamas, 2016; Devaskar et al., 1994; Kojima et al., 2004; Young, 1986). Insulin 4 expression has been measured in several brain regions, including cerebral cortex, choroid plexus, cerebellum, and the hippocampus (Mazucanti et al., 2019; Molnar et al., 2014; Nemoto et al., 2014). Insulin synthesis has been reported in neuronal progenitors of the hippocampus and olfactory bulbs (Kuwabara et al., 2011; Nemoto et al., 2014), and shows to be secreted by neurons only, not glial cells (Devaskar et al., 1994). Collectively, the evidence suggests that insulin is synthesized in the brain and that its production occurs in regions highly relevant for learning and memory. Due to methodological limitations in earlier years, it was not clear whether insulin produced in the brain makes any meaningful contribution to brain function as it is difficult to decipher whether the actual insulin protein in the brain stems from the CNS directly or was transported from the pancreas. Advancements in genetic engineering of mouse models, however, were able to establish direct evidence for brain-derived insulin (Mehran et al., 2012). The advantage of such models lies within the unique genetic makeup of mice which carry the Insulin 2 (Ins2) gene (human homolog) and an additional, pancreas-specific insulin coding gene Insulin 1 (Ins1) (Duvillie et al., 1997). Notably, Ins1 does not upregulate its gene expression in the brain to compensate for Ins2 loss (Mehran et al., 2012). Thus, exploiting such a dual gene system by selectively silencing insulin coding genes in the brain can have great potential to understanding the different roles of brain insulin in health and disease. 1.1.4 Brain-insulin action in disease Studies in humans and animals show that insulin plays a multifactorial role in brain function such as enhancing synaptic plasticity, dendritic spine formation, and increasing neurotransmitter turnover (Forner et al., 2017; Kellar & Craft, 2020). Insulin also influences the 5 clearance of the amyloid β peptide and phosphorylation of tau, which are hallmarks of Alzheimer’s Disease (Laws et al., 2017). Alzheimer’s Disease is the most prevalent form of dementia, more prevalent in women, and is on the rise due to an increasing aging population (Mielke et al., 2014). Thus, much research is dedicated to the treatment of Alzheimer’s Disease and insulin is gaining increasing attention in this field (Hallschmid, 2021; Hölscher, 2011; Suzanne, 2009). Not only do people with Alzheimer’s Disease often present with hyperglycemia and insulin dysfunction in peripheral organs, but they may also have insulin resistance in the brain (De Felice et al., 2014). Insulin applied to postmortem brain tissue activates insulin signalling pathways and patients with Alzheimer’s Disease showed a significant reduction in this effect within the cerebral cortex and hippocampus (Talbot et al., 2012). Insulin insensitivity in the brain is associated with reduced insulin receptor levels, binding affinity, and disrupts insulin signalling (Ma et al., 2015). Because diabetes, a chronic health condition in which patients are insulin resistant and hyperglycemic, is a strong risk factor for dementia (Hölscher, 2011), Alzheimer’s Disease is increasingly referred to as ‘Type 3 diabetes’ (De la Monte & Wands, 2008). Interestingly, people with type 2 diabetes (T2D) can present with cognitive impairment, specifically in learning and memory, similarly to early onset Alzheimer’s Disease (Ryan & Geckle, 2000). Insulin is used as therapeutic treatment in clinical trials via intranasal administration to improve cognitive function in Alzheimer’s Disease patients but also in obesity and T2D, highlighting the importance of local insulin action and its relevance to metabolic disorders (Freiherr et al., 2013; Zhang et al., 2015). Lifestyle interventions that increase insulin sensitivity, such as diet and exercise are also being used as preventative and therapeutic approaches for Alzheimer’s Disease (Kellar & Craft, 2020). Interestingly, the same lifestyle interventions are also known to improve learning and memory, the cognitive functions 6 to be first affected by Alzheimer’s Disease (Braak & Braak, 1995; Ko & Chye, 2020). A study examining brain insulin signalling in male mice fed a high fat diet revealed that a high fat diet induces brain insulin resistance and biochemical changes linked to increased amyloid beta deposition and decreased synaptic plasticity (Kothari et al., 2017). Another study on male rats found that exercise rescued the high fat diet-induced changes in brain insulin signalling (Jeong et al., 2018). Unfortunately, none of these studies included females. Overall, brain insulin dysfunction is indicated in both Alzheimer’s Disease and metabolic disorders and can be influenced by diet and exercise. However, not much is known about the brain insulin in females. 1.2 Learning and memory Animals, including humans, continuously respond to a changing environment-behavioural adaptation. This adaptation is rooted in the ability to learn from experience which requires remembering, encoding, and retrieving information of past events (Terry, 2017). This process is crucial for survival and thus, learning and memory evolved early in evolution and molecular mechanisms mediating these functions are evolutionarily conserved (Perry et al., 2013). Long-term memory is classified as explicit (declarative) and implicit (non-declarative) with further subcategorizations. Explicit memory encompasses a hippocampal-based memory system for events (episodic) and facts (semantic) requiring conscious participation for recall whereas implicit memory is largely under unconscious control e.g. reflexes (Eric et al., 2014). Interestingly, memory impairment in Alzheimer’s Disease and other neurodegenerative diseases are related to explicit memory, specifically explicit spatial memory, whereas unconscious (implicit) forms of memory remain relatively unaffected (Kessels et al., 2005). 7 Electrophysiological recording studies of hippocampal activity have revealed that one of the most dominate features of hippocampal neurons is their spatially specific firing (Moser et al., 2008; O'Keefe & Conway, 1978). Thus, understanding the structure, functions, and dynamics of hippocampal-dependent memory have been of large interest in biomedical research of neurodegenerative and related diseases. 1.2.1 The hippocampal formation The hippocampus is most famously known for its role in learning and memory (Milner et al., 1968) and is directly related to other important cognitive functions, such as decision making, sensory processing, and emotionality (LeBlanc et al., 2015; Tyng et al., 2017). The hippocampal formation is a c-shaped structure located in the medial temporal lobe and can be subdivided in separate regions (Figure 1A): dentate gyrus, subiculum, entorhinal cortex and the hippocampus proper (Schultz & Engelhardt, 2014). The hippocampus has been studied in the context of Alzheimer’s Disease, mood disorders. It is also receiving increasing attention in metabolic research due to its high sensitivity to environmental factors such as diet and exercise (Calvo‐Ochoa & Arias, 2015; Femenía et al., 2012; Jang et al., 2019; Laakso et al., 2000). An underlying feature of memory formation and hallmark trait of the hippocampus is its unique plastic ability (Kim et al., 2007). The synapses and dendrites of the hippocampus experience continues rearrangement, and unlike most areas of the brain, new neurons can be birthed throughout adulthood in this structure (Leuner & Gould, 2010). Hippocampal-dependent memory for space has shown to be connected to the dorsal region of the hippocampus, whereas ventral regions are more involved in emotionality and anxiety-like behaviours (Fanselow & Dong, 2010). 8 1.2.2 Dentate gyrus The dentate gyrus is a subregion of the hippocampus and is most recognized by its “V” or “U” shape appearance (Figure 1B). The dentate gyrus can be divided into three layers; the molecular cell layer (MCL) which comprises the outmost layer, the granule cell layer (GCL) as mid zone, and the polymorphic layer (PML) as innermost layer (Amaral et al., 2007). The MCL is largely cell body-free and occupies the dendrites of dentate granule cells, fibres from the entorhinal cortex, and a small number of interneurons and fibres that terminate from extrinsic inputs of other regions. The GCL is densely packed with granule cells and contains few other neurons at the boundary of the GCL and PML, like the dentate pyramidal basket cell. The PML is the region enclosed by the GCL and hosts a variety of different cell types with mossy fibres being the most abundant. The dentate gyrus plays a critical role in learning and memory as it is the first region to integrate sensory information to create spatial representations and memory (Kesner, 2013). It receives input from the entorhinal cortex and sends information to CA3 and CA1 regions of the hippocampus which are critical for memory and learning (Babcock et al., 2021). The dentate gyrus is functionally relevant to multiple memory processes such as spatial and context pattern separation, encoding processes, remote memory associated with neurogenesis, and odor separation (Kesner, 2007; Rolls, 2013; Sahay et al., 2011). The dentate gyrus can be functionally further divided into dorsal and ventral regions. Lesion studies of the dentate gyrus have shown that the complete dentate gyrus and dorsal dentate gyrus lesions result in spatial memory impairment, measured by the Morris Water Maze, whereas ventral dentate gyrus lesions did not show the same results (Jeltsch et al., 2001; Nanry et al., 1989). It is generally accepted that the 9 dorsal region of the dentate gyrus is related to spatial memory and ventral regions to odor memory and anxiety-like behaviors (Kesner, 2018; Weeden et al., 2015). Interestingly, the dentate gyrus is one of two regions during adulthood that retains the ability to generate new neurons and ablation experiments of neurogenesis in the dentate gyrus resulted in impaired spatial learning (Dupret et al., 2008). 1.3 Adult hippocampal neurogenesis New neurons form in the dentate gyrus of the hippocampus throughout life and play an important role in hippocampal function (Snyder & Drew, 2020). The formation of new neurons is a process that happens in three stages (proliferation, differentiation, and survival) and begins with neural progenitor cells (NPCs) in the subgranular zone of the dentate gyrus which provides a unique microenvironment (Figure 1B) (Cole et al., 2020; Morrens et al., 2012). At the proliferation stage two types of progenitors begin to develop. Type 1 progenitor cells or NPCs are slowly dividing and give rise to proliferating intermediate type 2a and type 2b progenitors under the influence of distinct transcription factors like Sox2 and NeuroD1, respectively (Steiner et al., 2006). Proliferation and fate specification are both under the influence of the canonical Wnt signaling pathway. Cell division during this stage is commonly determined by endogenous mitotic markers like Ki67, or incorporated markers such as 5-bromo-2’deoxyuridine (BrdU) and 5-ethynyl-2’-deoxyridine (EdU) (exogenous and integrated during S-phase) (Kee et al., 2002). After 2-3 days, type 2 cells begin differentiation and can give rise to neuroblasts (type 3) committing to neuronal fate and expressing endogenous immature marker doublecortin (DCX) (Brown et al., 2003). After about 3 weeks of differentiation maturing granule cells enter the 10 survival stage, begin morphogenesis, and develop into mature granule cells that are functionally integrated into the circuitry. Adult-born neurons present with unique properties suggesting that they have the largest impact on behavior during immature critical periods (Aimone et al., 2009; Kim et al., 2012; Snyder & Cameron, 2012). During such periods they recruit GABAergic interneurons at higher rates and undergo experience-dependent survival and innervation (Alvarez et al., 2016; Tashiro et al., 2007). Immature adult-born granule cells can be recruited by cortical inputs due to their greater excitability at this stage (Doetsch & Hen, 2005). Interestingly, studies that examined adult neurogenesis in Alzheimer’s Disease patients suggest the number of newly born neurons, specifically neuroblasts and immature neurons are reduced (Dard et al., 2019). Multiple studies using mouse models of Alzheimer’s Disease observed a reduction of proliferation and differentiation in adult neurogenesis (Hamilton et al., 2010; Verret et al., 2007; Zeng, 2016). Similarly, neurogenesis is reduced in diabetic mouse models (Ho et al., 2013; Zhang et al., 2008). 11 Figure 1: The dentate gyrus of the hippocampus and stages of adult neurogenesis Adult neurogenesis occurs in the dentate gyrus of the hippocampus. Stage 1 (Proliferation) creates type2a and type2 progenitor cells that can be labeled with mitotic marker EdU. Stage 2 (Differentiation) occurs after about 3 days and lasts up to 21 days. Neurons in this stage can be identified with cytoskeletal marker DCX. Neurons reach full maturity (Stage 3) after around 6 weeks and can be identified by staining for the neuronal protein NeuN. The experiment in this study used neurogenesis marker EdU and DCX. Schematic was adapted from “Stimulation of Synaptogenesis in Hippocampal Neurons”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates 12 1.3.1 Modulating factors of adult neurogenesis Given the highly plastic nature of the hippocampus, it is not surprising that adult neurogenesis and its behavioral effects can be heavily influenced by physiological (internal) and environmental (external) factors (Shohayeb et al., 2018). Internal factors like age and sex have shown to influence neuronal generation, maturation, and behavioral outcomes. As the age of an animal increases the proliferation and survival of adult-born neurons decreases (Kempermann, 2015). The decrease of hippocampal neurogenesis with aging is due to declining neural progenitor pool (Jessberger & Gage, 2008; Spalding et al., 2013; Villeda et al., 2011). In line with these observations of reduced neurogenesis, aged mice show impaired spatial memory and learning compared to young mice and decreased neurogenesis in adult rodents enunciates this effect (Kempermann, 2011; McDonald & Wojtowicz, 2005). A recent report summarized the use of males and females in studies on adult neurogenesis over the past 20 years and found that sex is often not reported or analyzed (Knudson et al., 2022). They reported that functional studies (manipulation of neurogenesis and measurement of behavioral outcomes) are mainly done in male animals. The studies that directly test male and female morphology and function of adult neurogenesis highlight the importance of needing to include both sexes (Yagi & Galea, 2019). A study using the Morris Water Maze, a hippocampal-dependent navigation task, shows that under regular conditions, males were faster in finding the hidden platform compared to females and showed greater cell survival in the dentate gyrus (Chow et al., 2013). Another study showed that under stress (cold water) males that lack neurogenesis were slower to escape the maze whereas female neurogenesis-deficient rats were faster (O’Leary et al., 2022). On a cellular level, evidence suggests significant differences in maturation between males and females (Yagi et al., 2020). Here, male adult-born dentate granule 13 cells reached maturity faster than females. Proliferation and density of new neurons is higher in males but declines after about 3 weeks whereas females show a more linear decline. Overall, there are few studies on adult neurogenesis that examine sex. The studies that do report significant differences between sexes that have yet to be fully understood. Apart from internal factors such as age and sex, adult neurogenesis is also well known to be regulated by environmental factors, such as exercise and diet (van Praag et al., 2005). Exercise increases neurogenesis in adult rodents and leads to improved memory and anti-depressant effects (Stangl & Thuret, 2009). In contrast, high fat diets reduce adult neurogenesis and lead to cognitive impairment (Park et al., 2010). Interestingly, high fat diet-induced impairment in spatial learning and reduction in neurogenesis can be alleviated by long-term exercises (Klein et al., 2016). Rodents with omega-3 fatty acid deficiency presented with impaired spatial memory and the effect could be rescued by dietary replenishment (Fedorova, 2006). Collectively, modulation of hippocampal neurogenesis takes place in context of internal (age and sex) and external (diet and exercise) factors. 1.3.2 Links to insulin synthesis Insulin synthesis and neuronal proliferation share common transcription factors and signaling pathways. The transcription factor Neurod1 is essential for neural differentiation and survival (Liu et al., 2000; Miyata, 1999). Conditional knockouts of the Neurod1 gene cause the complete absence of dentate gyrus and cerebellum whereas overexpression of Neurod1 causes premature neuronal maturation (Roybon et al., 2009). Neurod1 is a downstream target of the canonical Wnt signaling pathway, the same pathway that regulates the expression of insulin in pancreatic beta-cells (Papadopoulou & Edlund, 2005; Sander & German, 1997). Interestingly, a 14 study investigating brain-derived insulin found that the initiation of insulin production in the hypothalamus by Wnt signaling depends on Neurod1 (Lee et al., 2016). Studies on neurogenesis showed that activation of insulin in the neurogenic niche stimulates neural stem cell survival, proliferation, and differentiation (Åberg et al., 2003; Brooker et al., 2000). Overall, brain insulin and neurogenesis are closely linked. 1.4 Preliminary data Previous experiments in the Johnson Laboratory examined the effects of Ins2 loss in male and female mice on learning, memory, and anxiety-like behavior. These data shows that Ins2 knockout does not affect glucose tolerance or insulin sensitivity (Figure 2), likely due to compensation from the remaining Ins1 gene alleles (Leroux et al., 2001). Female mice had a small effect of Ins2 loss on the open-field test, but not other anxiety-like behavior tasks (Figure 3). Notably, female but not male, Ins2 knockout mice have impaired spatial memory and learning (Figure 3A). Bulk RNA sequencing analysis of female hippocampi revealed a downregulation of the Cyclin D1 (Ccnd1) gene in Ins2-/- mice (Figure 3B). Together with Ccnd2 and Ccnd3, Ccnd1 influences the transition between G1 and S mitotic phase and is thus a key player in cell cycle progression, and consequently proliferation (Herrup & Yang, 2007). These results could implicate a connection between brain-derived insulin and adult neurogenesis, and they inspired the aims of this study. 15 Figure 2: Ins2 knockout mice have the same peripheral metabolic profile as Ins2 controls. (A,B) There are no differences in body weight between Ins2 controls and knockouts in males and females (C,D) 16-18 months old male Ins2-/- had higher fasting blood glucose (FBG) levels. All other ages showed no difference and there was no difference between knockout and wildtype females. (C-F) Ins2-/- and Ins2+/+ mice showed no differences in glucose tolerance tests (GTT) or insulin tolerance tests (ITT). 16 Figure 3. Assessment of anxiety-like behaviour in male and female Ins2-/- mice. (A,B) Female but not male Ins2-/- mice completed fewer entries into the center of the OF. (C,D) Ins2-/- and Ins2+/+ mice did not spent significantly less time in the open arms but female Ins-/- mice show a trend for reduced time spent in open arms. (E,F) Ins2-/- and Ins2+/+ mice did not differ in their time spend in the light zone. 17 Figure 4. Female Ins2 knockouts show impaired spatial memory and downregulation of Cyclin D1. (A) Ins2-/- female mice showed impaired spatial memory assessed by the Morris Water Maze (MWM). (B) Bulk RNA sequencing analysis of female hippocampi confirmed the knockout of the Ins2 gene and revealed significant downregulation of cell cycle gene Ccnd1. 18 1.5 Study aims and hypothesis Insulin synthesis in the brain is a growing area of research and although gene expression has now been repeatedly observed in the brain, and specifically the hippocampus, whether insulin expression differs between adult males and females is unknown. To fill this knowledge gap, we aimed to measure brain-derived insulin expression across brain regions and compared hippocampal gene expression between sexes. Diet and exercise are potent modulators of pathological markers in Alzheimer’s Disease and metabolic disorder, and evidence shows they alter insulin signalling in male rodents. Not much is known if the same is true for females and whether diet and exercise influence brain insulin production itself. Thus, this study examines if brain insulin production is affected by diet and exercise in female mice. Insulin action in the brain plays a crucial role in cognitive function, neural growth, and brain health. Insulin impairment and cognitive decline are features of both diabetes and Alzheimer’s Disease. Insulin and insulin receptors are highly expressed in the hippocampus, the main region responsible for learning and memory, and hippocampal-dependent learning seems to be influenced by the loss of brain-derived insulin. In addition, adult neurogenesis is important for spatial learning and influenced by sex, age, exercise, and diet. Our lab’s previous data show that Ins2 loss impairs spatial learning in females who present with reduced gene expression of a cell division critical gene. Given these connections between insulin and adult hippocampal neurogenesis, this study’s overall aim is to examine whether the lack of Ins2 alleles influences adult neurogenesis. I hypothesized that 1) Ins2 mRNA is most highly expressed in the hippocampus, 2) Ins2 mRNA is lower in females than males, 3) Ins2 mRNA levels are lower in adult female mice fed a 19 western diet and higher in animals that exercised, 4) the loss of the Ins2 gene results in reduced neurogenesis in females but not in males. 20 Chapter 2: Materials and methods 2.1 Experimental animals and mouse model 2.1.1 Overview of experimental mice All mice used for the following experiments were group housed in the University of British Columbia’s Animal Care Facility and all procedures were conducted in line with the animal care protocol approved by the University of British Columbia Animal Care Committee. Unless otherwise specified, below the animals were fed a regular chow diet at libitum and housed on a 12-hour dark/light cycle. This study aimed to examine 1) mRNA levels of Ins2 across brain regions, 2) mRNA levels in diet and exercise conditions, 3) the effects of Ins2 loss on adult neurogenesis. To accomplish these aims we used wildtype (Strain # 000664 C57BL/6J, The Jackson Laboratory), isolated hippocampi from A/J mice provided by the Krystal laboratory as outlined elsewhere (Elisia et al., 2019), and transgenic knockout lacking one or two alleles of the Ins2 gene, respectively. 2.1.2 Transgenic knockout of Ins2 and genotyping We used Ins1-/-;Ins2+/- mice as previously generated and described elsewhere (Duvillie et al., 1997) and Ins1-/-;Ins2+/+ littermate controls. In these knockout mice, the Ins2 coding region was largely replaced by a LacZ/neomycin cassette. Ear notches were collected for genotyping using previously validated primers (Table 1) and DNA was isolated using the Sodium Hydroxide (NaOH) method. 10M NaOH was made with NaOH pellets (S80545, Sigma Aldrich) dissolved in distilled water. 1M Tris (15504-020, Invitrogen) was made in distilled water and pH adjusted 21 to 8. A stock of 50mM NaOH was made from the concentrated stock solution and 600µl of the 50mM NaOH solution was added to each ear notch in a microfuge tube. The tubes were incubated at 95°C in heat block for 10 minutes at 900 rpm. After incubation 50µl of 1M Tris was added and centrifuged for 6 minutes at 4°C (14000 rpm). Genotyping PCR Left Primer Sequence Right Primer Sequence Ins2 5’- TGC TCA GCT ACT CCT GAC TG -3’ 5’ – GTG CAG CAC TGA TCT ACA AT -3’ LacZ 5’- ACG GCA CGC TGA TTG AAG CA -3’ 5’- CCA GCG ACC AGA TGA TCA CA -3’ Table 1. Primer sequences for Ins2 knockout mice. 2.2 Ins2 expression experiments 2.2.1 Tissue preparation Brain tissue was extracted after mice were euthanized with CO2 and cervical dislocation as secondary method. The tissue was then micro-dissected in a dish with 1M PBS on ice for following brain regions; cerebral cortex (CTX), olfactory bulbs (OB), hippocampus (HIPP), hypothalamus (HYPO), cerebellum (CB), midbrain/residual (MID). Brain regions were flash frozen in dry ice and stored at -80°C until RNA isolation and DNA synthesis. RNA isolation was done as follows. First, tissue was homogenized in Trizol (Thermo Fisher) to protect against RNA degradation. After adding 50µl chloroform was added to the tissue and shaken for 20 seconds before incubation (15 minutes) at room temperature. Samples were centrifuged at 14000rpm for 15 minutes at 4 degrees and the aqueous phase was transferred 22 to a new tube with 70% ethanol. RNA extraction was performed using the Qiagen RNasy mini kit (cat #74108 Thermo Fisher). DNA was synthesized using the qScript cDNA Synthesis Kit (cat#101414-100, Quanta Bioscience). 2.2.2 Diet and exercise conditions Ins2 mRNA was measured in female A/J mice (12-weeks old) and tissue was obtained through a collaboration with the Krystal Lab at the B.C. Cancer Research Centre in Vancouver. Researchers at the Krystal lab conducted the experiment and isolated hippocampi as described above. I used the tissue for further processing (RNA isolation, DNA synthesis and qPCR analysis). Mice were exposed to four experimental conditions (Figure 3); 1) LC-FO diet + exercise 2) LC-FO diet + no exercise 3) Western diet + exercise 4) Western diet + no exercise. The LC-FO diet (low carbohydrate fish oil diet) was composed of amylose, soy protein, and fish oil. The Western diet was composed of amylopectin, casein, and western fats. We chose the specific diets because previous work from the Krystal lab showed that the LC-FO diet lowers circulating insulin and plasma glucose while the Western diet shows elevated plasma insulin, a precursor to insulin resistance. Further details on dietary composition and experimental design can be viewed in (Elisia et al., 2019). Our aim was to investigate if diet and exercise change brain insulin production. Exercise was defined by access to two running wheels (exercise condition) or no access to running wheels (no exercise). 23 A B Figure 5. Diet and exercise conditions for measuring Ins2 expression. (A) Diet composition of Western and LC-FO diet (15%Amylose/Soy/FO) as taken from (Elisia et al., 2019) (B) Experimental design for A/J female mice with and without running wheels. 24 2.2.3 Analysis of Ins2 gene expression Insulin expression was measured using quantitative polymerase chain reaction. The cDNA was subjected to Taqman real-time PCR (StepOnePlus, Applied Biosystems). Following primers were used; Ins2 Taqman reverse 280259: GAT CTA CAA TGC CAC GCT TCT G, Ins2 Taqman probe 224207: CCT GCT CCC GGG CCT CCA. PCR conditions were 2 min at 50 °C, 10 min at 95 °C and followed by 40 cycles of 15 sec at 95 °C, 1 min at 60 °C. Samples were normalized to the housekeeping gene beta actin by converting raw values to delta values. Reverse log of delta values was used for the analysis. Water and Ins2-/- tissue were used as negative controls in each plate. 2.3 Effects of Ins2 gene knockout on adult neurogenesis 2.3.1 Experimental design Variations of 20-month-old Ins2 genotypes (Ins2+/+: female n=9, male n=6; Ins2+/-: female n=7, male n=6; Ins2-/-; female n=5, male n=6) were given EdU drinking water (0.25mg/ml) for four weeks. Two liters of EdU drinking water was freshly prepared every week and divided to 120 ml bottles per cage. Body weight and water consumption were recorded every week. At the end of week 4 mice were transcardiacly perfused with 1M PBS followed by 4% paraformaldehyde (Sigma-Aldrich). Brains were extracted and postfixed in 45 paraformaldehyde overnight, then transferred to 30% sucrose (Fisher Scientific) solution for cryoprotection and remained in the solution until sectioning. Brains were sliced into 40 µm coronal sections using a cryostat at -20°C. Sections were collected in series of 6 throughout the entire rostral-caudal extent of the hippocampus and stored in anti-freeze solution consisting of ethylene glycol, glycerol, and 0.1M PBS at -20°C until immunostaining. 25 2.3.2 Measuring neurogenesis with Click-iT chemistry and immunostaining One series section was double stained for EdU and DCX. I used the Click-iT Plus EdU Alexa Fluor 647 Imaging Kit, Thermo Fisher Scientific (Figure 4) to examine cell proliferation and immunohistochemistry to stain for DCX to examine immature neurons with primary antibodies (Doublecortin polyclonal antibody, unconjucated, Host: Rabbit /IgG; Thermo Scientific) and secondary antibodies (Donkey anti Rabbit IgG (H+L), Alexa Fluor 488, Invitrogen). To test the accuracy of the staining I used gut sections from animals with EdU treatment as positive/internal control for cell proliferation and brain sections of 8-week-old B6 mice for immature neuron expression. Figure 6. Schematic of EdU Click-iT method for assessing cell proliferation. Click-iT technology uses a highly specific click reaction by binding the azide that is attached to the fluorophore to the alkyne group that is imbedded with the biomolecule EdU during mitosis. 26 2.3.3 Analysis Stained sections were analyzed with a Zeiss LSM 880 confocal microscope. Dorsal and ventral regions of the dentate gyrus were imaged using 20X objective. Stacks of images were stiched together and maximum projection was used to count EdU- and/or DCX-positive cells. The experimenter was blind to all conditions. The surface area of the dentate gyrus was traced, and cell count was normalized to surface area. Statistical analysis on comparing two groups were done using either a student’s t-test (equal sample size) or a Welch’s t-test (unequal sample size). I used a one-way ANOVA to determine if there was a significant difference amongst the three genotypes within females and males. 27 Chapter 3: Results 3.1 Ins2 mRNA levels across brain regions First, I focused on the general characterization of insulin levels across several brain regions in male and female B6 mice. The regions assessed were the cerebral cortex (CTX), olfactory bulbs (OB), hypothalamus (HYPO), the hippocampus (HIPP), cerebellum (CB), and the residual referred to as midbrain (MID). The expression of Ins2 was highest in the cerebellum and hippocampus in both males and females (Figure 1a). Females expressed higher levels of Ins2 mRNA in the hippocampus compared to males (Figure 2b). Figure 7. Increased levels of Ins2 mRNA in the hippocampus were sexually dimorphic. (A) Cerebellum (CB) and hippocampus (HIPP) showed highest levels of Ins2 mRNA (females n=10, males n=8). (B) Females (yellow) showed significantly higher expression of the Ins2 gene in the hippocampus compared to males (green); (females n=20, males n=19). Data are presented as mean ± SE; unpaired, two-tailed Student’s t-test with Welch’s correction. P-value < 0.5 is marked with *. 28 3.2 Ins2 mRNA Levels in exercise and diet conditions I next assessed Ins2 mRNA levels after exposure to different diets (low carb fish oil versus high carb western) and exercise conditions (no running wheel versus two running wheels). Under no exercise condition, the HC-Western diet group showed significantly higher expression of Ins2 mRNA compared with the LC-FO diet group (p=0.0176). This effect was not significant in animals that exercised (p=0.4460). Although a trend is indicated, there was no significant difference between no exercise and exercise conditions in animals eating a HC-Western diet (p=0.1903) or in animals eating a LC-FO diet (p=0.1825). Figure 8. Ins2 mRNA is significantly higher in mice that ate a High Carb Western (HC-Western) diet and did not exercise. Exercise conditions are defined by two available running wheels and no exercise conditions are defined by animals having no access to running wheels but are regularly housed. Low carb fish oil (LC-FO) diet is signified in blue and HC-Western in purple. Ins2 was normalized to housekeeping gene beta actin. Data are presented as mean ± SE; unpaired, two-tailed Student’s t-test. P-value < 0.5 is marked with *. 29 3.3 EdU and DCX staining in Ins2 knockout mice To assess neurogenesis, I stained the brain tissue with EdU and DCX. Internal controls for EdU were done by staining gut cross sections from mice treated with EdU water. Positive controls of DCX were done in 8-week-old (young) mice where DCX is known to be abundant. Negative control sections for EdU and DCX received all staining treatments except the Click-iT reaction and secondary antibody, respectively. Instead, sections were washed in 1X PBS. Figure 9. Staining controls for cellular proliferation (EdU) and immature neurons (DCX). (A) Positive expression of EdU in gut sections of animals that were treated with EdU. (B) Immature neuronal marker (DCX) expressing cells of young (8-week) old mice. (C) Sections that did not receive Click-iT chemistry or secondary antibody treatment of mice fed EdU in drinking water. 30 Figure 10. Staining of EdU and DCX in Ins2 genotypes of male and female mice. (A) Click-iT chemistry (EdU) and immunofluorescent (DCX) staining of Ins2+/+ male and female mice. (B) Click-iT chemistry (EdU) and immunofluorescent (DCX) staining of Ins2+/- male and female mice. (C) Click-iT chemistry (EdU) and immunofluorescent (DCX) staining of Ins2-/- male and female mice. Quantified results are discussed in figure 12 and 13. 31 Figure 11. Magnification of EdU+ and DCX+ cells. Example images of nuclei stain (Hoechst), Click-iT chemistry (EdU) and immunofluorescent (DCX) staining used for cell counting. 32 3.3.1 EdU+ cells are differentially expressed within sexes in Ins2 genotypes EdU+ cell count did not differ when comparing genotypes within each sex for the whole dentate gyrus. However, in the dorsal dentate gyrus Ins2 genotypes have different EdU+ cell count in males and females (Figure 4 A). Within females, Ins2+/- showed significantly higher EdU+ cell count compared to Ins2+/+; t(3.03)=8.24, p = 0.0159. There was no significant difference between Ins2+/+ and Ins2-/-; t(4.83)=1.38, p = 0.229. Within males, EdU+ cells were significantly higher in Ins2-/- compared to Ins2+/+; t(10)=2.36, p = 0.0399 but no difference between Ins2+/+ and Ins2+/-; t(10)=0.091, p = 0.929. When comparing males and females within each genotype, only heterozygous mice showed a significant difference, with females having higher EdU+ cell count than males; t(11)=2.542, p = 0.0274. In the ventral region of the dentate gyrus no differences in EdU+ cell count within sexes and between genotypes were detected (Figure 4 B). Males: F(2, 15) = 1.112, p = 0.3545; Females: F(2, 6.31) = 1.378, p = 0.3187. There were no differences in dentate gyrus volume between genotypes and sexes. Ins2+/ Ins2+/- Ins2-/- Figure 12. Cell proliferation in female and male mice responded differently to Ins2 loss. (A) Number of EdU+ cells normalized to surface area in dorsal regions of the dentate gyrus. (B) Number of EdU+ cells normalized to surface area in ventral regions of the dentate gyrus. Data are presented as mean ± SE; unpaired, two-tailed Student’s t-test. P-value < 0.5 is marked with *. 33 Figure 13. Immature neuron numbers are not affected by Ins2 loss in male and female mice. (A) Number of DCX+ cells normalized to surface area in dorsal regions of the dentate gyrus between genotypes. There were no significant differences within males (F(2, 15) = 2.75, p = 0.1727) and females (F(2, 8.6) = 2.17, p = 0.1727). (B) Number of DCX+ cells normalized to surface area in ventral regions of the dentate gyrus between genotypes. There were no differences within males (F(2, 15) = 1.912, p = 0.1821) and no significant differences within females (F(2, 9.24) = 1.06, p = 0.3846). Data are presented as mean ± SE; unpaired, one-way ANOVA. n.s., not significant. 3.3.2 No difference in DCX+ cells between sexes and Ins2 genotypes Immature neuronal marker DCX was assessed by counting positively expressed cells in dorsal and ventral regions of the dentate gyrus. No differences between genotypes and within sex was detected (Figure 5 A, B). Males dorsal F(2, 15) = 2.75, p = 0.1727 and ventral F(2, 15) = 1.912, p = 0.1821. Females dorsal F(2, 8.6) = 2.17, p = 0.1727 and ventral F(2, 9.24) = 1.06, p = 0.3846. Ins2+/ Ins2+/- Ins2-/- 34 Chapter 4: Conclusion 4.1 Brain-derived insulin characterization I aimed to confirm the expression of Ins2 in several brain regions and examine differences in mRNA levels between sexes. In line with previous studies, I confirmed that insulin is expressed in the several brain regions (Devaskar et al., 1994; Havrankova et al., 1978; Mehran et al., 2012). Studies that examined different regions of the brain for insulin synthesis are rare, vary in the type of regions examined, and in the animal models they used (Csajbok & Tamas, 2016; Havrankova et al., 1978; Mazucanti et al., 2019; Nemoto et al., 2014; Young III, 1986). However, studies that included hippocampal regions in their analysis consistently reported high levels of insulin expression (Kuwabara et al., 2011; Mehran et al., 2012). Studies investigating insulin synthesis used neuronal cultures from the hippocampus, indicating this area to be of high interest (Kuwabara et al., 2011; Nemoto et al., 2014). Interestingly, no study, to my knowledge, has specifically examined sex differences in insulin expression in these regions. Thus, these data show for the first time that adult female C57BL/6 mice show significantly higher brain-derived insulin than male littermates, highlighting that insulin synthesis in the brain is sexually dimorphic. Sex is a significant neuromodulator of adult neurogenesis and spatial memory (Chow et al., 2013). Neuroendocrine mechanisms are highly complex and dynamic in females, in great part due fluctuations of sex hormones during estrous cycle which can influence cell proliferation (Falconer & Galea, 2003). Studies on sex differences in adult neurogenesis after exposure to stress show that females may have a unique vulnerability to the duration of stress hormone exposure (Hillerer et al., 2013). Given the sensitivity to brain hormone levels in females, it is 35 possible that insulin, a neurotrophic factor, follows similar trends and may play a functionally more significant role in females than males and could explain that we see higher expression levels in the female hippocampus. It is possible that Ins2 mRNA levels are influenced by estrous cycle, or that females have a greater need for brain-derived insulin to co-regulate with other important, fluctuating neuroendocrine actions. Collectively, hormonal neuromodulation is highly plastic in females and future experiments should test these implications to understand the sex-specific role of brain-insulin production more fully. 4.2 Diet and exercise effect Ins2 expression Due to insulin’s integral role in metabolism, we were interested in how brain insulin levels may vary in response to diet and exercise. Low carbohydrate fish oil diets prevent high-fat diet induced insulin resistance, increases insulin sensitivity, and induce neurogenesis (Beltz et al., 2007; Fedorova, 2006; Hirabara et al., 2013; Storlien et al., 1987). In contrast, high carbohydrate or western diets increase plasma glucose levels, can induce insulin resistance, and when consumed chronically can lead to damage of insulin producing cells resulting in diabetes (Kopp, 2019). When looking through a neurobiological lens, past studies have shown that low carbohydrate studies improve cognitive function (Brinkworth, 2009). In fact, ketogenic diets are not only used to treat metabolic disorders, like obesity and diabetes (Augustin et al., 2018; Choi et al., 2020), but are increasingly used as lifestyle modification to treat people with neurological impairment, like Alzheimer’s Disease (McDonald & Cervenka, 2018). Studies that examined neuro-enhancing effects of ketogenic diets suggest that benefits are due to several factors, including ketone body esters and improved metabolic profile (e.g., reduced blood glucose) 36 (Włodarek, 2019). Diets high in carbohydrates and fats show opposing effects, impairing cognitive function, learning and memory (Greenwood et al., 2003). In mice, high fat diet-induced insulin resistance decreased dendritic spine abundance and spatial memory (Clegg et al., 2011). Collectively, diet composition can influence cell morphology and lead to changes in learning behaviour. Exercise is a potent modulator for neurogenesis and enhances cell proliferation and learning outcomes in older adults (Nilsson et al., 2020; van Praag et al., 2005). A study investigating voluntary exercise found that memory deficits decreased to a larger degree in older than younger rats in the Morris Water Maze (Siette et al., 2013). It also increased neurogenesis and restoration of presynaptic density in the hippocampus. In addition, exercise reverses negative effects of stress on learning and memory (Molteni et al., 2004). Overall, exercise is well established to have neuroprotective effects on memory and in Alzheimer’s diseases (Jahangiri et al., 2019). Multiple clinical trials reported significant improvements on cognitive function in exercise training groups compared with the control groups (Klusmann et al., 2010; Liu‐Ambrose et al., 2008; Vaughan et al., 2014). In contrast, physical inactivity in humans and rodents is linked to higher incidences of cognitive impairment and chronic illnesses (González et al., 2017; Park et al., 2022; Pauly et al., 2019). Collectively, diet and exercise are important modulators of health and can directly influence neuronal growth. Given the neurotrophic and memory enhancing effects of insulin administration to the brain, and the impairment of cognition due to insulin resistance, I aimed to understand whether diet and exercise change levels of brain-derived insulin. Contrary to my prediction that Ins2 expression would be reduced in animals that ate a Western diet and did not exercise, we found that Ins2 mRNA levels were increased under these conditions. Animals that exercised did not 37 show a difference between a LC-FO and Western diet. Given the current understanding of the literature, it may be that under no exercise conditions (absence of neuroprotection via exercise) the demands of a Western diet trigger a compensatory effect. Thus, increasing the transcription of the Ins2 gene in attempt to balance out the neurodegenerative effects of inactivity and a high carbohydrate diet. However, to confirm this interpretation and gain a full understanding of the reasons for Ins2 upregulation further experiments are required. 4.3 Considerations of sex differences in data interpretation There are several factors to consider when interpreting the data of this study. First, not much is known about the mechanisms of neurogenesis in females compared to males due to a long history of exclusively studying male animals (Galea et al., 2020). It is only in recent years that researchers have made a conscious effort to also investigate sex differences in rodent models (Garcia-Sifuentes & Maney, 2021). Thus, this study intends to add to the literature by specifically examining sex differences in the effects of Ins2 loss on adult neurogenesis. Second, we know that the process of proliferation and maturation of new neurons in adulthood behaves differently between sexes. In male rats (two-month-old), adult-born neurons mature faster than in females and show greater density of neural stem cells in dorsal but not ventral regions (Yagi et al., 2020). Interestingly, males showed a greater reduction in neurogenesis after 1 and 2 weeks of mitosis, while females did not show this variation in cell survival of new-born cells. The present data does not assess time points but rather a cumulative score of cell division across a four-week period and thus inferences to previous research examining changes in proliferation during specific time windows cannot be made. No differences in EdU+ cells between male and female Ins2+/+ mice were detected. In addition, 38 much research on sex differences in neurogenesis has been done in rats (Chow et al., 2013; Yagi & Galea, 2019). Thus, it is notable to point out that adult neurogenesis between rats and mice differs (Snyder et al., 2009). Adult-born neurons in male rats mature faster and survival rates of new granule cells are higher. Less is known about species differences in females. A third factor to consider when interpreting the data of this study is the influence of estrogen and age-related changes. It is generally accepted that estrogen plays a neuroprotective role and modulates hippocampus-dependent learning and memory (Barha & Galea, 2010). Estrous cycle in rodents has been shown to influence the number of new neurons in the dentate gyrus (Tanapat et al., 1999). Middle-aged female rats (14-month-old) but not young (3-month-old) females showed increased spine density in response to treatment with 17β-oestradiol, the predominant circulating estrogen in women (Morgenstern et al., 2008). The data of the current study is representative of advanced aged mice (20-month-old) that are considered post-menopausal, and we assume that the cyclic effects of estrogen do not play a critical role in the effects of our results. However, post-menopause itself could influence adult neurogenesis. 4.4 Brain-derived Insulin loss affects cell proliferation differently in females and males The data in the present study examined the effects of Ins2 gene loss on cell proliferation (measured via mitotic cell marker EdU). Cell proliferation is defined as any cell that underwent mitosis within the four weeks of EdU treatment. Preliminary data of the Johnson laboratory showed that Ins2-/- females experience spatial-memory impairment (Figure 2A), a hippocampal-dependent task. Studies on administration of insulin to the CNS found that it increases cognitive function in human and animals, while impaired insulin signalling negatively effects learning and memory (Lv et al., 2020). Since neurogenesis is a modulator of learning and memory, I 39 hypothesized that the loss of the Ins2 gene (lack of brain-derived insulin) will reduce cell proliferation in female but not male mice. We compared different gene-dosage (Ins2+/+; Ins2+/-; Ins2-/-) within females and male mice and counted the number of EdU+ cells. Contrary to my predictions, cell proliferation did either not differ (Ins2+/+ vs. Ins2-/- in females and Ins2+/+ vs. Ins2+/- in males) or was increased (Ins2+/+ vs. Ins2+/- in females and Ins2+/+ vs. Ins2-/- in males). Although surprising at first, in context of our current knowledge on insulin transport and IR binding, it is possible that the transport of circulating insulin into the brain increased to compensate for brain-derived insulin loss and consequentially stimulated cell proliferation. Insulin transport to the brain is slow and studies indicate that less than 1% of peripherally administered insulin reaches the CNS, indicating that circulating insulin levels may not be significantly affected by increased transport (Rhea & Banks, 2021). Insulin uptake is greater in lateral regions than medial regions e.g., hippocampus (Plum et al., 2005), highlighting a need for local insulin production. In addition, the amount of insulin transported to the brain is affected by several factors. Obesity (Kaiyala et al., 2000), Alzheimer’s Disease (Craft et al., 1996), and aging (Frölich et al., 1998) decrease insulin transport. Interestingly, fasting has the same effect (Strubbe et al., 1988). In contrast, a diabetic rodent model reported that insulin transport increases whereas hyperglycemia and hibernation abolished transport (Banks et al., 1997; Florant, 1991). In line with these observations, it has been suggested that BBB transport is a dynamic process that functions to allow brain insulin to act as a counter-regulatory hormone to peripheral insulin (Banks 2004). In addition, peripheral infusion of IGF-1 (structurally and functionally similar to insulin) showed that it induces adult neurogenesis (Åberg et al., 2000) and that the uptake of serum IGF-1 by the brain parenchyma mediates the increase in exercise-induced neurogenesis (Anderson et al., 2002). Thus, it could be possible that the absence of BDI 40 via Ins2 gene knockout elicits increased insulin transport in efforts to increase brain insulin levels in the hippocampus. The present data shows that female mice have increased proliferation when lacking one Ins2 allele, but do not show changes in proliferation with the loss of two copies. Given the dynamic relationship of peripheral and brain-derived insulin, it could be that the loss of one allele signals the need for increased insulin transport while producing enough local insulin to synergistically increase cellular proliferation. That no increase in proliferation was detected in Ins2-/- females may be because some brain-derived insulin synthesis is required or, more likely, because the excess influx of peripherally derived insulin lead to impaired insulin signalling. Insulin must bind to its receptor to elicit its effect. Insulin transport is an insulin receptors-mediated process, indicating that insulin signalling plays an important role in the communication between CNS and periphery. Interestingly, insulin receptors are highly abundant in the hippocampus and critical for cognition (Chen et al., 2022). Impaired insulin signaling in the brain is strongly linked to neurodegeneration and neuronal survival. Previous research showed that blocking of insulin receptors in the dentate gyrus reduces neurogenesis (Ronaghi et al., 2019). Reduced insulin sensitivity in the CNS can be characterized by decreased insulin receptor concentrations, decreased insulin binding affinity, and alterations in signaling cascades (Dutta et al., 2022). From studies on hyperinsulinemia, we know that too much insulin can lead to insulin receptor impairment (Cen et al., 2021; Kanety et al., 1994; Shanik et al., 2008). Therefore, if circulatory insulin attempts to compensate for brain-derived insulin loss, it is possible that Ins2-/- females, despite normal peripheral glucose homeostasis (Mehran et al., 2012), experienced insulin receptor impairment in the brain due to a chronically high turnover of 41 insulin transport. To test this interpretation further experiments examining IR abundance and insulin transport on Ins2 genotypes are needed. It is interesting that dentate gyrus proliferation of male mice responded differently to Ins2 loss. We observed that one copy of Ins2 did not change levels of cell proliferation but losing both copies did. In our experiment assessing Ins2 mRNA levels across brain regions, we saw that females have higher levels of hippocampal Ins2 mRNA compared with males, and previous unpublished data shows memory impairment in only Ins2-/- females, but not males. Together these results could indicate that brain insulin levels play a functionally more significant role in females than males, and that females are more sensitive to changes of BDI than males. This sex difference is in line with the observations that women are at higher risk for developing Alzheimer’s Disease despite its risk factor diabetes being more prevalent in men (Henderson, 1997; Patel, 2017; Skyler & Oddo, 2002). Because cell proliferation between Ins2-/- and Ins2+/+ did not differ in females but in males, we conclude that early-stage neurogenesis is not the mechanisms by which Ins2-/- females could experience impaired spatial memory as seen in preliminary experiments. However, if BDI plays a role in mediating insulin transport and insulin resistance, and the negative effects are only functionally relevant in Ins2-/- females but not males, it possible that behaviour effects were due to dysfunctions in insulin signalling which would be in line with the results of other studies (Zhao et al., 2004). 4.5 Brain-insulin loss does not affect the number of immature neurons Neurogenesis includes more than the proliferation of new cells. Mitotic markers do not reveal whether adult-born cells are in early or later stages of maturation unless specific time 42 windows were analyzed. EdU staining alone will also not answer whether new cells differentiated into neural or glial cells. In adult neurogenesis the a majority of new-born cells will differentiate into neurons (Cameron et al., 1993). To assess if the number of immature neurons differed between Ins2 genotypes, I counted DCX+ cells. I hypothesized that immature neurons are less abundant in mice lacking insulin. The data revealed no difference in the number of DCX+ cells between Ins2 genotypes within or between sex. The results could indicate that despite increased proliferation, new-born cells in female heterozygous and male complete knockouts did not progress further in their maturation. About 50% of progenitor cells die due to pressure of elimination by programmed cell death (Kempermann et al., 2003). Thus, it would be interesting to examine the effect of Ins2 loss on the rate of apoptosis between genotypes. On the other hand, immature neurons are in a transient state and by solely measuring DCX we do not know whether new-born cells in Ins2 knockouts may have progressed faster to later stages of development. Insulin can increase cell maturation, spine formation and dendrite complexity (Cheng et al., 2003; Chiu & Cline, 2010). This could be assessed by using a mature neuronal marker such as NeuN and categorizing its dendritic branching and spine formation. 4.6 Strengths and limitations My findings indicate that levels of brain-derived insulin differ between sexes and are involved in early stages of adult hippocampal neurogenesis. The Ins2 knockout mouse model in this study is most appropriate to directly test the effects of endogenous insulin of the brain to date. Silencing the Ins2 gene, while keeping Ins1 intact, allows to specifically examine the causal effects of insulin synthesis in the brain independent of peripheral insulin. Other methods of testing for local brain insulin ablated insulin-expressing neurons with β-cell toxin streptozotocin 43 (Grünblatt et al., 2007; Lester-Coll et al., 2006; Plaschke et al., 2010). However, this method also effects other areas of the brain, and it would be difficult to say whether effects on neurogenesis or behavior are due to BDI loss or toxicity. Thus, this mouse model can be considered as a powerful alternative method to examine endogenous insulin production. However, this model does not come without its own limitations. The Ins2 gene is also, in addition to the pancreas and brain, expressed in the thymus. The thymus is most known for its role in immunity and insulin deletion in the thymus can induce autoimmune diabetes (Fan et al., 2009). Thus, with a global Ins2 knockout, it cannot be entirely excluded that the results of this study are exclusively due to brain-insulin loss but may indirectly relate to autoimmune dysfunction. Generating a tissue-specific knockout could be used to test this limitation. 4.7 Overall conclusions Although increasingly more studies confirm the presence of insulin production in the brain, little is known about how BDI effects adult neurogenesis and even less about differences between sexes. 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Thesis/Dissertation
eng
Cell and Developmental Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Johnson, James D.
The effects of brain-derived insulin loss on adult neurogenesis in aged male and female Ins2 knockout mice
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