Mass Spectrometric based proteomics: Dynamic interplay of proteins and their complexes in progression of Alzheimer's Disease

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder and the major cause of dementia, affecting more than 50 million people worldwide. To characterize disease pathology of AD, we measured the protein changes in the prefrontal cortex, hippocampus and substantia nigra brain region of AD subjects and age matched controls using mass spectrometry (MS). Protein complexes play a dynamic role in diverse range of cellular functions. Thus, an in depth insight of protein complexes and their components is crucial for attaining a thorough understanding of their cellular activities. A key challenge is to determine novel protein complexes and to ascertain their role in neuronal physiology and behavior. A systematic approach was used, in which ubiquitous brain protein complexes and their components from human AD and control brain cortex were mapped and characterized that may impact AD pathology. BN-PAGE coupled SDS-PAGE and nano LC-MS/MS analysis of the 34 component factors constituting the 13 protein complexes exhibits differential expression of key subunits in AD neuronal cells. Increased expression of twenty protein components while decreased expression of nine protein components was observed in AD brain cortex in comparison with age matched control. Significant differential expression of the protein components interacting in the complexes includes FRIH, SLC4A4, ATP6V0A1, SLC1A2, MGST1, IGSF8, ACTN1, HSP90AA1, LDHB, APEH, GAPDH, PLP1, ACTB, MAP1B, STXBP1, ACO2, ATP5A1, ATP5B and TPPP. These proteins have been reported for the first time in human AD brain cortex working as interacting proteins in complexes and potentially indicates abnormal metabolic and cytoskeletal system perturbations in AD. By integrating new proteomic tools like IPA and Pathway Linker, our data illuminate canonical pathways mediated by upstream regulators like APP, presenilin, and MAPT dependent mechanism, proposing invariably these interacting molecular factors to be part of concomitant signaling cascades leading to neuronal degeneration and culminating in cell death. The second part of this study focused on nuclear fraction proteins as multiple in vivo localizations are affected during AD progression. The aim of study was to address alteration in multifaceted nuclear proteins and their interactions, responsible for orchestrating the nuclear structure and coordinating cellular activities leading them from physiology to pathology. Nuclear proteins from human AD brain cortex, hippocampus and substantia nigra and age matched controls were compared using two-dimensional gel electrophoresis (2DE) followed by mass spectrometry. The differentially expressed proteins were mapped to existing biological networks to elucidate the disease-related pathways and networks. Specific emphasis was made on hippocampus nuclear proteins considering the significance of this brain region in synaptic plasticity and higher cognitive functions. Expression analysis reveals 106 distinct proteins in 162 spots of nuclear proteins with a significant abundance difference between the experimental groups. ESI-Q-TOF MS/MS identification of selected spots detected increased occurrence of ACTB, GAPDH and TUBB4B whereas decreased expression of APEH, INA, ANXA5, H2B1B and H4 in brain hippocampus nuclear fraction of AD and age matched controls. The down regulation of H4 and upregulation of GAPDH and TUBB4B implicates their role in cell death. IPA analysis and Pathway Linker underscores the interactions of identified proteins with the nuclear proteome and its implications on AD pathology. The differentially expressed proteins were found to be associated with nucleic acid, protein and lipid metabolism, cellular assembly and transport. Furthermore, focused networks created by IPA indicates the regulation of identified proteins by APP, NFKB, ERK1/2 and AKT. Differential quantitative analysis of multi-localized proteins and their interaction networks were further explored through PTMs. In silico analysis reveals dysfunctional proteins due to their putative phosphorylation, glycosylation or nitrosylation. This study adds conceptual insights in AD research by underscoring the dynamism of interacting multifaceted proteins, their networks and pathways involved in AD pathology. The results obtained highlight the importance of protein modifications in neurodegenerative disorders. By detailed understanding of protein modifications in AD patients, new aspects of the pathogenesis and therapeutic targets may be revealed. This thesis demonstrates that proteomics is a promising approach for gaining deeper insight into mechanisms of nervous system disorders and find biomarkers for diagnosis of such diseases