Identification of Aberrant Molecular and Cellular Mechanisms Underlying the Pathogenesis of Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia which affects 50 million people worldwide and more than a million in Pakistan. It is primarily characterized by the formation of neurofibrillary tangles (NFTs) and amyloid beta (Aβ) plaques that progressively lead to cognitive decline. The Aβ toxicity plays a central role in AD progression and mediates several noxious mechanisms including mitochondrial damage, oxidative stress, inflammation, synaptic dysfunction, memory impairment and neuronal damage. In recent years, impaired adult hippocampal neurogenesis has gained much significance as a substantial contributing factor in AD pathology perhaps affected by Aβ-induced toxicity. However, no conclusive evidence exist that indicates the molecular regulation of adult hippocampal neurogenesis during the course of AD progression in relation to Aβ. Therefore, present work investigated the effects of Aβ (1-42) - induced toxicity on adult hippocampal neurogenesis. To assess the pattern of regulation of hippocampal neurogenesis, the expression of adult hippocampal neurogenesis markers, memory impairment, anxiety-like behavior and neuronal degeneration in BALB/c mice was examined following intracerebroventricular (ICV) injection of Aβ (1-42). The behavioral analysis using elevated plus maze, Morris water maze and novel object recognition tests exhibited the potential of Aβ (1-42) to induce anxiogenic effects and deficits in spatial memory and novelty preference, respectively. It also led to neuronal loss in the dentate gyrus (DG) and cornu ammonis (CA1, CA2 and CA3) regions of the hippocampus. Aβ (1-42) reduced the expression of the stage-dependent markers of neurogenesis i.e. proliferation marker protein Ki67, neuronal migration protein doublecortin (DCX) and differentiation marker, neuronal nuclear antigen (NeuN). These results provide the evidence that Aβ (1-42) - induced toxicity Abstract xx alters hippocampal neurogenesis, accompanied by memory impairment and neurodegeneration, however, further insight is warranted to explore the underlying molecular pathway(s). The study further explored the toxic effects of Aβ and its interaction with heparan sulfate (HS). The presence of heparan sulfate proteoglycans (HSPGs) and the side chains HS are evident in Aβ deposits in the brains of AD patients and transgenic animal models. Aβ interacts with HS and protects it from heparanase-mediated degradation while HS and HSPGs facilitate the conversion of non-fibrillar to fibrillar Aβ plaques thereby creating a positive feedback loop which is critical to the sustenance of AD pathology. Hence, the analysis of the changes in HS structure, biosynthesis genes, and subsequent alterations in its interactome/core proteins provide further understanding of the complex association. Therefore, SH-SY5Y neuroblastoma cells were treated with Aβ (1-42) that resulted in the death of about 50 percent of cells and caused a significant increase in the expression level of cleaved caspase-3, indicating apoptotic cell death via caspase-dependent pathway. Aβ (1-42) also produced a substantial change in the gene expression levels of HS biosynthetic enzymes (HS2ST1 and HS6ST2), HS core protein (GPC3), and a proteoglycan protease (ADAMTS4), however, HS composition was not significantly altered. Mass spectrometric analysis exhibited changes in HS interactome where seven of the extracellular and membrane proteins were differentially regulated in Aβ (1-42) treated cells including BCAP31, ITGA1, EEF1A1P5, GANAB, RPS4X, DSC1, and KRT17. Identified proteins are actively involved in various biological processes and deregulated expression of these proteins lead to synaptic damage, neuronal degeneration, compromised energy metabolism, lower cell viability and migration and impaired olfaction. These findings provide evidence that Aβ (1-42) may cause cytotoxic effects via alterations in Abstract xxi the expression pattern of HSPGs. Extracellular proteins can be easily accessible, therefore, may serve as potential drug targets, therefore, the study anticipates some novel AD-related targets that potentiate further research to establish the role of HS binding proteins in AD pathogenesis. Alongside Aβ, the genetic component also plays a critical role in AD progression. More than 20 loci have been associated with AD which contain a significant number of genetic variants. However, the plausible function of various non-coding variants is still unexplored. Consequently, this study unravels the regulatory role of AD-related single nucleotide polymorphisms (SNPs) and their proxy SNPs to determine the risk of developing AD. In silico analysis using SNAP web portal and RegulomeDB provided evidence of a high degree of potential regulatory function of 151 SNPs out of which 8 are novel AD-linked SNPs. The interactome analysis of the proteins affected by these novel SNPs through STRING 10.5, showed their functional relevance to AD pathology and revealed direct or indirect interaction with APP. It is postulated that these non-coding variants can be strongly associated with disease risk and the development of AD. Further validation through experimental studies will be helpful for the elucidation of the regulatory potential of the identified SNPs. In conclusion, the study comprehensively demonstrated that Aβ (1-42) impaired cognitive functions, decreased the process of hippocampal neurogenesis and altered HS biosynthesis and the expression of HS binding proteins. Moreover, non-coding SNPs regulate the expression of various proteins involved in AD pathogenesis. The present findings contribute to the existing knowledge of AD pathology and pave the way for further exploration and identification of underlying molecular mechanism(s) and therapeutic target(s) for AD through further validation of identified markers.