Alzheimer’s disease (AD) is a devastating neurodegenerative disease with growing prevalence in our society. Patients suffering from this debilitating disorder also develop neuropsychiatric symptoms. Depression is one of the most frequently conveyed comorbidity; moreover, depression is also a risk factor associated with AD development. There is a complex interplay between the neurobiology of depression and AD, but their concomitant disease mechanisms remain largely unknown. Retraction of axons and dendrites has been reported to be a common occurrence in both illnesses, proposing the involvement of cytoskeletal dysfunction. Tau is a microtubule-associated protein that undergoes aberrant processing to form neurofibrillary tangles in neurodegenerative diseases such as AD. However, the role of tau in depression has not been well studied. The elucidation of pathophysiological mechanisms in depression is important to provide a more holistic understanding of AD pathogenesis. This study proposes the potential participation of tau phosphorylation in the pathogenesis of depression. In addition, this study will also investigate tau modifications under concomitant models of depression and AD. Primary cultures of hippocampal neurons were exposed to independent and cotreatments of corticosterone and β-amyloid (Aβ), to induce in vitro models of depression and AD, respectively. Sprague Dawley rats were subcutaneously injected with corticosterone for 14 days to induce an in vivo model of depression. Tau phosphorylation, aggregation and interaction with microtubules were examined. Results demonstrated that in both in vitro and in vivo models of corticosterone-induce depression, tau underwent increased phosphorylation at residues S396 and S404. Phosphorylated tau showed decreased interactions with microtubules and increased vulnerability to aggregate. Furthermore, the in vivo model of depression illustrated an altered localization of tau in the CA3 region of the hippocampus. Co-treatment of corticosterone and Aβ exacerbated aberrant tau phosphorylation and aggregation. In conclusion, this study provides evidence for the role of tau in depression, suggesting the occurrence of abnormal tau phosphorylation as an early event in the pathogenesis. Additionally, the pathophysiology of depression and AD may involve similar mechanisms in tau phosphorylation and aggregation. This study provides insight into the neurobiological linkages between depression and AD, and emphasizes the importance of tau-targeted interventions in neuropsychiatric disorders. / published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Pan, Tak Yin. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Content --- p.v / Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease --- p.1 / Chapter 1.1.1 --- Cholinergic hypothesis --- p.2 / Chapter 1.1.2 --- p-amyloid hypothesis --- p.2 / Chapter 1.1.3 --- Taupathy hypothesis --- p.3 / Chapter 1.1.4 --- Current therapies --- p.4 / Chapter 1.2 --- Proteins Involved in Alzhemer's Disease --- p.5 / Chapter 1.2.1 --- Acetylcholinesterase (AChE) --- p.5 / Chapter 1.2.2 --- p-amyloid --- p.6 / Chapter 1.2.3 --- Paired helical filaments (PHF) --- p.7 / Chapter 1.2.4 --- Protein kinases --- p.8 / Chapter 1.2.4.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.9 / Chapter 1.2.4.2 --- Cyclin-dependent kinase-5 (CDK-5) --- p.9 / Chapter 1.2.5 --- Protein phosphatase (PP) --- p.10 / Chapter 1.2.5.1 --- Protein phosphatase 1 (PP-1) --- p.11 / Chapter 1.2.5.2 --- Protein phosphatise 2A (PP-2A) --- p.12 / Chapter 1.2.5.3 --- Protein phosphatise 2B (PP-2B) --- p.13 / Chapter 1.2.6 --- Apoptotic and Anti-apoptotic proteins --- p.14 / Chapter 1.2.6.1 --- Caspase-3 --- p.15 / Chapter 1.2.6.2 --- Bcl-2 --- p.15 / Chapter 1.3 --- Flavonoids --- p.16 / Chapter 1.3.1 --- Biosynthesis of flavonoids --- p.17 / Chapter 1.3.2 --- Biological functions of flavonoids in plants --- p.18 / Chapter 1.3.3 --- Beneficial effects of flavonoids on human health --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- Differentiation of SHSY-5Y cells --- p.20 / Chapter 2.1.1 --- SHSY-5Y cell culture --- p.20 / Chapter 2.1.2 --- Counting cells --- p.20 / Chapter 2.1.3 --- Retinoic acid differentiation --- p.21 / Chapter 2.2 --- Western blot analysis --- p.21 / Chapter 2.2.1 --- Extraction of proteins from mammalian cells --- p.21 / Chapter 2.2.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.22 / Chapter 2.2.3 --- Semi-dry protein transfer to nitrocellulose membrane --- p.23 / Chapter 2.2.4. --- Membrane blocking and immunostaining --- p.24 / Chapter 2.3 --- MTT assay --- p.25 / Chapter 2.4 --- Hoechst 33342 Nuclei staining --- p.25 / Chapter 2.5 --- Cell cycle analysis --- p.25 / Chapter 2.5.1 --- Ethanol fixation --- p.25 / Chapter 2.5.2 --- Propidium iodide staining --- p.26 / Chapter 2.6 --- Annexin V-FITC & Propidium iodide staining --- p.26 / Chapter 2.7 --- DNA fragmentation analysis --- p.26 / Chapter 2.7.1 --- Phenol/Chloroform extraction of DNA --- p.26 / Chapter 2.7.2 --- Ethanol precipitation of DNA --- p.27 / Chapter 2.7.3 --- Agarose gel electrophoresis of DNA --- p.27 / Chapter 2.8 --- Proteomic analysis --- p.28 / Chapter 2.8.1 --- First dimension: isoelectric focusing --- p.28 / Chapter 2.8.2 --- Second dimension: SDS PAGE --- p.29 / Chapter 2.8.3 --- Gel staining --- p.30 / Chapter 2.8.3.1 --- Silver staining --- p.30 / Chapter 2.8.3.2 --- SYBRO Ruby staining --- p.31 / Chapter 2.8.4 --- Gel scanning and image analysis --- p.31 / Chapter 2.8.5 --- ln-gel digestion --- p.32 / Chapter 2.8.6 --- Zip Tip for desalting the digested sample --- p.33 / Chapter 2.8.7 --- Protein identification with mass spectrometry and database search --- p.33 / Chapter Chapter 3: --- Characterization of Okadaic acid-induced tail hyperphosphorylation in SHSY-5Y cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Objectives --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Differentiation of SH-SY5Y cell --- p.38 / Chapter 3.3.2 --- Changes of protein expression after okadaic acid treatment --- p.40 / Chapter 3.3.3 --- Neurite Retraction Induced by okadaic acid --- p.42 / Chapter 3.3.4 --- Okadaic acid-induced Cell Death measured by MTT assay --- p.44 / Chapter 3.3.5 --- Hoechst 33342 Nuclei Staining --- p.44 / Chapter 3.3.6 --- Cell cycle analysis by propidium iodide staining --- p.47 / Chapter 3.3.7 --- Early Apoptotic cells detection by Annexin V/PI staini --- p.49 / Chapter 3.3.8 --- DNA fragmentation --- p.51 / Chapter 3.4 --- Discussion --- p.53 / Chapter Chapter 4: --- Flavonoids screening for protecting neuronal death by preventing tau hyperphosphorylation --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Objectives --- p.58 / Chapter 4.3 --- Tested flavonoids --- p.59 / Chapter 4.4 --- Results --- p.60 / Chapter 4.4.1 --- Toxicity of flavonoids --- p.60 / Chapter 4.4.2 --- Effects of flavonoid pre-treatment on OA-induced neu retractions and cell death --- p.62 / Chapter 4.4.3 --- Western blot analysis --- p.65 / Chapter 4.4.4 --- The effect of different concentrations of hesperidin or OA treatment --- p.70 / Chapter 4.4.5 --- Proteomic analysis --- p.74 / Chapter 4.5 --- Discussion --- p.78 / Chapter Chapter 5: --- General Discussion --- p.82 / References

Alzheimer’s disease is the most common form of dementia among older adults, and directly contributes to the third leading cause of death in the United States. Although amyloid plaques and tau-loaded neurofibrillary tangles have been identified as the main pathological features of Alzheimer’s disease for more than one hundred years, the molecular mechanism is still poorly understood and treatments are limited to palliative care. Oligomeric Amyloid beta plays a crucial synaptotoxic role in Alzheimer’s disease, and hyperphosphorylated tau facilitates Amyloid beta toxicity, but the link between the two remains controversial. Since tau is a microtubule associated protein and microtubules are critical for neuronal functions, regulation of dynamic microtubules may serve as the link between Amyloid beta and tau. Here I propose a model in which Amyloid beta can induce changes in MT dynamics in dendrites and axons that are primary to tau hyperphosphorylation, while these MT changes are sufficient to cause tau hyperphosphorylation and necessary for Amyloid beta synaptotoxicity through tau. My thesis work further characterizes mammalian excitatory presynaptic boutons as hotspots for activity-dependent dynamic microtubule nucleation that is required for synaptic transmission during neuronal activation or Amyloid beta-induced neuronal injury through tau.

Myeloid cells of the innate immune system have been strongly implicated in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Similarly, several lines of evidence call on the adaptive immune system as a critical driver of disease, particularly in PD. The immune dynamics in both of these diseases are complex, and span across not only the innate and adaptive immune systems, but also across the periphery local action in the central nervous system (CNS). This thesis aims to address critical gaps in our knowledge regarding molecular and functional alterations of immune cells in AD and PD. We apply tools including single nucleus RNA – and ATAC – sequencing as well as protein – level and functional studies to advance our understanding of molecular pathways involved in the innate and adaptive immune dysfunction in these diseases, including both immune cells in the CNS as well as in the periphery. Chapter 1 provides an overview of the evidence implicating myeloid cell dysfunction in AD and PD, including microglia as well as peripheral myeloid cells such as monocytes. It also describes the features of immune dysregulation in both diseases, and evidence implicating the adaptive immune system in PD. Chapter 2 aims to address our currently limited understanding of microglial molecular phenotypes and diversity in PD, by characterizing microglial transcriptomic and chromatin signatures in disease. We demonstrate microglial subpopulation-specific effects, including the focal depletion of a microglial population in the substantia nigra in PD, which open novel avenues for targeted neuroimmune interventions in PD. Chapter 3 aims to identify interactions regulating the infiltration and retention of peripheral immune cells into the CNS in PD; a process which is implicated in the progression of this disease, but the mechanisms of which are not fully understood. We characterized transcriptomic signatures of infiltrating lymphocytes and blood brain barrier cells, and found increased T cell infiltration in PD as well as fibroblast and endothelial populations associated with disease. We further identified transcriptional shifts suggestive of a proinflammatory and profibrotic milieu in disease, in which chemokines and extracellular matrix elements produced by fibroblasts may influence T cell trafficking and retention in the substantia nigra in PD. Chapter 4 aims to address the gap in our knowledge of how myeloid dysfunction in the periphery contributes to AD. While genetics implicate all myeloid cells in AD and PD, contributions of peripheral myeloid cells, such as monocytes, have been largely overlooked in place of microglia, which are resident in the CNS. We evaluate the convergence of the AD genetic risk loci on functional outcomes in monocytes, in the context of Aβ as an immune stimulus. We identified functional convergence of the CD33 and SPI1 AD risk variants in the context of Aβ stress, including reduced phagocytosis and loss of surface TREM2 expression, demonstrating an interaction between genetics and environment to reduce myeloid cell fitness. Finally, Chapter 5 concludes with a summary of key findings from this work, and discusses future directions for modulating innate and adaptive immune populations, both in the CNS and in the periphery, as therapeutic approaches for these neurodegenerative diseases.

Alzheimer’s Disease (AD) is a chronic neurodegenerative disease characterized by cognitive impairment, progressive memory loss, dementia, and behavioral disturbances that are associated with particular histological and molecular features, principally: neuritic plaques formed from deposits of amyloid beta protein (Aꞵ) and neurofibrillary tangles composed of accumulations of tau protein. Other factors such as lipid metabolism, neuroinflammation, protein homeostasis, cell death, and synaptic dysfunction also contribute to AD pathology. In addition to these factors, numerous studies have underlined the significant impact that miRNAs and the dysregulation of miRNAs can have in mediating multiple components of AD and tau pathology. In this thesis, we focused on the role of a highly-conserved, brain-enriched miRNA, miR-219, that our laboratory had previously found to be significantly downregulated in postmortem AD brain samples and could regulate the protein levels of tau and kinases that phosphorylate tau (GSK3ꞵ, CaMKIIɣ, and TTBK1) both in vitro and in vivo in D. melanogaster. Furthermore, we found that miR-219 could also mediate tau pathology, as evidenced by phosphorylated tau, in vitro and in D. melanogaster in vivo. This evidence led us to study whether these previously validated actions of miR-219 would be recapitulated in vivo in a mouse model of human tau pathology, htau, and illuminate whether or not miR-219 could be a potential therapeutic target or primary contributor for human AD and tau pathology. In order to do this, we overexpressed the levels of miR-219 in aged htau mice with tau pathology but unfortunately found no neuroprotective effect. Possibly due to the variability in behavioral results in this mouse model, we next provided an updated behavioral characterization of aged htau mice in a battery of useful memory tests often used in AD research. Lastly, we inhibited the levels of miR-219 in htau mice at an age before severe tau pathology occurs in order to see if miR-219 dysregulation could exacerbate tau pathology and associated cognitive impairment. We found that miR-219 inhibition led to severe deficits in short-term spatial memory in Y-Maze Novel Arm and long-term spatial and reference memory in Morris Water Maze. Furthermore, we performed biochemical analyses on the brains of these mice and found that miR-219 inhibition led to significantly increased protein levels of CaMKII, which has been extensively implicated in AD and could underlie the memory deficits seen in these mice. Upon immunofluorescence staining and analysis of brain sections taken from these mice, we found significantly higher levels of phosphorylated tau in cells transfected with our lentiviral miR-219 inhibitor in htau-Inh mice, indicating that inhibition of miR-219 leads to increased phosphorylated tau. Due to the design of our lentiviral vector, it is also possible that we inhibited miR-219 in other cell types in the brain (e.g., oligodendrocytes, microglia, astrocytes) whose function have been shown to be regulated by miR-219, and thus opens up many interesting future questions and research directions to fully analyze the effect that miR-219 inhibition may play in these cells and their contribution to cognitive impairment and tau pathology. We believe that our results demonstrate a critical role for miR-219 as an important contributor to both cognitive impairment and AD-related pathology, presumably through its regulation of CaMKIIɣ and the subsequent increase in phosphorylated tau.

Alzheimer's disease (AD) is the most common form of dementia, currently affecting around 17--25 million people worldwide. The typical neuropathological hallmarks of AD are amyloid beta (Abeta) deposition, presence of neurofibrillary tangles and neuronal cell death. Evidence from ongoing studies on the pathogenesis of AD, suggests that several different mechanisms are involved in neurons loss and thus decline of cognitive function. These include the metabolism of amyloid peptide, inflammation, cholesterol metabolism, and hormonal factors. / I have focused on the role of inflammation in the progression of AD. The inflammation hypothesis is based on findings of (1) elevated levels of inflammatory cytokines, such as IL-1, IL-6, TNFalpha, (2) the reduced levels of anti-inflammatory cytokines, like IL-10 in CSF and the blood of AD patients, and (3) activated microglia in the histological section of the patient's brain. On the other hand, the effects of the ApoE gene and differential age of onset between the two sexes suggested a modulation role for cholesterol and sex hormone like estrogen, which may influence the inflammatory response in the brain, so as to modulate the risk of AD. / In this project, the genetic risk factors predisposing to AD were investigated by genetic association studies of candidate genes. Candidate genes were shortlisted by two approaches. (I) Linkage-based candidate genes: Candidate genes were identified from reported loci with linkage to AD genome scan studies. Previous linkage studies of AD families revealed linked loci at 1p36, 1q23, 3p14, 4q32, 6p21, 6q27, 9q22, 10q24, 13q32, 15q26, 19q13 and 21q22. Several candidate genes from these loci including TNFalpha-related genes, TLR2, IGF-1, IFNalpha and MTHFR were selected for this project. (II) Hypothesis-based candidate genes: Candidate genes were selected according to their possible involvement in the inflammation hypothesis of AD. Under the hypothesis-based candidate gene approach, genes that might contribute to the inflammatory response of amyloid deposition were identified. These genes were validated by their expression level in the central nervous system. A further priorization step was carried out to select those genes showing a higher degree of inter-individual variation. Therefore, these genes were more likely to have a genetic/inherited variation at the population level. In other words, they are more likely to be the predisposition genes than genes without inter-individual variation (house-keeping genes are examples of genes showing little inter-individual variation). In this project, genes involved in the inflammatory pathway in the brain, such as IL-10 and HLA-A, and also genes that interact with the inflammatory pathway such as cholesterol related enzymes and estrogen receptors were investigated under the hypothesis-based approach. / This project is based on a case-control genetic association study which comprised of NINCDS-ADRDA diagnosed Chinese patients with AD (n=259) and age-matched non-demented subjects (n=248). Three genes PTGS2 (encoding for COX-2), MxA and ESR1 were selected for an intensive study by investigating their linkage disequilibrium pattern and using tagSNP strategy. TagSNPs selected for each gene were genotyped to investigate their association with the risk of AD. / This study showed that MTHFR, IL-10, HLA-A, CYP46A1, PTGS2 (COX-2) and ESR1 were associated with the risk of AD, and MxA, identified for the first time, was associated with the age of onset of AD. In conclusion, the results of my study further suggested the roles of inflammation in the pathogenesis of AD. / Ma Suk Ling. / "June 2006." / Advisers: Linda C. W. Lam; Nelson L. S. Tang. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1417. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 169-204). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The hypothesis to be tested states that the pathology of Alzheimer's disease (AD) involves elevated levels of oxidative stress, resulting in elevated levels of cellular oxidative defense mechanisms. If the premise is true, than AD pathologically afflicted cells should have a higher demand for glutathione (GSH) as an innate oxidative defense mechanism hence; greater GSH concentrations, increased GSH resynthesis capabilities, and increased levels of cystathionine gamma-lyase (CNase). Alzheimer diseased and age matched control lymphoblast cells, obtained from OHSU's Oregon Brain Aging Study, were cultured, and GSH biochemistry was subsequently evaluated. GSH was depleted by exposing cells to the GSH depleting agent diethylmaleate (DEM) and the resulting GSH concentrations were measured. GSH resynthesis was measured after depleting GSH with DEM, to a level of approximately half base GSH concentration, then removing the depleting agent, resuspending the cells in fresh medium (DEM-free), and subsequently measuring GSH levels. GSH concentrations were measured by HPLC, and all data was normalized to cellular protein concentration. Cellular CNase specific activity levels were measured by adding cytasthionine, the CNase substrate, and then measuring the amount of cysteine produced by means of the DTNB assay. The AD cell lines showed no increase in base levels of GSH as compared to control cell lines. The AD cell lines showed a statistically significant increase in GSH resynthesis capabilities and cystathionine gamma-lyase specific activity levels. These findings add further weight to the AD oxidative stress hypothesis, which is based on the premise that the causative agent of AD pathogenesis is an increase in the level of cellular free radicals produced.

One of the most foundational and personal philosophical questions one can ask is what makes you, you? In large part, you are made up of your relationships, experiences, and memories. The hippocampus, a brain region which is critical for the formation of memories, has been the focus of neuroscience research for decades due partially to this function, which is foundational to our individuality. In Alzheimer’s disease (AD), one of the most common and well-researched neurodegenerative diseases in the world, the hippocampus is one of the earliest targets. Despite extensive work on AD, we still lack a coherent understanding of what is causing the disease, the mechanisms by which it is causing neuronal dysfunction and death within the hippocampus and other brain regions, and how it ultimately causes deficits in cognition and behavior, leading to an erosion of our selves. In this thesis, I explore three independent but related questions: 1) what molecular mechanisms are causing early synaptic loss in AD, specifically within the hippocampus, 2) what molecular effectors are responsible for establishing and maintaining intracellular architecture in hippocampal neurons, which are exploited in early AD, and 3) how and when does the hippocampal circuit dysfunction in AD progression? Using a variety of experimental techniques, ranging from in utero and ex utero electroporation, primary murine and human neuronal cell culture, longitudinal confocal microscopy, immunohistochemistry, biochemistry, cell and molecular biology, in vivo two-photon calcium imaging, and behavioral assays, I have found that, within CA1 of the hippocampus, synapse loss requires degradation of the dendritic mitochondrial network, activity and input specificity are driving mitochondrial compartmentalization within CA1 neurons through the same pathway that is aberrantly overactivated in AD, and the hippocampal circuit is overly rigid in encoding the environment as the disease progresses.

The dysregulation of immune system function has been centrally implicated in numerous age-related and neurodegenerative disorders, including Alzheimer’s disease (AD). Genetic susceptibility studies have positioned microglia, brain-resident immune cells, as critical actors in the development and the progression of the disease. Microglia are highly plastic cells with diverse functions across many modalities, and the appropriate regulation of their activities are a prerequisite for central nervous system homeostasis and cognitive health. Aging and pathogenic contexts are posited to modify microglial behavior, inhibiting their neuroprotective function and promoting a dysfunctional state that drives disease. However, the mechanisms underlying these pathogenic alterations in microglial state and function are complex and poorly understood. This thesis identifies three elements that are altered in the AD brain and investigates how these mechanisms may serve as triggers producing microglial dysregulation in AD. Chapter 3 examines the role of expression of the transposable element LINE-1 in AD-related microglial dysfunction. Chapter 4 explores the regulation of PLCG2, which encodes a critical AD-associated signaling enzyme. Chapter 5 investigates the role of the AD-linked sorting receptor SORL1 in microglia. Together, these data expand our understanding of mechanisms driving altered microglial pathophysiology in AD and illuminate pathways of interest with potential therapeutic applications meriting deeper exploration.