UNIVERSITY OF CALGARY. An Investigation of the Role of Insulin Deficiency and Loss of PI3K-Akt Signaling Downstream. Tazrina Alrazi A THESIS

Transcription

1 UNIVERSITY OF CALGARY An Investigation of the Role of Insulin Deficiency and Loss of PI3K-Akt Signaling Downstream Regulators GSK3β -CREB Signaling in the Pathogenesis of the Diabetic Brain by Tazrina Alrazi A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN NEUROSCIENCE CALGARY, ALBERTA JULY, 2014 Tazrina Alrazi 2014

2 ABSTRACT Our lab has developed a robust streptozotocin-induced murine model with changes analogous to those observed in human diabetic brain, and our lab has shown that replacement of insulin in the brain via intranasal delivery prevents diabetes mellitus (DM) mediated neurodegeneration. Insulin is speculated to act through activation of PI3K-Akt signaling. We hypothesized that the neurodegenerative changes and cognitive impairments observed in the chronic type 1 DM brain occur through impaired insulin-mediated phosphorylation of CREB/GSK3β, critical neuronal signals downstream of PI3K-Akt pathway and direct support of these proteins will prevent these neurodegenerative changes. A transgenic murine model with downregulation of CREB in the forebrain was used to complement interventions to attempt to rescue the chronic type 1 DM brain. Intranasal TDZD-8 was used to inhibit GSK3β in wild type mice. Our results are consistent with the hypothesis that CREB is essential for brain insulin signaling but that GSK3β might not be directly involved in this pathway. ii

3 ACKNOWLEDGEMENTS This dissertation has been possible due to the kind support and assistance of several individuals whom I want to personally thank and acknowledge. First, I am eternally thankful to Dr. Cory Toth for being such an excellent mentor, supervisor and guide throughout my graduate study duration. I am grateful for the help he has provided with my western blot data analysis and additional information that made this study possible. His patience and guidance will always encourage me. Second, deepest gratitude to my supervisor Dr. Robert French for being such an excellent guide and also for helping me from time to time with my study; Dr. Vina Goghari for her unfailing and unselfish support; Dr. Dawn Pearson for her valuable comments and advice; Dr. Ursula Tour for being such an excellent guide for the MRI study; Dr. Alma Rosales for helping me with the western blot analysis. Special thanks to Dr. Zochodne for sharing his lab space and for providing such insightful comments during our joint lab meetings. I want to take the opportunity to thank all the members of the Toth and Zochodne labs. Special acknowledgement to summer student Kiara Reddy for helping me with the cognitive-behavioural studies; many thanks to David Kirk and Tad Foniok from the experimental imaging center for their valuable technical assistance. Third, utmost gratitude to Dr. Bill Stell for being an exceptional mentor and friend throughout my graduation studies. His contributions towards my well-being will always be remembered. I would like to thank my family for being there for me at all times. iii

4 Dedication Mom, dad, brother, Oreal, John, Rony and friends who made a foreign country home iv

5 TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS...iii DEDICATION iv TABLE OF CONTENTS v LIST OF FIGURES AND ILLUSTRATIONS. x LIST OF SYMBOLS, ABBREVIATIONS, NOMENCLATURES...xiii CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: BACKGROUND DIABETES MELLITUS HUMAN BRAIN WITH DIABETES COGNITIVE DYSFUNCTION IN DIABETIC PATIENTS COGNITION IN CHRONIC TYPE 2 DIABETES CHILDREN SUFFERING FROM TYPE 1 DM ADULTS WITH TYPE 1 DIABETES MILD COGNITIVE IMPAIRMENT, ALZHEIMER S DISEASE AND DIABETES WHITE MATTER ABNORMALITIES IN DIABETIC BRAIN DIABETIC BRAIN AND CEREBRAL ATROPHY DIABETIC BRAIN IMAGING DIABETIC BRAIN AND OLIGODENDROCYTES...18 v

6 2.3 EXPERIMENTAL ANIMAL MODELS WITH DIABETIC ENCEPHALOPATHY PATHOPHYSIOLOGY OF DIABETIC LEUKOENCEPHALOPATHY IN THE EXPERIMENTAL ANIMAL BRAIN INSULIN IN OUR BRAIN BRAIN INSULIN RECEPTOR EXPRESSION PATTERN THE GSK3β AND CREB PATHWAYS IN INSULIN SIGNALING MODIFYING THE GSK-3β AND CREB PATHWAY OF INSULIN SIGNALING INTRANASAL DELIVERY OF CHEMICALS TO THE CENTRAL NERVOUS SYSTEM OLFACTORY NERVE PATHWAYS IN INTRANASAL ADMINISTRATION OF DRUGS TRIGEMINAL NERVE PATHWAYS OF INTRANASAL INSULIN MOLECULAR STUDY PRELIMINARY DATA AND PREVIOUS WORK DONE IN OUR LAB.33 CHAPTER THREE: HYPOTHESIS AND OBJECTIVE MAIN RESEARCH HYPOTHESIS SPECIFIC OBJECTIVES/AIMS OBJECTIVE/AIM OBJECTIVE/AIM CHAPTER FOUR: PLAN AND METHODS DM AND NON-DM MICE CREB KNOCKDOWN TRANSGENIC MICE 38 vi

7 4.3 CD1 WILD TYPE MICE INTERVENTIONS BEHAVIOURAL TESTING MAGNETIC RESONANCE IMAGING (MRI) SACRIFICE OF ANIMALS MOLECULAR ANALYSIS ANALYSIS OF DATA TIMELINE.50 CHAPTER FIVE: RESULTS EXPERIMENTAL GROUP CHARACTERISTICS BLOOD GLUCOSE LEVELS IN CREB KNOCKDOWN MICE BODY WEIGHT IN CREB KNOCKDOWN MICE RESULTS OF PI3K-Akt/CREB PATHWAY PHARMACOLOGICAL MODULATION COGNITIVE BEHAVIOURAL ANALYSIS RESULTS FOR FOREBRAIN- SPECIFIC CREB KNOCKDOWN MICE BRAIN WEIGHT MRI RESULTS FOR FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE MRI BRAIN VOLUMES FOR CREB KNOCKDOWN MICE MRI DATA (DTI, MTR, T2) ANALYSES FOR FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE BRAIN WHITE MATTER TRACTS REGIONAL BRAIN VOLUME BY T2 WEIGHTED MRI IMAGES WESTERN BLOT RESULTS FOR CREB KNOCKDOWN MICE..75 vii

8 5.5 RESULTS FROM CD1 WILD TYPE MICE BLOOD SUGAR LEVELS FOR 2 ND AIM CD1 WILD TYPE MICE TREATED WITH TDZD-8/PLACEBO BODY WEIGHT IN CD1 WILD TYPE MICE TREATED WITH TDZD-8/PLACEBO RESULTS OF PI3K-Akt/GSK3β PATHWAY PHARMACOLOGICAL MODULATION BY TDZD BEHAVIOURAL TESTS RESULTS FOR CD1 WILD TYPE MICE TREATED WITH TDZD-8/PLACEBO BRAIN WEIGHT OF CD1 WILD TYPE MICE TREATED WITH TDZD8/PLACEBO WESTERN BLOT ANALYSIS OF CD1 MICE TREATED WITH PLACEBO/TDZD CHAPTER SIX: DISCUSSION AND CONCLUSION GENERAL REMARKS AND MAJOR FINDINGS METHODS AND LIMITATIONS TO BE CONSIDERED BATTERY OF COGNITIVE TESTS CONFOUNDING FACTORS FOR BEHAVIOURAL TESTING MRI STUDIES WESTERN BLOT ANALYSIS ANIMAL MODELS DRUG TREATMENT STATISTICAL ANALYSIS..99 viii

9 6.3 DIABETES AND WHITE MATTER ABNORMALITIES DIABETES AND GRAY MATTER ABNORMALITIES BODY WEIGHTS IN EXPERIMENTAL MICE BODY WEIGHT IN FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE BODY WEIGHT IN CD1 WILD TYPE MICE TREATED WITH TDZD- 8/PLACEBO BLOOD GLUCOSE LEVELS IN EXPERIMENTAL MICE BLOOD GLUCOSE LEVELS IN FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE BLOOD SUGAR LEVELS FOR 2 ND AIM CD1 WILD TYPE MICE TREATED WITH TDZD-8/PLACEBO DIABETES BRAIN AND INTRANASAL INSULIN DIABETES BRAIN AND INTRANASAL TDZD DIABETES BRAIN AND CREB SIGNALING DIABETES BRAIN AND GSK3β SIGNALING ALTERNATIVE INSULIN RECEPTOR SIGNALING PATHWAYS INTRANASAL DRUG DELIVERY COGNITIVE IMPAIRMENT IN MOUSE MODEL OF DIABETES SUMMARY AND CONCLUSION FUTURE DIRECTIONS.118 REFERENCES ix

10 LIST OF FIGURES AND ILLUSTRATIONS Figure 1: Insulin signaling pathways in brain showing the PI3K/Akt/CREB-GSK3 pathway, Ras mitogen-activated protein kinase (MAPK) pathway, the PI3K/Akt/mTOR pathway, and the PI3K/Akt/BAD pathway..6 Figure 2: Major insulin signaling pathway in brain (our hypothesis) 34 Figure 3: Flow chart showing study design with objective 1 and objective Figure 4.1: Showing timeline and methods for our study..51 Figure 4.2: On left, showing object recognition test with familiar object On right, showing object recognition test with novel and familiar object Figure 4.3: Showing hole board test.52 Figure 4.4: Showing radial arm test.. 52 Figure 4.5: Showing Morris water maze test Figure 5.1: Final experimental group numbers with intervention and mortality rates..55 Figure 5.1.1A: Monthly blood glucose levels 56 Figure 5.1.1B: Glycated hemoglobin level 56 Figure 5.1.2: Body weight for forebrain-specific CREB knockdown mice..57 Figure : Radial arm test for forebrain-specific CREB knockdown mice 59 x

11 Figure : Hole board test for forebrain-specific CREB knockdown mice 60 Figure : Object recognition test for CREB knockdown mice.61 Figure : Water maze results for CREB knockdown mice..62 Figure 5.2.2: Forebrain-specific CREB knockdown mice brain weights..63 Figure 5.3.1: MRI whole brain volumes for forebrain CREB knockdown mice...64 Figure : (a) Showing DTI MRI scans; (b) showing FA values obtained from the DTI scan analysis Figure : (c) Showing T2 weighted images of MRI scan; (d-e) showing 11 different brain regional values in mili seconds for T2 weighted images Figure : (f) Showing MTR MRI scans; (g-h) showing analysis of MTR scans for nine different brain regions Figure 5.3.3: (left) Hippocampal brain volume; (right) Cortex brain volume for forebrain-specific CREB knockdown mice...74 Figure 5.4: Showing western blots and results for forebrain-specific CREB knockdown mice hippocampal tissue 76 Figure 5.5.1(a): Showing monthly blood sugar levels for CD1 mice treated with TDZD- 8/placebo 77 Figure 5.5.1(b): Showing glycated hemoglobin levels for CD1 mice...78 Figure 5.5.2: Showing monthly weight of CD1 mice 79 xi

12 Figure : Showing hole board behavioural test results for CD1 mice.80 Figure : Showing radial arm test result for CD1 mice 81 Figure : Showing Morris water maze result for CD1 mice.82 Figure : Showing object recognition test result for CD1 mice...83 Figure 5.6.2: Showing brain weight in CD1 mice.84 Figure 5.7: (a) Western blot of cortex tissue from CD1 mice with statistical analysis; (b) Western blot for hippocampal tissue from CD1 mice with statistical analysis of data 86 xii

13 LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE Abbreviation Definition AC Anterior commissure AD Alzheimer's disease AGEs Advanced glycation end-products Akt Protein Kinase B BBB Blood Brain Barrier CAP Cbl-associated protein CA Cornu ammonis Cbl Casitas b-lineage lymphoma CC Corpus callosum CNS Central nervous system CPu Caudate/putamen CREB camp response element-binding DLE Diabetic leukoencephalopathy DM Diabetes mellitus DTI Diffusion tensor imaging xiii

14 FA Fractional anisotropy GSK3β Glycogen synthase kinase 3 beta HbA1c Glycated hemoglobin IC Internal capsule IGF Insulin-like Growth Factor I-I Intranasal insulin IR Insulin receptor IRS Insulin receptor substrate I-S Intranasal saline LTD Long-term depression LTP Long-term potentiation M1 Primary motor cortex MAPK Mitogen-activated protein kinase MCI Mild cognitive impairment MRI Magnetic resonance imaging MTR Magnetization transfer ratio mtor Mammalian target of rapamycin xiv

15 PC Posterior commissure PI3K Phosphoinositide 3-kinase PIP 2 Phosphatidylinositol (3,4)-bisphosphate PIP3 Phosphatidylinositol (3,4,5)- triphosphate RF Radiofrequency ROI Regions of interest S1 Primary somatosensory cortex STZ Streptozotocin TE Echo Time TK Tyrosine kinases TR Repetition time WMA White matter abnormalities xv

16 CHAPTER ONE: INTRODUCTION According to the World Health Organization (WHO) the world diabetic population in 2011 was 220 million 1. This is anticipated to double by the year 2030 due to urbanization, obesity, and aging 1,2. As diabetic patients live longer, morbidity due to diabetes mellitus (DM) complications are predicted to accumulate 37. DM is a group of metabolic disorders characterized by elevated blood sugar levels 1. DM is mainly classified into two types: type 1 diabetes and type 2 diabetes 1,6. Type 1 DM is typically caused by an autoimmune destruction of insulin-producing beta cells of the pancreas, leading to insulin deficiency. Type 2 DM is caused by insulin resistance. Clinical features are diverse and systemic with the most common complications of DM being diabetic neuropathy, nephropathy, retinopathy, heart disease, and vascular disease 6. Despite much knowledge on this metabolic disorder, the effects of DM in the brain, referred to as diabetic leukoencephalopathy (DLE), are often overlooked, with DM-mediated changes in the brain involving cognitive impairment 7-9. Cognitive changes occur over time, encompassing a slowing of information processing, reduced psychomotor efficiency, deficits in attention, and impaired mental flexibility 7-10.Experimental animal models of DM permit assessments of diabetic behaviours, treatment efficiency, disease prognosis and translational understanding to help interpret cognitive impairments occurring in human diabetic leukoencephalopathy, namely: learning and memory deficits, poor task comprehension, and a lack of problem-solving abilities 7-10,12,97. Our lab uses a murine model of streptozotocin (STZ)-induced type 1 DM brain. When given intraperitoneally during fasting conditions, STZ, a nitrosurea compound previously used as an antibiotic and chemotherapeutic, enters the pancreatic β cells through glucose transporter 2 (GLUT2) channels in the plasma membrane, causing toxicity in cells and locally observed 1

17 immune responses, resulting in abrupt onset of hypoinsulinemia and hyperglycemia 1,50. Thus, this is an animal model of untreated, yet sustainable, type 1 DM 12. The murine STZ model is well established for demonstrating cognitive dysfunction, brain atrophy (>20% loss of brain mass and volume), and brain structural changes 11-16,97 Type 2 diabetes murine models have also shown similar identifiable changes in behaviour and brain 1,15,16,69,90. The mechanism(s) by which DM affects the brain is uncertain and may be multifactorial. Hyperglycemia has neurotoxic effects like excessive production of advanced glycation end products (AGEs) 14,16,72,104,105,107 and oxidative stress. However, other studies have demonstrated cerebral blood flow and angiopathy like vascular changes in the DM brain 3,102,169. A deficiency of insulin, IR and associated signaling machinery, seen in rodent DM brains 3,5,12,20,21,25,26,28,29, may also indicate the role of insulin deficiency in the pathogenesis of DM brain changes. Interestingly some studies have recently demonstrated that direct insulin replacement to the brain minimizes such changes in the experimental type 1 DM brain 16,19,28,30,31. Therefore, a loss of insulin signaling could be a potential mechanism explaining diabetic changes in brain. At this time, there is no therapy to treat or cure the changes seen in DLE. My thesis examines the importance of the brain insulin pathway in type 1 DM. Previous work by Toth lab has revealed that insulin deficiency contributes significantly to neurodegenerative changes in the brains of a mouse model with type 1 DM 16,87. Long known for its ability to assist the cellular entry of glucose, insulin is also an important growth factor for the central nervous system and promotes synaptic connections between neurons 5,16,18,21,22,87,102,120,186. The Toth laboratory has developed a robust STZ-induced experimental murine model with changes analogous to the human diabetic brain 16,87, showing evidence of brain atrophy, white matter disease, and cognitive decline. Replacement of insulin in the brain via intranasal delivery 2

18 (thus directly targeting the brain and avoiding the systemic circulation) prevents DM-mediated neurodegeneration 16,28,30,31,87,115,117,142,145,146,148. Insulin is important in both peripheral and CNS glucose homeostasis; however the central action of insulin is not only glucose homeostasis but also neuroprotection 16. There are high concentrations of insulin receptors at the synapses, primarily within the hippocampus, frontal cortex and regions mostly concerned with memory and cognition which suggests a role in neuronal plasticity and normal neuronal well-being 87. Insulin binds to IR to exert its function in regeneration, neuroprotection or even neurogenesis in the hippocampus. IGF1 (insulin growth factor-1) also might bind to the insulin receptor and insulin might bind to IGFR (insulin growth factor receptor), suggesting there is cross-reactivity of insulin with IGF1 23,98,87,108. In the thesis I examine the roles of molecules downstream of PI3K-Akt specifically determining the importance of phosphorylation of camp response element-binding protein (CREB)/ glycogen synthase kinase 3 beta (GSK3β), both postulated to be critical neuronal signals in the insulin signaling pathway. Determination of the most important molecule(s), whether it be CREB, GSK3β, or otherwise, will permit more directly targeted therapies in the future for human translational work. We decided to look into CREB as our target molecule as it is a transcription factor mostly concerned with growth, proliferation, memory and toxicity when it is activated by phosphorylation 126, Our hypothesis was that if we knocked down this protein function in experimental mice forebrain (where it is highly active and abundant in wild type animals), chronic DM animals will not show any improvement of cognitive behaviour despite the application of insulin. We further decided to look into GSK3β protein as non-phosphorylated active GSK3β causes phosphorylation of tau protein leading to accumulation of phosphorylated 3

19 tau and β-amyloid plaques in brain which are highly neurodegenerative and lead to formation of neurofibrillary tangles, plaques, synaptic loss and down regulation of neuronal plasticity 4,15,70,122. We thought that inactivating this molecule by phosphorylation will prevent neuronal damage of brain tissue and improve cognitive behavior compared to animals with active GSK3 in their brain 125,187. Our model used intranasal delivery of insulin avoiding conventional subcutaneous routes, permitting bypass of the blood brain barrier and directly targeting the brain with negligible impact on peripheral blood glucose levels, which would otherwise result in hypoglycemia when delivered at the high doses needed 16,142 Insulin in the brain has its neuroprotective role and when administered in subcutaneous routes to treat type 1 DM, evidence shows that very little insulin actually reaches the brain hence subcutaneous routes of insulin administration may not a preferable route to treat brain demands of insulin. So the safest and most convenient way of insulin administration into brain is the intranasal route. The trigeminal and olfactory nerves connect the nasal cavity directly to many different areas of brain and insulin can travel through them into the brain without needing to cross the blood brain barrier, thus avoiding systemic side effects 24,87. Although how insulin travels through these nerve pathways is still unknown, studies show higher insulin concentration in brain following intranasal administration and it is believed to improve cognition and memory in experimental Alzheimer s disease (AD) 4,30,82,116,146. Our current work examined a transgenic model with down regulation of the target molecule CREB in the forebrain of C57BL/6 mice and another model where inhibition of GSK3β has occurred through delivery of intranasal TDZD-8 (a potent GSK3β inhibitor) in order to target these downstream molecules. We studied our mouse model using cognitive behavioral testing (Morris water maze, hole board, radial arm, and object recognition testing), in vivo magnetic resonance imaging (MRI) (to identify brain size and structure), and molecular 4

20 studies (western blot) to understand the diabetic disease process in murine brain and also to establish the importance of the downstream insulin signaling pathways. The STZ-induced diabetic transgenic murine model was an excellent tool for this project and the intranasal insulin delivery technique was a potent novel approach in the intervention of the diabetic disease process involving cognition. My main hypothesis was that neurodegenerative changes and cognitive impairments seen in the DM brain occur through impaired insulin-mediated phosphorylation of CREB/GSK3β, critical neuronal signals downstream in the insulin-pi3k-akt pathway. Furthermore, I postulated that direct support of these proteins will prevent cognitive dysfunction, cerebral atrophy and white matter abnormalities (WMA) in the diabetic mouse brain. I tested the hypothesis by sequentially monitoring changes in cognition through behavioral tests over 7 months, endpoint in vivo MRI, and molecular studies (western blot) in long-term type 1 DM or non-dm mice after 7 months of STZ injections (8-8 1/2 months of life). My rationale to carry out this project was that there is still a huge gap in knowledge in depicting the exact pathway for insulin signaling and there is a lot of cross talk between insulin and IGF in exerting their function by binding to their corresponding receptors. So, if my target proteins turn out to be the exact effector molecules for insulin signaling pathway downstream of the already known PI3K-Akt insulin pathway, it will help fill the gaps in our knowledge. Also by upregulating this pathway we may be able control the effect of insulin on the brain and prevent the neurological damage exerted by the lack of insulin signaling. 5

21 Figure 1: Proposed insulin-signaling pathways in brain showing the PI3K/Akt/CREB-GSK3 pathway, Ras mitogen-activated protein kinase (MAPK) pathway, the PI3K/Akt/mTOR pathway, and the PI3K/Akt/BAD pathway 135, ,188,196. 6

22 CHAPTER TWO: BACKGROUND 2.1 DIABETES MELLITUS Normal blood sugar level for mice is 6-8 mmol/l and a blood sugar level at or above 16 mmol/l is considered diabetic for these rodents. Type 1 DM was formerly known as insulin dependent diabetes mellitus (IDDM) or juvenile diabetes because it results from autoimmune destruction of insulin-producing beta cells of the pancreas 1,6 at an early age. The resultant total or subtotal insulin deficiency leads to hyperglycemia (elevated blood sugar level) and glycosuria (passage of glucose in urine). The classical symptoms are polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), and weight loss 6. Type 2 DM (formerly noninsulin-dependent diabetes mellitus, NIDDM or adult-onset diabetes) is characterized by hyperglycemia in the context of insulin resistance and relative insulin deficiency. This is in contrast to diabetes mellitus type 1, in which there is an absolute insulin deficiency due to destruction of islet cells in the pancreas 6. Type 1 DM represents approximately 5 10% of all diagnosed cases of diabetes 1 and the rest is type 2 DM. While the exact cause of type 1 DM is not known, it is believed that the etiology of type 1 DM involves a combination of genetic and environmental factors leading to an autoimmune defect. The treatment goal of type 1 DM is to keep blood glucose at normal levels by rigidly monitoring blood glucose level. The cardinal symptoms for type 2 are similar to those of type 1 DM, in addition to features of the metabolic syndrome including obesity. In type 1 DM, patients lose weight due to glycosuria, polyuria and inability of body cells to utilize glucose due to lack of insulin in blood. Primary complications of DM are peripheral and are characterized by end organ damage like diabetic 7

23 retinopathy, nephropathy and renal failure, neurological complications and death 1. Centrally DM causes DLE. However MRI studies have revealed that these changes are a result of long standing uncontrolled diabetes irrespective of vascular damage (stroke) 16,85. People suffering from chronic diabetes face a lifetime of complications, which is a burden both socially and economically. Our health-care system battles with tremendous challenges in coping with the needs of diabetes complicated patients. It has become a concern of modern society to find a cure for the chronic debilitating complications of this metabolic disorder and to protect our generation from its lethal effects. 2.2 HUMAN BRAIN WITH DIABETES: Both type 1 and type 2 DM impose progressive detrimental changes in human brain in the form of DLE 78. Scientists have demonstrated a high propensity for cortical thinning and disruption of white matter tracts in brain during chronic type 1 diabetes 41,55,57,84,161. The aims of our present project were to identify cognitive impairment in type 1 diabetic encephalopathy and the underlying pathophysiology of WMA and cerebral atrophy, and were evaluated by MRI and molecular studies COGNITIVE DYSFUNCTION IN DIABETIC PATIENTS: Cognition is a vast group of complex mental processes that includes attention, language, learning, memory, and beyond. One of the most common diabetic brain complications is cognitive decline measured by behavioural tests and its incidence may be as high as 40% in people with diabetes 1,4,8,9,10,12,18,45,145. A systematic review of longitudinal studies reported an overall % increase in the occurrence of dementia in individuals with diabetes compared to 8

24 non-diabetic control individuals 10. Evidence from neurocognitive testing confirms that diabetesassociated cognitive deficits are marked and many studies suggest that the structural and functional cerebral changes in diabetes are related to hyperglycemia-induced end-organ damage, macrovascular disease, hypoglycemia, insulin resistance and amyloid lesions 1,10. It is now well established that experimental type 1 DM has detrimental effects on proliferation of hippocampal cells throughout life, synaptic transmission in pyramidal cells and survival 1,16,25,29,49,60,141. Neurogenesis can also be impaired by STZ treatment which might lead to dysfunction in learning and memory in type 1 diabetic mice 25,33,49,60,90. However, amelioration of cognitive deficits may not always involve restoration of neurogenesis to control levels. The brain deterioration seen in DM can be compared with senile brain atrophy 48 but it is not limited to the elderly; diabetic brain changes have been described in both children and adults with either type (1 or 2) of DM 1,9,10,12,58,59,67,71,106, COGNITION IN CHRONIC TYPE 2 DIABETES: Although type 2 diabetic patients are diagnosed to have moderate degrees of cognitive impairments, mostly involving verbal memory or information processing speed 1,52, the underlying mechanisms are still unknown. It is assumed that the pathogenesis of cognitive impairment in type 2 DM is largely due to metabolic syndrome with or without hyperglycemia, which encompasses hypertension, dyslipidemia, obesity and is associated with atherosclerotic cardiovascular disease, ischemic stroke and with cognitive decline and dementia 1,6,52,63. According to some recent studies, impaired glucose metabolism and associated disease conditions have global effects causing structural and functional changes in brain leading 9

25 to loss of global cognition and brain atrophy 52. Also improper diabetic treatment may lead to worsening cognitive function as we age compared to properly treated diabetic individuals due to abnormal blood glucose levels 52. Features of DM might resemble features of aging, however both type 1 and type 2 diabetic populations be it young or old, suffer from cognitive deficits equally 69. There is no direct correlation between hypoglycemia and cognitive deficits so far described 1. In chronic diabetes, it has been shown that specific regions of the brain get highly affected by the metabolic insult, mostly involving the hippocampus and frontal lobe which are areas highly studied for their role in memory, executive function and learning 1,52. Type 2 DM mostly affects the elderly population and studies have concluded that it plays an important role in the development of dementia depending on the extent of the disease and associated complications 17,52,53,55. One of the major problems seen in chronic diabetic patients is AD which is the most common form of dementia seen in chronically ill patients. AD is irreversible and only rehabilitation measures 4,11,87,190 are available CHILDREN SUFFERING FROM TYPE 1 DM: Type 1 has chronic neurodegenerative effects on children. Although there are some controversies about how diabetes affects the brain in children, neuropsychological test performance and school achievement show that diabetic children demonstrate inattention, distractibility and poor reaction time that is worse with earlier onset of the disease 44,51,58,59, Incidence of lower IQ in children suffering from type 1 DM compared to normal children is 10

26 prevalent and as they grow older the effect persists. It is anticipated that the degree of hyperglycemia is associated with a loss of executive functions, and to a lesser extent with a decline in learning and memory 17,55,190. It is important to avoid both hypoglycemia and chronic hyperglycemia during childhood to preserve specific cognitive skills 51,55. Type 2 DM complications in youth (microalbuminuria, hypertension etc.) need early diagnosis and aggressive treatment compared to type 1 DM in the same age group despite shorter duration and lower HbA1C ADULTS WITH TYPE 1 DIABETES: Adult patients with type 1 DM suffer from mild to moderate cognitive impairments which include slowing of mental speed and diminished mental flexibility but learning and memory are unaffected 55,190. Impaired cognitive performance affects both older (>57 years) and younger (< 58 years) age groups but the impairments are chronic and progressive 8,46,68. The pathogenesis of cognitive impairment and diabetic brain atrophy is still unknown to the scientific community and we are trying to pinpoint the exact mechanism to look into the pathways directly related to this question. There is a direct mechanistic link between diabetes and development of cognitive impairment and an early onset of Alzheimer s disease symptoms, and it is suggested that insulin therapy improves cognition 4,30,62,76,112,116. It is observed that patients treated with insulin have a better outcome in preserving adequate brain health represented by cognitive evaluations, compared to those who do not receive insulin therapy or have inadequate treatment. Exercise, 11

27 enriched environment, and hormones like estrogen are known to improve memory in neurodegenerative diseases 34,40,42,81. Depression is another comorbid factor with type 1 diabetic patient, which is often observed but ignored or left untreated. Depression occurs not only in older age groups with type 1 DM but also young adults. Both type 1 and type 2 DM impose a great amount of stress on patients because of the disease complications 4,72,83 and it must be treated with vigorously in order to prevent stress from propagating into many other health issues like loss of or increased appetite, depression, psychosis, immobility, fear, anxiety and decreased quality of life 1,39,47. For my MSc project I am examined the brain white matter pathways in order to evaluate the effects of type 1 DM on brain white matter tracts, and its correlation with cognitive impairment and cerebral atrophy. Further, I have performed a molecular analysis of brain tissue from chronic type 1 diabetic animals to see in vivo changes after giving the animals different treatments for a long period of time MILD COGNITIVE IMPAIRMENT, ALZHEIMER S DISEASE AND DIABETES: Many studies have shown a direct link between people/animals suffering from chronic DM and varying degrees of cognitive impairment. AD is the most common form of dementia (loss of memory), characterized by formation of phosphorylated tau and amyloid plaques in the brain and numerous studies have shown that DM increases the risks of developing AD in patients 4,11,109,125,190. Researchers have seen that DM leads to formation of increased phosphorylated tau and accumulation of β-amyloid due to increased phosphorylation of amyloid precursor protein (APP), and there is also lack of insulin degrading enzyme (IDE) 75 necessary 12

28 for the prevention of β-amyloid plaque accumulation in the brain. In AD, phosphorylated tau leads to formation of neurofibrillary tangles and neurodegeneration, whereas β-amyloid forms extracellular amyloid plaques which are the earliest cardinal features of AD 125,187. DM leads to formation of the cardinal features in the form of neuropathological markers of AD. Although the apparent pathophysiology of DM leads to the macrovascular and microvascular insults caused by elevated blood sugar levels or lack of insulin/insulin resistance, the overall mechanism causing DLE is still an area that is unknown to the scientific community. The initial cognitive impairments observed in DM, later lead to more complicated dementia. This conversion of mild cognitive impairment to dementia is inevitable in absence of intervention. Mild cognitive impairment (MCI) refers to the deterioration of memory, cognition, attention and other neuropsychological behaviour that results in a lower IQ than is normal for the individual s age and level of education, without interfering with daily chores 43,76,79,80,82,170,173. The pathophysiology behind MCI includes resistance to insulin, defeective glucose metabolism, lack of insulin, oxidative stress, and these are similar with DM pathophysiology 172. The fasting blood glucose levels and amyloid beta 42 levels in MCI patients have been found to be linearly correlated with each other, suggesting a link that is perhaps causative 82. To evaluate the cognitive impairment over time in MCI, the association between DLE and WMA can be used as a general guideline WHITE MATTER ABNORMALITIES IN DIABETIC BRAIN: The Rotterdam study was one of the pioneers in establishing a strong link between WMA and cognitive impairment 17,55,190. The study determined that WMA elevates the risk of incident dementia. Many other population-based studies also support this data 84. WMA is a condition which can be detected pathologically, but during life, it can be demonstrated using neuroimaging 13

29 techniques such as fractional anisotropy using diffusion tensor imaging (DTI) or with T2- weighted MRI of the brain 92. Moreover, it is a risk factor for abnormalities in gait and balance, and stroke 11, DIABETIC BRAIN AND CEREBRAL ATROPHY: Cerebral atrophy is a proven pathological manifestation of chronic diabetic brain with a 10% higher prevalence in diabetic patients compared to non-diabetics 85. It occurs in both type 1 and type 2 DM and worsens with poor diabetic control 1,85. Both animal and human studies have demonstrated significant brain loss as a consequence of impaired glucose metabolism in diabetes. Brain weights of diabetic animals show significant loss of weight compared to nondiabetic animals and MRI data could suggest possible correlation with the brain weight findings. Histological studies show microstructural changes that demonstrate the processes of synaptic loss, demyelination and ultimately loss of brain volume 20,190. Many forms of cognitive impairment are associated with changes similar to those in diabetic brain, with the obvious reason being impaired insulin signaling in the DM brain. Insulin receptors localized in brain are mostly concentrated in areas such as the hippocampus, frontal cortex, hypothalamus, and many other regions important for memory and cognition 87. Based upon its actions and the localization of its receptors, insulin plays important roles in maintaining neuronal health and function 16,87. However, the exact reason for DM-mediated cerebral atrophy is still unknown in clinical studies; both cortical and subcortical microscopic and macroscopic atrophy are present in DM. This atrophy may relate to loss of synapses or neurons in the DM brain. DM in contrast to early Alzheimer s disease, shows medial temporal lobe atrophy 1,87. Evidently there are insulin 14

30 receptors at central synapses which demonstrate the potential role for neuronal insulin in the inhibition of synaptic loss and consequent neuronal loss 38. Synaptic loss is a feature of diabetes, which may correspond to brain atrophy observed in DM mouse/rat brains. However, neuronal loss is not an established feature of diabetes; it could be a late temporal consequence of synaptic loss 38,120, DIABETIC BRAIN IMAGING: Neuroimaging studies have been a significant benefit for further understanding of brain structure and function. Magnetic resonance imaging (MRI) and computerized tomography (CT) scans are invaluable non-invasive techniques. Previously any structural study in brain had to be done after harvesting brain tissue of animals and there could be no follow up after preliminary studies, however functional MRI (fmri) examines brain function in vivo, permitting more efficient studies of cognition. There are many different forms of MR images and associated software available now which can permit assessment of both white matter and gray matter tracts in brain 16,73,86,92-94,189. These new tools permit identification of disease processes such as infarction, oedema, blood circulation, impact of different oxygen concentrations in blood on brain and many more conditions. As there is no radioactivity used in MRI, it is safe and is useful for regular serial use in animals and humans. We have used three sequences of MRI scans for our current study, namely: diffusion tensor imaging (DTI), T2 weighted images and magnetization transfer ratio (MTR) to detect white matter changes and brain volumes in diabetic brain. A brief overview of these imaging techniques with their principle of application is provided hereafter. 15

31 The principle of MRI scans is to detect the radio frequency signal emitted by the excited hydrogen atoms present in brain tissue. When applied at the appropriate resonant frequency, the water molecules in body are excited by a strong oscillating magnetic field. Gradient coil magnetic fields are used for maintaining the orientation of image. As the magnetic field changes, the atoms get excited and different tissue contrast is detected by determining the rate at which the atoms return to their equilibrium state. In T2 images, the local difference in hydrogen nuclei per unit tissue volume is used as the source of MR scans. T2 is the transverse relaxation time of hydrogen protons after being excited by the magnetic field induced by the gradient coils at a specific radio frequency (RF). T2 values represent the local de-phasing rate of hydrogen ions within a specific number of nuclei. T2 maps are usually obtained from multi-spin-echo images at repetition time (TR) = 1200 ms, 12 echoes and echo time (TE) = 12.5 ms. The field of view usually used is 2ˣ2 cm, with an acquisition matrix of 256ˣ256 and a slice thickness of 0.75 mm 16. The more structured the brain tissue, the more rapidly the de-phasing occurs, so we get shorter T2. T2 values are an excellent guide for determining demyelination, axonal loss, gliosis and increased free water in brain tissue as all of these produce high T2 signals in T2 weighted images. T2 images are excellent for determining brain atrophy as these scans could be used for brain volumetric measurements 73,93. T2 relaxation time is shortened (short T2) by intact biological tissues, like brain, due to their bonds with macromolecular structures or it can be longer when protons may have free movement (e.g. in water, long T2). For this reason, it is wiser to detect early brain injuries by histology rather than using conventional MRIs as the scans would be insensitive at the earliest insult. Magnetization transfer (MT) protocol uses imaging sequences by sending off-resonance RF pulses to saturate the restricted proton pool, resulting in an exchange of magnetization 16

32 between the free and restricted protons causing changes in the intensity of MR signals which depend on the rate and degree of this exchange. Both T2 weighted images and MTR can be analyzed by locally available Marevisi software (Marevisi, IBD). MTR gives information in evaluating integrity of different tissue structures, particularly white matter. Reduction in MTR values is a useful guideline in disease conditions like multiple sclerosis within white matter lesions and regions of demyelination. Tissue changes observed in cerebral ischemia or infarction following stroke could be detected by lower MTR values. Lower brain tissue MTR values are associated with diabetes both in human and rodents 94. Diffusion tensor imaging (DTI) measures the degree and directionality of movement of water molecules within brain tissue and fractional anisotrophy (FA) values can be calculated from these images. FA values measure this restriction, which varies from 0 to 1. When protons in water molecules move along a structure (like sipping water through a straw) the FA value is 1 and when the protons in water move in every direction, without any specific directionality, the vector of movement is zero, FA value is 0 in that case (imagine throwing a stone in water and the water cannot move at all). Thus, for isotropic diffusion in cerebrospinal fluid, FA would be 0, and alongside a properly myelinated white matter tract the FA would be 1. Reduction in FA refers to an increase in water diffusion in the direction perpendicular to the white matter tracts and this means demyelination (degeneration of white matter tracts) and/or loss of axonal fibers by other cells. DTI measures the mean diffusivity, in other terms the overall diffusion of protons in one particular direction as well as the degree of diffusion of protons in multiple directions, the direction of the greatest amount of diffusion (which is regarded parallel to the orientation of white matter tracts in that MRI voxel). White matter tracts in brain are consisted of densely packed axons (neuronal projections) as well as many neuroglial cells and few other cell types 17

33 and water molecules are dispersed in between these intracellular and extracellular spaces. As a general rule water should run parallel to intact myelinated white matter tracts rather than across or through it. So FA used in DTI is well suited to measure the white matter microstructure in brain. DTI is used to measure brain microarchitecture changes in stroke, Alzheimer s disease, brain tumor and multiple sclerosis DIABETIC BRAIN AND OLIGODENDROCYTES: Oligodendrocytes are the central myelinating cells. Diabetic brain atrophy, neuronal losses are closely linked to loss of central myelin 98,136. There is higher content of (NF)κB in oligodendrocytes found in the DM rodent brain as compared to non-dm rodent oligodendrocytes 16. This can be reversed by intranasal insulin delivery which causes insulin receptor ligation and probably acts through the PI3K-Akt pathway causing activation of Akt which leads to oligodendrocyte progenitor proliferation 16,147,98. GSK3β is another downstream regulator of insulin receptor ligation. GSK3β phosphorylation is imperative for oligodendrocyte stability and regulation of gene expression 125,141,130,178,179,187. Consequently, insulin may promote myelination, protecting against WMA development 141, EXPERIMENTAL ANIMAL MODELS WITH DIABETIC ENCEPHALOPATHY: DM research uses many animal models, but rodents are the most often examined animals 12,16,97,125. Other than mice and rats, larger animals like rabbits and dogs are also used for these studies. Animals (mice, rats) are usually injected with toxins like streptozotocin (STZ) or 18

34 alloxan that induce hyperglycemia through pancreatic toxicity. Inbreeding of animals (transgenic/wild type) produce strains that are considered good models for type 1 or type 2 DM with the desired clinical manifestations of obesity and/or insulin resistance 16,97,164. Although murine models are used extensively for diabetes research, they do not totally mimic human diabetic conditions and complications 12. Keeping these limitations in mind, researchers prefer using the murine models as they are by far the best and most economical models for non-human type 1 DM. Also, these models are robust, permitting several months of study. Animal models allow us direct access to tissues, breeding control and gene alteration, and the testing of novel long-term therapies throughout the lifespan of the rodents 12,16. In humans, type 1 DM is characterized by autoimmune destruction of the insulinproducing pancreatic β-cells in the islets of Langerhans. Initially there is gradual partial onset of insulinopenia which leads to subtotal or total insulinopenia. Experimentally we have both spontaneous and artificially induced type 1 DM models. Genetic manipulation leads to spontaneous diabetic rodent model, however artificial diabetic models must be induced by destroying pancreatic islet cells either with chemical toxins or with surgical ablation. The most established drug for inducing type 1 DM in mouse models is STZ, it is a β-cell cytotoxic drug injected intraperitoneally at a measured dosage as overdose might lead to instant death (it is very toxic). STZ is derived from Streptomyces achromogenes (a species of gram-positive bacterium that belongs in the genus Streptomyces) and is an alkylating agent. It is a nitrosourea compound with broad-spectrum antibiotic and anti-neoplastic activity. Its mechanism of action is very powerful for living organisms and it exerts its function by interfering with glucokinase function and glucose transport, and by inducing DNA damage which leads to cell death or malfunction. The glucose transporter GLUT-2 takes up STZ inside the cell and is found in high concentration 19

35 in the insulin producing pancreatic β-cells 50. These cells have a high nicotinamide adenine dinucleotide (NAD + ) content, which is highly sensitive to STZ, hence administration of this drug leads to destruction of islets of Langerhans cells leading to insulinopenia. The blood-brainbarrier lacks GLUT-2 transporters; hence brain is partially immune from the direct toxic effects of this drug 100,101. To inject STZ in lab animals and achieve diabetic status there are reliable protocols for avoiding toxicity/overdose 16. The safest way is to give multiple low doses over consecutive days to induce destruction of the pancreatic β-cells by STZ with only a small amount of residual insulin persisting 16. Typically, these STZ mice would have hyperglycemia and low levels of endogenous insulin which correlates with type 1 DM in humans. Like type 1 DM in humans, STZ-injected mice develop diabetic retinopathy, nephropathy, neuropathy and many other metabolic disorders seen in diabetic populations. Diabetic animal models have similar cognitive impairments to chronic diabetic human patients, and a battery of behavioural tests can be done to evaluate the difference from controls. These tests evaluate the loss of problem solving skills and poor comprehension, impaired procedural memory; and deficits in learning. Memory is assessed by the latency to refrain from crossing into the punished compartment 114. Type 1 DM mice have been studied in our lab and Dr. Toth found that over time these animals lost their cognitive function in terms of visuospatial and procedural tasks 16. The performance in different behavioural tasks was dependent on the duration of DM and severity of hyperglycemia and intensive insulin therapy helped preventing them. The cognitive behavioural tests used in our study have been used in many previous studies for evaluating cognition in mice. STZ-models of type 1 DM chronically loss insulin signaling in brain due to downregulation of insulin receptors 16,33,49,60,119,129. Our lab uses mice injected with STZ to induce experimental type 20

36 1 DM in them. In our experimental models sustained hyperglycemia is maintained over 8 months (equivalent to about human years) (Personal communication, Dr. Cory Toth). 2.4 PATHOPHYSIOLOGY OF DIABETIC LEUKOENCEPHALOPATHY IN THE EXPERIMENTAL ANIMAL BRAIN: The pathophysiology of diabetic brain is not yet understood; however, many pathways have been postulated to be potentially significant for the pathological and pathophysiological changes occurring in diabetic encephalopathy. Studies have looked into the PI3K (phosphoinositide-3-kinase)/akt (protein kinase B) /CREB pathway 98,99,126, AGE-RAGE pathway (advanced glycation end products binding to its receptor, RAGE) 14,16,72,104,105,107, MARK (mitogen activated protein kinase) pathway 98,185, mtor (mammalian target of rapamycin) pathway and the PI3K/Akt/Bad pathway 180 (figure 1). Insulin is believed to be the central activator for each of these pathways. It is anticipated that all these pathways converge; causing the chronic diabetic brain changes and contributing to cognitive impairments. Many recent studies have indicated a direct link between insulin and its improper signaling to be associated with these chronic brain changes leading to DLE and AD from chronic diabetes 16,33,125,187. The insulin pathways in brain involve many protein kinases which lead to phosphorylation or activation of critical transcription factors. Insulin binds to insulin receptor (IR) and phosphorylates IR substrate (IRS) which activates the above mentioned insulin pathways in brain. The major nodes in insulin signaling would be IR/IRS, PI3K, and Akt 111,186. In diabetic rodent brain the downstream regulators of the insulin pathway are downregulated which include PI3K, Akt, pakt, pcreb, and pgsk3β 16. Preliminary data from our lab have not 21

37 suggested the possibility of mitogen activated protein kinases to be involved in chronic changes in the DM brain (personal communication: Dr. Toth). The brain requires proper insulin signaling. Insulin, IR, IDE and the signaling pathway itself can be targeted by many other molecules actively functioning in brain. IDE is a protease that degrades amyloid beta protein; accumulation of which is a cardinal feature of AD and thus it could be the possible link between AD and DM; it has been shown that there is a reduction of IDE in DM compared non-dm brain 75,112,124. Further research is needed to clarify the role of IDE in DLE and its association with chronic brain deterioration leading to memory deficits. Previously insulin was regarded as an endogenous hormone that principally regulates blood glucose levels only, but recent findings have broadened our knowledge revealing that insulin and insulin receptors (IRs) are important for many other cellular mechanisms. The central nervous system has a high concentration of IRs located mainly at the neurons, including the synapses, and upon glia, particularly in the areas proven to be associated with memory and learning, namely: cerebral cortex, olfactory bulb, hippocampus, amygdala and septum 16,87. Activation of hippocampal IRs can lead to enhanced memory and learning. In fact, chronic learning leads to specific increases in the expression of IRs and insulin-signaling pathways in the hippocampus 123,171. Hippocampal neurons undergo neurogenesis, synaptic plasticity and insulin might play a role in this. Passive-avoidance task memory can be enhanced by intracerebroventricular insulin injections 114, and cognitive impairments in STZ-injected mice can be prevented by intranasal insulin supplementation 16. As well, human memory can be improved by intravenous or intranasal delivery of insulin 87,115. So it is possible that insulin is an important neurotrophic factor in the management of central diabetic complications. Nevertheless, the exact 22

38 mechanism for the neuroprotective role of insulin for diabetic brain is not yet known and most of the information available is focused on the glycemic regulatory role of insulin only INSULIN IN OUR BRAIN: The discovery that both human and rodent brains have IRs and insulin can cross the blood-brain-barrier by a receptor mediated transcytosis, drew the attention of the scientific community back to the central roles of brain insulin and insulin receptors 118. We now know that brain is sensitive to insulin. The specific localization of brain insulin receptors have helped establish specific roles designated for insulin in the brain. The rate of insulin entry into the brain is altered by many pathological and physiological mechanisms. For example, insulin transportation in brain is high at neonatal periods, with decreases during aging, starvation, obesity, and in chronic neurodegenerative diseases like AD 87. So, insulin transport is clearly an important aspect of DLE that needs further research BRAIN INSULIN RECEPTOR EXPRESSION PATTERN: Insulin signaling in brain begins with binding of insulin to IRs. IRs are a family of tyrosine kinases (TK) including insulin-like growth factor (IGF) receptor and the insulin receptor-related receptor (IRR) 87. The central IRs have similar properties to those in the periphery. These receptors are tetrameric proteins with two α and two β subunits acting as allosteric enzymes. Insulin binds to the α subunit resulting in autophosphorylation and activation 23

39 of the TK moiety of the β subunit. There are some structural and functional differences between central and peripheral IRs. Both the α and β subunits of peripheral and glial insulin receptors have slightly higher molecular weight than central subunits. Also, the central receptors do not show downregulation such as is seen with the peripheral receptors subject to hyperinsulinemia 87. The distribution of IRs in brain is highly concentrated in areas like the hypothalamus, olfactory bulbs, and hippocampus; however, these receptors could be found in other brain areas with variable concentrations. The IRs have high concentrations at synaptic densities, suggesting their possible role in synaptic plasticity, neuronal functioning, neurogenesis and synaptic transmissions 87. The IR concentration depends on the stage of brain development, for instance it has been shown that IR concentration increases in the thalamus during neurogenesis, with down regulation in adulthood, suggesting its role in early embryonic development and synaptic plasticity and synapse formation 87. The insulin signaling cascade is the same in both central and peripheral IRs. First, these receptors are phosphorylated by the binding of insulin which is followed by phosphorylation of the specialized adapter protein, insulin receptor substrate (IRS) on tyrosine residues, and it activates downstream pathways and regulates many different biological responses like glucose transport, mitogenesis, protein synthesis and cell survival 26. The IRS protein family consists of six similarly structured proteins (IRS1-6) with many different functions and tissue distributions in the conduction of insulin hormonal response 121,122. IRS1 and IRS2 are mostly located universally, IRS3 is distributed upon adipocytes 87. IRS4 is found in embryonic tissues or cell lines, and IRS5 and IRS6 are specific to their restricted distribution, function and insulin signaling 87. IRS1 is the most important in the context of brain as it is widely distributed in both brain and spinal cord 121. The important structures worth mentioning in the context of IRS1 are 24

40 hippocampus, thalamus and hypothalamic nuclei, basal ganglion, cerebral cortex and brainstem nuclei. Chronic hyperinsulinemia leads to reduction of IRS1 expression in cultured neurons as well as in the peripheral tissues in mice, which suggests that IRS1 might be a crucial factor for regulating the development of neuronal insulin resistance THE GSK3β AND CREB PATHWAYS IN INSULIN SIGNALING: There are many insulin signaling pathways each with their own unique characteristics, however they all share several signaling components that affect one another in complex manners. The PI3K-Akt pathway is the major pathway responsible for metabolic action of insulin 98,126. MAPK pathway regulates cell growth and proliferation via acting through activation of the ERK/MEK, Raf, CDC42, and JNK ,98,110,185, mtor pathway is involved in activation of Akt and protein synthesis 181,183,184, and BAD is a pro-apoptotic protein of the Bcl-2 family which promotes apoptosis in its non-phosphorylated state 180,196 (figure 1). In the PI3K/Akt pathway of insulin signaling activated PI3K phosphorylates Akt 74. In mouse models, phosphorylated Akt (pakt) activates/inactivates other kinases, signaling proteins and transcription factors, including GSK-3 (glycogen synthase kinase-3) 187 and CREB (camp response element binding protein) 126, Activation of the PI3K-Akt pathway inactivates its downstream kinase GSK-3 by phosphorylation. Akt phosphorylates isoforms of GSK-3 (serine-9 of GSK-3β; serine-21 of GSK-3α) and inhibits GSK-3 activity 125. Activated GSK3 phosphorylates transcription factors for cell proliferation, differentiation and apoptosis. Inactivation of GSK-3β protects brain from tau protein hyperphosphorylation and intracellular accumulation of tau. Phosphorylated tau accumulates and forms neurofibrillary tangles that disrupt cytoskeletal (microtubule) integrity of neurons 125. Active GSK-3 helps in amyloid beta 25

41 (β-amyloid) protein accumulation and brain damage, along with phosphorylated tau formation, both of which are the cardinal findings of AD. This suggests that impaired insulin signaling can be directly linked to early AD 125,187. In addition, phosphorylated GSK-3 may assist in regulation of protein synthesis 125. β-amyloid plaques are also associated with increased insulin resistance in association with obesity 131. Therefore, higher β-amyloid plaque frequency at the periphery will prevent cells from utilizing glucose due to insulin resistance, in turn causing hyperglycaemia. β- amyloid plaques are also known to destroy pancreatic β cells which also leads to insulinopenia and in turn to diabetic metabolic disorders 131. Thus, inhibition of GSK3β activation by phosphorylation may be neuroprotective by acting as the main modulator of insulin signaling, preventing accumulation of tau protein and β-amyloid plaques both in brain and in the periphery 187. CREB is another important downstream transcription factor associated with the insulin/pi3k-akt pathway. It is activated by phosphorylation at Ser133 by the PI3K-Akt pathway. CREB is localized within the nucleus and is essential for stimulus-transcription coupling and activated CREB leads to series of cellular events happening on the cell membrane which assists with a change in gene expression. CREB can regulate individual neurons or the entire neuronal circuit by controlling neuronal protein synthesis. It interacts with numerous other intracellular pathways in order to transmit information initiated by membrane receptor-mediated actions to the cell nucleus. Among them, the effects of signaling pathways involving camp, Ca 2+, MAPK 98,185, Mtor 181,183,184, BAD 180 on CREB and CREB-regulated gene transcription have been well studied 132,135. CREB may be responsible for memory processes but the mechanism is still unclear - this could involve processes such as the induction of long-term potentiation or depression of synaptic strength, the growth and formation of new synaptic 26

42 connections 113,138, or protein synthesis-dependent processes involved in the retrieval and reconsolidation of memory 127. The precise target genes of CREB to help learning and memory within various brain regions of animals require further study. Our focus on PI3K-Akt pathway has been persisted because of its role in synaptic plasticity and maintaining normal neuronal efficacy in terms of insulin signaling. Our ability to store and recall memories depends highly on synaptic plasticity; including the processes of long term potentiation (LTP) and long term depression (LTD) of synaptic transmission which performs strengthening or reduction of synaptic efficacy respectively. LTP is directly maintained by PI3K signaling and LTD can be highly influenced by PI3K signaling. Prior work by Toth lab has demonstrated that downregulating the upstream regulatory factors of PI3K-Akt pathway (PI3K and Akt) directly influences memory, learning, and cognitive behaviour (work done by Derakhshan). Upregulating this pathway has just the opposite effect on the chronic diabetic brain. The downstream regulators of PI3K-Akt pathway, pcreb, and pgsk3β are known to diminish in the experimental diabetic animal brain models and it might be corrected by administering intranasal insulin. Our study will evaluate the function of downstream of PI3K-Akt pathway modulators GSK3β and CREB in the pathogenesis of chronic type 1 DM brain, in order to determine if controlled modulation of these two protein molecules can assist with maintenance of normal neuronal housekeeping function in the DM brain MODIFYING THE GSK-3β AND CREB PATHWAY OF INSULIN SIGNALING: 27

43 GSK3β and CREB are thought to be the downstream molecules most proximal to insulinmediated transcription factor modulation. For our research, we wish to have molecular disruption or enhancement of these molecules. TDZD-8 is a small, soluble, highly selective, non-atp competitive inhibitor of GSK3β which selectively binds to GSK3β 140.Protein phosphatase 1 (PP1), the main dephosphorylator (inactivator) of CREB, can be highly, selectively and specifically inhibited by tautomycin (previous work in Toth lab).there are some other modulators of CREB/ GSK3β, but they have insolubility, poor specificity, or intolerable side effects or toxicity limiting their use. Systemic effect of insulin upon blood glucose control is not entirely dependent on the modulation of PI3K-Akt pathway. We chose to work with intranasal TDZD-8 because it is a well-established molecule that has been used in lab experiments for inhibiting GSK3β activation without any major side effects 140,141. For our experiments with CREB, we used forebrain CREB knockdown transgenic mice and administered intranasal insulin as treatment of chronic diabetic brain. Mice having lower expression of CREB (forebrain CREB knockdown transgenic) should be lacking the overall beneficial effects of insulin administration in the brain if CREB is critical to insulin s actions in the DM brain. 2.5 INTRANASAL DELIVERY OF CHEMICALS TO THE CENTRAL NERVOUS SYSTEM: 28

44 Intranasal delivery of non-toxic drugs is a noninvasive method to avoid systemic side effects of insulin or other peptides and therapeutics 16,28,30,31,87,115,117,142,146,148 that can be directly delivered to the CNS, avoiding systemic effects by bypassing the blood-brain-barrier. In 1989, William H. Frey II first proposed intranasal delivery of drugs as a novel route to bypass conventional systemic routes. After this many studies reported intranasal route as a potential pathway for delivering therapeutics effectively to the brain target regions directly without any systemic side effects. This route has been proven beneficial for the treatment of many neurological disorders and diseases concerned with brain. Intranasal route of insulin administration was actually proposed to avoid painful subcutaneous injections but later it was discovered that to get the appropriate bioavailability in systemic circulation insulin needs to be administered along with mucoadhesives, enzyme inhibitors or absorption enhancers because of the natural barriers in the nasal passage 87. But these additives caused nasal irritation and repeated large dosages were needed to maintain normoglycemia (normal blood glucose levels) which forced studies to abandon this route for the treatment of diabetes. Many years later this route was suggested as a novel method for delivering therapeutics for the treatment of AD and it has been shown to improve memory, attention and cognitive function within 21 days of initiation of intranasal insulin (I-I) treatment 30,117,146. We know that severe hypoglycemia might lead to coma and death and its consequences are more severe than hyperglycemia in most cases, hence treating brain insulin deficiency through systemic administration of insulin is unacceptable as it might lead to uncontrollable hypoglycemia leading to devastating coma and potential brain injury 27,32. Repeated administration of I-I did not cause hypoglycemia which made it effective for treating specific CNS disorders only; without any significant systemic disasters. Our lab has also found that I-I is more effective in treating CNS disorders compared to systemic routes as it avoids 29

45 mortality by lowering risks of systemic hypoglycemia and achieves higher brain concentrations than systemic route. It shields diabetic brain from neurodegeneration and thus protects behavioral detriment 16. The exact pathway of insulin from nasal cavity to CNS is still unclear but supporting evidence suggests that pathways that connect nasal passage to brain, especially the Olfactory and Trigeminal nerve pathways, are most important 87,149,150. To administer insulin directly into the brain, intracerebroventricular injections could be another way but this is highly invasive, requires high degree of aseptic precautions and extremely inconvenient for regular prolonged use. Keeping these in mind, I-I might be the best way to avoid most of the side effects and maintain normal brain function OLFACTORY NERVE PATHWAYS IN INTRANASAL ADMINISTRATION OF DRUGS: Insulin travels to central brain following nasal administration by olfactory and trigeminal neurons. The dendrites of olfactory neurons extend into the olfactory epithelium mucous layer within the nose, while their axons pass centrally through the subarachnoid space containing CSF and synapse with mitral cells in the olfactory bulbs. From here, the neural projections extend non-specifically and diffusely to the anterior olfactory nucleus, olfactory tract, piriform cortex, amygdala, and hypothalamus 87,149. It is thought that the intranasal delivery of drugs follows either an extraneuronal or intraneuronal pathway but the exact mechanism is still a puzzle. When rats were given intranasal fluorescent tracers; the tracers underwent transcellular absorption across the olfactory epithelium and they were transferred to the olfactory bulb, within only several minutes. However, it has been observed that intraneuronal drug transport within the nerve tract is slow and takes several hours 142,149,150. Hence the fast transmit (within minutes) of 30

46 intranasal administration of drugs could not be explained with the already available experiments in intraneuronal pathways. Interestingly it has been observed that extracellular mechanism of drug transport takes only several minutes. Hence it is suggested that the intranasal drugs might travel in the extraneuronal pathway. There is monthly regeneration of the olfactory receptors as they have direct contact with toxins externally-which gives us a leaky blood-brain-barrier due to their persistent turnover. However, the olfactory neuron ensheathing cells, which are oligodendroglia-like cells, have minimum turnover keeping the axons of olfactory receptor neurons surrounded creating continuous, fluid-filled perineurial channels which remain exposed, allowing travel along their length as well TRIGEMINAL NERVE PATHWAYS OF INTRANASAL INSULIN: Following intranasal administration of drugs they travel via trigeminal nerve as this nerve innervates the respiratory and olfactory areas of nasal passage 87,142,150. The trigeminal nerve also sends some branches which terminate in the olfactory bulbs. Thorne et al. used I-IGF-I to exhibit intranasal conveyance of drugs to the brain along trigeminal pathways and observed high concentrations of radioactivity in the nerve branches, cervical spinal cord, trigeminal ganglion, pons and medulla 87. Intranasal studies with other drugs, including interferon-β1b, hypocretin-1, and peptides, also showed high levels of radioactivity in the trigeminal nerve

47 2.6 MOLECULAR STUDY: Western blot analysis was used for molecular study. It is a semi-quantitative, protein immunoblot technique, and critical for its ability to detect specific proteins in a sample of tissue homogenate or extract. Gel electrophoresis is the method used in western blot to separate denatured proteins by their weight and length of the polypeptide molecule. Many commercially available monoclonal or polyclonal antibodies are available now-a-days to detect specific proteins 162,192. We sacrificed our animals to harvest brain tissue for molecular analysis. Whole brain was dissected two halves (right and left cerebrum) and each half was separated into four regions, namely: cortex, hippocampus, brain stem and thalamus, and cerebellum; and stored in -80 C. The goal of our molecular study was to evaluate the brain insulin pathway players and also to look for our proteins of interest. We used cortex and hippocampus to look for our desired proteins as the treatments used were anticipated to have the highest desired effects in those two areas of mouse brain and also the proteins of interest were anticipated to have high concentrations in those areas. I performed western blot analysis with the brain tissue collected from CD1 mice treated with either TDZD-8/placebo (2 nd aim) to look for the proteins: GSK3β, pgsk3β, CREB, pcreb; and also actin to use as control for normalization and comparative comparison. The tissue samples from forebrain CREB-Knockdown mice (1 st aim) were ran for western blot analysis by Dr. Alma Rosales (Toth lab technician) to look for the similar proteins mentioned above as well as PI3K, Akt, and pakt. All western blot data collected from both the aims were analyzed by Dr. Cory Toth. 32

48 2.7 PRELIMINARY DATA AND PREVIOUS WORK DONE IN OUR LAB: Dr. Cory Toth ran the preliminary data in our lab during 1 month. Non-DM young mice were divided into groups of 5 and given the GSK3β inhibitor TDZD-8 (50µg/day)/placebo for equal volumes. No morbidity, mortality or glycemic changes were identified. Mice brains were harvested for cortex and hippocampus portion, and tissues were processed for protein blotting and electrophoretic mobility shift assays (EMSA). In each case, 6 lanes of protein were analyzed for each grouped intervention. Mouse brains receiving intranasal TDZD-8 (50µg/day) demonstrated elevated levels of pgsk3β compared to placebo treated group. No chronic change was observed in alternative insulin-mediated MAPK pathway in the diabetic brain consisting of ERK, MEK and JNK. Other unpublished research projects were done by two other students under Dr. Cory Toth s supervision and contribute to the hypotheses here within: (a) An investigation of the role of insulin deficiency and loss of PI3K-AKT pathway in the pathogenesis of diabetic brain by Fatemeh Derakhshan. The role of insulin was found to be dependent on both PI3K and Akt activity in the brain. (b) The effect of promoting CREB phosphorylation in the in the diabetic brain using tautomycin was described by Bryan Duong (BSc honors thesis). Here, there was a small effect on prevention of cognitive decline by enhancing CREB phosphorylation in the diabetic brain. The effects noted were smaller in magnitude than with Derakhshan s work supporting PI3K and Akt signaling. 33

49 Figure 2: Major insulin signaling pathway in brain (our hypothesis) Figure 2 showing major insulin signaling pathway in brain and drug (TDZD-8) that can be used to upregulate the pathway, thereby modulating effects of this pathway function in brain. CHAPTER THREE: HYPOTHESIS AND OBJECTIVE Insulin s role in normal neuronal well-being and neuronal plasticity is dependent on insulin signaling pathways and the pathway we are interested in is PI3K-Akt pathway of insulin signaling. For my thesis project, I was interested to explore downstream of this PI3K-Akt pathway and explore more if there is a direct correlation between the downstream molecules of this pathway and the neuroprotective effects of insulin s mechanism of action. My proteins of interest were phosphorylated/non-phosphorylated CREB and GSK3β. C57BL/6 transgenic mice 34

50 with downregulated forebrain CREB activity were used for my first aim and we gave them I- I/placebo (intranasal saline, I-S) as treatment. For the second aim, wild type CD1 mice were given intranasal TDZD-8 (a potent GSK3β inhibitor)/placebo (DMSO). In both the aims, the evaluating factors for effective insulin signaling in brain were a battery of cognitive behavioural tests, end point MRI studies and molecular analysis of brain tissue (western blot analysis).my main research hypothesis and objectives were as follows: 3.1 Main research hypothesis: The neurodegenerative changes and cognitive impairments seen in the DM brain occur through impaired insulin-mediated phosphorylation of CREB/GSK3β, critical neuronal signals downstream in the insulin-pi3k-akt pathway, and direct support of these proteins will prevent cognitive dysfunction, cerebral atrophy and WMA in the diabetic mouse brain (figure 2). 3.2 SPECIFIC OBJECTIVES/AIMS: OBJECTIVE/AIM 1: To examine transgenic mice deficient of forebrain specific CREB activity in the type 1 diabetic mice brain receiving intranasal insulin.for this study, I separated the C57BL/6 transgenic mice into three different groups, each receiving different treatment protocols: (a)11 STZ (type 1 DM) CREB knockdown mice were treated with I-I, (b)11 STZ (type 1 DM) CREB knockdown mice were treated with I-S; and (c)10 non-stz (non-dm) CREB knockdown mice 35

51 were treated with I-S. These mice were forebrain CREB knockdown and they had intact CREB activity in rest of the body. All the mice were monitored for changes in cognitive function along with endpoint MRI studies. No significant behavioral or structural improvement in brain was expected in the three mice groups because they were all CREB knockdown in the forebrain and insulin was not expected to exert significant beneficial effect without activated CREB (figure 3) OBJECTIVE/AIM 2: To determine if GSK3β inactivation by TDZD-8 has neuroprotective effects in respect of cognition in type 1 diabetic mouse brain. For this study wild type mice with CD1 background were divided into 3 main groups with different treatment protocols: (a)13 STZ (type 1 DM) CD1 mice were treated with TDZD-8, (b)15 STZ (type 1 DM) CD1 mice were treated with Placebo; and (c)12 non-stz (non-dm) CD1 mice were treated with Placebo. Similar to objective one, mice were monitored for behavioral changes with endpoint MRI studies. TDZD-8 is a potent GSK3β inactivator, hence we anticipated potential cognitive and structural improvements in the STZ group receiving TDZD-8 compared to the STZ group treated with placebo only. After harvesting brain tissue, I used western blot analysis technique for molecular analysis of my desired proteins; namely: GSK3β, pgsk3β, CREB, pcreb and actin as control (figure 3). 36

52 Figure 3: Flow chart showing study design with objective 1 and objective 2 CHAPTER FOUR: PLAN AND METHODS 4.1 DM and non-dm Mice: Mice with an initial body weight of 20-30g were enrolled in the studies and all mice, DM/non-DM, were tested monthly for body weights for 7 months after treatments ensue. For first objective, C57BL/6 background of transgenic mice were used, ordered from Jackson Laboratory, Bar Harbour, Maine and for second objective, CD1 background mice from Charles River, PQ were ordered. All mice, at 1/1½ month of age, were injected with intraperitoneal STZ 37

53 (Sigma, St. Louis, MO); or equal volumes of citrate buffer as placebo after fasting for eight hours on each day for three consecutive days. STZ was dissolved in a citrate buffer solution (ph=4.8) (Sigma-Aldrich Co., St.Louis, Missouri) minutes prior to injections for each of three consecutive day injections with once daily doses of 60 mg/kg, 50 mg/kg, 40 mg/kg to induce DM. Mice in non-dm groups were injected with citrate buffer solution. Important precautions were taken to not to injury any vital organs like kidney, liver, gut or any major arteries or veins during intraperitoneal injections. Mice well-being was assured by monitoring them for about 8 hours post injections. A 8 hours overnight fasting whole blood glucose level of 16 mmol/l, was our criterion for experimental diabetes 13,16. Blood glucose levels were assayed using the Accu-Chek Active Blood Glucose Meter by pricking tail veins. Considering that about 5% of STZ mice might not develop DM; mice that developed hyperglycemic state within 30 days post STZ injections were continued in the study; with other mice excluded. Monthly blood glucose levels for 7 months and endpoint glycosylated hemoglobin (HbA1C) values (indicator of high blood glucose concentrations over maximum 3 months period of time) were checked to ensure chronic DM. 4.2 CREB KNOCKDOWN TRANSGENIC MICE: For my first aim, we used one commercially available transgenic model of C57BL/6 mice. These animals were forebrain knockdowns. B6.Cg-Tg(Camk2a-Crebbp*)1364Tabe/J mice are hemizygous for the CaMKIIa-FLAG-CBP 1 transgene (CREB knockdown), leading to a dominant negative truncation mutation of the CREB-binding protein (FLAG-CBP 1), spatially directed to forebrain neurons (including hippocampus) and temporally directed to postnatal 38

54 development by the CaMKIIa promoter ((C57BL/6 x SJL)F1 phenotype, Jackson Laboratory, Bar Harbour, Maine. These mice are believed to have normal short-term memory, but had abnormalities in contextual conditioning, spatial learning, and reduced hippocampal long term potentiation. CREB knockdown mice were obtained as breeding pairs for colony establishment, with hemizygote mice bred together and identified through genotyping. 4.3 CD1 WILD TYPE MICE: For my 2 nd aim, we used CD1 background of wild type mice. These mice were ordered from Charles River, PQ and were used to block the downstream of PI3K-Akt pathway of insulin signaling by TDZD-8 which is a potent GSK3β inhibitor. TDZD-8 administrations was supposed to upregulate insulin signaling and improve brain function, including memory, learning, cognitive behaviours and other paradigms of brain proteins observed in molecular studies in chronic type 1 DM. 4.4 INTERVENTIONS: A sample size for intervention groups was calculated based upon convenience and differences in brain atrophy observed in DM and non-dm mice to date 16. We decided on a minimum sample size of n= 8 per group considering an α of 0.05 and β of 0.5 using observed mean and standard deviation. Bearing in mind the possible mortality of STZ-induced DM mice after seven months of DM, we decided to study mice in each DM group and mice in each non-dm group

55 I-I (Humulin R 100U/ml By Lilly Eli & Co) or commercially available DPBS (Dulbecco's Phosphate-Buffered Saline) was used in our 1st aim and we used commercially available TDZD-8/DMSO (Dimethyl sulfoxide) for our 2 nd aim. DPBS/DMSO was used as placebo/solvent for insulin/tdzd-8 respectively. All intervention dosages were decided based upon the success of our previous studies done by Dr. Toth. No other management of diabetes occurred throughout the mice lifetime in our experiments 16. Observer was blinded about all the experimental groups to avoid biases, with blinding removed only after all data assessments were performed. 0.87U/d I-I or equal volume I-S (DPBS) was given to each of the first objective group mice. 6 drops of insulin/saline 5μ each at 6 consecutive minute intervals were given using a 20- µl pipettor in alternate nostrils. Equipment like ViaNase electronic atomizer or needleless syringe is currently used in AD to deliver insulin to the brain via intranasal route in humans, but these are neither available nor practical in mice 87. During intranasal delivery of treatments, positioning of the head is very important to avoid ingestion or entry into the respiratory tract leading to respiratory depression. Other confounding factors for intranasal approach might be method of delivery, delivery volume and surgical interventions for lab animals. The nasal cavity has high vascularity and the maximum chance of absorption occurs with delivery of the drug in the upper third of the cavity and olfactory epithelium. Keeping the head at 70 degrees with the spinal axis while body is in a supine position gives the best positioning. Although this might cause drug to be drained into esophagus/and trachea. Considering the limitations we kept our mice supine with fully extended neck. To immobilize un-anesthetized mice while dosing, they were lifted gently by the scruff of their necks, holding them firm in the palm of our hands to prevent unnecessary movement which might lead to treatment failure due to failure to administer 40

56 at the right route/improper dosing. The efficacy of drug absorption also depends on anatomical conditions like deviated nasal septum, nasal polyp or infectious conditions like allergic rhinitis regardless the appropriate amount or position. Also, continuous nasal administration of different formulations may lead to nasal mucosal irritation; anosmia and scratching for irritation might lead to epistaxis in rodents. The solvents used in different nasal formulations should be nonirritant, like normal saline, DPBS or DMSO to avoid complications. Olfactory regions in the rodent nasal cavity are more extensive (about 50% of the nasal cavity surface area) as compared to humans (8%). This has to be considered while translating rodent work into human studies. For our second aim, 50µg/day TDZD-8 dissolved in DMSO/equal volume DMSO was administered in the same methods mentioned above. 4.5 BEHAVIOURAL TESTING: A battery of four different cognitive behavioral tests was performed to add validity to our result interpretations. The goal of these tests was to study visuospatial and procedural memory in mice. These four tests were: Morris water maze, radial arm, hole board, and object recognition. The tests chosen had been used previously to investigate learning and memory in mice 1,16,88,151,152,155, each test evaluated different skills to account for the potential confounding factors of diabetic complications. Spatial information processing and reference memory was evaluated by hole board and radial arm tests 1,16, while the Morris water maze was used to assess spatial information processing, procedural memory and aversive motivation 1,13,16,88,151. On the other hand, the object recognition test evaluated novelty seeking and exploratory behaviour 1,13,152,155. Mice were trained at age 1/1½ months for 10 days prior to their testing and before DM was established in them. The tests were continued for 7 months in 1 st aim and 5 41

57 months in 2 nd aim. Specific instruments, same visual cues and designated lab space were used for the behavioral tests. The following order was maintained while testing: Hole board test, radial arm test, object recognition test (each performed while fasting over night for 8-10 hours) followed by feeding and then the Morris water maze test 1 h later. All mice were tested once weekly during day time in between 7am-6pm 16. All the instruments were cleaned in between each animal trial with 70% ethanol to prevent the possibility of scent traces forming an olfactory cue. Tasteless, odourless and colourless Cheerios were used as food reward. Overnight fasting was done to encourage food seeking behavior in mice. Age, diabetic complications, stress etc. reduced their exploring performance over time. The hole board test is based on exploratory/seeking behavior, and a rectangular open field (60 X 90 cm) made of cardboard walls of 60 cm height and an opaque black floor with eight holes (2.5 cm diameter) which were placed in two lines of four, equidistant from each other and from the walls; was used 13,16. The mouse was placed always in the same corner of the box (close to hole #4 and the longest distance from hole # 8) every time this test was run keeping the food reward always in the same hole (hole # 7). The mouse had to go to the hole containing the food reward and the latency to find food hole was timed. Also recorded were the number of incorrect visits (mistakes) and the sequence of exploration. The maximum time for training was 10 minutes and for tests was 3 minutes 13. One of the confounding factors of this test could be that mice are nocturnal animals and their activity might be hampered during day time due to change in circadian rhythms. Also mice are scared of the open field used in this test and hid in corners. We observed mice to be exploring mostly at the corners (especially the holes# 4,5,8 were visited maximally by them in an attempt to escape/find food reward) (figure 4.3). According to our,observations while training, hole #7 was usually the least visited by the mice and we anticipated 42

58 that while testing if a mouse actually remembered where the food was placed, it will directly go to that hole without making mistakes (exploring all the other holes). The radial arm test is based on natural foraging tendencies of mice. The test instrument consisted of a central platform with 8 radiating arms (each 76 cm long and 12 cm wide) with adjacent arms separated by 45, which served as runways 13,16 (figure 4.4). Latency to find the food containing arm was recorded including mistakes and sequence of exploration. A visit was considered if a mouse entered half-way through a runway. Food reward was located in a single runway 180 from the initial constant starting point. The maximum time for training and for test was 5 minutes. Both the radial arm and the hole board tests evaluated similar brain regions including the hippocampal complex for initial spatial information processing and learning, the dorsal striatum for procedure comprehension, and the neocortex for long-term memory storage 13,16. Same confounding factors as mentioned before were observed and over time mice were found sitting in any non-specific runways without exploring the maze. Considering the limitations for hole board test and radial arm test, we discarded number of mistakes and sequence of visits data from our analysis and repeat visit of a previously visited hole/runway was regarded as reference memory error 13,16. The Morris water maze test assesses hippocampal-based learning and memory in diabetic mice 13,16. We trained the mice for 10 consecutive days, 3 minutes/day, to be adapted to the pool water and the water temperature (25-26 C) 13,16 prior to testing. A coloured circular pool (88 cm in diameter and 20 cm in height) was filled with water keeping a clear 10 cm radius platform hidden 1cm below the surface of the water (figure 4.5). The mouse was always placed in the pool 90 opposite to the platform at the same position. Latency to reach the hidden platform was recorded. Brain regions involved included the hippocampal complex for initial spatial 43

59 information processing and learning, the dorsal striatum for procedure comprehension, the amygdala for escape behaviour, and the neocortex for long-term memory storage. The object recognition test was performed in a square wooden box (60 x 60 x 60 cm) which was colored black from inside with a transparent plastic floor and children s lego blocks were used as objects (figure 4.2). This test was done in two steps each for 3 minutes at more than 30 minute interval. In the first step, two identical colourful lego blocks were placed inside the box close to the center and mice were let to explore them for 3 minutes. In the 2 nd step, one of the identical object was replace by a new lego object of different shape, size and colour. Time spent at each object (T= novel object exploration time, t= familiar object exploration time) was recorded for the 2nd step. A visit was considered to occur when a mouse directed its nose to the object at a distance 2 m or when it touched the object 13. Usually the idea was that, in the 1 st step mice would explore both the objects equally as they were identical and they will not be biased to explore any one of them more over the other. When new object was introduced in the 2 nd step, it was expected that mice would explore the new object more compared to the familiar one as novelty seeking is an inborn behavior with intact memory. Failure to explore novel object more over the familiar object would represent a lack of short-term memory. The main brain regions involved in this test were the perirhinal cortex located in parahippocampus that was important for visual perception and visual memory and the hippocampal complex for short-term memory processing. 4.6 MAGNETIC RESONANCE IMAGING (MRI): 44

60 Endpoint MRI scans were performed on four mice from each cohort from the first aim at the Experimental Imaging Centre (EIC) at University of Calgary using quadrature volume coil and a Bruker 9.4 Tesla and 21 cm bore MR imaging system. Isoflurane anesthesia was used before scan and respiration rate and temperature were monitored during the procedure. Unfortunately one mouse from non-dm cohort died while scanning. All the scans were performed by David Kirk and Tad Foniok at the EIC using their standard protocol. T2-weighted images were obtained in the same way as described before in the background section and were used to analyze white matter tracts and whole brain volume. Volumetric brain measurements were calculated as a summation of cross sectional areas for each slice multiplied by the thickness of the MR slices 16,93. MT ratio (MTR) was calculated as MTR= {(Mo Ms)/Mo)} 100%, where Ms and Mo were the signal intensities obtained with and without MT saturation, respectively 94. For DTI, six different orientations were applied as diffusion sensitizing gradients. DTI used a TR of 6.5 s, an TE of 35 ms, and 6 signal averages to acquire a series of diffusion-weighted images at b = s/mm2 in 30 different directions including the acquisition of five Ao images (b=0). DTI images were acquired for 0.5 mm thick slices, using a 2x2 cm 2 field of view and a 128x128 matrix 90,95,159,160. All the MRI scans were analyzed by software mentioned before. Brain regions of interest (ROI) were used bilaterally for measurements for each of the control and DM animals. Brain areas like whole brain, primary motor cortex (M1), hippocampal regions (CA1-CA3), caudate/putamen (CPu), primary somatosensory cortex (S1), corpus callosum (CC), internal capsule (IC), posterior commissure (PC), anterior commissure (AC), pons, and cerebellum were 45

61 evaluated using ROI. Representing regions are well recognized as regions that become abnormal within diabetic brains, as well as cortical and subcortical regions important in memory and cognition 16,36,73,86,92,154. Results between groups were compared using one way ANOVA with significant p SACRIFICE OF ANIMALS: All mice were sacrificed at the end of behavioural studies; followed by immediate cardiocentesis (collection of blood from the heart). After that we did immediate decapitation followed by decerebration. Brains were harvested and weighed followed by dissecting into regions, kept in separate specifically identified tubes, and put in either dry ice or stored in a -80 C freezer until molecular analysis. Collected blood samples were sent for HbA1C levels (Calgary Laboratory Services). 2% vaporized isoflurane anesthesia was used prior to sacrifice and regular 1ml sterilized syringe was used for blood collection. 4.8 MOLECULAR ANALYSIS: Western blot analysis technique was used for molecular analysis of our harvested brain tissue 192. Tissue preparation was done at ice cold temperature to prevent protein denaturing. The western blot protocol followed by our lab was: A) Protein extraction and quantification 46

62 B) Casting of gels C) Sample preparation for electrophoresis D) Electrophoresis (separation of proteins) E) Transfer (transfer of proteins from gel to membrane) F) Blocking G) Detection H) Analysis Protein extraction and quantification Samples were weighed and then broken down manually by forceps tips. We used 20-30mg of brain tissue from each animal. Usually the lysis buffer RIPA, protease, phosphatase and glass beads are added to the samples and put in a bullet blender for mechanically breaking them down into smaller pieces. For our specific proteins namely: CREB, pcreb, GSK, pgsk and actin; we replaced RIPA and phosphatase with phosphosafe to prevent degradation of tissue phosphorylation and protect our proteins of interest. After that, samples were cooled down in a cold room in -4 C at 1300 rpm (ramp per minute) for 13 minutes, followed by collection of the supernatant. Next, cuvettes were arranged accordingly, commercially available reagents A and B were added and BCA (protein analysis kit, known strength) was used as standard. After this, the cuvettes were put at 37 C in anoven for 30 minutes, followed by analysis of the samples within 10 minutes for protein by detecting absorbances in a spectrophotometer. The absorbances were used in a designated chart to determine protein levels in the samples. 47

63 Gel Preparation: We used 10% APS (ammonium persulfate), TEMED (tetramethylethylenediamine), 8% acrylamide, sodium dodecyl sulfate (SDS) and Tris buffers for gel preparation. After washing plates, cast holder, combs, gaskets and glass plate holders very well with soap and water, we wiped them with ethanol and set the instruments for casting gels. TEMED+APS were added to running gel and poured immediately into glass plates avoiding bubbles. 20% ethyl alcohol was added to flatten the gel and eliminate bubbles. The gel was let to set for about 25mins and ethyl alcohol was removed. Next we added stacking gel, comb and let it set for about 20 minutes. Later we removed comb, wrapped it with wet towel and stored in plastic bag in 4 C. Sample preparation for electrophoresis and running gel: Samples were mixed with loading buffer in a proportion 1:1 in 0.5ml tubes. We boiled samples in 95 C for 3 minutes, followed by spinning them down and let them cool. Next we prepared 700 ml running buffer, assembled tank with gels and buffer, loaded 9µl of marker and sample into the wells and ran electrophoresis at room temperature with 70V(volt) for 20-25mins, then changed to 100V until the desired separation. Transfer of proteins from gel to membrane: For this step, we prepared 800ml transfer buffer, stored it in 4 C, assembled the tank and added a stirring bar. After soaking the polyvinylidene difluoride (PVDF) membrane in methanol, we prepared the cassette, inserted it into the tank and transferred protein for 2hours at 100V in the cold room. After transfer we stored the membrane either in room temperature after drying with blotting paper or blocked the membrane with milk solution. For transfer electroblotting was used as the primary method. 48

64 Blocking Our blocking solution consisted of 3-5% Bovine serum albumin (BSA) in Tris-Buffered Saline (TBS), with a minute percentage (0.1%) of detergent Tween 20. Probing membrane with primary and secondary antibodies: Probing of the membrane was done after blocking it for 1 hour at room temperature, post activation with 50% methanol. We used primary antibody and incubated for 1 hour, rinsed and washed membrane 3 times each 14 minutes for this step. Next, secondary antibody was added at recommended dilutions and membrane was incubated another 1 hour followed by washing it 3 times for 10,9,8 minutes each. Following this step, we added reagent PerkinElmer to develop bands. Membrane stripping: In between detecting two different proteins by specific antibodies, we added Pierce stripping buffer to our membrane followed by 3 times wash with TBS and blocked it for 1hr. This ensured clarity of detection, reduced noise and made detection more specific avoiding false positive/negative results. Detection: 49

65 We used the dark room to develop our western blot films to identify the bands present. Different exposure times were used to ensure correct protein bands for CREB, pcreb, GSK3β, pgsk3β and actin. This detection can be discussed in two steps:. 4.9 ANALYSIS OF DATA: Cognitive behavioural data were analyzed by two-way repeated measure ANOVA with post hoc Bonferroni correction (significance with p value 0.05) using software SPSS. To determine difference at each monthly time point, one way ANOVA (significance with p value 0.05) was done using the software GraphPad Prism. All other data were analyzed with one way ANOVA (significance with p value 0.05) by the software GraphPad Prism. Difference between group means was compared using a t test. All error bars in our study represent SEM TIMELINE: For the first aim with forebrain CREB knockdown mice, weekly behavioural tests were performed for 7 months starting from age 1/1½ month. Monthly body weight and blood glucose levels were recorded. At about 6 th month of the behavioural testing, in vivo MRI scans were done. After 7 th month of cognitive tests, we sacrificed our animals, collected blood, harvested brain tissue and did molecular analysis of the tissue samples (figure 4.1). For the second aim with CD1 wild type mice receiving intranasal TDZD-8/placebo, we performed 5 months of cognitive behavioural tests from age 1/1½ month of the mice and sacrificed them after the 6 th month of behavioural tests. Brain tissue and blood were collected. 50

66 Following tissue collection, western blot analysis was done with the tissue samples from the 2 nd aim (figure 4.1). Figure 4.1: Timeline and methods for our study Figure 4.2: Left, object recognition test with familiar object. Right, object recognition test with novel and familiar object (courtesy: Dr. C. Toth Dr. Toth) 51

67 Figure 4.3: Hole board test (courtesy: Dr. C. Toth) Figure 4.4: Radial arm test (courtesy: Dr. C. Toth) Figure 4.5: Morris water maze test (courtesy: Dr. C. Toth) 52

68 CHAPTER FIVE: RESULTS 5.1 EXPERIMENTAL GROUP CHARACTERISTICS Our initial protocol was to examine three different cohorts of mice with different interventions. In my first aim, forebrain-specific CREB knockdown mice were tested. Forebrain-specific CREB knockdown non-dm (control, n = 10) mice were injected with citrate solution as placebo using I-S intervention. STZ-injected (DM) forebrain-specific CREB knockdown mice received either I-I (n = 11) or I-S (n = 11). Mice were injected using 60, 50, 40 mg/kg body weight STZ/citrate solution for 3 consecutive days. About 7 days following the last injection mice reached the required glycemic effect ( 16mmol/l for DM and 6-8mmol/l for non- DM). Mice were fasted overnight (~8-10 hours), with blood sugar levels determined 7-10 days following final STZ/citrate injection. The mortality rate for non-dm forebrain-specific CREB knockdown mice was 10%, the mortality rate for both DM with I-I/I-S was 18% (figure 5.1). All mortality rates were below our expected mortality rate (20-25%), hence we proceeded with the numbers we had in each cohort and collected data. For the 2 nd aim, CD1 wild type mice were tested. CD1 wild type non-dm (control, n = 12) mice were injected with citrate solution as placebo using intranasal placebo (DMSO) intervention. STZ injected (DM) CD1 mice received either intranasal TDZD-8 (n = 13) or placebo (DMSO) (n = 15). The same procedure as above was observed for ensuring desired glycemic effects in all 3 cohorts. The mortality rate for non-dm placebo mice was 0%, the mortality rate for both DM groups receiving either intranasal TDZD-8 or placebo was 26.67%, which was slightly higher than our expected mortality rate (20-25%) but negligible (figure 5.1). Hence we proceeded with the numbers we had in each cohort and collected data. Data from any 53

69 animal that experienced mortality after the 12-week point of the cognitive studies were carried through using the last obtainable data point. All DM mice appeared very sick, emaciated and cachexic but the CD1 mice had more grave diabetic metabolic complications compared to C57BL/6 mice. The initial protocol for both the aims was to run cognitive behavioural tests for 7 months, end point MRI at 6 th month of study, and sacrifice of the animals after 7months of study followed by blood and brain tissue collection. 1 st aim was completed according to the initial protocol (figure 4.1). But for our 2 nd aim, after the 5 th month, due to sudden break out of a lethal virus at the mouse halfway house (the affected animals were operated by a different lab) where we used to house our mice, our animals were shifted to bio-safety facility and we were no longer allowed to bring the mice outside the bio-safety facility to perform our regular behavioural tests and MRI scans. Hence, we had to sacrifice our animals and harvest the tissue and collect blood on the 6 th month. We could not perform MRI scans because of the mentioned situation which was beyond our control. Our mice were not affected by the virus, only by the situation. We had a 3 rd aim for which we ordered GSK3β knockout CD1 mice from Jackson s laboratory, but unfortunately we did not receive genetically modified mice as were ordered. After extensive genotyping in our laboratory, there was a significant delay of over 8 months before we could receive the correct mice. Although these mice continued to be studied, it could not be performed within the scope of my MSc project. As a result, my project was modified with the approval of the committee to include more molecular work, including western blots of my harvested brain tissues from the prior works (figure 4.1). 54

70 Figure 5.1: Final experimental group numbers with intervention and mortality rates 55

71 5.1.1 BLOOD GLUCOSE LEVELS IN CREB KNOCKDOWN MICE (1 ST AIM): Fig 5.1.1A shows the monthly blood glucose levels for forebrain-specific CREB knockdown mice. The green line represents non-dm control I-S group with normoglycemia (6-8momo/l) throughout 7 months of study. The blue line in fig: 5.1.1A represents DM with I-I group with hyperglycemia maintained throughout the study. The red line in fig: 5.1.1A refers to DM with I-S group with hyperglycemia throughout the time of study. The data represents significant difference among control vs. DM with I-S and control vs. DM with I-I and no difference among DM with I-I and DM with I-S. The data in fig 5.1.1A was analyzed as group mean with SEM and difference between groups was analyzed by one way ANOVA (p <0.05). At endpoint, HbA1c (glycated hemoglobin) levels for all diabetic mice were significantly higher than the non-diabetic mice (fig: 5.1.1B). Time Fig 5.1.1A: Monthly blood glucose levels (ANOVA, p <0.05) Fig 5.1.1B: Glycated hemoglobin level (ANOVA, p <0.05) 56

72 5.1.2 BODY WEIGHT IN CREB KNOCKDOWN MICE (1 ST AIM): The initial body weight for each C57BL/6 forebrain CREB knockdown mouse was in the range grams (fig 5.1.2). However, after STZ injection, the diabetic mice showed maintained lower body weights compared to non-dm controls which consistently gained weight throughout study. There was no significant difference between the two diabetes cohorts (one way ANOVA, p<0.05). Figure 5.1.2: Body weights for forebrain-specific CREB knockdown mice 57

73 5.2 RESULTS OF PI3K-Akt/CREB PATHWAY PHARMACOLOGICAL MODULATION (1 ST AIM: FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE): We used forebrain-specific CREB knockdown mice to evaluate the efficiency of administering I-I in absence of CREB as we hypothesized that CREB activation is necessary for proper insulin signaling and knocking down CREB specifically at the forebrain will prevent the beneficial effects of I-I intervention. As per figure 5.1, we divided our CREB knockdown mice into 3 groups with specific interventions. The observations of the experiment will be discussed hereby with data diagrams. 58

74 5.2.1 COGNITIVE BEHAVIOURAL ANALYSIS RESULTS (1 ST AIM) FOR FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE: RADIAL ARM TEST RESULT: Fig : Radial arm test for forebrain-specific CREB knockdown mice Figure shows radial arm test results for forebrain-specific CREB knockdown mice. The green line represents DM with I-S and Red line shows DM with I-I, both of which did not retain memory compared to non-dm control with I-S in Blue line. A significant difference was seen between DM mice and control in that retention of memory was independent of administration of insulin. Differences between groups were analyzed by a two-way repeated measure ANOVA, with post hoc test Bonferroni correction (significance with p <0.05). 59

75 HOLE BOARD TEST: Figure : Hole board test for forebrain-specific CREB knockdown mice Figure shows hole board test results for forebrain-specific CREB knockdown mice. The green line represents DM with I-S and red line shows DM with I-I, both of which did not retain memory as well as non-dm with I-S (blue line). Significant difference was seen between DM mice and control in respect of retention of memory and administering insulin had no beneficial effect. Data were analyzed between groups as mean and SEM by two way repeated measure ANOVA with post hoc test Bonferroni correction (p <0.05). 60

76 OBJECT RECOGNITION: Figure : Object recognition test for CREB knockdown mice Figure shows object recognition test results for forebrain-specific CREB knockdown mice. The green line represents DM with I-I and red line shows DM with I-S, both of which did not retain memory compared to controls in blue line. Significant difference was seen between DM mice and control in respect of retention of memory and administering insulin had no beneficial effect in improving memory for forebrain-specific CREB knockout DM mice over 7 months. Data were analyzed between groups as mean with SEM by two way repeated measure ANOVA with post hoc test Bonferroni correction (p <0.05). Here, T= novel object exploration time and t= familiar object exploration time. 61

77 MORRISON WATER MAZE TEST: Figure : Water maze results for CREB knockdown mice Figure shows Morris water maze test results for CREB knockdown mice. The green line represents DM with I-S and Red line shows DM with I-I, both of which did not retain memory compared to non-dm control with I-S in Blue line. A significant difference was seen between DM mice and control in that retention of memory was independent of administration of insulin. Differences between groups were analyzed by a two-way repeated measure ANOVA, with post hoc test Bonferroni correction (significance with p <0.05). 62

78 5.2.2 BRAIN WEIGHT: Whole brain weights from each cohort at the final endpoint demonstrated that control group with I-S had higher brain weights compared to both diabetic groups. There was no significant difference between diabetic cohorts with I-I or I-S with respect to wet brain weight (fig 5.2.2). To investigate the possible differences,, the group means (±SEM) by one way ANOVA (significance with p value <0.05). Figure 5.2.2: Forebrain-specific CREB knockdown mice bain weights (1 st aim) 63

79 5.3 MRI RESULTS FOR FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE (1 ST AIM): MRI BRAIN VOLUMES FOR CREB KNOCKDOWN MICE: Figure 5.3.1: MRI whole brain volumes for forebrain CREB knockdown mice We used MRI T2 weighted image values to measure whole brain volumes. No significant difference was observed in DM groups receiving either I-I or I-S, in respect of brain volumes. The DM mice had lower whole brain volumes compared to non-dm I-S controls. We randomly selected and analyzed 4 mice data from each cohort of DM mice and 3 mice from control group. No oedema was observed. To compare between groups we used mean with SEM and one-way ANOVA (significance with p value <0.05) (fig: 5.3.1). 64

80 5.3.2 MRI DATA (DTI, MTR, T2) ANALYSES FOR FOREBRAIN-SPECIFIC CREB KNOCKDOWN MICE BRAIN WHITE MATTER TRACTS: We had four mice from each of the DM mice cohorts and three mice from control non- DM cohort MRI scans for DTI, MTR and T2 weighted image analysis with the cardinal aim to explore mice brain white matter tracts and regional brain volume for gray matter regions. Fractional anisotropy (FA) values were used to explore DTI. As explained previously, in house software (MedINRIA, INRIA-Asclepios Research Team, v1.8.0) was used to analyze MRI scans {figure (a)} for FA values. General idea is if we have higher FA values in DTI, we anticipate that we have intact white matter tracts and the less the value, the more the damage to white matter tracts. The highest value for FA is 1 and the lowest is 0. The data in figure (b) shows that there was no difference in all three groups, namely: control non-dm receiving I-S, DM receiving I-I or DM receiving I-S; in respect of white matter tracts while exploring corpus callosum (CC), internal capsule (IC), CA1 region and CA2 regions (hippocampal regions) of the brain (these areas were chosen as they are expected to have highest white matter tracts and are the first to get affected usually by neurodegenerative disorders). To investigate possible differences, the data were analyzed by one-way ANOVA (significance with p value <0.05). 65

81 (a) Figure : (a) Showing DTI MRI scans 66

82 (b) Figure : (b) showing FA values obtained from the DTI scan analysis. Locally available software Marevisi (Marevisi, IBD) was used to evaluate T2 and MTR values. For T2-weighted image values with MRI scans, higher the values more the degeneration of white matter and lower the values less damage to the white matter tracts. We analyzed 11 brain regions {fig (c)} looking for lesions and the areas were: hippocampal regions (CA1- CA3), primary somatosensory cortex (S1), primary motor cortex (M1), caudate/putamen (CPu), corpus callosum (CC), internal capsule (IC), Anterior commissure (AC), pons, and cerebellum. We observed no significant differences in white matter tracts in all the three different cohorts {fig (d-e)}. To investigate difference the data was analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05). 67

83 (c) Figure : (c) Showing T2 weighted images of MRI scan 68

84 (d) Figure : (d) showing 5 out of 11 different brain regional values in milliseconds for T2 weighted images. 69

85 (e) Figure : (e) showing 6 out of 11 different brain regional values in milliseconds for T2 weighted images. 70

86 (f) Figure : (f) Showing MTR MRI scans 71

87 (g) Figure : (g) showing analysis of MTR scans for five out of nine different brain regions. 72

88 (h) Figure : (h) showing analysis of MTR scans for four out of nine different brain regions. For MTR, we analyzed nine different regions from MRI scans {figure (f)}, namely: hippocampal regions (CA1-CA3), caudate/putamen (CPu), corpus callosum (CC), internal capsule (IC), Anterior commissure (AC), posterior commissure (PC) and cortex. As found with T2 imaging, there were no significant differences between the three cohorts with respect to MTR values across these selected regions of interest {figure (g-h}. 73

89 Our results show that, despite the presence of diabetes, the forebrain-specific CREB knockdown diabetic mice maintained brain white matter structural integrity, unexpectedly, as well as forebrain-specific CREB knockdown non-diabetic mice. To investigate difference, the data were analyzed one-way ANOVA (significance, p <0.05) REGIONAL BRAIN VOLUME BY T2 WEIGHTED MRI IMAGES: Figure showing that the regional brain volumes (hippocampus and cortex) obtained analyzing T2 weighted images were different among three cohorts; higher volume in non-dm controls and significantly lower volume in both the DM cohorts. Administration of insulin had no influence over brain volume among the diabetic cohorts. These areas are highest in gray matter concentrations in brain, indicating that perhaps the loss of brain volumes among DM mice was due to loss of gray matter volumes. To investigate possible differences, the data were analyzed by one-way ANOVA (significance, with <0.05). Figure 5.3.3: (left) Hippocampal brain volume; (right) Cortex brain volume for forebrainspecific CREB knockdown mice. 74

90 5.4 WESTERN BLOT RESULTS FOR CREB KNOCKDOWN MICE: Figure 5.4 shows western blots that we obtained from our CREB knockdown hippocampal tissue analysis. We analyzed the blots using NIH imagej software and normalization was done with actin values. No significant difference was observed among all three mice groups in PI3K, Akt, pakt, CREB, pcreb, GSK and pgsk protein expressions when normalized by actin values. We observed very low expression of CREB proteins in all the mice hippocampal brain tissue samples, indicating our model for forebrain-specific CREB knockdown mice was valid. To investigate difference the data was analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05). 75

91 Figure 5.4: Showing western blots and results for forebrain-specific CREB knockdown mice hippocampal tissue (courtesy: Alma Rosales for western blot and Dr. Cory Toth for analyzing the data). 5.5 RESULTS FROM CD1 WILD TYPE MICE (2 ND AIM): BLOOD SUGAR LEVELS FOR 2 ND AIM CD1 WILD TYPE MICE TREATED WITH TDZD-8/PLACEBO (2 ND AIM): Monthly blood sugar levels revealed that the control non-dm with placebo group shown in blue line always maintained normal blood sugar levels which is 6-8mmo/L compared to both DM groups. The DM group treated with placebo (in green line) had significantly higher blood glucose levels (mostly at or above 25mmol/L) compared to the other diabetic group treated with TDZD-8 (in red line) (maintaining blood glucose levels very close to at or above 16mmo/L) (fig 76

92 5.5.1a). To investigate difference the data was analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05) at every monthly data point. Figure 5.5.1(a): Showing monthly blood sugar levels for CD1 mice treated with TDZD- 8/placebo 77

93 Figure 5.5.1(b): Showing glycated hemoglobin levels for CD1 mice End point HbA1C levels for all three groups of mice (figure 5.5.1b) showed control group with placebo was within the non-diabetic level and both DM groups had high glycated hemoglobin levels ensuring they were chronically diabetic. To investigate difference the data was analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05). 78

94 5.5.2 BODY WEIGHT IN CD1 WILD TYPE MICE TREATED WITH TDZD- 8/PLACEBO (2 ND AIM): Monthly body weight analysis (figure 5.5.2) of all three groups concluded that the control non-dm group gained body weight persistently over 5 months, whereas DM-placebo did not gain any body weight. On the other hand, interestingly, DM-TDZD-8 group did gain some weight which was significantly higher than other DM-placebo group; but not as high as control non-dm group. Significant differences were observed in weight between DM-TDZD-8 group and DM-placebo group as well as between DM-TDZD-8 group and control non-dm group. To investigate difference the data was analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05) at every monthly data point. Figure 5.5.2: Showing monthly weight of CD1 mice 79

95 5.6 RESULTS OF PI3K-AKT/GSK3Β PATHWAY PHARMACOLOGICAL MODULATION BY TDZD-8 (2 ND AIM): BEHAVIOURAL TESTS RESULTS FOR CD1 WILD TYPE MICE TREATED WITH TDZD-8/PLACEBO (2 ND AIM): We performed the same battery of behavioural tests (hole board, radial arm, object recognition and water maze) and analyzed the data as mentioned before. The mice were tested weekly for 5 months Hole board test: Figure : Showing hole board behavioural test results for CD1 mice Fig showing that control group receiving placebo (blue line) took significantly less time to find the food compared to DM placebo group (green line), indicating the latency to 80

96 reach food hole was shorter for controls and they retained the memory to find the food. DM TDZD-8 group (red line) performed as good as the control group and they took same amount of time to reach the food hole. Data analyzed between groups as mean with SEM in two-way repeated measure ANOVA with post hoc test Bonferroni correction (significance with p value <0.05) RADIAL ARM Figure : Showing radial arm test result for CD1 mice Fig showing that control group receiving placebo (blue line) took significantly less time to find the food arm compared to DM placebo group (green line), indicating the latency to reach the food arm was shorter for controls, and that they retained the memory to find the 81

97 food. DM TDZD-8 group (red line) performed as good as the control group and they took same amount of time to reach the food arm. Data analyzed between groups as mean with SEM by two way repeated measure ANOVA with post hoc test Bonferroni correction (significance with p <0.05) MORRIS WATER MAZE TEST: Figure : Showing Morris water maze result for CD1 mice Latencies to find hidden platform in water maze test were indifferent and shorter for both control non-dm group (blue line) and DM-TDZD-8 group (red line) compared to DM with placebo group (green line) who performed worse (fig ). Data analyzed between groups as mean with SEM by two way repeated measure ANOVA with post hoc test Bonferroni correction (significance with p <0.05). 82

98 : OBJECT RECOGNITION TEST: Figure : Showing object recognition test result for CD1 mice Fig shows that control (blue line) and DM-TDZD-8 (red line) mice retained novelty seeking behavior as compared to DM-placebo group (green line) who showed significantly poor performance in this test. Data analyzed between groups as mean with SEM in two way repeated measure ANOVA with post hoc test Bonferroni correction (significant with p value <0.05). 83

99 5.6.2 BRAIN WEIGHT OF CD1 WILD TYPE MICE TREATED WITH TDZD- 8/PLACEBO: Figure 5.6.2: Showing brain weight in CD1 mice Data analysis showed that, control group had higher brain weights compared to placebo diabetic group. On The other hand, DM-TDZD-8 had similar brain weights as the controls, denoting to the fact that TDZD-8 administration was neuroprotective for the diabetic animals (fig 5.6.2). To investigate difference the data was analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05). 5.7 WESTERN BLOT ANALYSIS OF CD1 MICE TREATED WITH PLACEBO/TDZD-8 (2 ND AIM): I performed western blot technique for molecular analysis of our proteins of interest (creb, pcreb, GSK, pgsk) for our 2 nd aim CD1 mice brain tissue. I used cortical (fig 5.7a) and 84

100 hippocampal (fig 5.7b) tissue samples for running the western blots and Dr. Toth helped with the software NIH ImageJ for analyzing the western blot films and also statistical analysis of the obtained data. Actin was used as control. We can observe visually from both the figures that there were high degree of discrepancies among the groups and no specific pattern could be observed. NIH ImageJ software measured the blots based on its optical density and area occupied in the films. All data were normalized first based on the lowest value from each group and later these values were again normalized by actin to prevent error and complications. The standard error of mean observed during manipulation of data made us believe that the results were not consistent among animals from the same group which made it ultimately non-conclusive data. After running the analysis, we concluded that, the results from running the western blot were highly variable and there was no pattern observed to come to a conclusion with the results. Administering TDZD-8 did not increase pgsk3β levels as we expected, which indicates that the improved behaviour/brain weight observed in DM TDZD-8 mice compared to the DM placebo mice, may not be due to blockade of GSK3β activation by phosphorylation but some other pathways through which TDZD-8 exerted its neuroprotective beneficial effects. TDZD-8 might be involved in inactivating GSK3 in some other ways rather than via phosphorylation. TDZD-8 had no significant effect on CREB or pcreb protein expressions. To investigate difference, the data were analyzed as group mean with SEM by one way ANOVA (significance with p value <0.05). 85

101 (a) (b) Figure 5.7: (a) Western blot of cortex tissue from CD1 mice with statistical analysis; (b) Western blot for hippocampal tissue from CD1 mice with statistical analysis of data (western blot done by T. Alrazi, data analysis done by Dr. C. Toth ) CHAPTER SIX: DISCUSSION AND CONCLUSION 86