Alzheimer disease is characterized by the initial degeneration of a subset of cholinergic neurons. This pattern of degeneration can be triggered by overexpression of the amyloid precursor protein (APP) gene in humans. Interestingly, APP is widely expressed; it is therefore unclear why only certain cholinergic neurons are vulnerable to degeneration. We show that widespread expression of the human APP gene in the nematode Caenorhabditis elegans also induces age-dependent apoptotic degeneration of select cholinergic neurons. Identical results were obtained by overexpressing the orthologous worm gene apl-1. The pattern of neurodegeneration matched the cell-autonomous accumulation of APP protein in vulnerable neurons and could be activated cell-non-autonomously by distinct portions of APP. Vulnerability to APP accumulation and degeneration depended inversely on the level of ASK1/p38MAPK innate-immune signaling in cholinergic neurons. Lastly, we identify a compound P7C3 that blocks entrance to apoptosis caused by APP or immunodeficiency. Our results suggest that immunosenescence sculpts the cellular pattern of neurodegeneration by APP. / text

published_or_final_version / Medicine / Master / Master of Philosophy

Alzheimer’s disease (AD) is the most frequent neurodegenerative disorder which is characterized by impaired mental functions such as memory, language, perception, behavior and personality, as well as cognitive skills. The molecular mechanisms underlying this disease is still largely unknown, but numerous evidence emerge to support a cell cycle hypothesis which implicates the deregulation of cell cycle proteins as key mediators of neuronal dysfunction and loss in AD brains. One of these signals in Aβ-induced neuronal death model is Cdk/Rb/E2F pathway, where Aβ insult evokes activation of Cdk4/6, which subsequently phosphorylates pRb protein, resulting in activation of E2F transcription factors. However, the mechanism(s) by which Cdk/Rb/E2F mediates neuronal death remains elusive. Therefore, the goal of this project is to characterize the downstream events of cell cycle pathway, which include the involvement of transcription factors c-Myb, Pax6 and Patz1 in Aβ-induced neuronal death signaling. In this study, we showed that Pax6 is a direct target gene for Both E2F1 and c-Myb. Both Pax6 and c-Myb are up-regulated by Aβ insults in cultured cortical neurons. And with E2F1 silencing by siRNA, Aβ-induced Pax6 and c-Myb expression is blocked, suggesting E2F1 is responsible for their elevation. Importantly, siRNA-mediated downregulation of either c-Myb or Pax6 protects neurons from death evoked by Aβ peptide, suggesting they are proapoptotic proteins, delivering death signals sent from upstream E2F1. Next, though ChIP assay, we identified two target genes for Pax6. One is Patz1, another transcription factor that is Aβ-induced pro-apoptotic protein. The other one is GSK3β, which is a pathogenic kinase involved in Tau protein hyperphosphorylation and NFT formation. In conclusion, this dissertation shows that cell cycle regulators Cdk/Rb/E2F modulate neuronal death signals by activating downstream transcription factors c-Myb and Pax6, further upregulating GSK3β. We provided evidence suggesting that Aβ induced neurotoxicity leads to Tau hyperphosphorylation through a mechanism involving cell cycle activation and subsequent activation of c-Myb/Pax6/GSK3β. In brief, in the present study, we delineate a transcriptional cascade downstream of cell cycle pathway leads to neuronal apoptosis as well as Tau/NFT pathology. The characterization of this novel pathway lends support for development of new therapeutic agents and for better experimental models for AD. Lastly, the cascade between cell cycle activation and tauopathy in Aβ-induced neuronal death needs to be further researched in the future. / HKU 3 Minute Thesis Award, Champion (2011) / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Presenilin (PS) is the catalytic subunit of the gamma-secretase which is responsible for the cleavage of amyloid precursor protein to form beta amyloid (Aβ). Mutations in PS cause familial Alzheimer’s disease (FAD) by increasing the Aβ plaques formation in the brain and thereby induce neurodegeneration. Apart from this, FAD-linked PS mutations have been demonstrated to disrupt cellular calcium (Ca2+) homeostasis. Ca2+is a vital secondary messenger that involved in various neurophysiological functions, including memory, learning, and neuroplasticity and mounting evidence suggesting that Ca2+dysregulation associated with PS mutations may play a proximal role in the AD pathogenesis. Yet, the molecular mechanism for Ca2+dysregulation in AD remains debatable. It has been reported that cellular Ca2+homeostatsis can be disrupted in various ways. On the one hand, mutant PS has been demonstrated to exaggerate Ca2+release from the endoplasmic reticulum (ER) through different pathways. On the other hand, attenuatedCa2+influx from the extracellular medium through the capacitative Ca2+entry (CCE) pathway has also been reported to bring about cellular Ca2+disruption. However, the molecular mechanism for the PS mutation-mediated CCE deficits is largely unknown. For this reason, the objective of the current study is to elucidate the underlying molecular mechanism for attenuated CCE in AD. In this study, human neuronal cell line SH-SY5Y is employed as a cellular model to investigate the effect of wild-type or FAD-linked PS1 mutation on CCE pathway. Using single cell Ca2+imaging technique, significant CCE deficits was observed in SH-SY5Y stably expressing FAD-linked PS1mutation, PS1M146L. Interestingly, this CCE attenuation in PS1 mutant expressing cells was not mediated by the down-regulation of STIM1 and Orai1 expression, the known essential molecular players in the CCE pathway. Instead, co-immunoprecipitation and proximity ligation assay have suggested a physical interaction between PS1 and STIM1 proteins. Moreover, a putative gamma-secretase mediated STIM1 cleavage was discovered by western blotting. In addition, confocal imaging showed that PS1M146L significantlyreduceSTIM1 puncta formation and ER translocation followed by the activation of CCE pathway by ER Ca2+store depletion with thapsigargin. This indicated that mutant PS1 attenuates CCE by affecting STIM1 oligomerization or its recruitment with Orai1. Taken together, our results suggested the negative regulatory role of PS on CCE pathway and hypothesized the molecular mechanism of CCE where FAD-linked PS mutation is perceived as a gain-of-function mutation and enhanced the negative impact on STIM1 to inhibit Ca2+entry.This hypothetic model provides new insights into the molecular regulation for CCE pathway and the identification of the interacting domains between PS1 and STIM1 may suggest novel targets for the development of therapeutic agents that help to treat the disease. / published_or_final_version / Physiology / Master / Master of Philosophy

Alzheimer’s disease (AD) is characterized pathologically by proteinaceous aggregates composed primarily of amyloid \(\beta (A \beta)\) and tau. Diseases characterized by abnormal deposition of tau are collectively termed “tauopathies.” \(A \beta\) acts upstream of tau in the AD pathogenesis pathway, but tau expression is required for the neurodegenerative effects of \(A \beta\). Mitochondrial abnormalities have been documented in Alzheimer’s disease and related tauopathies, but the causal relationship between mitochondrial changes and neurodegeneration, as well as specific mechanisms promoting mitochondrial dysfunction, are unclear. Mitochondrial morphology is regulated by fission and fusion events within and between individual mitochondria, and misregulation of this process has been observed in several neurodegenerative diseases. The contribution of mitochondrial dynamics to the pathogenesis of Alzheimer’s disease and tauopathy has not yet been determined. We have found that expression of tau promotes elongation of mitochondria in Drosophila and vertebrate neurons. Elongation is followed by mitochondrial dysfunction, aberrant cell cycle reactivation, and cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. Tau induces mitochondrial elongation by inhibiting mitochondrial localization of DRP1, the primary effector of fission. We have previously demonstrated that direct tau-mediated stabilization of filamentous (F)-actin is critical for neurotoxicity. Here we show that actin stabilization is responsible for the mislocalization of DRP1 following tau expression. Additionally, we identify regulatory roles for F-actin and myosin II in DRP1 localization. Similarly to overexpression of human tau, loss of endogenous Drosophila tau (dtau) induces mitochondrial elongation, but through distinct mechanisms. Expression of human \(A \beta\)in Drosophila induces mitochondrial fragmentation and neuronal toxicity, which are reversed by depletion of dtau. Together, we demonstrate that human disease-associated tau induces neurotoxicity through disruption of mitochondrial dynamics, which can be mediated by enhanced actin stabilization. We also observe a novel role for dtau in the regulation of mitochondrial dynamics, a function critical to the ability of endogenous tau to mediate the effects of \(A \beta\). These findings offer new insights into the contribution of mitochondrial dysfunction to AD and tauopathy, and highlight the emerging role of mitochondrial dynamics in the pathogenesis of neurodegenerative disease.

Conformational ensembles are models of proteins that capture variations in conformation that result from thermal fluctuations. Ensemble based models are important tools for studying Intrinsically Disordered Proteins (IDPs), which adopt a heterogeneous set of conformations in solution. In order to construct an ensemble that provides an accurate model for a protein, one must identify a set of conformations, and their relative stabilities, that agree with experimental data. Inferring the characteristics of an ensemble for an IDP is a problem plagued by degeneracy; that is, one can typically construct many different ensembles that agree with any given set of experimental measurements. In light of this problem, this thesis will introduce three tools for characterizing ensembles: (1) an algorithm for modeling ensembles that provides estimates for the uncertainty in the resulting model, (2) a fast algorithm for constructing ensembles for large or complex IDPs and (3) a measure of the degree of disorder in an ensemble. Our hypothesis is that a protein can be accurately modeled as an ensemble only when the degeneracy of the model is appropriately accounted for. We demonstrate these methods by constructing ensembles for K18 tau protein, \(\alpha\)-synuclein and amyloid beta - IDPs that are implicated in the pathogenesis of Alzheimer's and Parkinson's diseases.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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