Mitochondrial Dynamic Abnormalities in Alzheimer's Disease Sirui Jiang Case Western Reserve University

MITOCHONDRIAL DYNAMIC ABNORMALITIES IN ALZHEIMER’S DISEASE by SIRUI JIANG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Xiongwei Zhu Department of Pathology CASE WESTERN RESERVE UNIVERSITY January 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of SIRUI JIANG Candidate for the degree of Doctor of Philosophy* Dr. Shu Chen (Committee Chair) Dr. Xiongwei Zhu Dr. Xinglong Wang Dr. George Dubyak Dr. Charles Hoppel August 15, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein Table of Contents Table of Contents 1 List of Figures 3 Acknowledgements 5 List of Abbreviations 7 Abstract 10 Chapter 1. Introduction 12 Introduction to Alzheimer’s Disease 13 General Information 13 Pathology 14 Pathogenesis 15 Introduction to Mitochondrial Dynamics 20 Mitochondrial Function and Neuronal Health 20 Mitochondrial Dynamics 21 Mitochondrial Dynamics and Mitochondrial Function 23 Mitochondrial Dynamics and Mitochondrial Transport 24 Mitochondrial Deficits in AD 26 Mitochondrial Dysfunction in AD 26 Aβ and Mitochondrial Dysfunction 27 Mitochondrial Dynamic Abnormalities in AD: Recent Advances 28 Conclusion 34 1 Chapter 2. Mfn2 ablation causes an oxidative stress response and eventual neuronal death in the hippocampus and cortex 36 Abstract 37 Background 39 Methods 43 Results 47 Discussion 54 Figures 60 Chapter 3. DLP1 Cleavage by Calpain in Alzheimer’s Disease 71 Abstract 72 Background 73 Methods 77 Results 80 Discussion 85 Figures 89 Chapter 4. Summary, Discussion and Future Directions 96 References 108 2 List of Figures Figure 2.1 Cre-mediated ablation of Mfn2 expression in the hippocampus and cortex of Mfn2 cKO mice 60 Figure 2.2 Quantification of DLP1 and OPA1 in cKO mice 61 Figure 2.3 Mfn2 ablation caused mitochondrial fragmentation and ultrastructural damage in the hippocampus in vivo as evidenced by electron microscopic analysis. 62 Figure 2.4 Mfn2 ablation caused abnormal mitochondrial distribution in vivo. 63 Figure 2.5 Mfn2 ablation caused mitochondrial dysfunction in the brain of Mfn2 cKO mice. 64 Figure 2.6 Mfn2 ablation caused neurodegeneration in the hippocampus and cortex in vivo. 65 Figure 2.7 Nissl staining of the cortex 66 Figure 2.8 Mfn2 ablation caused increased oxidative stress in the hippocampus and cortex in vivo. 67 Figure 2.9 Mfn2 ablation caused increased neuroinflammation in hippocampus and cortex in vivo. 68 Figure 2.10 Quantification of immunostaining of GFAP, IBA-1, and MAP2 69 Figure 2.11 Mfn2 ablation caused abnormal cytoskeletal alterations in hippocampus and cortex in vivo. 70 Figure 3.1 Dose- and time-dependent cleavage of recombinant DLP1 by calpain-1. 89 3 Figure 3.2 DLP1 is cleaved by calpain in M17 neuroblastoma cell lysates after incubation with calpain-1. 90 Figure 3.3 Calpain-dependent cleavage of spectrin and DLP1 in glutamate-treated rat primary cortical neurons. 91 Figure 3.4 Calpain-dependent cleavage of spectrin and DLP1 in rat primary cortical neurons treated with soluble Aβ oligomers. 92 Figure 3.5 Calpain-dependent cleavage of spectrin and DLP1 in rat primary cortical neurons treated with okadaic acid. 93 Figure 3.6 Calpain-dependent cleavage of spectrin and DLP1 in primary cortical neurons isolated from CRND8 APP transgenic mice. 94 Figure 3.7 Decreased level of DLP1 in Alzheimer’s Disease (AD) brain. 95 4 Acknowledgements First and foremost, I would sincerely like to thank my thesis advisor, Dr. Xiongwei Zhu, for his never ending support throughout my training in this doctoral program. Dr. Zhu has always been a source of great assistance and advice whether it is science or my future career. He has not only taught me how to think as a scientist but also encouraged me to approach scientific questions with a well thought out and critical methodology. This thesis would not be possible without his extensive expertise and immense amount of patience and support. I would also like to thank my co-mentor, Dr. Xinglong Wang, for his impeccable advice and guidance through my training. He is always approachable for any technical or methodological questions in regards to my research. He has also taught me how to approach a scientific problem from many angles and develop the proper methods in order to tackle any kind of problem. Of course this work could not have been completed without the help and assistance of my laboratory colleagues such as Dr. Wenzhang Wang, Dr. Xiaopin Ma, Sandra Siedlak, Sandy Torres, Priya Nandy, and many others who have contributed to my learning and research. 5 I would like to thank my thesis committee: Dr. Robert Petersen, Dr. Shu Chen, Dr. George Dubyak, Dr. Xinglong Wang, and Dr. Charles Hoppel for their continued guidance and advice through my doctoral training. I would like to acknowledge the financial support of the National Institute of Health grant T32 GM007250, T32 NS077888, R01 NS083385, Dr. Robert M. Kohrman Memorial Fund, and the Department of Pathology at Case Western Reserve University. Finally, I would like to thank my parents and sister who have always been a source of unconditional love, comfort, and support for me throughout this long journey. They have always inspired me to pursue my dreams and have been with me every step of the way and for that, I am forever grateful. 6 List of Abbreviations AD Alzheimer's disease ADDLs amyloid-beta derived diffusible ligands ApoE Apolipoprotein E APP Amyloid precursor protein Aβ Amyloid-β C83 C-terminal fragment of 83 amino acids C99 C-terminal fragment of 99 amino acids CaMKII-Cre+/-/Mfn2loxP/loxP Mfn2 conditional knockout mouse Cdk5 cyclin-dependent protein kinase 5 CHOP C/EBP homologous protein cKO Conditional knockout CNS Central nervous system CREB cAMP response element-binding protein DIV Days in vitro DLP1 Dynamin-like protein DNP 2,4-Dinitrophenol EM Electron microscopy ER Endoplasmic Reticulum fAD Familial AD Fis1 Mitochondrial fission 1 protein GDAP1 Ganglioside induced differentiation associated protein 1 GFAP Glial fibrillary acidic protein 7 GSK-3β Glycogen-synthase kinase-3β GWAS Genome wide association studies HBSS HEPES-buffered salt solution IBA-1 Ionized calcium-binding adapter molecule 1 LOAD Late-onset AD LTP Long-term potentiation MAM Mitochondria associated membrane MAP1/2 Microtubule associated proteins ½ MAP2 Microtubule-associated protein-2 MAPT Microtubule associated protein tau Mff Mitochondrial fission factor Mfn1 Mitofusin 1 Mfn2 Mitofusin 2 Mid49 Mitochondrial elongation factor 2 Mid51 Mitochondrial elongation factor 1 mtDNA Mitochondrial DNA NeuN Neuronal nuclei NFTs Neurofibrillary tangles NO Nitric oxide OCR Oxygen consumption rate OPA1 Optic atrophy protein 1 OXPHOS Oxidative phosphorylation PBS Phosphate-buffered saline 8 PET Positron emission tomography PHFs Paired helical filaments PKA Protein kinase A PP-2A Protein phosphatase-2A PS1 Presenilin 1 PS2 Presenilin 2 ROS Reactive oxygen species RyR Ryanodine receptor sAD Sporadic AD SNPs Single nucleotide polymorphisms SP Senile plaques TPR Tetratricopeptide TUNEL Terminal deoxynucleotidyl transferase nick-end labeling 9 Mitochondrial Dynamic Abnormalities in Alzheimer’s Disease Abstract by SIRUI JIANG Alzheimer’s Disease (AD) is the most common cause of dementia leading to progressive memory loss and neurodegeneration in the hippocampus and frontal cortex. While it has been shown that mitochondrial dysfunction is an early and prominent feature in the progression of AD, it is unclear whether mitochondrial dysfunction itself can lead to neurodegeneration in AD-affected brain regions. Evidence has already suggested that various mitochondrial dynamic proteins (DLP1, OPA1, Mfn1, Mfn2, Fis1) are altered in AD and that there is an imbalance of mitochondrial fission and fusion yet there is a knowledge gap of whether this altered dynamics leads to neurodegeneration and what mechanisms lead to altered mitochondrial proteins in AD. To answer these questions, we created an Mfn2 conditional knockout mouse to recapitulate mitochondrial fragmentation phenotype in the hippocampus and frontal cortex. We found that indeed loss of mitochondrial fusion leads to mitochondrial morphological and bioenergetics abnormalities. These early changes lead to a series of events including oxidative stress, inflammation, and microtubule abnormalities that precede neurodegeneration. To understand the underlying mechanisms leading to loss of important mitochondrial dynamic proteins in AD, we treated primary neurons with amyloid-beta derived diffusible ligands to mimic AD and we found that the loss of DLP1 and Mfn2 is attributed to the calcium-activated protease, calpain. We also found that calpain specifically cleaves DLP1 leading the appearance of several cleavage fragments in both AD transgenic mice as well 10 as AD patient brains. Altogether, these studies show that loss of mitochondrial dynamics could lead to neurodegeneration and that the loss of mitochondrial dynamic proteins in AD could be through the activity of calcium-activated proteases such as calpain. 11 Chapter 1. Introduction 12 Alzheimer’s Disease General Information Alzheimer’s Disease (AD) is the most common neurodegenerative disorder of the elderly that leads to progressive memory loss, impairments in behavior and language, and ultimately death. As the most prevalent form of dementia, AD affects nearly 5.7 million Americans and over 50 million people worldwide (Alzheimer’s Association, 2018). This disease was first observed

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