Insulin Actions on Hippocampal Neurons

University of Kentucky UKnowledge

Theses and Dissertations--Pharmacology and Nutritional Sciences Pharmacology and Nutritional Sciences

2017

INSULIN ACTIONS ON HIPPOCAMPAL NEURONS

Shaniya Maimaiti University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2017.366

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Recommended Citation Maimaiti, Shaniya, "INSULIN ACTIONS ON HIPPOCAMPAL NEURONS" (2017). Theses and Dissertations-- Pharmacology and Nutritional Sciences. 20. https://uknowledge.uky.edu/pharmacol_etds/20

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REVIEW, APPROVAL AND ACCEPTANCE

The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above.

Shaniya Maimaiti, Student

Dr. Olivier Thibault, Major Professor

Dr. Rolf Craven, Director of Graduate Studies INSULIN ACTIONS ON HIPPOCAMPAL NEURONS

______

DISSERTATION ______

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky

By Shaniya Maimaiti

Lexington, Kentucky

Director: Dr. Olivier Thibault, Professor of Pharmacology and Nutritional Sciences Lexington, Kentucky 2017

ABSTRACT OF DISSERTATION

INSULIN ACTIONS ON HIPPOCAMPAL NEURONS Aging is the main risk factor for cognitive decline. The hippocampus, a brain region critical for learning and memory formation, is especially vulnerable to normal and pathological age-related cognitive decline. Dysregulation of both insulin and intracellular Ca2+ signaling appear to coexist and their compromised actions may synergistically contribute to neuronal dysfunction with aging. This dissertation focused on the interaction between insulin, Ca2+ dysregulation, and cognition in hippocampal neurons by examining the contributions of insulin to Ca2+ signaling events that influence memory formation. I tested the hypothesis that insulin would increase cognition in aged animals by altering Ca2+- dependent physiological mechanisms involved in learning. The possible effects of insulin on learning and memory in young and aged rats were studied. In addition, the effects of insulin on the Ca2+-dependent afterhyperpolarization in CA1 pyramidal hippocampal neurons from young and aged animals were compared. Further, primary hippocampal cultures were used to examine the possible effects of insulin on voltage-gated Ca2+ channel activity and Ca2+- induced Ca2+-release; mechanisms known to influence the AHP. We found that intranasal insulin improved memory in aged F344 rats. Young and aged F344 rats were treated with Humalog®, a short-acting insulin analog, or Levemir®, a long-acting insulin analog. The aged rats performed similar to young rats in the Morris Water Maze, a hippocampal dependent spatial learning and memory task. Electrophysiological recordings from CA1 hippocampal neurons revealed that insulin reduced the age-related increase in the Ca2+-dependent afterhyperpolarization, a prominent biomarker of brain aging that is associated with cognitive decline. Patch clamping recording from hippocampal cultured neurons showed that insulin reduced Ca2+ channel currents. Intracellular Ca2+ levels were also monitored using Fura-2 in response to cellular depolarization. Results indicated that a reduction in Ca2+-induced Ca2+-release from intracellular stores occurred in the presence of insulin. These results suggest that increasing brain insulin levels in aged rats may have improved memory by reducing the AHP and intracellular Ca2+concentrations. This study indicates a possible mechanism responsible for the beneficial effects of intranasal insulin on cognitive function absorbed in selective Alzheimer’s patients. Thus, insulin therapy may reduce or prevent age-related compromises to Ca2+ regulatory pathways typically associated with cognitive decline.

KEY WORDS: Insulin, Aging, Cognition, Intracellular Ca2+, Ca2+ imaging, Whole-cell patch clamping

Shaniya Maimaiti Student’s Signature 08/04/2017 Date INSULIN ACTIONS ON HIPPOCAMPAL NEURONS

Shaniya Maimaiti

Dr. Olivier Thibault Director of Dissertation

Dr. Rolf Craven Director of Graduate Studies

08/04/2017 I dedicate this thesis to my daughter. Alina you are my pride and joy. ACKNOWLEDGEMENTS

Foremost, I would like to thank my extraordinary mentor Professor Olivier

Thibault for his patience, motivation, enthusiasm, and wide range of knowledge.

He is the funniest advisor and a smart, experienced scientist. I thank him for always focusing on my best interest and working closely with me on my research projects, for giving me the guidance, insight, and skills I needed.

Thank Dr. Thibault for guiding me through all the challenges I have faced in research and personal life. I learned from Dr. Thibault 1) to do good science 2) to be a good team player 3) to fix things 4) to help others 5) give back to society by becoming involved with local community services. Dr. Thibault’s excellent mentorship was the key for all my learning progress and accomplishments in graduate school.

I would also like to thank Professor Bret N. Smith. He served as my primary advisor before I started in the graduate program. Dr. Smith introduced me to neuroscience when I was undergrad, and without his encouragement, enthusiasm, and support I would not be where I am today. I will always be grateful for him for believing in me and seeing me as a potential neuroscientist.

I would also like to thank Dr. Nada M Porter, Chair of Pharmacology and

Nutritional Sciences, for encouraging me and advising me during one of the most difficult times of my life. I never forgot her and I hope to be as kind, lively, enthusiastic, and energetic as she is. I am also very grateful for her direction

iii and support on my research projects.

I would also like to thank Dr. Robert W. Hadley for teaching me in graduate

courses and serving as one of my committee members. I am grateful for his

immense knowledge, guidance, and constructive feedback on my graduate training. I also very thankful for his fun jokes during our meetings which made

them less intense and more enjoyable.

I would also like to thank Dr. Alexandre Martin for being my outside examiner.

I would also like to thank Dr. Rolf J. Craven, Director of Graduate Studies for the

Department of Pharmacology and Nutritional Sciences, for keeping his office

door open for me and keeping me on track.

I would also like to thank Dr. Donna Weber, former Director of Graduate Studies

for the Department of Pharmacology and Nutritional Sciences, for providing so

much support and guidance. She always goes the extra mile and beyond to

help students.

I would also like to thank Veronique Thibault for being there for me and guiding

me thought the hardest time in my life. Importantly, she helped me bring my

daughter Alina into my life. Without her help and encouragement, I couldn’t put

myself together and move forward. When nothing worked in my life and I felt

lost, she guided me through the darkness and “pumped me up”. I thank her for

being someone I can trust and depend on. This dissertation could not be

completed without her help and support.

iv I would also like to thank Dr. Lawrence Brewer. I truly appreciate the mentorship and friendship he gave me all these years. Whenever things did not work out well in the lab or in my personal life, he was the first person I talk to. Because he always said right thing and knew how to pick me up. He didn’t fail me once.

Importantly, whenever I had questions related to research or life, he always has the answers for me, and I will always be grateful for his help and guidance.

I would also like to thank Dr. Chris Gant for teaching me the techniques, knowledge in the field, and for helping me troubleshoot any problems I had in the lab. Also, I thank him for sharing with me his hard times as a graduate student to ease my frustrations.

I would also like to thank past and present members of the Thibault Lab (Dr.

Tristano Pancani, Chris DeMoll, Katie L Anderson, Ben Rauh, Hilaree N Frazier,

Sarah Sternbach, Adam O Ghoweri, and Grant A Fox) for providing me help and friendship.

I also thank department for creating a great atmosphere for research and graduate training.

Thank to Miss Kelley M Secrest and Miss Deborah J Turner, for helping me through various problems related to my training as well as their guidance and friendship throughout graduate school.

I would also like to thank my friends, who made these years so much exciting and fun, for your friendship through both good and bad times.

v I would also like to thank Miss Toni and Miss Alison, for taking great care of

Alina while I have completed my education.

I would also like to thank my parents for encouraging me to pursue my dream.

Also, I am grateful to them for giving me everything I needed in life. I am also grateful for my in-laws for taking care of Alina while I worked and studied.

I like to thank my husband and daughter for nurturing me and pushing me hard to graduate as soon as possible. I deeply appreciate their comfort and love.

vi TABLE OF CONTENTS

Acknowledgement ...... iii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1 Thesis Overview and Significanc ...... 1

1.1 Brain Aging and cognition ...... 2

1.2 Insulin ...... 3

1.3 Zinc ...... 4

1.4 Insulin formulations and alternative routes for insulin administration...... 4

1.5 Insulin signaling in the periphery ...... 7

1.6 Insulin signaling in the brain ...... 11

1.7 Insulin receptors in the brain vs. in the periphery ...... 13

1.8 Insulin synthesis in the brain ...... 15

1.8.1 Peripheral source of insulin ...... 15

1.8.2 Central source of insulin ...... 16

1.9 Metabolism and the brain ...... 17

1.10 Intracellular calcium levels ...... 20

1.10.1 Voltage-gated Ca2+ channels ...... 21

1.10.2 Structure and classification of voltage-gated Ca2+ channels ...... 23

1.10.3 Voltage-gated Ca2+ channels activation and inactivation ...... 26

1.10.4 Ryanodine receptors ...... 27

1.10.5 Ryanodine receptor isoforms and expression ...... 29

vii 1.10.6 Structure of ryanodine receptors ...... 30

1.11 Hippocampus ...... 30

1.12 Insulin actions on neurons ...... 32

1.13 Thesis significance ...... 33

Chapter 2 Intranasal insulin improves age-related cognitive deficits and reverses electrophysiological correlates of brain aging ...... 36

2.1 Introduction ...... 37

2.2 Methods ...... 40

Animals: ...... 40

In vivo insulin delivery and doses: ...... 40

Blood glucose levels ...... 41

Animal behavior treatment groups and Morris water maze (MWM)

procedures: ...... 42

Electrophysiology ...... 44

Field potentials ...... 52

AHP-Intracellular recordings ...... 53

Statistics...... 55

2.3 Results ...... 55

Blood analyses ...... 55

Behavioral analyses...... 56

Electrophysiology analyses ...... 57

2.4 Discussion ...... 61

viii Chapter 3 Link between studies ...... 68

3.1 Intranasal insulin and cognition ...... 68

3.2 Insulin and Ca2+-dependent after hyperpolarization ...... 70

3.3 Voltage-gated Ca2+ channels dysfunction with aging ...... 71

3.4 Ryanodine receptors dysregulation with aging ...... 72

Chapter 4 Novel Ca2+-related targets of insulin in hippocampal neurons ...... 75

4.1 Introduction ...... 77

4.2 Experimental procedures ...... 79

Cell culture ...... 79

VGCC recording solution ...... 80

Drugs and solution application: ...... 81

Whole-cell recording and analysis: ...... 81

Ca2+ imaging and analysis ...... 82

Statistics...... 83

4.3 Results ...... 84

4.3.1 Effect of insulin on whole-cell Ca2+currents...... 84

4.3.2 Effect of insulin on spontaneous Ca2+ transients...... 87

4.3.3 Effect of insulin on KCl- mediated Ca2+ transients...... 89

4.3.4 Insulin appears to reduce CICR function...... 93

4.3.5 Insulin actions occur via occupation of the insulin receptor...... 95

4.4 Discussion ...... 97

4.5 Conclusions ...... 103

ix Chapter 5 Discussion and conclusion ...... 105

5.1 Effects of insulin on memory and Ca2+-dependent AHP ...... 105

5.2 Whole-cell patch clamping and Ca2+ imaging in hippocampal neurons 108

5.3 Insulin improves memory by regulating intracellular Ca2+ levels ...... 109

5.4 Aging-related changes in memory and hippocampal Ca2+ levels ...... 110

5.5 Potential relevance to healthy and pathological aging ...... 111

5.6 Study limitations ...... 112

5.7 Future directions...... 114

References ...... 116

Vita ...... 143

x LIST OF TABLES

Table 1. Insulin formulations ...... 6

Table 2. Ca2+ channels ...... 25

Table 3. Blood glucose analysis ...... 52

Table 4. Electrophysiological parameters ...... 86

LIST OF FIGURES

Figure 1.1 Mechanisms of Insulin Actions in the Periphery...... 8

Figure 1.2 Insulin signaling pathways and translocation of glucose transporter

(GLUT) to the plasma membrane by insulin...... 10

Figure 1.3 Actions of insulin in the brain...... 12

Figure 1. 4 The mechanism of insulin secretion by the pancreatic beta cell...... 16

Figure 1.5 Voltage-gated Ca2+ channels structure...... 23

Figure 1.6 Ryanodine receptors and cognitive function with aging ...... 28

Figure 2.1 Morris water maze spatial learning and memory task...... 46

Figure 2.2 Impact of in vivo intranasal Humalog® on intracellular and extracellular physiology...... 47

Figure 2.3 Impact of ex vivo Humalog® and zinc on the AHP...... 49

Figure 2.4 Zinc-free Apidra® reduces the AHP ex vivo...... 50

Figure 2.5 Intranasal insulin delivery and experimental timeline...... 51

Figure 4.1 Ca2+ channel currents are reduced in response to acute insulin application in mixed hippocampal cultured neurons...... 85

Figure 4.2 Spontaneous Ca2+ transients are not altered in response to acute insulin exposure...... 88

Figure 4. 3 Effects of insulin on KCl-induced depolarization...... 91

Figure 4.4 Ryanodine receptors are likely targets of insulin actions...... 94

Figure 4.5 Anti-insulin affibody peptide and anti-insulin receptor antibody inhibited insulin actions on KCl-mediated Ca2+ transients...... 96

xii

Chapter 1 Thesis Overview and Significance

Aging, an inexorable natural process, often presents many challenges ranging from ambulatory to neuronal function. One of these challenges, age-related cognitive decline has received significant public attention because of its profound social and economic impact. A well-recognized mechanism contributing to age- related cognitive decline involves dysregulation of intracellular calcium. Although

Insulin has been recognized as the key hormone regulating cellular glucose metabolism, its downstream effects on learning and memory have only recently been found. Some evidence suggests that insulin undergoes age-related changes that ultimately influence calcium regulatory mechanisms in various brain regions, including the hippocampus, an area of the brain closely related to learning and memory. Thus, the negative impact of brain aging on cognitive function may be due to impaired insulin signaling and intracellular calcium homeostasis. However, the link between insulin and intracellular calcium in the context of cognitive decline with aging is not understood. This dissertation focused on the effects of insulin on memory and its underlying neuronal mechanisms. I specifically focused on studying1) the effects of insulin treatment on learning and memory in aged animals as well as 2) the effects of insulin on intracellular calcium regulation. Understanding the links between insulin and neuronal mechanisms involving hippocampal calcium processes may help develop therapeutic strategies to decrease complications associated with age- related cognitive decline.

1.1 Brain Aging and cognition

Aging leads to a function decline of many organs, including the brain. Age-related changes in the brain such as reduced insulin signaling, reduced neuronal glucose uptake, increased glucocorticoid levels, and increased intracellular calcium concentration may give rise to pathological brain aging. One of the hallmarks of brain aging is cognitive decline [1, 2]. Many factors could influence the age-related decline of cognitive function, including dysfunctions of circadian clock [3], and oxidative stress [4-9]. Growing evidence supports the concept that brain insulin and calcium may play an important role in the mechanisms behind memory decline in advanced age and Alzheimer’s disease (AD) [10-12]. At the cellular level, brain insulin deficiency and insulin resistance may be one of the fundamental changes associated with altered memory function during pathological aging [13-20]. Brain insulin signaling disruption has been closely associated with cognitive decline and AD [14, 21-25]. At the receptor level, the amount of insulin receptors and their functions are decreased in aging and AD animal models [26-28]. At the clinical level, studies suggest that memory improved in AD patients are due to changes in their insulin levels [29]. In aged animals, it has also been shown that intranasal delivery of insulin to the central nervous system (CNS) positively correlates with increased brain cognitive function [30]. The intranasal route of insulin administration increases insulin acutely in the brain without risk of peripheral hypoglycemia. Importantly, it significantly enhances the memory in both aged and AD patients [31-35].

Although intranasal insulin enhances memory in AD patients, varying outcomes

arise with its clinical setting usage. Men and women with AD respond differently

to intranasal insulin dosage [36]. In addition, some clinical studies reported that

patients carrying at least one copy of the Apolipoprotein E4 (the strongest genetic risk factor for late onset Alzheimer’s disease) did not benefit from intranasal insulin treatment [37]. Therefore, gender and ApoE status in AD

patients increase outcome variability in intranasal insulin treatment. These

varying outcomes highlighted the need to understand the underlying neuronal

mechanism the effects of insulin on cognitive function with aging.

1.2 Insulin

Identifying the link between the pancreas and diabetes by Von Morning and

Minkowski in 1890 opened the door for the discovery of insulin as a therapy. This

life-sustaining hormone saved an enormous number of lives since its discovery. In

the early twentieth century, Sharpey-Schafer hypothesized that pancreas secretes

a hormone that controls glucose levels [38]. In 192, this hypothesis was confirmed

and a novel protocol to purify insulin was developed. In 1922, insulin therapy was

successful on a type 1 diabetic patient. Since the introduction of human insulin in

1982 and insulin analogs in 1996, insulin therapy become the standard treatment

options for diabetic patients [39, 40] by mimicking physiological insulin secretion in

the body to regulate blood glucose levels. After almost 100 years of research, it is

clear that Insulin is a natural peptide hormone secreted by the pancreatic β-cell in

response to elevated blood glucose concentration, and that it plays an indispensable role in maintaining blood glucose concentration and regulating metabolism [41]. In insulin therapy, to enhance the insulin’s effects and reduce the number of injection different routes of insulin delivery, as well as different insulin formulations were designed by combining insulin with zinc and/or basic proteins protamine [42].

1.3 Zinc

Zinc is an essential ion involved in many physiological processes ranging from cell proliferation to neuronal function. Zinc plays a critical role in regulating insulin synthesis, storage, and secretion in the pancreatic beta cells [43-45]. Zinc combined with insulin and induces proinsulin hexamerization, also co-releases with insulin from the beta cells [46]. It is well documented that zinc, pancreatic beta cell, and diabetes have a strong cause and effect relationship. Zinc is highly abundant in the pancreatic islet and zinc deficiency has been associated with diabetes [47]. High levels of zinc can be toxic to the cells, specifically to the neurons [48, 49]. Zinc present in the brain with its highest concentration in the olfactory bulb, hippocampus, hypothalamus, and cortex. Increased zinc stimulates synaptic events via AMPA receptors and improves synaptic summation [50] and influences LTP induction and memory [51].

1.4 Insulin formulations and alternative routes for insulin administration

Insulin formulations (Table 1.1), also different routes of insulin delivery (e.g., nasal, pulmonary inhaled, intravenous insulin infusion, insulin pumps) were developed [52, 53]. Currently, insulin cannot take orally because it will break down in stomach before it regulates glucose levels. Earlier research has focused on understanding the molecular mechanism through which insulin regulates glucose to uncovered insulin’s potential effects on memory enhancement. Although insulin is effective and widely used, researchers continue to study the molecular mechanisms by which insulin regulates glucose uptake in fat, muscle, and neuronal cells to obtain even more benefits from this life-sustaining hormone.

Table 1. Insulin Types by Brand Name, Generic Name, Onset, Peak, and Duration of Action.

Type of Insulin Brand Name Generic Name Onset Peak Duration _Insulin 15 30 to 90 3 to 5 _NovoLog aspart minutes minutes hours _Insulin 15 30 to 90 3 to 5 Rapid-Acting _Apidra glulisine minutes minutes hours _Insulin 15 30 to 90 3 to 5 _Humalog lispro minutes minutes hours _Humulin R 30 to 60 2 to 4 5 to 8 Short-Acting _Regular ( R) _Novolin R minutes hours hours Intermediate- _Humulin N 1 to 3 12 to 16 _NPH (N) 8 hours Acting _Novolin N hours hours _Insulin _Levemir determir 20 to 26 Long-Acting 1 hour Peakless _Insulin hours _Lantus glargine _Humulin _70% NPH Pre-mixed NPH 70/30, 30 to 60 10 to 16 and 30% Varies (Intermediate- _Novolin minutes hours regular acting) and 70/30 regular (short- _50% NPH _Humulin 30 to 60 10 to 16 acting) and 30% Varies 50/50 minutes hours regular _75% insulin Pre-mixed lispro _Humalog 10 to 15 10 to 16 insulin lispro protamine Varies Mix 75/25 minutes hours protamine and 25% suspension insulin lispro (Intermediate- _50% insulin acting) and lispro _Humalog 10 to 15 10 to 16 insulin lispro protamine Varies Mix 50/50 minutes hours (rapid-acting) and 50% insulin lispro Pre-mixed insulin aspart _70% insulin protamine aspart suspension _NovoLog 5 to 15 10 to 16 protamine Varies (Intermediate- Mix 70/30 minutes hours and 30% acting) and aspart insulin aspart (rapid-acting)

1.5 Insulin signaling in the periphery

Insulin plays a vital role in maintaining blood glucose levels in the periphery.

Insulin in the muscle and fat tissues promote glucose uptake. Insulin stimulates glucose storage, and inhibits glucose secretion in the liver [41, 54-61].

Insulin regulates glucose uptake in fat and muscle cells by inducing translocation of glucose transporters to the plasma membrane [62-68] by PI3K/AKT pathway

[69]. Insulin also regulates glucose concentration via suppression of hepatic glucose production (HGP) [61, 70, 71]. Previous studies suggest that insulin regulates HGP by binding to the insulin receptor and activating serine/threonine kinases AKT [72, 73], which further inhibits Foxo1 phosphorylation [74-76].

Therefore, insulin prevents large alteration in blood glucose levels during the day by enhancing glucose metabolism in the cells and suppressing hepatic glucose production.

Figure 1.1 Mechanisms of Insulin Actions in the Periphery.

In healthy individuals, blood glucose levels are tightly controlled by insulin, a natural hormone produced by the pancreatic beta cells in response to high blood glucose levels. In fat tissues, insulin increases triglyceride synthesis/ storage and decreases triglyceride breakdown. In the liver, insulin increases glycogen deposition and glucose use while decreasing glycogenolysis and gluconeogenesis. In muscle cells, insulin increases glucose uptake and protein synthesis while decreasing protein degradation.

Insulin promotes glucose uptake by cells through four major glucose transporter

isoforms are widely distributed throughout the body. The glucose transporter 4

(Glut-4) is highly expressed in fat and muscle cells [77]; Glut-1 and Glut-2 are

expressed in the endothelial cells lining the blood vessels and liver, intestine,

kidney, pancreatic cells; and Glut-3 is expressed in the neurons [78]. When insulin binds to the insulin receptors on the surface of target cells, it promotes translocation of glucose transporters to the plasma membrane, which moves glucose into the cell. Once glucose move inside the cell, it delivers energy to it.

Altered insulin levels, insulin receptors and/ or glucose transporters all can lead to obesity, insulin resistance, and diabetes [79-83]. Although insulin is recognized as glucose regulating hormone, it is increasingly clear now that insulin also plays a critical role in the regulation of neuroplasticity, neuromodulation, neurotrophism, neuronal growth and survival [84-92].

Therefore, insulin is important not only for regulating sugar, but also for learning and memory. To initiate insulin signaling, insulin has to bind to the insulin receptor and activates the insulin receptor tyrosine kinase.

Together with human insulin receptor characterizations, studies of insulin receptor tyrosine kinase activity have enhanced our understanding of the mechanisms of insulin action in various organs. Indeed, insulin acts on the fat, muscle, liver, and brain through the insulin receptor substrate pathways.

Figure 1.2 Insulin signaling pathways and translocation of glucose transporter (GLUT) to the plasma membrane by insulin.

To initiate insulin signaling, insulin must bind to insulin receptors on the plasma membrane. Insulin receptors contain two α subunits outside the cell and two β subunits inside the cell. α subunits have an insulin binding site, and β subunits have a tyrosine kinase phosphorylation site. Once insulin binds to insulin receptors, it triggers autophosphorylation of β subunits of insulin receptors, which leads to phosphorylation of insulin receptor substrate (IRS), which further phosphorylates PI3K and AKT. Next, downstream signaling pathways promote translocation of glucose transporters (GLUT) to the plasma membrane, which brings glucose inside the cell modified from [93].

1.6 Insulin signaling in the brain

While insulin plays an essential role in the brain, brain insulin itself is important for regulating insulin secretion from the pancreatic beta cells [94]. Important actions of insulin in the brain are now gradually becoming clear, even through the brain has been considered an insulin-independent organ for several decades. Insulin and insulin-like growth factor (IGF1-2) bind to insulin receptor and IGF-1R and initiate insulin signaling. Insulin receptor and IGF-1R co-express in the brain and share the intracellular signaling pathways; however, each of them has a unique function. This view has been further tested using neuron-specific insulin receptor and IGF-1R knockout mice. Deleting insulin receptors in the brain leads to mild obesity and insulin resistance without influencing brain size and development [95].

However, deleting IGF-1R affects brain size and development and contributes to behavior changes [96]. All the insulin receptor substrates (IRS1-4) present in the brain, and IRS2 play a vital role in the brain since their deletion results in impaired brain development [97, 98]. IRS mediate the phosphorylation of AKT/PI3K, which further activates several downstream signaling pathways [99]. Insulin downstream signaling promotes translocation of glucose transporters to the plasma membrane both in peripheral and brain tissues [62-68, 77, 100, 101]. Taken together, peripheral and brain insulin share similar signaling pathways.

Figure 1.3 Actions of insulin in the brain.

The brain has been traditionally considered insulin-insensitive. However, now it is clear that normal brain functions depend on insulin, and insulin receptors are widely expressed in the brain. Insulin activates insulin signaling via binding insulin receptors and regulates various neuronal functions: it enhances learning and memory and regulates energy homeostasis, whole body metabolism, body temperature, mood, and appetite modified from [93].

This year we are celebrating the 96th anniversary of the discovery of insulin by

Frederick Banting. Yet only over the past few years has interest increased in brain insulin’s role in cognition, food intake, body weight, and whole-body metabolism. Insulin in the brain has been detected at high levels in mouse embryos (E9) at early developmental stages and reduces with age [102]. Insulin receptors are located at brain synapses, and highly concentrated at the postsynaptic density [103]. Therefore, insulin may regulate synaptic function and excitatory synaptic transmission stability during neuronal development via insulin receptors. Furthermore, the insulin receptor substrate 53 (highly expressed in the postsynaptic density) also plays a role in regulating spine density in hippocampal cultured neurons [104].

1.7 Insulin receptors in the brain vs. in the periphery

Insulin receptors belong to a tyrosine kinase family and activate through insulin binding [105]. The insulin receptor is a dimeric transmembrane protein encoded by a single gene, INSR, and alternative splicing of INSR gives rise to α and

β subunits. The insulin receptor is composed of two extracellular α subunits that bind to insulin and two intracellular β subunits with tyrosine kinase activity [106].

Insulin binds to α subunits of the insulin receptor, leading to autophosphorylation of β subunits and activating the receptor, which further phosphorylates and recruits different substrate (IRS) adaptor proteins [41, 107]. Insulin and insulin receptor signaling play an important role both in the periphery and the brain and

mediate many cell functions ranging from glucose metabolism to learning and memory. Furthermore, insulin receptors are expressed in almost all tissues in the

body. However, researchers have found that the anti-insulin receptor that blocked the insulin receptor in the periphery had no effect on brain insulin receptors [108]. This study strongly supports that some structural and molecular differences exist between the peripheral and brain insulin receptors. Moreover, the difference between peripheral and brain insulin receptors is primarily due to alternative mRNA splicing of the exon-11.

Brain insulin receptors lack exon-11, and therefore, brain insulin receptors have smaller subunits compared to peripheral insulin receptors [108-111]. Brain insulin receptors do not down-regulate in response to prolonged and/or a high

concentration of insulin exposure [112, 113]. Brain insulin receptors reduce with

aging and influence learning and memory, and learning improves insulin receptor

functions [28]. These studies along with studies from the NIRKO mice model (a

neuron-specific insulin receptor gene deletion [95] provide clear understanding of

role of brain insulin receptor in brain function (food intake, metabolism,

reproduction, cognition [114]. However, it is important to understand how insulin

gets into the brain.

1.8 Insulin synthesis in the brain

1.8.1 Peripheral source of insulin

It is widely believed that insulin is synthesized and released mainly from the

pancreatic beta cells in response to blood glucose levels. Insulin is initially

synthesized as preproinsulin, and removal of its amino terminal single peptide

generates proinsulin. Excision of C peptide within the endoplasmic reticulum

gives rise to α (21 amino acids) and β (30 amino acids) of insulin. C peptide is a

biomarker of insulin secretion. Although various factors may influence insulin

secretion, one key factor is blood glucose level. When glucose concentration

rises, glucose moves into the cell by a glucose transport. Then, glucose is

metabolized to generate ATP, which blocks ATP- dependent K+ channels in the

beta cell membrane, which results in the depolarization of the cell. Depolarization causes the Ca2+ ion channels to open, and Ca2+ ions to move in, which triggers vesicles containing insulin to move to the membrane fuse, and release the insulin by exocytosis.

Figure 1. 4 The mechanism of insulin secretion by the pancreatic beta cell.

Increases in blood glucose levels trigger insulin release. Modified from Molecular biology of the cell (3rd ed.) by Alberts B, et al., 1994.

1.8.2 Central source of insulin

Previous studies show that insulin produced by the pancreas could enter the brain by crossing the blood brain barrier to maintain energy balance [107, 115-

120]. Furthermore, insulin permeability changes during aging and in patients with diabetes and AD [121, 122]. Based on different experimental evidence, the brain also produces its own supply of insulin [10]. In 1983, Dorn et al. confirmed the hypothesis that part of the brain insulin is produced in the central nervous system (CNS). Results from the study of human cadaver brains demonstrated that insulin and C-peptide were present in most nerve cells [123]. Further, C- peptide and insulin receptors in the brain decreased with aging [124]. Unlike mammals, in drosophila and C.elegans, neurons are the primary cells to 16

produce insulin [99]. Together with animal studies and observations from hippocampal cultured neurons studies, it is clear that insulin can be synthesized locally in the brain and released from the neurons in response to depolarization

[125, 126]. While brain insulin and insulin receptor signaling undergo changes with aging, insulin resistance may contribute to pathological brain aging [127-

133].

Insulin also is valuable for treating a wide variety of non-diabetic diseases, such as schizophrenia, anxiety, nervousness, cancer, and cognitive function. While insulin in the brain enhances learning and memory, insulin resistance is linked to Alzheimer’s disease and other neurodegenerative disorders [134-136].

Currently, increasing insulin signaling in the brain is one of the best treatment options to improve memory in aged and AD populations [137].

1.9 Metabolism and the brain

The brain is a complex organ and needs lots of energy to run properly [138].

Glucose is the main substrate that satisfies brain high-energy demands. The brain (rich with neurons) weighs only 2% of body weight, but neurons use 25 percent of all the glucose energy in the body while its energy store capacity is small. Hence, the brain requires a constant glucose supply from the blood, and glucose is transported into the brain via the glucose transporters through the blood brain barrier. Although the brain is an energy-intensive organ, it has traditionally been considered insulin-insensitive. However, recently this view has

been challenged, and now it is clear that normal brain function depends on

insulin. Moreover, brain functions such as learning and memory are positively correlated with effective glucose metabolism in the brain. Dysregulation of brain glucose metabolism can lead to loss of energy for brain functions, which further

influences the neurotransmitters, neuronal communication, and cognitive

function. Therefore, the traditional role of Insulin in the brain is regulating glucose

metabolism and enhancing neuronal survival. It is also known that insulin and

insulin receptors in the brain reduce with aging [124]. Additionally, the link

between metabolic disturbances and neurodegenerative diseases have been

well documented. Researchers have suggested that Alzheimer’s disease is

positively correlated with obesity and diabetes [16, 139-145]. Previous studies

suggest that type 2 diabetes increases the risk of developing AD [146-149]. Also,

some studies suggest that AD is a brain- specific form of diabetes (type 3

diabetes) [150-155]. Furthermore, clinical studies suggest that AD patients

present abnormal brain glucose metabolism [26, 156]. Normal brain glucose

metabolism is vital for brain function and controlled by brain insulin/insulin

receptor signaling pathways [157].

Insulin in the brain is the key to many neurological disorders. In fact, brain insulin

regulates both periphery and central glucose metabolism, learning, and memory

[87, 107, 158, 159]. Insulin exerts its effects though insulin receptors, which are

widely distributed in various regions of the brain [107, 160-162]. More

specifically, insulin receptors are highly expressed in the hippocampus [107,

163], a brain region that is critical in synaptic plasticity, learning, memory, and cognition. Brain insulin and insulin receptor levels decrease with aging and AD, which is linked to age-related memory impairment [152, 164-166]. Insulin receptors are also expressed in the hypothalamus, the widely studied region of insulin action in the brain. Insulin circulates at the level proportionate to body fat mass, inhibits food intake through its actions on the hypothalamic neurons [167,

168], and regulates glucose metabolism [169] and energy expenditure [170] both in the periphery and the brain [171]. The highest concentration of insulin and insulin receptors are detected in the olfactory bulb [172, 173], and insulin regulates olfactory functions, which are important for food intake and appetite

[174]. Insulin receptors are also present in the cerebral cortex and cerebellum

[28] and plays an important role for neuronal survival [158]. Furthermore, Kahn et al created neuron-specific insulin receptor knockout mice (NIRKO) to study insulin actions in the brain. The study results suggest that depletion of insulin receptors in neurons lead to obesity and dysregulation of a reproduction hormone [95]. Insulin has diverse effects in the brain. Insulin may promote better nerve cell communication, but the disruption of brain insulin signaling may lead to metabolic dysregulation and cognitive impairment [87, 134, 175]. Insulin has also been viewed as the potential missing link between memory and intracellular

Ca2+ dysregulation with aging. Insulin regulation of glucose homeostasis is an important modulator of neuronal plasticity. Also, maintenance of intracellular

Ca2+ homeostasis is vital for normal synaptic plasticity as well as cell excitability.

1.10 Intracellular calcium levels

Calcium (Ca2+) is an essential ion that rules many aspects of cellular life, including cellular excitability, exocytosis, apoptosis, toxicity, and transcription, and mediates many cell functions, including muscle contraction, chemical sense, cell proliferation, fertilization, immune response, learning and memory. For this reason, intracellular Ca2+ levels are precisely regulated in young healthy individuals. However, failure of this regulation occurs with aging and AD. The first proposal of a “Ca2+ hypothesis of brain aging” by Khachaturian in 1984 provides a simple cellular mechanism that alters Ca2+ homeostasis underlying many age- related changes. After two more decades of studies, the relationship between dysregulation of intracellular Ca2+ and pathological brain aging has become clear and is now the key therapeutic target to treat many neurodegenerative diseases.

Thanks to the excellent work of Sydney Ringer, who in 1883 discovered the importance of Ca2+ signaling in the survival of fish, heart, and muscle contraction. After 50 years, in the early 1940s, Lewis Victor Heilbrun confirmed the theory that Ca2+ is a universal intracellular messenger able to regulate all cellular reactions and that Ca2+ also involves neurotransmitter release into nerve terminals [176, 177]. After several decades of research, it is clear that in the brain, Ca2+ plays a critical role for synaptic activity, neuronal development, neuronal survival, and cognition [178-180]. Discovery of patch clamping and

Ca2+ imaging techniques advanced our understanding of pathology associated

with dysfunctions of Ca2+ signaling cascades [181]. At rest, intracellular Ca2+ concentration is very low (~100 nM free) and extracellular Ca2+ concentration (~

2 mM) is high, about a 20,000–fold concentration gradient. Intracellular [Ca2+] is maintained within a specific limit by the different Ca2+ handling mechanisms

[182]. However, brain aging contributes to the loss of intracellular Ca2+ homeostasis [183, 184]. In healthy individuals, intracellular Ca2+ homeostasis is tightly regulated and plays a vital role in neuronal survival and function. In contrast, intracellular Ca2+ levels increase with aging, which contributes to memory decline and other neurodegenerative disorders such as AD [180, 185,

186]. Specifically, an age-related increase in hippocampus Ca2+ levels has been much investigated during the last several decades because Ca2+-dependent after hyperpolarization is larger (AHP) in aged animals and links to cognitive impairment with aging and AD [180, 182, 185-192]. In addition, high intracellular

Ca2+ levels and larger Ca2+ dependent AHP are seen in the hippocampus of diabetic rats [193, 194].

It appears that the increase in hippocampal intracellular Ca2+ levels are mainly mediated through voltage gated Ca2+ channels and N-methyl-D- aspartate

(NMDA) receptors on the plasma membrane, and intracellular Ca2+ release via ryanodine receptors on the endoplasmic reticulum [195].

1.10.1 Voltage-gated Ca2+ channels

Voltage-gated Ca2+ channels (VGCCs) are found in the membrane of neurons,

muscles, glial cells (excitable and non-excitable cells), and are permeable to

Ca2+ ions [196]. VGCCs are voltage-sensitive and mediate the fastest Ca2+

influx in response to membrane depolarization, thus, changing intracellular Ca2+

levels greatly within milliseconds [197-201]. As a result, Ca2+ enters the cell

through voltage-gated Ca2+ channels and serves as intracellular second

messengers to control a wide variety of cell functions. In cardiac, smooth, and

skeletal muscles, activation of VGCCs mediates contraction by increasing

intracellular Ca2+ levels and by further activating ryanodine dependent Ca2+-

induced Ca2+-release in the sarcoplasmic reticulum [202-206]. In the endocrine

cells, VGCCs activation influences the hormone secretion [207]. In the neurons,

Ca2+–mediated process via VGCCs include synaptic transmission,

neurotransmitter release, and gene transcription; it also activates calmodulin-

dependent protein kinase ll (CaMKll) and protein kinase C (PKC) which are

important for neuronal survival, learning and memory [198, 208-217]. While Ca2+

channels serve as a key pathway for entry of Ca2+ into the neuron and initiate

many physiological events, inappropriate expression or dysfunction of VGCCs

can result is pathophysiological changes leading to neuronal Ca2+ dysregulation

and toxicity in the brain, which have been linked to neurodegenerative diseases

[218-221]. Furthermore, VGCCs density increases with aging, and this

contributes to cognitive decline with aging and AD [184, 222-224]. Therefore,

voltage-gated Ca2+ channels are important therapeutic targets for age-related

cognitive decline and other conditions.

1.10.2 Structure and classification of voltage-gated Ca2+ channels

The five-polypeptide subunit that forms voltage-gated Ca2+ channels is a central

pore-forming α1 subunit, dimer of α2 and δ subunits and intracellular β subunit,

and transmembrane γ subunit. There are ten Cavα1 subunits that share four

homologous domains, each with six membrane-spain helices (S1-S6). S4

(Positively charged and hydrophobic) is the key controller for voltage-dependent

activation [197, 198, 208, 225-227] and P loop motif (negatively charged) is

located between S5 and S6. Together they form a pore that is permeable to

Ca2+ and barium [197, 228].

Figure 1.5 Voltage-gated Ca2+ channels structure.

High voltage activated (HVA) channels (P, Q, N, L, R-type) contain Caα1, Caβ,

Caγ, and Caα2δ subunits. Low voltage activated channels (T-type) contain

Caα1subunit modified from [220].

The Caα1 subunit has three main families (Cav1, Cav2, Cav3) [226, 229]. The

Cav1 family includes four members (Cav1.1, Cav1.2, Cav1.3, Cav1.4) and all four members encode neuronal L-type Ca2+ channels and Cav1.1 encodes skeletal muscle Ca2+ channels [230-233]. Further, Cav1.1 knockout mice cannot contract their diaphragms and die at birth [234]. Mice that lack Cav1.2 die in utero because the heart muscle cannot contract [235]. Cav1.2 is also important for neuronal function and mutations in this subunit links to bipolar disorder,

Schizophrenia, and depression [236-239]. Cav1.3 null mice are deaf due to loss of Ca2+ current [240] and loss of normal auditory brain stem development [241].

Mice deficient in Cav1.4 are blind due to loss of normal function in the outer retina [242, 243].

The Cav2 family has three members (Cav2.1, Cav2.2, Cav2.2) and gives rise to

P/Q-type [244], N-type, and R-type Ca2+ channels [233]. The Cav3 family has three members (Cav3.1, Cav3.2, Cav3.3) that encode T-type channels [245].

Mice with a lack of Cav2.1, Cav3.1, and Cav3.2 display a range of dysfunctions

(seizures, slow heart rate, compromised coronary, epilepsies) [246-250].

The Cavβ subunit has four members (Cavβ1- Cavβ4) and links to Cavα1 domain l-ll [251, 252]. The Cavα2δ subunit has four different members (Cavα2δ1−

Cavα2δ4) and is anchored to extracellular plasma membrane via glycophosphatidylinositol (GPI) [253-255]. Earlier studies suggest that the

Cavα2δ subunit plays an important role in regulating synaptic targeting and

release probability of voltage-gated Ca2+ channels in neurons [256, 257].

Table 2. Types of voltage gated Ca2+ channels (VGCCs) activation and inactivation profiles modified from [258].

Ca2+- α1-Subunit Activation Inactivation Native current dependent subtypes profile profile inactivation

P/Q-type α1A (CaV2.1) High voltage Moderate/slow Yes/no

N-type α1B (CaV2.2) High voltage Fast Yes/no

α1C (CaV1.2)

α1D (CaV1.3) L-type High voltage Moderate/slow Yes α1F (CaV1.4)

α1S (CaV1.1)

R-type α1E (CaV2.3) High voltage Fast/moderate No

α1G (CaV3.1)

T-type α1H (CaV3.2) Low voltage Very fast No

α1I (CaV3.3)

There are several members of the VGCCs in the brain due to different genes that

encode ten α1 subunits of Ca2+ channel, including L-type, N-type, P/Q-type, R- type, and T-type channels [214, 259]. Based on physiological and

pharmacological properties, they are categorized as high voltage (HVA) and low voltage (LVA). HVA channels (L- type, P/Q-type, N-type) formed through a combination of Cavα1, Cavβ, and Cavα2δ subunits activate in response to strong and large depolarization. On the other hand, Low voltage-activated (T-type) channels only contain the Cavα1 subunit and open in response to weak depolarization [226, 260-263]. The R-type is intermediate voltage activated channels. In neurons, both HVA and LVA channels can control neuronal firing

properties [264-268].

1.10.3 Voltage-gated Ca2+ channels activation and inactivation

Activation of voltage-gated Ca2+ channels results in Ca2+ influx and mediates neurotransmitter release, neuronal firing, hormone secretion, muscle contraction, and gene expression. However, prolonged activation of Ca2+ channels contributes to elevation of intracellular Ca2+ levels, which is cytotoxic [221].

Therefore, there are two types of Ca2+ channel inactivation, voltage- and/or

Ca2+-dependent inactivation, to limit intracellular Ca2+ overload. All voltage- gated Ca2+ channels can be inactivated by voltage-dependent manner [269].

The underlying mechanism responsible for voltage-dependent Ca2+ channel inactivation is different from voltage-gated K+ and Na+ channels (“ball and chain” and “Hinged-lid”) mechanisms [270]. Many studies suggest that prolonged depolarization triggers structural changes in four S6 of the α1 subunits, and further exposes docking site for the domain l-ll linker. Thus, it modulates inactivation of voltage-gated Ca2+ channels [269, 271-273]. Other subunits of

Ca2+ channels also influence the kinetics and rate of inactivation.

Voltage-gated Ca2+ channels inactivate not only in response to voltage, but also to the influx of Ca2+ [274]. Ca2+-dependent inactivation becomes important when studying the effects of drugs on Ca2+ channel currents. A rise in intracellular Ca2+ triggers binding of Ca2+ to the channel and initiates inactivation [275] and Calmodulin plays a vital role in Ca2+-dependent

inactivation [276]. Therefore, barium was used as a charge carrier when obtaining Ca2+ channel currents while studying insulin effects on VGCCs.

1.10.4 Ryanodine receptors

Ryanodine receptors (RyRs), intracellular Ca2+ release channels, are located in the endoplasmic (ER) reticulum membrane [136, 277], and mediate rapid intracellular Ca2+ release into the cytosol, which allows the amplification of intracellular Ca2+ concentration. RyRs are widely expressed in almost all tissues

(muscle, myocardium, and neurons) and are involved in a variety of key Ca2+ signaling events [278], ranging from excitation-contraction coupling in muscle cells to learning and memory in nerve cells. There are three major RyRs isoforms

(RyR1-3) and they are named after plant alkaloid ryanodine [279]. Ryanodine is a natural agonist at low concentration, which allows efflux of Ca2+ from ER and increases intracellular Ca2+ levels. At high concentrations (~20 uM), Ryanodine is an antagonist, which inhibits the Ca2+ flow out of the ER.

RyRs are the largest ion channels and the highest-conductance intracellular

Ca2+ channel, with a molecular mass that is greater than megadalton [280].

RyRs are regulated by the Ca2+ ion and VGCCs [281]. Ca2+ influx through voltage-gated Ca2+ channels triggers second messenger signals that open the

RyRs channels and give rise to large Ca2+ release from internal Ca2+ stores

(endoplasmic reticulum) [282, 283]. Thus, Ca2+ activation of the RyRs on the endoplasmic reticulum amplifies the intracellular [Ca2+] through Ca2+-induce

Ca2+-release (CICR) process [284]. Additionally, various ions, small molecules,

and proteins may also regulate the RyRs function, including protein kinase A

(PKA), FK506 binding proteins, and calmodulin (CaM) [285-287]. In skeletal and

cardiac muscle, RyR1 and RyR2 play a critical role in excitation and contraction coupling [288, 289]. High intracellular Ca2+ concentration induces muscle contraction and Ca2+ ATPase dependent Ca2+ uptake by sarcoplasmic reticulum results in relaxation [281, 290]. In neurons, RyRs are crucial for signal transduction and influence hormone secretion, synaptic plasticity, and learning

[291-293].

Figure 1.6 Ryanodine receptors and cognitive function with aging

Ryanodine receptors (RYRs) on the endoplasmic reticulum (ER) undergo functional changes with aging. Furthermore, the RYRs mediated increase in

Ca2+-induced Ca2+-release (CICR) is one of the major sources of Ca2+

dysregulation with aging. Moreover, amplified CICR influences the size of AHP

and cognitive function. As a result, dysfunction of RYRs leads to an increase in

intracellular Ca2+ via CICR, which further contributes to larger AHP, Ca2+

dysregulation, and memory decline.

1.10.5 Ryanodine receptor isoforms and expression

Ryanodine receptor isoforms are RyR1, RyR2, and RyR3 and are expressed in

various tissues. RyR1 is the most intensely studied type and its main function is

to trigger Ca2+ efflux from the sarcoplasmic reticulum membrane of skeletal

muscle. RyR1 is highly expressed in the skeletal muscle [294, 295]. Also, it is widely distributed in cardiac muscle, smooth muscle, stomach, kidney, thymus, cerebellum, purkinje cells, adrenal glands, ovaries, testis, and B-lymphocytes at low levels [294, 296-299]. Dysfunction of the RyR1 gene is associated with life- threatening muscle diseases such as malignant hyperthermia [300] and multiminicore disease [301].

RyR2 is dominantly expressed in the cardiac muscles [277]. Different concentrations of RyR2 are also detected in purkinje cells of the cerebellum, cerebral cortex, stomach, kidney, adrenal glands, ovaries, thymus, and lungs

[302-305]. Mutation in the RyR2 gene may cause arrhythmogenic right ventricular dysplasia type 2 and Ventricular arrhythmias by changing Ca2+ homeostasis [306-308].

RyR3 is present in the brain and mainly found in the hypothalamus, corpus striatum, and hippocampal neurons [296, 304, 309]. Different studies suggest

that inappropriate expression or dysfunction of RyR3 links to Alzheimer’s disease

[136, 186].

1.10.6 Structure of ryanodine receptors

All three isoforms (RyR1, RyR2, RyR3) share similar structure. The structure of

RyR1 (Skeletal muscle ryanodine receptors) is widely studied and it includes four

identical subunits and is divided into two regions: a transmembrane (TM) region

and a cytoplasmic (CY) region [294, 295]. The CY region plays a critical role in

regulating RyR1 channel gating properties via interacting with intracellular

messengers. Dysregulation of the CY region of RyR1changes its gating and links

to many diseases [310].

RyR2 is dominantly expressed in the heart and brain and is activated by Ca2+

influx through plasma voltage-gated Ca2+ channels in response to

depolarization. Peng et al. used electron microscopy to map the structure of open

and closed states of RyR2 from porcine hearts. The results suggest that RyR2

has different cytoplasmic domain compare to RyR1 due to rotation of the central

domain, which influence the RyR2 channel gating [311].

1.11 Hippocampus

Hippocampus is critical for learning and memory [312] and is highly vulnerable

to damage and loss during aging and AD [111, 191, 313-315]. The

hippocampus, located in the medial temporal lobe, belongs to the limbic family,

one on each side of the brain. The human hippocampus has a curved shape

resembling a seahorse; it appears as a ‘C’ shape in hippocampal cultured neurons. In addition to the hippocampus, the cerebellum, amygdala, and other systems are also involved in learning and memory.

The hippocampus is responsible for the formation and consolidation of short- term memory and long-term memory, as well as spatial navigation. The H.M study is the starting point for understanding the importance of the hippocampus in memory. H.M is a famous patient whose case provided an understanding of

how our brain works. After the neurosurgeon removed H.M’s hippocampus,

patient was unable to form new memories, and, as a result, he forgot daily

events as fast as they occurred [316]. The H.M case study continued for five

decades until his death, and the study results shed light on the relationship

between cognitive and neuronal organization of memory [317]. Since H.M

became amnesic after bilateral hippocampus lesion, the removal of

hippocampus is responsible for his memory impairment; moreover, magnetic

resonance imaging study further confirmed that lesion of the hippocampus can

result in global and enduring amnesia [318]. This study, along with cumulative

animal studies, made it clear that the hippocampus plays a critical role in the

formation of immediate memory, long-term memory, and memory retention [319].

In addition, earlier studies suggest that the hippocampus volume decreased with

aging [320]. Furthermore, in neurodegenerative diseases such as Alzheimer’s

disease, the hippocampus is one of the first brain regions to become damaged.

Also, an increase in hippocampal neurogenesis is one of the hallmarks of early

AD [321, 322].

On the other hand, in healthy individuals, high levels of insulin receptors have

been seen in CA1, a hippocampal region that is important in learning and

memory [159, 323]. The interruption of insulin production and insulin receptor

activity may cause cognitive decline [28]. Also, neuron loss in the CA1 region of

the hippocampus differentiates the healthy individual from AD patients [313, 324-

326]. Therefore, losing and/or damaging the hippocampal neurons causes significant cognitive problems.

1.12 Insulin actions on neurons

Insulin has wide raging effects in neurons of various brain regions, including

regulating neuronal activity and synaptic function. Insulin and insulin receptors

are present in the fetal central and peripheral nervous system, where it

promotes axonal growth and brain development in the hippocampal cultured

neurons [327]. Insulin can be produced and released from the hippocampal

cultured neurons in response to depolarization [125, 126]. Insulin inhibits

norepinephrine uptake by the hippocampal cultured neurons [328]. In the

hypothalamus, insulin reduces the activity of POMC, AgRP, and steroidogenic

factor 1 (SF-1) neurons through KATP channel activation [329, 330]. In the hippocampus, insulin increases insertion of NMDA receptors into the cell membrane [89] via tyrosine phosphorylation of NR2A and NR2B subunits [85].

Thus, insulin may promote long-term potentiation, which is important for learning

and memory. Insulin-dependent internalization of the AMPA receptor [88, 331,

332] is important for long-term depression, which is another important

mechanism for consolidation of navigational memory in the hippocampus. Insulin

also regulates inhibitory synapse by increasing cell surface GABAA receptor

expression [91, 92, 333]. In addition, insulin enhances synaptic plasticity by increasing the expression of dendritic postsynaptic density scaffolding protein

(PSD-95), which is important for the formation of the postsynaptic junction [334]

and Ca2+ homeostasis in hippocampal neurons (Maimaiti et al., 2017). Insulin

stimulates the translocation of glucose transporter-4 (Glut-4) to hippocampal

plasma membranes [100]. Glut-4 is important for glucose uptake and utilization

in the brain. As a result, insulin regulates neuronal metabolism and energy

needed for learning and memory via Glut-4. In addition, insulin also regulates

visual system function by controlling synapse density [84]. Insulin modulates L-

type Ca2+ current and intracellular Ca2+ levels in rod photoreceptors [335].

1.13 Thesis significance

Hippocampus-dependent memory loss is one of the hallmarks of brain aging and

Alzheimer’s disease (AD). This growing epidemic includes an estimated 5.3

million people in the United States, with the economic burden on healthcare

estimated at 259 billion dollars (http://www.alz.org/facts/). Most importantly, the

day-to-day lives of those affected by AD and their families must be considered

as we investigate new therapeutic targets and new approaches for the treatment of this devastating disease. Indeed, AD therapeutic options are very limited in their ability to improve the lives of those impacted by this disease.

Therefore, innovative and effective therapeutic strategies are necessary to address the needs of this patient population.

In recent years, the study of insulin as a cognitive enhancer has gained momentum. Insulin and its receptors have been detected in the hippocampus, one of the major brain regions that is damaged and/or lost in Alzheimer’s disease [155, 336]. In addition, insulin regulates energy for proper brain function and survival [158], and insulin signaling has been shown to be deficient in postmortem brain tissue of AD patients [337, 338]. Thus, increasing insulin signaling in the brain appears to be a valid therapeutic target for improving cognitive function in aging and/or AD. Earlier clinical trials have shown that intranasal insulin treatment significantly improves memory in AD patients [31-

35] .Therefore, based on these clinical studies as well as animal studies, intranasal insulin administration appears to be one of the best methods to treat cognitive decline due to its minimal effect on systemic glucose levels and noted improvements in memory processes. However, there have been only a few studies focusing on identifying the neuronal molecular mechanisms underlying these effects. My dissertation is designed to identify the potential mechanisms responsible for insulin-mediated memory improvement as those seen in human and animal studies. Specifically, I focus on identifying whether insulin can offset

cognitive decline with aging through a mechanism dependent on reestablishing

Ca2+ homeostasis.

Intracellular Ca2+ signaling may be a key neuronal molecular regulator of hippocampal-dependent memory. Intracellular Ca2+ levels increase associates with memory decline in aging and AD [156, 314, 339, 340]. Thus, we examined the relationship between insulin, memory and intracellular Ca2+ levels to better understand the mechanism(s) underlying insulin action on hippocampal neurons. This information will enable to us to better understand how insulin therapy may affect patients and may also shine light on the underlying neuronal mechanisms.

The following manuscript has been published in the Journals of Gerontology:

Series A: J Gerontology A Bio Sci Med Sci. 2016 Jan;71(1):30-9. Epub 2015

Feb 6. As an initial step, we explored the role of intranasal insulin in improving hippocampal-dependent memory in experimental aged animals. Additionally, we delineated insulin’s effects on Ca2+-dependent after hyperpolarization in the

CA1 region of the dorsal hippocampus using hippocampal slices recordings in young and aged rats. Because brain aging is the greatest risk factor for AD, studies into how insulin may alter biomarkers of brain aging could improve current therapeutic strategies for memory loss with aging and AD.

Chapter 2 Intranasal insulin improves age-related cognitive deficits and reverses electrophysiological correlates of brain aging

Authors: Shaniya Maimaiti, Katie L Anderson, Chris DeMoll, Lawrence L Brewer, Ben Rauh, Chris J Gant, Erick Blalock, Nada M. Porter, Olivier Thibault

Synopsis

Peripheral insulin resistance is a key component of metabolic syndrome

associated with obesity, dyslipidemia, hypertension, and Type 2 diabetes. While

the impact of insulin resistance is well recognized in the periphery, it is also

becoming apparent in the brain. Recent studies suggest that insulin resistance

may be a factor in brain aging and Alzheimer’s disease (AD) whereby intranasal

insulin therapy, which delivers insulin to the brain, improves cognition and

memory in AD patients. Here, we tested a clinically-relevant delivery method to

determine the impact of two forms of insulin, short-acting insulin lispro

(Humalog®) or long-acting insulin detemir (Levemir®), on cognitive functions in

aged F344 rats. We also explored insulin effects on the Ca2+-dependent

hippocampal afterhyperpolarization (AHP), a well-characterized

neurophysiological marker of aging which is increased in the aged, memory

impaired animal. Low-dose intranasal insulin improved memory recall in aged

animals such that their performance was similar to that seen in younger animals.

Further, because ex vivo insulin also reduced the AHP, our results suggest that

the AHP may be a novel cellular target of insulin in the brain, and improved

cognitive performance following intranasal insulin therapy may be the result of insulin actions on the AHP.

2.1 Introduction

Results from preclinical settings argue that peripheral insulin resistance, a component of type 2 diabetes and/ or the metabolic syndrome, may have a negative impact on cognitive function. Emerging studies report that in diabetic models, insulin responses are attenuated in the brain, possibly indicating the presence of insulin resistance in this tissue. Clinical and preclinical studies show that insulin and insulin-like growth factor-1 (IGF-1) receptors, their message and their function, are often reduced in the hippocampus during normal and pathological aging [27, 111, 124, 150, 341]. Similarly, in animal models of aging and Alzheimer’s disease (AD), brain insulin receptor signaling is reduced, often in the presence of peripheral metabolic dysregulation [28, 342-348]. Importantly, several studies provide evidence that brain insulin therapy can greatly reduce cognitive decline with age and/ or AD, and even improve memory in young adults

[reviewed in 33].

In recent studies examining the effects of insulin on cognition, insulin has been administered via the intranasal route to bypass the blood brain barrier. Notable,

Born and colleagues have shown that intranasal insulin is capable of entering the human brain rapidly [349]. However, the underlying mechanism(s) leading to cognitive improvement are not yet clear. Potential molecular/ cellular targets

affected by insulin in the brain and capable of offsetting cognitive decline include

changes in neuronal firing [350], Ca2+-dependent K+ channels [351], GABA

currents [352] and tau and Aβ metabolism [353-355]. Insulin also alters synaptic

structures [356, 357], modifies synaptic plasticity [88, 358, 359] and increases

delivery of NMDA receptors to the plasma membrane [89]. Similar effects have

been observed in response to IGF-1 replacement, showing increased AMPA-

mediated excitatory postsynaptic potentials [360, 361], and increased NMDA

receptor trafficking [362], effects that appear capable of offsetting age-dependent

cognitive decline [363]. Further, MRI data in men and women has provided

evidence that intranasal insulin can have an impact on brain blood flow [364],

and on whole-body insulin sensitivity via regulation of hypothalamic activity [365,

366]. Whether this later effect is purely mediated by central insulin effects or

reflects permeation of small insulin amounts into the bloodstream is currently

being investigated [367-369].

Another important candidate mechanism that may underlie the effects of

intranasal insulin pertains to Ca2+ homeostasis. Ca2+ dysregulation is prominent

in models of diabetes [370]. In the dorsal root ganglion and the hippocampus of

animals with STZ-induced diabetes [193, 371], broader Ca2+ action potentials, larger Ca2+-dependent afterhyperpolarizations (AHPs), and aberrant intracellular

Ca2+ release are seen [193, 372, 373]. Impaired memory and long-term

potentiation maintenance is also present [374]. Clinical data also supports a role for Ca2+ dysregulation in peripheral insulin resistance as evidenced by the

presence of abnormal and sustained adipocyte Ca2+ elevations in hyperinsulinemic, obese subjects [375]. Similar evidence for an increase in Ca2+ levels in skin fibroblasts from Type 2 diabetics has also been reported [376]. In recent work, we have shown that the Ca2+-dependent AHP, which increases in amplitude and duration with aging [377-379], is sensitive to insulin (ex vivo), and that this sensitivity is reduced by a high fat diet [380].

While some studies in humans demonstrate the efficacy of intranasal insulin as a cognitive enhancer, studies in animal models are required to understand the potential mechanisms underlying the beneficial effects of intranasal insulin.

However, only one prior study has measured the impact of intranasal insulin on brain function in the awake animal, focusing on olfactory discrimination in the young mouse [381]. Because the greatest risk factor for AD is aging, research into how central nervous system insulin signaling affects brain aging may improve understanding of the biological conditions. Further, because of the sustained growth in the aging population, implementing strategies to reduce the impact of brain aging will likely benefit the aging population, and by extension, may reduce the incidence of age-related neurodegenerative diseases that share cellular mechanisms with brain aging (e.g., AD).

We, therefore, assessed learning and memory performance in aged rats in response to two different insulin formulations (short-acting insulin Humalog® and long-acting insulin Levemir®), and quantified Ca2+ homeostasis. Insulin is

available primarily in shorter- and longer-acting forms, and intranasal longer- acting insulin formulations (e.g., Levemir®) may potentially be superior to shorter-acting ones (e.g., Humalog®) especially in APOE-ε4 carriers [382]. In

addition, because insulin is often formulated with zinc, to more specifically

address the impact of insulin, we studied a zinc-free insulin formulation (Apidra®

), as well as zinc alone on the AHP. Overall, our goals were to test whether

intranasal insulin could reduce cognitive decline in aged animals, as well as

redress the accompanying neuronal Ca2+ dysregulation seen in the

hippocampus of aged animals.

2.2 Methods

Animals: Young male F344 (3 months old), aged male F344 (21 months old), and young male Sprague-Dawley rats (2-6 months old) were used for this study.

Young F344 animals were not treated with insulin, and were used to help gauge

the impact of intranasal insulin on memory function in aged F344. All animals

were fed an 18% protein rodent diet (Harlan Teklad, diet #2018; Madison, WI).

In vivo insulin delivery and doses: Levemir® doses were approximately

0.0715, 0.143, or 0.286 IU/day/rat delivered in two 5 uL applications applied to

the tip of the right naris using a P10 Eppendorf pipette (see supplemental figure

1) [adapted from 383]. Humalog® dose was chosen to deliver 0.0715 IU/day/rat

for behavioral characterization. An intranasal 0.286 IU/rat dose was also

administered once to aged animals (n = 3) to monitor the impact on peripheral

glucose (supplemental Table 1). Each application was separated by 1 min,

during which the animal was held supine and immobile in a DecapiCone®

(Braintree Scientific Inc., Braintree, MA). Each animal was acclimated to this procedure for 4 days prior to initiation of the Morris water maze protocol. All animal groups were exposed to the DecapiCone® for the same duration, and no signs of stress from the bagging procedure were noted. The insulin doses were chosen to approximate several clinically-relevant doses used in past studies ranging from 10 IU/day to 40 IU/day and were adjusted by brain weights.

Assuming a 70 year old human brain weighs 1360 g [384], 10, 20 and 40 IU/day are equivalent to 0.0074, 0.0147, and 0.0294 IU/day/g of brain, respectively.

Assuming a 21 month old F344 brain weighs 2.06 g [385], our equivalent doses were 0.035, 0.069 and 0.138 IU/day/g of brain. It should be noted that these values may underestimate final insulin concentration given the greater olfactory epithelium surface in the nasal passages of the rat (50%), compared to man (8%)

[386]. Insulin solutions were made fresh weekly from 100 U/mL vials (Humalog®, insulin lispro, Lilly; Levemir®, insulin detemir, Novo Nordisk) and diluted into sterile 0.9% saline.

Blood glucose levels: To ensure that the intranasal delivery did not result in reduced peripheral glucose levels, and to test whether stress from the acute restraint procedures (DecapiCone®) could elevate blood glucose levels, we measured blood glucose (FreeStyle Lite glucometer; Abbott Laboratories, Abbott

Park, IL) from dorsal tail veins in a subset of aged animals either exposed with

intranasal Levemir® (n = 5 from each treatment group) or Humalog® (n = 3),

both before and approximately 1 hour after dosing (see supplemental Table 1).

Animal behavior treatment groups and Morris water maze (MWM) procedures: Two cohorts of F344 animals were used for behavioral characterization (supplemental Fig. 1B). One cohort of animals (n = 50) was used to test the impact of long-acting insulin Levemir® at 3 different doses (low, medium and high; n = 10 per group) compared to an intranasal saline young group (3 months; n = 10) and an intranasal saline aged group (21 months old; n

= 10). A second cohort of aged animals (n = 20) was used to test the impact of short-acting insulin Humalog® against saline (n = 10 per group). For analysis,

behavioral data from the two aged saline control groups in both cohorts were

combined because memory performance on probe day was similar (path length

to goal, p = 0.89 and cumulative search error, p = 0.913). One aged animal in the

saline group died during acclimation prior to initiation of behavioral testing. The

remaining 69 animals where trained on the MWM. Animals were considered

visually impaired or unable to learn the task if they could not find the platform on

all 3 of the trials on the cue day (visual platform) and at least one of the

remaining 3 training days. Based on these conditions, 2 aged Humalog® animals

and 3 aged low-dose Levemir® animals were removed from the analysis.

Behavioral results are therefore presented on 64 animals, 10 young saline, 19

aged saline, 8 aged low-dose Humalog® (10 IU/day), 7 aged low-dose Levemir®

(10 IU/day), 10 aged medium-dose Levemir® (20 IU/day), and 10 aged high-

dose Levemir® (40 IU/day). On cue day, no significant differences were found across any of the groups on measures of path length (1-way ANOVA, p>0.05) and swim speed across insulin dose (1-way ANOVA, post-hoc p>0.05), indicating motivation and motor activity were likely similar.

Insulin or saline treatment in both cohorts was initiated 4 days prior to the initiation of training (sup. Fig. 1B) and was delivered 1-3 hours before testing on the MWM. The MWM protocol has been published elsewhere [380, 387], but briefly, water temperature was maintained at 26-27oC, and black liquid tempera paint was used to make the water opaque and hide the submerged escape platform (~1.5 cm below water surface). A Videomex-V acquisition system and water maze analysis software (version 4.64, Columbus Instruments, Columbus,

OH, USA) were used to track and measure animal movements. Animals were allowed 60 s to find the platform, after which they were guided to it. Each animal stayed on the platform for 30 s before returning to a heated holding chamber for about 2 min. On cue day, a white cup hanging above the partially submerged platform helped orient the animals. Animals were subjected to 3 trials per day

(semi-random drop location for each trial) for cue and training days. Twenty-four hours after the last training day, a probe trial was initiated with the platform removed (60 s max swim time). Swim speed was derived from the distance travelled over time on the last trial of the third training day and on the first trial of the cue day. On all cue and training days the inter-trial interval was approximately 150 s. All behavioral experiments were conducted with the

experimenters blind to the treatment groups using color coding of the insulin and

saline vials.

Electrophysiology: Hippocampal slices were used across two electrophysiological setups, one monitoring extracellular field potentials and, the other measuring intracellular potentials (e.g., AHP). All data were acquired using pClamp 8.0 (Molecular Devices MDS, Toronto, Canada) through a Digidata

1320A A/D converter (Molecular Devices). Potentials (e.g., amplitude and duration of AHPs, field EPSPs) were quantified off-line using Clampfit (Molecular

Devices). Nineteen of the twenty behaviorally characterized aged animals

(second cohort used to test the impact of short-acting insulin Humalog® against saline) were used 2-5 weeks after the last day of behavioral characterization to determine the impact of single intranasal insulin dose in vivo. From those, a total of 30 slices used for extracellular field potential recordings (Fig. 2D and 2E) and

13 cells used for intracellular recordings (Fig. 2B and 2C) were used for statistical analyses and are presented using an n of 1 per cell/slice. In data presented in figure 3, 16 Sprague-Dawley animals (2-6 months old) were used to monitor the impact of zinc and Humalog® ex-vivo both extracellularly and intracellularly (30 slices and 23 cells, respectively). Work presented in figure 4 (intracellular physiology only) is compiled from a total of 17 F344 male rats split into young (n

= 8; 3 months) and aged (n = 9; 22 months). This last group of animals yielded recordings from 6 cells for young ACSF, 8 cells from young Apidra®, 6 cells for aged ACSF and 5 cells for aged Apidra®.

Figure 2.1 Morris water maze spatial learning and memory task.

(A) Path length to submerged goal platform on the initial 3 days of training

showed improved performance across all groups tested (RM ANOVA p<0.0001),

however, no group effect was seen. On probe day, with platform removed,

animals receiving low doses of insulin (Humalog® or Levemir®) showed reduced

path lengths to goal (B) and improved memory performance based on reductions

in cumulative search error (C). Analysis of swim speed measured on the third training day shows aged F344 animals are slower (note no effect of insulin dose on speed (D)). Means ± SEM are shown. Asterisks indicate significant

differences from the young saline group at p<0.05; pound sign indicates a trend

(p=0.059) in comparison to the young saline group.

Figure 2.2 Impact of in vivo intranasal Humalog® on intracellular and extracellular physiology.

(A) Representative intracellular recording of the AHP following a series of 4 action potentials (truncated to emphasize the AHP). The mAHP (B) and sAHP

(C) amplitudes recorded 3-7 hours after intranasal Humalog® in F344 rats were not significantly different. EPSP amplitudes (D) and I/O slopes (E) when recorded extracellularly 3-7 hours after intranasal Humalog® also were not significantly altered. Means ± SEM are shown. 47

Figure 2.3 Impact of ex vivo Humalog® and zinc on the AHP.

(A) Representative voltage trace of the AHP recorded in a cell from an ACSF treated slice (black) and a Humalog® treated slice (1 nM, grey). Data are derived from young Sprague-Dawley animals. Traces are truncated to emphasize the

AHP. Significant reductions in the sAHP amplitude (C) and duration (D) were seen in response to Humalog® as well as zinc. Note that zinc and low concentration Humalog® seem to work more robustly on the sAHP (only a trend was noted for the mAHP (B)). No significant effect was detected during extracellular recording of field EPSP amplitudes (E) or slopes of the I/O curve

(F). Means ± SEM are shown.

Figure 2.4 Zinc-free Apidra® reduces the AHP ex vivo.

The mAHP amplitude is shown in (A) and the sAHP amplitude is shown in (B).

Means ± SEM are presented in response to 1nM Apidra®. These data are taken from young and aged F344 rats. Asterisks indicate significant age-dependent increases in the mAHP and sAHP, and pound signs illustrate significant insulin- mediated reductions in the AHP. Again, the impact of insulin seems more robust on the sAHP compared to the mAHP. These recordings were obtained with 4.5 mM glucose in the ACSF.

Figure 2.5 Intranasal insulin delivery and experimental timeline.

(A) This photograph shows a supine aged F344 rat during intranasal insulin delivery. The animal is held in place temporarily in a decapicone® with the head gently restrained. Note that the pipette tip is not inserted into the naris but presented at the entrance of the right naris. (B) Timeline used for behavioral

(Morris water maze – MWM) and electrophysiological characterization presented in figures 1 and 2 using two cohorts of F344 rats. T1, T2 and T3 are initial training days on the MWM and are followed with a 24 h memory recall task

(Probe). Each red triangle represents a consecutive day for intranasal insulin delivery in 2 different cohorts of animals. All animals were treated/ handled similarly across cohorts, and received 11 saline or insulin doses. Animals in cohort 2 were also used 2-5 weeks after behavioral characterization, to test the

electrophysiological impact of intranasal Humalog® in vivo. Electrophysiology data presented in figures 3 and 4 are derived from Spraque-Dawley and F344 animals, respectively.

Table 3. Blood glucose analysis.

10 IU 20 IU 40 IU 40 IU Saline Levemir® Levemir® Levemir® Humalog® (n=5) (n=5) (n=5) (n=5) (n=3)

Pre-Dose 73.2 ± 2.9 72.6 ± 6.2 70.8 ± 7.2 75.2 ± 3.8 65.3 ± 2.0

Post-Dose 82.0 ± 3.7 82.2 ± 4.0 74.2 ± 6.5 78.4 ± 5.3 82.3 ± 6.0

Numbers represent mean ± SEM peripheral glucose levels (mg/dL) taken before

(Pre-Dose) and 30-90 min after intranasal insulin Levemir® or Humalog® delivery (Post-Dose). The number in parentheses represents the number of aged animal tested in each group. Intranasal insulin (Levemir® or Humalog®) did not reduce peripheral blood glucose levels.

Field potentials: Rats were anesthetized with Isoflurane® prior to decapitation.

Brains were quickly removed and placed in oxygenated artificial cerebro-spinal fluid (ACSF) of the following composition (in mM): 114 NaCl, 3 KCl, 10 Glucose,

1.25 KH2PO4, 26 NaHCO3, 8 MgCl2, 0.1 CaCl2. Hippocampi were sectioned

(coronal) with a Vibratome® 3000 (Bannockburn, IL) set at 400 µm. Slices were transferred to an interface-type recording chamber (static) containing oxygenated recording ACSF of the following composition (in mM): 114 NaCl, 3 KCl, 10

Glucose, 1.25 KH2PO4, 26 NaHCO3, 2.5 CaCl2 and 1.3 MgCl2. The recording

chamber was kept at 32° C and warm moist 95 % O2/ 5 % CO2 was continually

delivered to the chamber. Bipolar synaptic stimulation was delivered through a

SD9K stimulator (Astro Med Inc., Grass Instr., Warwick, RI). Input/output curves

(I/O) were recorded using a series of increasing voltage steps. The relationship between stimulus intensity and EPSP amplitudes (prior to dendritic spike contamination) was fit (linear, r2>0.9) to yield a measure on the slope of the I/O.

Data are also reported as the absolute amplitudes of the EPSPs measured as

the maximum deflection from baseline. Insulin (1, 100, 500 nM Humalog®) and/or zinc treatment (130 nM – see AHP-intracellular recordings) under these conditions was initiated 1 hour after placing the slices in the chamber

(compounds remained in the chamber for the duration of the experiment.

AHP-Intracellular recordings: Hippocampal slices (350 µm) were transferred from the static chamber and placed in a perfusion chamber (RC22C, Warner

Instruments, Co., Hamden, CT) and maintained in a continuous flow of oxygenated ACSF pre-heated at 32°C using a TC2Bip/HPRE2 in-line heating system (Cell Micro Controls, Northfolk, VA). The ACSF contained 2 mM CaCl2

and 2 mM MgCl2 and in some experiments (Figure 4 only), the traditional 10 mM

glucose concentration was replaced with 4.5 mM glucose. This was done to test

whether higher glucose concentrations altered insulin sensitivity. We chose 4.5

mM to help maintain tissue health and physiology for extended hours [388], to

approximate values in anesthetized preparations [389], and to consider

fluctuations in brain glucose levels across a day [390]. As previously described,

cells were impaled with sharp microelectrodes filled with 2M KMeSO4 and 10mM

HEPES, pH 7.4 (tip resistance 90-120 MΩ) [377]. Recordings of membrane input

resistance (IR) were obtained in response to 800 ms, 200 pA hyperpolarizing

current injections using an Axoclamp 2B amplifier (Molecular Devices, MDS,

Toronto, Canada) while holding the cell at -70 mV. To generate an

afterhyperpolarization (AHP) cells were held at -65 mV (baseline) and

depolarized with a 100 ms current injection to generate four Na+ action potentials. The medium AHP (mAHP) was measured as the peak hyperpolarization immediately after the offset of the depolarizing current injection, the slow AHP (sAHP) was measured 800 ms after the end of the current injection. The AHP duration was measured from the end of the depolarizing step until return to baseline.

Several concentrations and formulations of insulin were tested ex vivo by addition to the solution bathing the hippocampal slices. Humalog® concentrations of 1, 100, and 500 nM were used. A zinc (ZnCl2) concentration of

130 nM was used to mirror levels of free zinc attained in 250 nM Humalog®.

Apidra® (insulin glulisine, Sanofi, a zinc-free insulin formulation) was used at 1

nM and results were compared to ACSF. Under these experiments, Apidra® was

used to treat slices both in the static chamber and during recording of the AHP.

Apidra® was continuously perfused into the flow of the oxygenated recording

ACSF using a precision perfusion pump (Orion Research, Model 341B, Boston,

MA) and resulting in a final concentration of 1 nM (diluted in sterile saline once a week from the 600 uM stock). Only neurons with input resistance > 25 MΩ, holding current < 500 pA and action potentials reaching above zero mV were included in the analysis.

Statistics: Statistical analyses testing for significance (p<0.05) of main factors and interactions used a 1-way or a 2-way ANOVA as well as a t-test design

(some electrophysiology). Fisher’s PLSD was used for post-hoc comparisons. All tests were conducted using StatView statistical package (version 5, Cary, NC) or

GraphPad Prism V5 (San Diego, CA). Together the behavioral and electrophysiological data presented here are derived from a total of 106 animals.

2.3 Results

Blood analyses. Several studies show that intranasal insulin either has no effect, or a moderate lowering effect (within the euglycemic range) on peripheral blood glucose [31, 367-369, 383, 391, 392]. A 2-way RM ANOVA shows intranasal insulin did not alter peripheral blood glucose levels in a subset of the aged animals (n = 5/group) 30-90 min after intranasal insulin Levemir® delivery

(supplemental Table 1). In another group of aged animals, 40 IU Humalog® also was unable to alter blood glucose levels. This result is consistent with transport of insulin to the perivascular space around blood vessels, indicating the hormone may not have crossed into the blood stream [34, 386].

Behavioral analyses. All groups learned to find the platform in the MWM as

demonstrated by a significant decrease in path length to goal across the first 3

days of training (F(2,116)=27.2; p<0.0001). During this learning phase, path length

(Fig. 1A) however, showed no significant differences across groups with age or

intranasal insulin dose (2-way RM ANOVA). As shown previously [387, 393],

analysis of swim speed revealed a significant group effect (F(5,58)=12.5;

p<0.0001) with post-hoc tests indicating aged animals swam at slower speeds

compared to young animals, but no difference between the insulin treatment

groups was noted (Fig. 1D). Latency to target, as predicted from slower speeds

in aged animals, was significant across 3 days of training (F(2,116)=28.9; p<0.0001), with increased latency seen in all aged animals compared to young saline animals (data not shown). Because of this decrease in swim speed we chose to assess search errors and quantify memory performance on the probe day (no platform) using a proximity measure analysis. This approach is less dependent on swim speed than latency and is tabulated using a cumulative search error derived from the subject’s distance to the platform summed over the time to reach the platform and is then subtracted from an ideal path (straight line); smaller values are indicative of better performance [394].

Compared to the learning component of the task, the 24 h memory recall

component measured during the probe trial (1-way ANOVA) revealed a

significant group effect on path length to goal (F(5,58)=2.9; p<0.05) and on

cumulative search error (F(5,58)=3.3; p<0.05). Post-hoc analyses identified a significant increase on path length and cumulative search error in the aged saline

(p<0.05) and aged medium-dose Levemir® (p<0.005) groups compared to the young saline group (Figs. 1B and 1C). A trend (p=0.059) for improved performance was found for the high-dose Levemir® animals on path length measures only. Notably, no significant differences were found between young saline treated and aged animals treated with either low-dose Humalog® or low- dose Levemir® on path length or cumulative proximal distance (Figs. 1B and

1C), indicating that the lower dose intranasal insulin significantly reduced age- dependent memory loss. Additional post-hoc analyses on memory performance in aged animals, comparing combined low dose insulins (Humalog® and

Levemir®) with combined medium- and high-dose Levemir®, revealed significant

improvement in the low-dose insulin group on path length (F(1,33)=5.25; p<0.05)

and cumulative search error (F(1,33)=5.91; p<0.05). Thus, each of the different

insulin formulations (Humalog® and Levemir® at low-doses) improved

performance despite being administered to two separate cohorts of aged

animals, indicating reproducibility of the insulin effect, and it appears lower,

rather than higher doses of insulin are able to redress cognitive decline with

aging.

Electrophysiology analyses. Based on improved memory recall observed in

aged animals treated with low-dose insulins, we next characterized the effects of

in vivo intranasal insulin on hippocampal electrophysiology in a subset of the

Humalog® treated animals that were behaviorally characterized. Supplemental

Table 2 shows that passive membrane properties were not different between

groups and suggests cells with comparable baseline characteristics were

analyzed. Intracellular recording of CA1 pyramidal neurons were obtained from

animals treated with intranasal saline or Humalog® (see Methods).

Approximately 3-7 hours following intranasal insulin delivery the Ca2+-dependent

AHP was not significantly different between groups (Figs. 2B and 2C; t-test). This was true for measures of AHP amplitude (medium and slow AHP) and duration

(not shown). Extracellular synaptic potentials also showed no significant difference across groups (Figs. 2D and 2E; t-test). EPSP amplitudes measured at 33% of the I/O were slightly larger in the Humalog® treated animals and I/O slopes based on EPSP amplitude measures were somewhat reduced (Figs. 2D and 2E, respectively, p<0.1). These results are surprising given our prior report that insulin application to hippocampal slices reduces the AHP [380]. In the prior study, however, hippocampal slices were perfused for 15 minutes with insulin and then studied electrophysiologically. The results obtained in the present study raise the possibility that the delay between intranasal insulin exposure and electrophysiological recording (3-7 h) was too long and that the effect of insulin on the AHP may have dissipated. Thus, to confirm that insulin must be present in order to alter hippocampal physiology, we tested a range of ex-vivo insulin

concentrations and formulations on hippocampal slices from a separate group of

animals.

We used young animals in the next series of experiments, and examined three

different doses of Humalog®, comparing Humalog®’s actions to zinc and ACSF.

In most insulin formulations, zinc is used to promote hexamer formation and

prolong stability and duration of action in vivo. However, zinc is also known to

modify ion flux across neuronal membranes, reducing activity at NMDA and

GABAA receptors, ion channels associated with memory-related processes

[reviewed in 395]. Extracellular recordings in the presence of zinc or Humalog® showed that neither EPSP amplitudes, nor I/O slopes were significantly altered when compared to ACSF controls (Figs. 3E and 3F; 1-way ANOVA). Similarly to

data shown in figure 3, these results indicate little if any, effects of insulin on

extracellular postsynaptic potentials.

Intracellular recordings obtained from the same group of animals showed that the

Ca2+-dependent AHP was sensitive to treatment with zinc or insulin. As previously reported, measures of postsynaptic excitability demonstrated the sensitivity of the AHP to both zinc [396] and insulin [380]. A main effect of

treatment on the slow AHP (F(4,18)=3.34; p<0.05) and its duration (F(4,18)= 4.49;

p<0.02) was seen across 5 groups of recorded cells (Figs. 3C and 3D). Albeit

being somewhat reduced, the mAHP was not significantly altered by insulin (Fig.

3B). These results suggest a component of the insulin effect on the AHP could

well be mediated by zinc found in this insulin formulation. To test the direct

impact of ex vivo insulin on the AHP, we next investigated a zinc-free formulation

of insulin (Apidra®).

In this next series of experiments we obtained hippocampal slices from young

(n=8) and aged (n=9) F344 rats and focused exclusively on intracellular

potentials and the response to ex vivo Apidra® exposure (1 nM). Further, a

recent study identified that 0.1-100 nM exogenous insulin concentrations could

reliably elicit dose-dependent activation of the insulin signaling pathway in human

brain tissues [20], and approximated physiological levels near 1 nM. For these

reasons, we used 1 nM Apidra®. As previously reported [377-379, 397] and

shown here (Fig. 4), mAHP and sAHP amplitudes recorded in CA1 pyramidal

neurons were increased (F(1,24)=17.6, p<0.0005; F(1,24)=4.7, p<0.05, respectively)

in aged, compared to young animals (2-way ANOVA). Treatment with the zinc- free Apidra® insulin significantly reduced the AHP in slices from both young and aged rats as indicated by a main effect of treatment on the mAHP (F(1,24)=32,

P<0.0001) and the sAHP (F(1,24)=29.1, p<0.0001). However, the Apidra® -

mediated AHP reduction was larger in aged animals compared to young animals

(Figs. 4A and 4B) as evidenced by a significant interaction term in the 2-way

ANOVA (F(1,24)=10.9, p<0.005 for the mAHP). These results are consistent with

our prior work using Humalog® showing that the hippocampal AHP is sensitive to

insulin [380]. In the experiments presented here however, it is clear that insulin

alone has a direct effect on the AHP, and that this effect persists even when

lower, more physiological glucose levels are used in the ACSF (experiments in

Figure 4 were conducted with 4.5 rather than the typical 10 mM glucose

concentration - see Methods). Finally, it is interesting to note that Apidra® did not

alter holding current when compared to control cells (sup. Table 2), indicating insulin is neither activating nor inhibiting a tonic current near resting potential.

2.4 Discussion

Studies in humans have shown that intranasal insulin has promising cognition enhancing effects, alleviating, and perhaps even slowing the progression of age- related neurodegenerative disorders. However, the mechanisms underlying insulin’s effects in the brain are unclear. In order to identify potential mechanisms, we treated aged rodents with intranasal insulin, attempting to model aspects of the clinical trials reporting on the beneficial impact of intranasal insulin. Here, we describe physiological actions of insulin in the brain, highlighting a potential new mechanism of action of insulin on hippocampal function in both young and aged rats. This first analysis of intranasal insulin in aged animals demonstrates that intranasal insulin improved memory recall in aged F344 rats.

We show that the effect on memory was observed in two separate cohorts of animals, using two different insulin formulations (Humalog® and Levemir®)

delivered at relatively low dose (0.0715 IU/day/rat). Thus, these studies also

demonstrate reproducibility of the effect of insulin on behavior. We also show that

the Ca2+-dependent afterhyperpolarization (AHP), and in particular the slow

AHP, a Ca2+-related biomarker of brain aging which increases with age and

cognitive decline, is reduced by acute insulin exposure in aged animals and

could well represent a novel target of brain insulin underlying the improvement

seen in memory recall.

Long-acting insulin (Levemir®) was as effective as short-acting insulin

(Humalog®) on memory recall in aged animals, inducing levels of performance

indistinguishable from those seen in young animals. Levemir®’s long duration of action is dependent on the presence of albumin in the periphery and a fatty acid modification of the insulin structure, increasing its’ affinity for albumin.

Humalog®’s shorter duration and faster onset are due to amino acid modifications in the β-chain of the insulin molecule. Because both versions of insulin were able to reverse cognitive decline to a similar degree in aged animals, our results suggest both insulins were able to gain access to the brain following intranasal administration despite structural differences. While results from clinical studies in memory-impaired older adults have reported improved word recall within 15 min post intranasal delivery [398], a prior clinical study on younger

adults demonstrated the superiority of a faster acting insulin (Novolog®)

compared to regular insulin (Actrapid®) on delayed word recall after 7 weeks of

intranasal 160 IU/day Novolog® [392]. Using shorter delays (i.e., 10 min) a

recent study reveals 40 IU intranasal insulin delivery to young adult males

improves odor-cued spatial memory [399]. Clearly, the memory enhancing

effects of insulin are depend on treatment regimen (acute vs. subchronic), but

also on the age and gender of the subject [400], as well as on the insulin

formulation used [382, 401]. As mentioned above, a single, acute intranasal dose of insulin is capable of enhancing memory in humans and our electrophysiological studies showing an acute action of insulin on the AHP indicate that insulin can have rapid effects on neuronal processes critical to

memory (see below). However, in the current study, it is unclear whether the

effects on cognition observed here were due to an acute action of insulin (i.e., the

dose received 1-3 h prior to memory testing), or the result of the cumulative daily

treatments received over the course of 8-11 days.

he decrease in the AHP by insulin was observed in field CA1 of the

hippocampus, a synaptic zone which plays a key role in memory processing.

Decreasing the AHP in this structure would be expected to increase neuronal

excitability and facilitate throughput during physiological activation. Consistent

with this, exogenous insulin increases excitatory neurotransmitter receptor

trafficking including NMDA and AMPA receptors [88, 358, 359, 402], thus

promoting network activation. Furthermore, pure insulin has been shown to

activate an inward current and depolarize hypothalamic neurons via activation of

transient receptor potential channels, providing supporting evidence that insulin

raises excitability [403]. However, recent results provide evidence that insulin or

other therapies that raise insulin (exendin-4 and glucagon-like peptide-1),

increase functional GABAA conductances in CA1 [352] as well as in CA3 pyramidal neurons [404]. This observation is in line with prior reports of enhanced GABAA function in response to insulin, a resulting decrease in synaptic

activity [350, 351] and a long-lasting enhancement of inhibitory post-synaptic currents (IPSCs) [405]. The net result, therefore, of enhancing both steady-state

hyperpolarizing (GABA-mediated) and short term depolarizing forces (e.g.,

reduced AHP or direct depolarization) could well increase synaptic signal-to-

noise, thereby improving throughput and neuronal communication. Clearly, as

seen in hypothalamic arcuate nucleus neurons, it is important to note that

insulin’s modulatory influence on membrane potential/ excitability, is insulin

formulation dependent, as well as cell-type specific [403].

To extend slice health and allow for extended neuronal recordings, high glucose

concentrations (10 mM) are commonly used in the ex-vivo hippocampal slice.

However, this superphysiological glucose concentration may alter insulin

sensitivity in the brain. For this reason, we used lower glucose levels in some

experiments while maintaining tissue health (see Table 2), and more closely

approximating physiological glucose levels. Importantly, significant AHP

reduction was seen irrespective of recording conditions (10 or 4.5 mM glucose).

Thus, the results presented here appear compatible with potential effects of

insulin on hippocampal neurons in vivo.

The placebo group in almost all clinical trials studying the impact of intranasal

insulin on memory function is generally exposed to either saline or HOE-31, a dilution buffer that does not contain zinc. Given that all insulin formulations except Apidra® contain zinc, our results showing that Apidra® is capable of reducing the AHP in hippocampal neurons ex vivo could have clinical significance. These data are consistent with insulin per se being able to alter hippocampal physiology, and suggest that insulin and not zinc may be responsible for the CNS effects of intranasal insulin on memory. Clearly further clinical trials with Apidra® appear warranted.

Because the AHP is dependent on a Ca2+-activated K+ conductance, the insulin-

mediated reductions in the AHP may be due to inhibition of Ca2+ influx [406] or

inhibition of L-type Ca2+ channel currents as seen in pinealocytes [407]. Taken

together, these results suggest insulin acts through the insulin receptor to reduce

Ca2+-mediated functions possibly through PI3 kinase- or tyrosine kinase-

mediated phosphorylation of Ca2+ channel proteins. Indeed, insulin can enhance

mitochondrial function and therefore, Ca2+ homeostasis through PI3 kinase

mechanisms in the periphery [193]. Insulin also increases sarco-endoplasmic

reticulum Ca2+-ATPase (SERCA) function in heart cells via an Akt (protein

kinase B) pathway, and in neurons, insulin has been shown to reduce KV1.3

potassium channel function [408]. Notably, all of these potential mechanisms are

consistent with reductions in available Ca2+, and hence, reductions in the

amplitude of Ca2+-dependent hyperpolarizing potentials.

Our data cannot address whether the beneficial actions of intranasal insulin on memory recall are mediated, in parts or in whole, by the effects of insulin on the

AHP. While suggestive that this may be the case, we do not provide evidence to support this conclusion. Further, because young animals were not tested for the

impact of intranasal insulin, future studies will be needed to test the hypothesis

that the observed effect is age-selective, or generally nootropic. Finally, although a similar insulin dose yielded quantitatively similar enhancement on memory recall in two cohorts of aged animals, it is not clear that higher Humalog® doses might also be ineffective, similarly to higher Levemir® doses.

The present results shed new light on a previously unrecognized insulin mechanism in the brain. Reductions in the AHP could enhance neuronal communication and might represent a pathway through which low dose

Humalog® or Levemir® improve memory recall in aged animals. We show that

insulin targeted the AHP, a Ca2+-mediated conductance, and reduced AHP amplitude and duration, elevations of which are characteristic of brain aging. We propose that facilitating insulin signaling restores Ca2+ homeostasis in aged

animals, resulting in optimal levels of membrane excitability and synaptic

plasticity, in part by limiting the amplitude and duration of the AHP in CA1

neurons (Figs. 4 and 5). During aging, reductions in brain insulin levels and/or

insulin receptor function (see Introduction) may help promote neuronal Ca2+

dysregulation, resulting in impaired membrane excitability and reduced synaptic

plasticity (e.g., larger AHP). Clearly, given the reductions in insulin receptor RNA

and protein in the aged rodent [27, 111], greater occupation of the leftover insulin

receptors with insulin could well offset reductions in signaling through these

receptors, or may even slow the age-related reduction in insulin receptor density.

Collectively, these results indicate robust associations between brain insulin,

Ca2+ homeostasis, and aging-related cognitive decline, and suggest the value of further developing “insulin-raising” strategies for treating cognitive decline in age and disease, perhaps curbing the development of AD.

Chapter 3 Link between studies

3.1 Intranasal insulin and cognition

In the late1990s, several clinical studies suggested that diabetes is a risk factor for cognitive decline and AD [142, 409, 410]. Also, through population-based studies, it has become clear that dysregulation in insulin signaling increases the risk for cognitive decline with aging and AD [139, 411-417]. In addition, imaging studies have shown that diabetes patients have smaller hippocampi and that brain functional connectivity also changed compared to healthy individuals [418-

422]. Furthermore, studies have found that insulin receptors are widely expressed in different regions of the brain and play a vital role in regulating whole-body glucose metabolism, energy balance, and neurotransmission in the brain [423]. In addition, aging can alter insulin and insulin receptor function, reduce pancreatic beta cells function, and reduce insulin secretion by pancreatic beta cells [424, 425]. These studies ignited interest in the effects of insulin in the brain in the context of cognition and led the way for later research confirming that insulin is an important cognitive enhancer.

Interest in the study of intranasal insulin has also stemmed from a number of clinical studies demonstrating that intravenous insulin infusion during clamped glucose conditions improved memory [15, 29, 137, 149]. Clearly, due to the danger of hypoglycemia, insulin infusion peripherally is not a feasible approach for treating AD. On the other hand, it has been shown that intranasal insulin

delivery targets the brain, bypassing the blood brain barrier, and without the risk of hypoglycemia [349, 426]. There are several ongoing intranasal insulin clinical trials, including study of nasal insulin to fight forgetfulness (SNIFF), intranasal insulin in children and young adults at risk of type 1 diabetes, intranasal insulin for the HAND (HIV-associated neurocognitive disorder), and intranasal insulin for stroke patients. My research is focus on the neuronal effects of intranasal insulin on the context of cognitive decline with aging.

Intranasal administration of insulin appears to be one of the best methods to treat cognitive decline due to its minimal effect on systemic glucose levels and its acute improvements on hippocampal-dependent memory. Intranasal insulin enters the brain mainly through olfactory and trigeminal pathways [427-429].

Insulin binds to insulin receptors in various brain regions, including the hippocampus [430]. The hippocampus is well-known as a principle brain region for learning and memory. Normal insulin signaling of neurons within the hippocampus area is typically associated with healthy brain aging. Furthermore, deficiency in brain insulin signaling accelerates pathological brain aging and cognitive decline. The goal of intranasal insulin study in our lab is to achieve a greater understanding of the effects of insulin and insulin signaling pathways that might slow or reverse age-related memory decline and pathologies.

We found that a repeated low dose of intranasal insulin had a positive effect on memory recall in aged F344 rats. However, medium and high doses of insulin did not have a beneficial effect on memory in aged F344 rats. This varying

outcome is in line with clinical intranasal insulin studies. Clinical studies show that intranasal insulin therapy improves memory maintainability in healthy and

AD patients [32, 431]. Although intranasal insulin enhances memory in AD patients, varying outcomes arise from its clinical setting usage [431]. Men and women with AD respond differently to intranasal insulin dosages [36]. In addition, some clinical studies have reported that patients carrying at least one copy of the Apolipoprotein E4 (the strongest genetic risk factor for late onset

Alzheimer’s disease) did not benefit from intranasal insulin treatment [37].

Hence, these clinical trials revealed that not everybody responds equally to intranasal insulin therapy. Based on both clinical and animal studies, different formulations and dosages of insulin, as well as gender and ApoE status in AD patients increase the outcome variability in intranasal insulin treatment. A clear understanding of the mechanisms underlying insulin actions in the brain will help address these varying outcomes and increase the efficacy and safety of using intranasal insulin to treat cognitive impairment.

3.2 Insulin and Ca2+-dependent after hyperpolarization

It is also well documented that the Ca2+-dependent AHP (Ca2+ related biomarker of brain aging) increases in aged and memory-impaired animals. The greater AHP is correlated with reduced neuronal excitability and learning ability

[30, 184, 377, 379, 380, 432-437]. It is widely accepted that reducing the AHP is one viable pathway to enhance learning and memory in aging animals. Data

from our lab suggests that insulin reduces the AHP in CA1 neurons from young and aged rat hippocampal slices [30, 380]. These findings are in line with previous behavioral studies, suggesting that insulin therapy improves memory by reducing the Ca2+ dependent AHP [30]. However, the molecular mechanism(s) that result in such an interaction has not been studied in depth.

Ca2+ and potassium are the main ions that influence the amplitude and duration of AHP. Here, I focused on the relationship between insulin and intracellular

Ca2+ homeostasis.

Another factor besides insulin that is related to cognitive decline in aging and AD is elevated intracellular Ca2+ concentration [187-191]. The two main sources of intracellular Ca2+ are Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) on the plasma membrane and Ca2+ efflux via ryanodine receptors (RyRs) mediated

Ca2+-induced Ca2+-release (CICR) on the endoplasmic reticulum.

3.3 Voltage-gated Ca2+ channels dysfunction with aging

Voltage-gated Ca2+ channels (VGCCs) are responsible for Ca2+ influx into the cytosol in response to membrane depolarization. VGCCs play a critical role in neuronal excitability, neurotransmission and Ca2+ signaling, which is important for learning and memory in healthy and disease states. All types of VGCCs are expressed in the hippocampus and involved in neuronal firing, action potential, and synaptic plasticity [438]. Furthermore, the density of plasma membrane

VGCCs increases with aging and results in elevated intracellular Ca2+ levels,

which is toxic to neurons and leads to cell death [226, 439-441]. Earlier studies showed that elevated Ca2+ influx via VGCCs contribute to bigger AHP and cognitive decline in aged animals [184, 224, 437, 442, 444-446]. In addition, voltage-gated Ca2+ channel blockers have been shown to improve cognition in aged animals, as well as in dementia and AD patients [447-450]. Moreover, knockout of voltage-gated Ca2+ channels have a great impact on memory in aged animals [451, 452]. In hippocampal neurons, Ca2+ entry though VGCCs during membrane potential triggers Ca2+-induced Ca2+-release from endoplasmic reticulum via the ryanodine receptors, causing amplification of intracellular Ca2+ levels [220, 377, 435, 453].

3.4 Ryanodine receptors dysregulation with aging

Ryanodine receptors (RyRs), an intracellular Ca2+ release channel, is responsible for Ca2+ release from endoplasmic reticulum (ER) and triggers amplification of intracellular Ca2+ levels through RyR-medicate Ca2+-induced

Ca2+-release (CICR). RyRs plays a vital role in maintaining intracellular Ca2+ homeostasis. Thus, RyR-mediated Ca2+ release is critical to cellular physiological events ranging from muscle contraction to learning and memory. All

RyRs isoforms (RyR1-3) are expressed in the various regions of the brain, including the hippocampus, cerebellum, olfactory region and cerebral cortex, which are located within the presynaptic terminals of neurons. The RyRs channels have been found to show changes in functional efficacy during

aging/AD [136, 377, 435, 453-458] and influence the amplitude and duration of

Ca2+ dependent afterhyperpolarization (AHP) [192, 377]. Additionally, increases in RyRs-mediated intracellular Ca2+ release through CICR mechanisms during aging results in loss of intracellular Ca2+ homeostasis, thus greatly influencing cognitive function [192, 377, 459].

Taken together, age-related increases in RyRs-mediated intracellular Ca2+ release mechanisms are also responsible for larger AHP seen in memory impaired aged animals. Ultimately, lowering Ca2+ efflux via RyRs is important for reducing the AHP.

However, a central question of whether insulin reduces VGCCs currents remains unanswered. Furthermore, it is not clear whether insulin affects RyRs function. To address these questions, the activity of VGCCs was recorded from neurons that were treated with acute (10 minute) insulin vs. control solutions. In parallel studies, Ca2+ imaging was used in cultured hippocampal neurons to evaluate the RyRs-dependent intracellular Ca2+ level changes in insulin treated neurons. Our approach helped us to delineate one of the many neuronal molecular mechanisms underlying insulin’s effect on memory through the reduction of AHP via regulating intracellular Ca2+.

The following manuscript has been submitted to Neuroscience.

In this manuscript, I continue to study the underlying mechanisms responsible

for the memory enhancing effects of intranasal insulin in aged animals. It is

clear that Ca2+-dependent AHP is one of insulin’s neuronal targets from the

previous study presented in this dissertation (Chapter 2). Those data led me to

focus further on the relationships between insulin and hippocampal intracellular

Ca2+, which influence the amplitude and duration of AHP. Voltage-gated Ca2+

channels and ryanodine receptors are major factors that contribute to

dysregulation of intracellular Ca2+ homeostasis present in aged animals.

Hence, I tested acute insulin’s effect on single live hippocampal neurons in

culture using whole-cell patch clamping. In addition, insulin effects on resting

and depolarization mediated intracellular Ca2+ transients were examined in the single live neurons using Ca2+ imaging techniques.

Chapter 4 Novel Ca2+-related targets of insulin in hippocampal neurons

Authors: Shaniya Maimaiti, Hilaree N. Frazier, Katie L. Anderson, Adam O. Ghoweri, Lawrence B. Brewer, Nada M. Porter, Olivier Thibault

Department of Pharmacology and Nutritional Sciences, University of Kentucky Medical Center, Lexington, KY 40536

Synopsis

Both insulin signaling disruption and Ca2+ dysregulation are closely related to memory loss during aging and increase the vulnerability to Alzheimer’s disease

(AD). In hippocampal neurons, aging-related changes in Ca2+ regulatory pathways have been shown to lead to higher intracellular Ca2+ levels and an increase in the Ca2+-dependent afterhyperpolarization (AHP), which is associated with cognitive decline. Recent studies suggest that insulin reduces the Ca2+-dependent AHP. Given the sensitivity of neurons to insulin and evidence that brain insulin levels are reduced with age, insulin-mediated alterations in Ca2+ homeostasis may underlie the beneficial actions of insulin in the brain. Indeed, increasing insulin signaling in the brain via intranasal delivery has yielded promising results such as improving memory in both clinical and animal studies. However, while several mechanisms have been proposed, few have focused on regulation on intracellular Ca2+. In the present study, we further examined the effects of acute insulin on Ca2+ pathways in primary hippocampal neurons in culture. Using the whole-cell patch-clamp technique, we found that acute insulin delivery reduced voltage-gated Ca2+ currents. Fura-2 imaging was

used to also address acute insulin effects on spontaneous and depolarization-

mediated Ca2+ transients. Results indicated that insulin reduced Ca2+ transients,

which appears to have involved a reduction in ryanodine receptor function.

Together, these results indicate insulin regulates pathways to control intracellular

Ca2+ which may be responsible for reducing the AHP and improving memory.

This may be one mechanism contributing to improved memory recall in response

to intranasal insulin therapy in the clinic.

Keywords: diabetes, intranasal, excitability, imaging, electrophysiology, aging

4.1 Introduction

Aging is a major risk factor for Alzheimer’s disease (AD), and both brain aging and AD are characterized by a progressive decline in cognitive and memory function. A promising treatment for cognitive impairment seen in AD is to maintain or enhance insulin signaling in the brain. Insulin is a cognitive and neural modulator that is synthesized by pancreatic β-cells and enters the brain through the blood brain barrier [460-463]. In the periphery and in the brain, insulin has been shown to bind to insulin receptors and to regulate glucose uptake by inducing translocation of glucose transporters to the plasma membrane [464]. In aging or AD, declining insulin levels, insulin receptor numbers, and/or glucose transporters can lead to dysregulation in glucose uptake. This is evidenced by nearly two decades of research showing strong associations between diabetes, cognitive decline, and AD [16, 141-144]. At the cellular level, brain insulin deficiency and reduction in insulin signaling, perhaps mediated by insulin resistance, could represent one of the altered pathways linked to altered memory function or synaptic communication during aging and

AD [15-20, 30]. Underlying this relationship is evidence that insulin receptor numbers and their functions are decreased in aging and AD animal models [111,

346, 465, 466]. As such, interruption of insulin production or in insulin receptor activity may cause cognitive decline [323]. One approach designed to combat this reduction in insulin signaling that has received much interest in the clinic is the use of intranasal insulin delivery to selectively increase ligand concentration in the brain [349].

Intranasal insulin therapy has been shown to improve memory function in AD

patients [32, 431]. The intranasal route of insulin administration raises insulin

acutely in the central nervous system without much risk of peripheral

hypoglycemia [30, 367, 426, 467, 468]. Importantly, it significantly enhances

memory in both healthy individuals and AD patients [31, 33-35, 37]. In aging and

AD animal models, the positive impact of intranasal insulin in combating cognitive

decline has also been reported [30, 381, 467, 469-473]. The mechanisms of

action in the brain, and specifically on neurons of the hippocampus where insulin

plays a recognizable role in learning and memory [28, 111], remain however,

largely unknown.

Because previous work on mechanisms of cognitive aging has focused on Ca2+

dysregulation in hippocampal neurons providing evidence of enhanced Ca2+

levels [377, 439, 445, 474-476] and voltage-gated Ca2+ channels (VGCC)

activity [224, 432, 445, 477], we focused on studying two well-characterized

Ca2+ sources in hippocampal neurons in culture. The rationale for this approach was based on recent evidence from our lab that insulin acutely reduces the

Ca2+-dependent afterhyperpolarization (AHP) in neurons recorded from hippocampal slices [30, 380]. The AHP is a hyperpolarization potential that is enhanced in aging, limits neuronal firing, and is associated with cognitive decline

[379, 380, 433, 478]. The larger AHP seen in aging is mediated in part by an

increase in the density of L-type voltage gated Ca2+ channels (L-VGCC) [224]

and by Ca2+-induced Ca2+-release (CICR) through activation of RyRs [377,

479, 480]. Together, these two sources of Ca2+ contribute to elevated

intracellular Ca2+ levels, larger AHPs and altered synaptic communication.

Thus, here, we tested the hypothesis that acute insulin (glulisine, zinc-free and

fast acting) could reduce Ca2+ levels, or stabilize Ca2+ homeostasis by altering

VGCC function and/ or ryanodine receptor (RyR) function. To address these

questions, we used patch clamp recording of VGCCs with rapid drug delivery,

as well as Fura-2 imaging during depolarization in the presence or absence of

RyR blocker. Our results indicate that insulin is able to reduce Ca2+ levels

during periods of neuronal depolarization and that this is mediated, at least in

part, by reductions in VGCC and RyR function. These two aging-sensitive

neuronal Ca2+ targets are therefore also sensitive to the actions of insulin and

could represent novel therapeutic targets for cognitive decline in aging and/ or

AD. Re-establishing Ca2+ homeostasis represents a mechanism by which

insulin and by extension, intranasal insulin, may offset learning and memory

dysregulation in vivo.

4.2 Experimental procedures

Cell culture: Hippocampal mixed (neuron/glia) cultures were prepared as

described previously [439, 481, 482] and established from (E18) Sprague-

Dawley rats. E18 pups and hippocampi were dissected under a microscope in

ice-cold Hank’s balanced salt solution (Thermo Fisher Scientific Inc., MA, USA)

supplemented with 4.2 mM NaHCO3, 10 mg/L gentamicin and 12 mM HEPES

(pH 7.3). Hippocampi were transferred to a 37oC 0.25 % Trypsin EDTA solution

(Thermo Fisher) and left at room temperature for 11 minutes. Trypsin was

removed and the hippocampi were washed three times with Minimum Essential

Medium (MEM). Hippocampi were then titrated, and diluted with MEM to the desired final concentration (5-7 x 105 neurons/ml) before being plated onto 35

mm poly-L-lysine coated dishes. Cultured neurons were incubated (36°C, 5%

CO2, 95% O2) for 24 h before the first medium exchange. At this time, half of the

medium was replaced with 90% SMEM supplemented with 10% Horse serum.

After three days in vitro (DIV), half of the medium was replaced with SMEM,

horse serum, 5-Fluoro-2-Dioxyuridine and uridine to stop glial cell growth. At DIV

10, a sodium bicarbonate solution (200 uL) was added to help maintain pH and

limit evaporation.

For whole-cell recording experiments, plastic culture dishes were used (Corning

Inc., Corning, NY, USA) and for Ca2+ imaging experiments, glass bottom culture

dishes (Mattek Crop., Ashland MA, USA) were used. All data presented were

collected between DIV 13 and 17, and experiments were conducted following a

24 h exposure to lower glucose-containing MEM (5.5 mM; MEM with no added

glucose). This was done to maintain normal glucose oxidation rates and insulin

sensitivity [482]. All data presented were obtained at room temperature.

VGCC recording solution: For whole-cell, external recording solution of VGCC

currents, was as follows (in mM): 111 NaCl, 5 BaCl.H2O, 5 CsCl, 2 MgCl2, 10

glucose, 10 HEPES, 20 TEA.Cl.H2O, pH 7.35 with NaOH, and 500 nM tetrodotoxin (TTX) was added before recording to inhibit Na+ channels. The

internal pipette solution (in mM): 145 CH4O3S-methanesulfonic acid, 10 HEPES,

3 MgCl2, 11 EGTA, 1 CaCl2, 13 TEA.Cl.H2O, 14 phosphocreatine Tris-salt, 4

Tris-ATP, 0.3 Tris-GTP, pH 7.3 with CsOH. All solutions were sterile filtered.

Drugs and solution application: External solutions were delivered using a rapid

solution exchange system (SF77A - Warner Instruments Corp. Hamden, CT,

USA) positioned approximately 400 um above the cell being recorded. Flow rate

was set to 0.3 mL/min and a control solution supplemented with TTX was used to establish baseline recording (~10 min) during which passive cell membrane properties were obtained (e.g., membrane resistance, capacitance, access resistance), and currents were allowed to run up. Rapidly switching the position of the tubing delivering the solutions above the recorded or imaged cell allowed us to tightly control the environment and deliver either insulin, KCl (50 mM), ryanodine (20 uM), a high-affinity small peptide interacting with insulin (affibody, ab31906, Abcam, Cambridge, MA, USA; 100 ng/mL, 500 ng/mL, or 1 ug/mL), or an insulin receptor antibody (S961, Phoenix Pharmaceuticals Inc., Burlingame,

CA., USA; 500 ng/mL). Insulin glulisine (10 nM, a fast acting, zinc-free insulin) was prepared weekly in external recording solution from a 6 uM stock (Apidra®

600 uM, Sanofi-Avantis US. LLC, diluted in sterile saline). This concentration was chosen based on our previous results from our lab [30, 380].

Whole-cell recording and analysis: To minimize capacitance artifacts, whole-cell

patch-clamp electrodes were coated with polystyrene Q-dope before recording

(see Table 1 for membrane and electrode properties). The culture dish was

rinsed with external solution twice and then supplemented with TTX. To allow for

currents to stabilize, all data were recorded 5-10 min after the whole-cell

configuration was achieved. Current/voltage (I-V) relationships (-60 to + 30 mV)

were used to identify the voltage step eliciting the maximal current amplitude.

This voltage was used for each cell in the study in order to compare data at the

peak of the I-V relationship (150 ms depolarization). Cells were either held at -70

mV (150 ms) or -40 mV (350 ms) and currents were elicited at the maximal peak

response. All currents were leak subtracted using 5-8 scaled hyperpolarizing

sub-pulses. Because insulin may alter cell size, we report on measures of current

densities (pA/pF), derived from dividing peak current amplitude by membrane

capacitance (measured in pClamp) for each cell. All recordings were conducted

on the stage of an epifluorescence microscope (E600FN - Nikon Inc., Melville,

NY, USA) placed on an anti-vibration table. An amplifier (Axopatch 1D -

Molecular Devices, Sunnyvale, CA, USA) in combination with an A/D board

(Digidata 1200 - Molecular Devices) and acquisition software (pClamp 7 -

Molecular Devices) were used for electrophysiology acquisition. Data were

digitized at 5-10 KHz and low-pass filtered at 2-5 KHz and were quantified in

Clampfit 7 (Molecular Devices).

Ca2+ imaging and analysis: Cultures were incubated in Ca2+ imaging solution

(in mM): 145 NaCl, 2.5 KCl, 10 HEPES, 10 D-glucose, 2 CaCl2, 1MgCl2, 0.01 glycine, pH 7.3 with NaOH for 30 min in the dark. This solution contained 2 uM

Fura-2 AM (F1221 – Invitrogen) and was made monthly with fresh DMSO

(0.085%) and Pluronic® F-127 (0.015%, weight/ volume). Each culture dish was then rinsed three times with Ca2+ imaging solution containing no indicator and placed in the dark for 20 min (de-esterification period). The dish was then placed

on the stage of the microscope. Intracellular Ca2+ transients were visualized by

exciting Fura-2 at 340 +/-20 nm and 380 +/- 20 nm using a high-speed filter changer (Lambda DG4, Sutter instruments, Novato, CA, USA) to obtain a ratiometric value independent of indicator concentration. Emitted light was passed through a dichroic filter (400 nm high pass) and an emitter filter (520 +/-

30 nm; Chroma Technology) and was digitized onto the sensor of an EMCCD

camera (iXon-Andor Technology, Belfast, Ireland). Ca2+ transients were

measured and analyzed using Imaging Workbench 5.0 (INDEC BioSystems

Santa Clara, CA) as previously published [481, 482]. The greater ratio (340/ 380

nm) values reported reflect higher Ca2+ levels. The gray value for both excitation

wavelengths of each cell measured (region-of-interest - ROI drawn around soma)

was background subtracted from an area devoid of cellular components. When

imaging Ca2+ during periods of depolarization (50 mM KCl), the Ca2+ imaging

solution was modified with a reduction in NaCl to 97 mM in order to control for

osmolarity changes.

Statistics: Data were analyzed using Prism 5.0 (GraphPad Software, La Jolla,

CA, USA). Paired t-tests and repeated measures ANOVA were used to test for

group differences with significance set at p< 0.05. For electrophysiology

experiments, data from each recorded cell (perfusion conditions precluded

recording 2 cells per dish) was considered a single data point (i.e., n=1). For

Ca2+ imaging data, 5-10 cells in the field of view were averaged and considered

a single data point (i.e., n=1).

4.3 Results

4.3.1 Effect of insulin on whole-cell Ca2+currents.

Ca2+ currents were recorded before and after a 10 mins perfusion of either

external solution (time control; n=19), or external solution supplemented with 10

nM insulin (n=17) using the SF77A fast perfusion system. Cells were held at -70

mV and were stepped to the peak voltage determined from the I-V relationship.

Peak and late currents (obtained immediately prior to termination of the voltage

step) were quantified and are presented normalized to cell size (pA/pF). A

significant decrease in peak (-20%; p<0.0001) and late (-31%, p<0.002) Ca2+

currents in response to acute insulin application was seen (Figure 1A-C). Based

on a subset of the cells recorded (n=7 per group), there was no change in the I-V

relationship (Figure 1D).

To determine if the actions of insulin were selective for L-type Ca2+ channels,

cells were held at -40 mV and stepped (350 ms) to peak voltage. Whole-cell

recording analysis indicated that similarly to reductions seen from a holding

potential of -70 mV, L-type-enriched Ca2+ currents were significantly reduced

(Figure 1E and F) both at the peak (-22%; p<0.0004) and during the late current

phase (-33%; p<0.005). The data suggest that insulin did not alter the inactivation

rate of currents (Figure 1A), and did not show a greater inhibitory effect on

currents elicited from -40 mV. This indicates insulin does not selectively reduce

L-VGCCs but instead, may reduce all VGCCs.

Figure 4.1 Ca2+ channel currents are reduced in response to acute insulin application in mixed hippocampal cultured neurons.

A. Example of VGCC currents recorded during recording solution perfusion

(black) and after 10 min insulin (grey) during maximal step depolarization from -

70 mV. B.-C. Quantification across groups of cells following 10 min of insulin perfusion (Insulin) versus recording solution perfusion (Time Control) shows peak and late Ca2+ currents were significantly reduced (p<0.05). D. In a subgroup of cells, current-voltage relationships were not significantly altered. E. Example of the effects of insulin on L-VGCCs recorded from a holding potential of -40 mV during recording solution perfusion (black) and after 10 min insulin perfusion

(gray). F.-G. Compared to time control, recordings from -40 mV, max peak (F) and late Ca2+ current (G) were significantly reduced by insulin (p<0.05). The effect of insulin on VGCC does not appear to be L-VGCC selective. All data represent mean +/- SEM and are normalized to cell size (density, pA/pF), asterisks indicate significance at the p<0.05 level.

Table 4. Patch clamping parameters.

Group Cm (pF) Rm (MΩ) Ra (MΩ) HC (pA) Bath (n=17) 50.8 ± 3.89 432.9 ± 32.0 10.6 ± 0.85 -102 ± 8.6 10 nM Insulin 56.2 ± 3.5 415 ± 34.8 10.7 ± 0.77 -100 ± 8.7 (n=19)

All data presented as means± SEM on measurements of patched neurons membrane capacitance (Cm), membrane resistance (Rm), access resistance

(Ra), and holding current (HC) (control n= 17, insulin n=19). No significant

difference was found between control and insulin treated neurons in any parameters measurements (p>0.05). Comparisons made using unpaired student t-test.

4.3.2 Effect of insulin on spontaneous Ca2+ transients.

We then used ratiometric Ca2+ imaging to test whether insulin could alter spontaneous activity in cultured neurons. We used the similar experimental time frame as presented in Figure 1 and monitored Ca2+ oscillations during 10 min of

Ca2+ imaging solution perfusion (time control; n=11) and during the next 10 minutes when the solution was supplemented with 10 nM insulin (n=14). We show here that resting Ca2+ levels were not altered by acute insulin exposure

(Figure 2D; p>0.05). Similarly, insulin did not change spontaneous network activity (no TTX) derived from measures of spontaneous Ca2+ events (Figure 2A

– right). Those include measures of area-under-the curve (AUC; Figure 2 B; p>0.05), peak amplitude (Figure 2C; p>0.05), and the number of spontaneous events detected (Figure 2E; p>0.05) in control conditions and in insulin-treated experiments. These spontaneous Ca2+ transients were blocked by TTX (data not shown).

Figure 4.2 Spontaneous Ca2+ transients are not altered in response to acute

insulin exposure.

A. Example of neurons and ROI selection (green circles and numbered arrows-

left). Right shows the three ROIs measuring somatic Ca2+ across time. This approach was used to quantify changes during 10 min of imaging solution perfusion (Time Control) as well as during 10 min of insulin perfusion (Insulin).

B.-E. Insulin perfusion did not change spontaneous Ca2+ activity based on measures of AUC, peak transient amplitude, or the number of events detected.

Resting Ca2+ levels also were not altered by insulin. All data represent mean

+/- SEM.

4.3.3 Effect of insulin on KCl- mediated Ca2+ transients.

We used potassium-mediated depolarization (30 s) in Ca2+ imaging

experimental conditions to investigate the impact of insulin on overall neuronal

Ca2+ homeostasis. This approach causes membrane depolarization with

subsequent activation of VGCCs and Ca2+ influx through the plasma membrane,

which in turn, activates Ca2+-induced Ca2+-release (CICR) from the

endoplasmic reticulum through activation of RyRs [482-484]. Potassium-induced

Ca2+ transients were reduced by 10 min of insulin perfusion (n=23) compared to

10 min of Ca2+ imaging perfusion (time control; n=14; Figure 3A and B).

Consistent with the results on spontaneous Ca2+ fluctuations, insulin did not

reduce resting Ca2+ levels measured immediately prior to the depolarization

(Figure 3D). While a trend in reduction of the peak Ca2+ transient amplitude was

noted in response to insulin (Figure 3C; p=0.08), measures of the AUC of the

depolarization envelope unmasked a significant insulin-mediate reduction (Figure

3B; p<0.0001). Notably, the reduction in the potassium-mediated Ca2+ response

seemed to selectively reduce the delayed Ca2+ component or “hump”, present

after the peak response (Figure 3A, bottom right). This brought to our attention a

potential link to CICR, given that this response by nature, is often delayed

compared to an initiating Ca2+ spike.

Figure 4. 3 Effects of insulin on KCl-induced depolarization.

A. Pseudocolored images of cells in culture during Fura-2 imaged at rest (left)

and during peak depolarization (right). Red colors indicate greater levels of Ca2+

(higher ratios). Two sequential KCl depolarizations were separated by 10

minutes of either imaging perfusion or insulin. Representative Ca2+ transients are shown and highlight measures of resting Ca2+, peak Ca2+ and AUC. Note the selective effect of insulin on reducing the late phase of the Ca2+ transient, essentially eliminating the “hump”. B. Ratiometric quantification during time

control experiments or after insulin, shows the AUC resulting from KCl-induced

depolarization was significantly decreased by insulin. C.-D. Peak ratio or resting ratio measures were not altered by insulin. A trend for peak ratio levels in response to insulin was noted. All data represent mean +/- SEM. Asterisks indicate significance at the p<0.05 level.

4.3.4 Insulin appears to reduce CICR function.

To test whether the reduction in the “hump” during potassium-induced

depolarization reflected inhibition of the CICR component, we used high

concentrations of ryanodine (20 uM) which significantly reduce CICR [377, 474].

These experiments required three 30 s repeated potassium depolarizations each

separated by 10 min (time control; Figure 4). To test for the impact of ryanodine

and/ or insulin, the first depolarization was triggered after 2 min of imaging

solution perfusion, the second depolarization occurred after 10 min of ryanodine

perfusion, and the third depolarization was after an additional 10 min of insulin

perfusion (Figure 4A). As shown in Figure 4A and B, blocking CICR with

ryanodine during potassium mediated depolarization (n=10) significantly reduced

the “hump” after the initial high amplitude transient (Figure 4B, F(2,29) = 53.8; p

<0.0001). As consistently shown throughout our studies, time control

experiments show no significant difference in the variables measured (n=11).

Post-hoc analyses revealed no significant difference in the AUC obtained under ryanodine or insulin conditions. Thus, addition of insulin had no further impact on this Ca2+ plateau, indicating that high ryanodine concentration may have occluded the impact of insulin. The lack of an additive effect between insulin and ryanodine actions suggests insulin may reduce CICR by inhibiting ryanodine

receptors.

Figure 4.4 Ryanodine receptors are likely targets of insulin actions.

A. Three sequential KCl-mediated depolarizations were used in these

experiments and were triggered under imaging solution perfusion (left) and

during perfusion with ryanodine, or insulin (right). Representative ratiometric

Ca2+ transients highlight the impact of inhibiting CICR on the “hump”. Addition of

insulin did not further reduce the “hump”. B. Quantification across the groups of

cells imaged shows the significant insulin-mediated reduction in the AUC as in

Figure 3. As expected, ryanodine (Rya) also significantly reduced the “hump”, indicating ryanodine and insulin may both be working on inhibition of RyRs. All data represent mean +/- SEM. Asterisks indicate significance at the p<0.05 level.

4.3.5 Insulin actions occur via occupation of the insulin receptor.

To investigate whether 1) components other than insulin in the glulisine

formulation, or whether 2) the actions of insulin were mediated via occupation of

the insulin receptor, we used an insulin binding peptide (i.e., affibody) and an anti-insulin receptor antibody, respectively. For these experiments, we used the same experimental protocol as presented in Figure 3 with two potassium- mediated depolarization 10 min apart. Either the affibody peptide (n=6-16 per condition) or the antibody (n=9) was used. The results are again compared to time controls with perfusion of imaging solution (n=16). While lower doses of the affibody (100 ng and 500 ng/mL) did not prevent the insulin-mediated reductions in the Ca2+ AUC (Figure 5A, p<0.01 and p<0.05, respectively), a higher dose (1 ug/ml) was able to completely inhibit this effect such that the reduction in AUC was no longer present (p>0.05). Doses used are modelled after in vivo studies

[485, 486]. These results suggest that the effects of insulin on hippocampal Ca2+ levels were mediated through insulin receptors (Figure 5B - right).

Figure 4.5 Anti-insulin affibody peptide and anti-insulin receptor antibody inhibited insulin actions on KCl-mediated Ca2+ transients.

Experiments were conducted as in Figure 3, with 2 sequential KCl

depolarizations. A. High-dose (1 ug/mL) anti-insulin affibody neutralized the effect of insulin on KCl-induced Ca2+ transients while lower concentrations (100 ng/mL and 500 ng/mL) did not. B. 500 ng/mL insulin receptor antibody also was able to inhibit insulin actions on the KCl-mediated Ca2+ transients. These results indicate the actions reported here on Ca2+ homeostasis are mediated by insulin working on insulin receptors. All data represent mean +/- SEM. Asterisks indicate

significance at the p<0.05 level.

4.4 Discussion

The experiments described here were intended to characterize actions of acute insulin exposure on neuronal Ca2+ currents and Ca2+ levels. The techniques used provide the most direct approach for analyses on the impact of insulin on hippocampal neurons without the confounding interactions of blood vessels and other cell types. Cultured neurons are detached from hormonal and perfusion influences, providing for a simplified environment where acute drug exposure is rapid and direct, free from the diffusion hindrance of the brain parenchyma or extra-neuronal interactions. Similar approaches in our lab and others have revealed significant connections between the actions of several hormones and neuronal function [474, 482]. Our results provide evidence that Apidra®, a zinc- free insulin formulation, is able to reduce VGCC currents and Ca2+ levels attained during neuronal depolarization. The mechanism is dependent on insulin receptor activation, and also appears to engage ryanodine receptors as CICR contributions were reduced in response to acute insulin treatment.

Ca2+ currents are a target of acute insulin in neurons

The insulin-mediated reduction in whole-cell currents does not appear to be specific to one subtype VGCC. This is supported by evidence of currents recorded from -70 mV before and approximately 10 minutes after the addition of

10 nM insulin, showing the same degree of inhibition for the peak and late currents (i.e., similar inactivation rates in Figure 1A). Furthermore, the reduction in currents elicited from a holding potential of -40 mV, which favors L-type Ca2+

currents, was not significantly greater in amplitude when compared to the

inhibition seen from -70 mV, arguing against a selective effect of insulin on L-type

Ca2+ channels. Previous work on regulation of VGCC in different cell types also notes that the effect of insulin may not be selective for a particular type of VGCC

(e.g., N- or L-type VGCCs; [487, 488].

This insulin mediated reduction in VGCC was not accompanied by a shift in the I-

V relationship (Figure 1D), indicating the mechanism likely impacts flux of the

charge carrier through the channels rather than alterations in the voltage

sensitivity or inactivation properties of the channels. This result is aligned with

prior work in pinealocytes and photoreceptors, showing that tyrosine

phosphorylation can reduce L-VGCC function [335, 407]. Evidence also suggests

that the pore-forming alpha subunit of some VGCCs can be regulated directly by

tyrosine residue phosphorylation [487-489]. Prior work has shown that acute

insulin can alter other ion channels including large-conductance Ca2+-activated

potassium channels (BK-type). Under these conditions, insulin reduced somatic

Ca2+ levels during spontaneous oscillations in cultured hippocampal neurons

[406]. Further, given the evidence that reductions in Src binding to the alpha 1

subunit of the L-VGCC significantly reduces channel current amplitude in cardiac

myocytes [490] and that insulin can reduce Src activity [491], this signaling

pathway likely highlights a potential mechanism by which insulin reduces VGCC

current flux.

Still, it should be noted that some cells respond to tyrosine kinase

phosphorylation with enhanced VGCC function and potentiated Ca2+ levels [492,

493], and that given the complexity of second messenger systems working

through many kinases and phosphatases [494], the exact impact of insulin on

VGCC likely depends on the presence and state of activation of other interacting

pathways (i.e., PKA, PKC, AKAP, phosphatases, etc.). Irrespective of the mechanism of inhibition of VGCC by insulin, it is clear that VGCC are a target of insulin in neurons. The effect described here, therefore, is likely to mediate the beneficial impact of the hormone on cognitive function following intranasal delivery in animal models [30, 467, 469, 470] and in early AD subjects [32, 33,

35, 398, 401, 495]. Indeed, according to the Ca2+ hypothesis of aging and

dementia [188, 443, 496], reducing Ca2+ levels in postsynaptic neurons is a

favorable approach to offset cognitive decline in aging and AD (see below).

Other potential targets of insulin actions

Previous work shows insulin can increase cell surface expression of NMDA

receptors [89] and can reduce surface expression of AMPA receptors [88, 332].

These studies employed similar insulin exposure time as used in the current

study, but concentrations were 50-1000-fold higher. Thus, it is not clear whether

transient changes in NMDA and AMPA surface expression would manifest using

10 nM insulin in the absence of TTX. As reported in Figure 2, the nearly

physiological insulin concentration used here does not necessarily alter network

excitability. This is evidenced by a lack of insulin action on resting Ca2+ levels,

peak amplitude of the spontaneous Ca2+ responses, the AUC of the

spontaneous events, as well as the number of spontaneous events. Our work,

therefore, may suggest that higher concentrations of insulin are needed to alter

network oscillations, overall excitability and spontaneous activity. Although not

tested directly here, our results do not provide evidence that insulin can increase

surface expression of GABAA receptors [92, 405] which should have reduced

excitability. Within the time frame tested here (~10 minutes) and at the low

concentration used, we did not see evidence of depression in spontaneous Ca2+

events (Figure 2). Thus, further studies focusing on insulin concentration-

responses and time of exposure are needed to clarify these discrepancies. Of

course, it will also be important to address the different types of insulin

formulations used and to include the adequate zinc controls, given that almost all

insulin formulations are zinc-based.

While insulin did not alter spontaneous Ca2+ activity and Ca2+ events amplitudes within the network of cells in culture (Figure 2), we did notice a significant alteration in the shape of the potassium-mediated Ca2+ responses following insulin treatment (Figure 3). The disappearance of the “hump” following the peak Ca2+ rise revealed the possibility that RyRs might be sensitive to insulin. We tested this hypothesis by pretreating the cultures with high concentrations of ryanodine to inhibit Ca2+-induced Ca2+ release (CICR). As shown here, ryanodine pretreatment precluded further insulin-mediated inhibition of the Ca2+ response to depolarization (Figure 4). This suggests insulin is capable of reducing CICR likely through tyrosine mediated inhibition of the RyR.

This result is in line with prior evidence that neuronal store-operated Ca2+ channels (SOCCs) responsible for a small but significant secondary Ca2+ entry

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pathway for replenishing the endoplasmic reticulum, are also a target of protein tyrosine phosphorylation [497]. It should be noted, however, that this result is contrary to what is seen in striated muscle cells, where insulin elicits an increase in Ca2+ release through activation of RyR [498]. Future studies are necessary to identify whether the different RyR subtypes can respond differently to insulin, or whether these effects are tissue specific, and dependent on particular signaling pathways.

We suggest that the novel finding of insulin actions on CICR could be highly beneficial to neurons and to Ca2+ dysregulation seen in aging and/ or AD, and that this new target potentially offers a secondary level of protection from sustained Ca2+-related actions in neurons. While it is not clear that the reduction in the “hump” (Figure 4) may provide a neuroprotective function to neurons, the insulin-mediated reduction in depolarization observed during this Ca2+ envelope could well keep neurons engaged during periods of activation.

Learning and memory selective actions of insulin in the brain

Past work studying insulin function in the brain identified critical links to learning and memory. Whether exogenous insulin on hippocampal neurons worked through changes in GABA, NMDA, AMPA, or even glucose transporters, it was understood that insulin could have a specific and selective impact on learning and memory and conversely, that learning also could have an impact on insulin receptors [28, 499]. More recent analyses of insulin action provide evidence that large and diverse areas of the CNS are indeed sensitive to insulin. Previous work shows insulin is a dynamic hormone with a rich influence on brain function.

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Insulin stimulates the translocation of glucose transporter-4 to plasma membranes of hippocampal neurons within 15-30 min [100]. This rapid process

is likely to regulate neuronal metabolic demands and the energy needed for

learning and memory processing. In addition, insulin enhances synaptic plasticity

by increasing the expression of dendritic postsynaptic density scaffolding protein

(PSD-95), a key element of postsynaptic junctions in hippocampal neurons [334].

Together these functions and their sensitivity to insulin undoubtedly contribute to

improved encoding of synaptic information for the storage, and perhaps, the

retrieval of memories.

Equally concrete examples of the links between insulin and memory come from

evidence that insulin can acutely reduce the hippocampal AHP, a Ca2+-

dependent hyperpolarizing potential, which maintains hyperpolarization and

precludes action potential threshold from being reached [30, 380]. The AHP is

larger in aged compared to young animals [184, 379, 433, 453, 476, 500, 501],

thus reducing the amplitude or duration of this hyperpolarizing potential may help

combat age-related memory dysregulation by allowing cells to participate within a

functional network [502]. Indeed, it has been shown that elevating potassium

channel activity impairs learning [503], while lowering L-VGCC reduces the AHP

[432, 452] and enhances learning [432, 504]. Moreover, increasing cholinergic

neurotransmission which enhances cellular excitability also can enhance learning

[436, 505-507].

Still, the evidence of robust insulin actions linked to memory processes or

processes capable of facilitating memory encoding (e.g., metabolism, blood

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flow), does not detract from the traditional role of insulin in homeostasis [508,

509]. Indeed, it is becoming very clear that once insulin in the brain, the hormone is not limited in function to a single or even a few target areas [366, 510],

strengthening its position as a potential therapeutic target which requires greater

research emphasis.

4.5 Conclusions

We used imaging and electrophysiology techniques to show that zinc-free insulin

could have rapid and selective effects on Ca2+ sensitive functions in neurons,

and that the effects are dependent on insulin activating the insulin receptor

(Figure 5). This clearly engages a series of intracellular signals that through

uncharacterized pathways or mechanisms, lead to rapid Ca2+ reductions during

neuronal depolarization. Clearly, more research will need to be conducted to fully

characterize the mechanism underlying this phenotype and to address the

selectivity of the effect using different insulin formulations. Importantly, we remark

that reducing Ca2+ dysregulation in brain aging, AD and in neuronal cultures has

generally been associated with positive and neuroprotective outcomes, from

improving cognitive functions to reducing cellular toxicity. We present evidence

here that insulin could well be an endogenous modulator of these functions.

Reductions in insulin levels or in insulin signaling in the brain during aging and in

AD may highlight one of the mechanisms that could reduce metabolism, thereby

reducing cognitive function. However, our results also suggest that replacing

insulin or enhancing insulin signaling in the brain of aging, or early AD subjects

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may be a valuable approach. We emphasize that the novel impact of insulin on neurons presented here could well underline the beneficial impact of intranasal insulin on cognitive enhancement in the clinic and in animal studies. If insulin is able to reduce Ca2+ levels in vivo, the noted reductions in insulin-sensitive Ca2+ responses may offset brain aging processes and ultimately, offset the impact of dementia on brain function.

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Chapter 5 Discussion and conclusion

The data collected for this dissertation project is presented in the form of two

manuscripts. The purpose was to test the hypothesis that intranasal insulin could offset cognitive decline in aged animals and to characterize the impact of insulin on neuronal calcium status. The first manuscript (see chapter two) has been published in the Journals of Gerontology Series A in 2016 [30] and the second

manuscript (see chapter four) has been submitted to Neuroscience (Maimaiti et

al., 2017).

5.1 Effects of insulin on memory and Ca2+-dependent AHP

In the first study, to test whether intranasal insulin could reverse aging-related

memory decline, we used the Morris water maze, a hippocampal dependent

spatial learning and memory task, to evaluate insulin effects on memory in young

and aged F344 rats. Aged animals were divided into five groups; each group

repeatedly received saline, a low dose of Humalog® and a low, medium, and

high dose of Levemir®. Also, the young control group received saline, used as a

normal young baseline. The behavioral study results showed that intranasal

administration of two different formulations of insulin (Humalog®, a short-acting

insulin analogue, or Levemir®, a long-acting insulin analogue) reversed the

memory decline in aged F344 rats compared to aged rats in the saline controls.

We found that while a low dose of Humalog® and Levemir® enhanced the

memory, a medium dose of Levemir® worsened the memory, and a high dose of

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Levemir® did not change the memory. Therefore, it is clear that selective doses and formulations of insulin have beneficial effects on the memory in the aged animals. In addition, controversial clinical study results exist regarding insulin

dosages and formulations for use in AD patients. In animal and clinical studies,

different dosages and formulations of insulin have various outcomes. These

varied results led to further investigation of the underlying mechanisms

responsible for intranasal insulin’s beneficial effects on memory as seen in select

animals and human studies, highlighting the importance of enhancing insulin

action in the brain.

So far, three main methods (insulin sensitizers, intravenous insulin infusion, and

intranasal insulin) have been used for increasing brain insulin levels. Due to the

poor permeability of insulin sensitizer (Thiazolidinedione) to the blood brain

barrier [511, 512], and the high risk of hypoglycemia from intravenous insulin

infusion (see chapter three), however, intranasal insulin appears to be the best

method to increase brain insulin levels. Nevertheless, the underlying

mechanisms responsible for intranasal insulin’s positive effects are still poorly

understood. One mechanism responsible for memory decline with aging is

hippocampal Ca2+ dysregulation.

It has been more than 30 years since researchers have focused on Ca2+

dysregulation in the aging brain, which is measured by recording Ca2+- dependent afterhyperpolarization (AHP) in the hippocampus neurons. Therefore, we examined the effects of insulin on Ca2+-dependent AHP, one of the well- known mechanisms that increases with aging and contributes to memory decline.

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Our results show that treatment of hippocampal slices with insulin reduced Ca2+- dependent AHP in hippocampal neurons to the levels seen in young neurons. It is well documented that Ca2+-dependent AHP (a key biomarker of brain aging) increases in aged, and memory-impaired animals [30, 184, 377, 379, 380, 432-

434]. Thus, insulin-mediated reduction in Ca2+-dependent AHP may be responsible for some of the beneficial effects of insulin on cognition.

Most insulin formulations were designed by combining insulin with zinc. In fact, zinc itself plays a vital role in the physiological functions of cells, and is like Ca2+, an essential ion to regulate intracellular signaling. Therefore, it is important to test insulin and zinc effects separately on brain Ca2+ regulatory processes. Thus, when the effects of Apidra® (fast acting zinc-free human insulin) and zinc on the

AHP were tested separately, both were found to reduce the AHP. Therefore, we used the zinc-free human insulin Apidra® to study specific insulin actions in hippocampal neurons without the influence of zinc.

The next question we asked was how does insulin reduce the Ca2+-dependent

AHP? The greater AHP seen in aged memory-impaired animals positively correlated with elevated intracellular Ca2+ concentration. This alteration results from increased Ca2+ influx via voltage-gated Ca2+ channels and Ca2+-induce

Ca2+-release via RyRs (see chapter three). Conversely, it is possible that a reduction in AHP with insulin treatment may mediate through the reduction in intracellular Ca2+ levels via VGCCs and RyRs.

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5.2 Whole-cell patch clamping and Ca2+ imaging in hippocampal neurons

In the second manuscript, we aimed to identify the effects of insulin on hippocampal Ca2+ levels in cultured neurons. The significance of this data is showing that insulin reestablishes hippocampal Ca2+ homeostasis. Aging and

AD animal models have shown that elevated intracellular Ca2+ levels in the hippocampus contribute to poor spatial memory [180, 453]. Reestablishment of intracellular Ca2+ homeostasis might be one of the possible neuronal mechanisms responsible for the beneficial effects of insulin on cognition. Two main sources of intracellular Ca2+ are Ca2+ influx through voltage-gated Ca2+ channels on the plasma membrane and internal Ca2+ efflux from the endoplasmic reticulum via ryanodine-mediated Ca2+ release in the hippocampus. It is well documented that both voltage-gated Ca2+ channels and ryanodine receptors undergo functional changes with aging and that lead to elevated intracellular Ca2+ levels, which is toxic for neurons (see chapter three).

It is widely accepted that reducing the elevated intracellular Ca2+ levels is one of the established approaches to improving memory in the aging and AD population

[377, 435, 453]. Therefore, insulin effects on both voltage-gated Ca2+ channels and ryanodine receptors were tested. Insulin affected two main intracellular Ca2+ sources, including Ca2+ influx via voltage-gated Ca2+ channels and RyRs- mediated Ca2+ release. After 10 minutes of insulin perfusion to patched single neurons, whole-cell patch clamp recording results show that both VGCCs and L- type VGCCs currents decreased in hippocampal neurons. We found that acute insulin perfusion to neurons resulted in the same level of reduction in both

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VGCCs and L-VGCCs. This data suggests that insulin uniformly affects all type

of voltage-gated Ca2+ channels. It is well documented that an elevated

intracellular Ca2+ results from increased L-VGCCs, which has been linked to memory decline in aged animals (see chapter three). Therefore, insulin-mediated reduction in Ca2+ influx via this channel may be an underlying mechanism partially responsible for insulin’s memory-enhancing effects seen in animal and clinical studies. In addition, Ca2+ imaging data show that acute insulin perfusion did not change resting Ca2+ transients, and significantly reduced ryanodine- mediated Ca2+ transients. Intracellular Ca2+ concentration is amplified due to enhanced ryanodine-mediated Ca2+-induced Ca2+-release from the endoplasmic reticulum in animal models of aging and AD [377, 435, 513].

Reduction in RyRs-mediated Ca2+ transients may underlie neuronal mechanisms responsible for positive actions of insulin in hippocampal neurons.

Furthermore, this result indicates that RyRs inhibitors will improve the memory in

AD patients.

5.3 Insulin improves memory by regulating intracellular Ca2+ levels

Together, these two studies combine intranasal insulin approaches,

hippocampal-dependent memory and learning task, sharp electrophysiology,

whole-cell patch clamp, spontaneous and depolarization mediated Ca2+ imaging

to highlight potential neuronal mechanisms of insulin-mediated cognitive

improvement seen in clinical studies. We show that hippocampal Ca2+-

dependent AHP, voltage-gated Ca2+ channel currents, and Ca2+ transients are

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increased in aged rats—all major factors influencing memory decline with

aging—are significantly reduced by insulin treatment. Furthermore, this reduction

in voltage-gated Ca2+ channel currents and ryanodine-mediated Ca2+ transients

were correlated with reversal of Ca2+ dysregulation and memory impairment

observed in aged rats.

These study results support the concept that brain insulin resistance/ deficiency

present in aged, diabetic and AD models of rats could be a major contributor to

aging-related cognitive decline. Indeed, increasing brain insulin levels using the

intranasal route improves memory by reducing AHP via regulating intracellular

Ca2+ levels. Thus, taken together, these findings suggest that insulin is a key

hormone influence over neuronal Ca2+ regulation and strengthen the brain

insulin-deficiency mechanistic hypothesis of pathological aging.

5.4 Aging-related changes in memory and hippocampal Ca2+ levels

Insulin treatment reversed both aged-related memory decline and Ca2+

dysregulation in hippocampal neurons. It has been decades since our lab and others have focused on the relationship between cognitive decline and increased

Ca2+ dysregulation in aged animals [30, 224, 433, 435, 514]. Ca2+ dysregulation

means elevated intracellular Ca2+ concentration, that gives rise to a larger AHP

in aged animals and is considered one of the neuronal mechanisms underlying

memory decline with aging. In addition, researchers identified pharmacological

agents such as Ca2+ channel blockers (inhibiting the elevation of intracellular

Ca2+ concentration) that reduce the amplitude and duration of the AHP to

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improve cognitive function in aged animals [515]. Furthermore, previous studies from our research group using gene transfer approaches to increase and/or decrease FKBP1b (a small immunophilin that stabilizes RyR-mediated Ca2+ release) expression in the hippocampus showed a similar correlation between memory impairment and Ca2+ dysregulation [435]. In the present study, we found a consistent association between age-related changes in hippocampal- dependent memory, and Ca2+ levels. Most importantly, insulin treatment improved memory in aged rats, reduced the AHP, Ca2+ channel currents, and

Ca2+ transients. Insulin in the brain may be a key element for maintaining hippocampal Ca2+ homeostasis and cognitive function during aging.

5.5 Potential relevance to healthy and pathological aging

Biologists suggest that brain function starts to decline from the third decade of life leading to either healthy or pathological aging, suggesting that some changes in natural brain aging give rise to pathological neurodegeneration and AD. These changes include brain insulin deficiency and loss of neuronal Ca2+ homeostasis.

Disruption of brain insulin signaling contributes to the AD clinical symptoms

(memory impairment) and pathology [137, 149]. In addition, increased intracellular Ca2+ concentration from the dysfunction of VGCCs and RyR receptors markedly influence neuronal survival, communications, and function, which have been considered major risk factors for AD [185, 435, 516, 517].

Conversely, brain-specific enhancing insulin signaling improved cognitive function in selective AD patients, and improved memory in experimental aged

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animals. In addition, our study results show that intracellular Ca2+ levels

decreased with insulin treatment in hippocampal-cultured neurons. Importantly,

brain insulin deficiency during aging together with other factors may accelerate

Ca2+ dysregulation and stimulate amplification of intracellular Ca2+ levels,

increasing the vulnerability of AD. Thus, studies presented here support that

increasing brain insulin levels may be a vital approach for promoting healthy

aging and, possibly, reversing cognitive decline present in aging and AD

populations.

5.6 Study limitations

In the second study, all the whole-cell patch clamping and Ca2+ imaging data

were obtained from the hippocampal cultured neurons in response to acute insulin treatment. Cells in culture are not expose to same multitude of signals environment that is present in functional human body. Thus, those data cannot

directly translate into a clinical AD study. However, it provides potential

mechanisms for insulin actions on hippocampal neurons. Primary hippocampal cultured neurons have been used as a valid model for studying the neuronal molecular mechanisms underlying the age-related changes in Ca2+ channels and neuronal survival [439], as well as for visualizing protein expression, trafficking, and localization [518]. We used primary hippocampal cultured

neurons to study insulin action on hippocampal Ca2+ levels. Intracellular Ca2+

homeostasis is important for proper neuronal function throughout life, and it

influences a wide range of neuronal development processes, including

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formation of neurotransmitters, ion channels, neurite outgrowth,

synaptogenesis, and intrinsic firing patterns [519-522]. Intracellular Ca2+

homeostasis is dependent on the proper function of voltage-gated Ca2+

channels on the plasma membrane and ryanodine receptors on the

endoplasmic reticulum. In addition, both these channels have shown functional changes during aging. The voltage-gated Ca2+ channel density increases with aging, and this negatively correlates with neuronal survival in cultured hippocampal neurons [439]. Hippocampal neurons in the primary culture undergo time-dependent development and changes as well [183, 523]. From day in vitro (DIV) 1 to DIV 6, neurons rapidly attach to the dish, grow, and establish networks. From age DIV 10 to DIV 28, unhealthy and dead neurons increase as a function of age in the culture. In addition, a whole-cell Ca2+ currents recording from hippocampal neurons at different ages in the culture show that Ca2+ current density increases as neurons are aged in the culture

[439]. Hippocampal cultured neurons help us to study the effects of insulin on hippocampal neurons in a single isolated environment that eliminates other factors. Thus, hippocampal cultured neurons seem to be an appropriate model for studying the link between insulin and hippocampal Ca2+ levels.

As I mentioned earlier, Ca2+-dependent AHP depends on both Ca2+ and K+ ions. In my dissertation, I focus only on the Ca2+; therefore, the K+ component still needs to be studied. Therefore, those data partially explain the mechanism of insulin actions on the AHP and memory.

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5.7 Future directions

These studies have shown that insulin reduces the Ca2+-dependent, K+-

mediated slow afterhyperpolarization (sAHP), reduces the VGCCs currents, and

reduces RyRs-mediated Ca2+ transients. This study provided a clear link

between insulin and intracellular Ca2+ concentration (mainly through voltage- gated Ca2+ channels and RyRs mediated Ca2+-induced Ca2+-release) in hippocampal neurons. However, the effects of insulin on potassium channels in hippocampal neurons in the context of cognitive functions have not yet been studied. There are a few studies showing a connection between insulin and potassium channels in the kidney [524] but not in the brain. In fact, “Ca2+- activated K+ channels play an important role in the control of neuronal excitability via the generation of the AHP” (Davies et al., 2006). Furthermore, earlier studies suggest that potassium channels contribute to the generation of sAHP in hippocampal CA1 neurons [525-529]. In addition, potassium channels undergo functional changes with aging and influence neuronal function and sAHP [530, 531]. From these findings arose the idea that insulin may be able to rebalance potassium channel functions, which may further reduce amplitude and duration of the sAHP. Other graduate students in the lab could look at the relationship between insulin and potassium channels in the hippocampal neurons.

Earlier research intensely focused on RyRs’ role in heart and skeletal muscle excitation-contraction. Some studies suggest that the cardiac ryanodine

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receptor (RyR2) is a promising drug target for treating arrhythmogenesis [532].

Moreover, RycalsTM is a drug available on the market to inhibit cardiac and skeletal muscle specific RyRs [533]. In addition, dantrolene, an RyRs blocker, is a widely used drug to treat malignant hyperthermia in clinic. This dissertation showed that insulin reduced the RyRs-mediated Ca2+ transients in hippocampal neurons. This data suggests that the inhibition of RyRs channels positively correlates with memory improvement in aged animals. Therefore,

RyRs’ role is not limited to the periphery, but also plays a vital role in the brain in the context of memory. Specifically, in the hippocampal, RyRs plays an important role in regulating intracellular Ca2+ levels and maintaining cognitive function [435, 453]. A few researchers are promoting the idea that hippocampus-specific RyRs inhibition is a therapeutic target for AD.

Furthermore, this study strongly supports the hypothesis formulated by Dr.

Grace Stutzmann that RyRs blockers are an effective anti-AD therapy. Thus,

RyRs could be a drug target for inhibiting pathological intracellular Ca2+ leaks in

the hippocampal neurons, and, more importantly, for reversing Ca2+

dysregulation and memory impairment with aging and AD.

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Vita

Shaniya Maimaiti

EDUCATION

University of Kentucky Ph.D. in Pharmacology and Nutritional Sciences University of Kentucky Bachelor of Science in Mathematics December 2011 AWARDS Academic Honor 2010 Academic Honor 2011 International student scholarship 2011 Graduate student fellowship 2012 4th grade science fair judge - SCAPA Middle School, Lexington KY 2014 7th grade science fair judge - SCAPA Middle School, Lexington KY 2017

PRESENTATIONS 1) Maimaiti, S, Halmos, K, and Smith, B.N. Altered glucokinase expression and function in identified GABA neurons in the nucleus tractus solitarius in mice with type 1 diabetes. SFN, New Orleans, LA, 2012. 2) Shaniya Maimaiti, Chris DeMoll, Katie L. Anderson, Ryan Griggs, Bradley K. Taylor. Nada M. Porter, Olivier Thibault. Ca2+-dependent cellular mechanism of aging and diabetes: focus on insulin. Barnstable Brown diabetes research day, Lexington, KY, 2013. 3) Shaniya Maimaiti, Katie L. Anderson, Ben Rauh, Nada M. Porter and Olivier Thibault. Brain insulin exposure: acute and intranasal effects on Ca2+ electrophysiological biomarkers of aging and memory. BGSFN, Lexington, KY, 2013. 4) Shaniya Maimaiti Katie L. Anderson, Chris DeMoll, , Benjamin Rauh, Eric M Blalock, Nada M. Porter, Olivier Thibault. Brain insulin exposure: acute and intranasal effects on Ca2+ electrophysiological biomarkers of aging and memory. Marksberry Symposium, Lexington, KY, 2013. 5) Olivier Thibault, Shaniya Maimaiti, Katie L. Anderson, Lawrence D. Brewer, Joseph R. Calabrese1, and David E. Kemp1. The effect of PPAR alpha/gamma modulator DSP-8658 on reversing cognitive decline in aging. SFN, Washington DC, 2014. 6) Shaniya Maimaiti, Lawrence D. Brewer, Katie L. Anderson, Eric M. Blalock, Nada M. Porter and Olivier Thibault. Intranasal adiponectin versus insulin on behavioral and electrophysiological biomarkers of aging and memory decline. SFN, Washington DC, 2014.

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7) S Maimaiti, K. Anderson, J.Popovic, L.Brewer, O.Thibault. Mechanisms of Insulin Actions on Hippocampal Neurons. BGSFN, Lexington, KY, 2015. 8) K. L. Anderson, S. Maimaiti, Z.R. Majeed, L.D. Brewer, O. Thibault. Region and Time Specific Intranasal Insulin Signaling in Young and Aged F344 Rats. BGSFN, Lexington, KY, 2015. 9) S Maimaiti, K. Anderson, J.Popovic, L.Brewer, O.Thibault. Mechanisms of Insulin Actions on Hippocampal Neurons. Marksberry Symposium, Lexington, KY, 2015. 10) K. L. Anderson, S. Maimaiti, Z.R. Majeed, L.D. Brewer, O. Thibault. Region and Time Specific Intranasal Insulin Signaling in Young and Aged F344 Rats. Marksberry Symposium, Lexington, KY, 2015. 11) S Maimaiti, K. Anderson, J.Popovic, L.Brewer, O.Thibault. Mechanisms of Insulin Actions on Hippocampal Neurons.SFN, Chicago, Illinois, 2015. 12) Anderson K.L, Frazier H, Maimaiti S, Bakshi V.V, Majeed Z, Brewer L.D, Porter N.M, Lin A-L, and Thibault O. Impact of single and repeated dose intrabasal zinc-free insulin in young and aged F344 on cognition, signaling, and brain metabolism. SFN, Chicago, Illinois, 2015. 13) Z.R.Majeed, H.N. Frazier, K. Hampton, S. Maimaiti, K.L. Anderson, J.Popovic, L.B. Brewer, S.D. Kraner, C.M.Norris, N. Porter, R.J. Craven, O. Thibault. SFN, Chicago, Illinois, 2015. 14) S. Maimaiti, H. Frazier, K. Anderson, J.Popovic, L.Brewer, N.M. Porter, P.W. Landfield, O.Thibault. Mechanisms of Insulin Actions on Hippocampal Neurons. Marksberry Symposium, Lexington, KY, 2015 15) Anderson K.L, Frazier H, Maimaiti S, Bakshi V.V, Majeed Z, Brewer L.D, Porter N.M, Lin A-L, and Thibault O. Impact of single and repeated dose intrabasal zinc-free insulin in young and aged F344 on cognition, signaling, and brain metabolism. Marksberry Symposium, Lexington, KY, 2015 16) Z.R.Majeed, H.N. Frazier, K. Hampton, S. Maimaiti, K.L. Anderson, J.Popovic, L.B. Brewer, S.D. Kraner, C.M.Norris, N. Porter, R.J. Craven, O. Thibault. Marksberry Symposium, Lexington, KY, 2015 17) Hilaree Frazier, Kaia Hampton, Shaniya Maimaiti, Katie Anderson, Lawrence Brewer, Susan Kraner, Christopher Norris, Nada Porter, Rolf Craven, Olivier Thibault. Signaling and Expression of a Truncated, Constitutively Active Human Insulin Receptor. American Society for Neurochemistry, Denver, Colorado, 2016. 18) S. Maimaiti, H. Frazier, K. Anderson, J.Popovic, L.Brewer, N.M. Porter, P.W. Landfield, O.Thibault. Acute Insulin Treatment on Hippocampal Neurons Highlights New Mechanisms of Action. BGSFN, Lexington, KY, 2016 19) S. Maimaiti, H. Frazier, K. Anderson, J.Popovic, L.Brewer, N.M. Porter, P.W. Landfield, O.Thibault. Acute Insulin Treatment on Hippocampal Neurons Highlights New Mechanisms of Action. SFN, San Diego, California, 2016 20) Hilaree Frazier, Kaia Hampton, Shaniya Maimaiti, Katie Anderson, Lawrence Brewer, Susan Kraner, Christopher Norris, Nada Porter, Rolf Craven, Olivier Thibault. Signaling and Expression of a Truncated, Constitutively Active Human Insulin Receptor. SFN, San Diego, California, 2016 21) S. Maimaiti, H. Frazier, K. Anderson, J.Popovic, L.Brewer, N.M. Porter, P.W. Landfield, O.Thibault. Acute Insulin Treatment on Hippocampal Neurons Highlights New

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Mechanisms of Action. International Society of Neurogastronomy Symposium, Lexington, KY, 2016

SEMINARS • University of Kentucky College of Medicine department of Pharmacology and Nutritional Sciences “Insulin actions in the hippocampal CA1 neurons”. May 2014 • University of Kentucky College of Medicine department of Pharmacology and Nutritional Sciences “Acute Insulin actions in the hippocampal CA1 neurons”. May 2015 • University of Kentucky College of Medicine department of Pharmacology and Nutritional Sciences “ Insulin actions in the hippocampal CA1 neurons”. Feb 2016 • University of Kentucky College of Medicine defense seminar “ Insulin actions in the hippocampal CA1 neurons”. July 2016

PROFESSIONAL SOCIETY AFFILIATIONS • Local bluegrass chapter for the Society for Neuroscience 2012 - Present

• National member of the Society for Neuroscience 2014 – Present

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PUBLICATIONS 1. Bruce A. Berkowitza,b,1, Jacob Lenninga*, Nikita Khetarpala*, Catherine Trana*, Johnny Y. Wua, Ali M. Berria, Kristin Dernaya, E. Mark Haackec, Fatema Shafie-Khorassanid, Robert H. Podolskyd, John C. Gante, Shaniya Maimaitie, Olivier Thibaute, Geoffrey G. Murphyf, Brian M. Bennettg, and Robin Robertsa . In Vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer’s disease and angelman syndrome. Research communication for FASEB J. 2017 2. Frazier HN, Maimaiti S, Anderson KL, Brewer LD, Gant JC, Porter NM, Thibault O. Ca2+’s role as nuanced modulator of cellular physiology in the brain. Biochem Biophys Commun. 2016 Aug 20. Pii: S0006-291X(16)31361-4.doi:10.1016/j.bbrc.2016.08.105. PMID: 27553276 3. Anderson K.L, Frazier H, Maimaiti S, Bakshi V.V, Majeed Z, Brewer L.D, Porter N.M, Lin A.-L, and Thibault O. Impact of single and repeated dose intranasal zinc-free insulin in young and aged F344 on cognition, signaling, and brain metabolism. J GerontoL A Biol Sci Med Sci. 2016 Apr 10. pii: glw065. PMID: 27069097 4. Shaniya Maimaiti, Katie L. Anderson, Chris DeMoll, Benjamin Rauh, Eric M Blalock, Nada M. Porter, Olivier Thibault. Brain insulin exposure: acute and intranasal effects on Ca2+ electrophysiological biomarkers of aging and memory. J Gerontol A Biol Sci Med Sci. 2016 Jan;71(1):30-9. doi: 10.1093/gerona/glu314. Epub 2015 Feb 6. PMID: 25659889

5. Shaniya Maimaiti, Chris DeMoll, Katie L. Anderson, Ryan B.Griggs, Bradley K.Taylor, Nada M. Porter, Olivier Thibault. Short-lived diabetes in the young-adult ZDF rat does not exacerbate neuronal Ca2+ biomarkers of aging. Brain Res. 2015 Sep 24;1621:214- 21. doi: 10.1016/j.brainres.2014.10.052. Epub 2014 Nov 6. PMID: 25451110 6. Halmos KC, Gyarmati P, Xu H, Maimaiti S, Jancso G, Benedek G, Smith, B.N. Altered glucokinase expression and function in identified GABA neurons in the nucleus tractus solitarius in mice with type 1 diabetes. Neuroscience. 2015 Oct 15;306: 115-22. doi: 10.1016/j. Epub 2015 Aug 20. PMID: 26297899 7. Pancani T., Maimaiti S, Anderson K.L., Frazier H., Gant J.C., Landfield P.W. and Thibault O. (2015) Translational Neuroscience: Models of aging, a book in the series entitled Neuromethods, published by Springer Protocols, Humana Press Wolfgang Walz, Ph.D., Editor-in-chief. Submitted. 8. S. Maimaiti, H. Frazier, K. Anderson, L.Brewer, N.M. Porter, O.Thibault. Novel Ca2+- sensitive targets of insulin actions in neurons. Submitted to neuroscience. Jun 2017.

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