Theses (MSc. Molecular Medicine)

Alzheimer’s disease is a neurodegenerative disease that encounters more than half of the elderly population worldwide. The existing Alzheimer’s disease drugs are associated with limitations that warrant the discovery of novel drugs. Previous studies suggested orientin could be the potential Alzheimer’s disease therapeutic agent due to its neuroprotective, antioxidant, and anti- inflammatory properties. In this study, molecular dynamics simulations were performed to study and compare the binding patterns of orientin and the control ligand, MY9. AmberTools22 was used to perform molecular dynamics simulations. The 3D structure of BACE1 was retrieved from Protein Data Bank. Two stages of minimization were performed to remove the steric hindrance of the starting structure. The simulation started with heating up to 300 K, followed by the equilibration stage, and finally data collection for 50 ns. Thermodynamic properties were monitored throughout the simulation time and structural analysis was performed to analyse the conformation changes of the simulation systems. As the result, all simulation systems are stable throughout the simulation time and the result of the structural analysis indicates that the complex is stable as no significant deviation is detected in the analysis output. The binding interaction of orientin to BACE1 stabilised by hydrogen bond interaction is favourable, although it is weaker compared to MY9 and the compactness is similar to the BACE1-MY9 complex, indicating favourable binding interaction. Lastly, orientin binding also induces lesser conformation changes to BACE1 due to higher solvent accessibility. In accordance with the comparative analysis, it can be concluded that orientin has better therapeutic potential compared to MY9 which depicted the significance of orientin as a novel therapeutic approach in molecular dynamics treatment. Keywords: Alzheimer’s disease, orientin, BACE1, beta-amyloid biosynthesis, molecular dynamics simulation

Background: The histopathological hallmarks of Alzheimer’s disease (AD) are aggregation of senile plaques (SPs) and neurofibrillary tangles (NFTs) which resulting in progressive dysfunction of synaptic activity and eventually neurodegeneration. The aggregation of amyloid-β peptides and deposition of tau oligomers contributes to the pathological formation of SPs and NFTs respectively. Besides, neuroinflammation also contribute to pathogenesis and progression of AD. Neural stem cells (NSCs) have the ability to self-proliferate and protect the nervous system. Thus, this regenerative characteristic of NSCs provide great promise in regenerating neuronal cells in the brain for aging, neuroinflammation and neurodegeneration. This study aims to investigate the protective effect of NSCs (NE4C) against microglial-mediated neurotoxicity. Methods: Cell viability assay was used to study the protective effect of NE-4C on the viability of SH-SY5Y. Western blot was used to study the expression of tau and its signalling pathway: glycogen synthase kinase 3β (GSK3β) and p38α mitogen-activated protein kinase (p38α MAPK); also amyloid-β processing protein: amyloid-β precursor protein (APP) and beta-site APP cleaving enzyme 1 (BACE1). The expression of beta-amyloid (Aβ) was determined by enzyme-linked immunosorbent assay (ELISA). Results: The protective effect of NE4C on SH-SY5Y cells against microglia-mediated neurotoxicity was observed in cell viability studies. The expression of phosphorylated tau, Aβ, GSK3β and phosphorylated p38α MAPK in SH-SY5Y cells were downregulated when cultured in the conditioned media of co-culture of NE-4C and BV2 cells. However, LPS stimulation do not affect the expression of APP and BACE1 in SH-SY5Y cells. Co-cultured of NE-4C cells with BV2 cells resulted in decrease in APP and BACE1 expression regardless of microglial activation by LPS. Conclusion: The NE-4C cells confer protective effect on SH-SY5Y cells against microglia-mediated neurotoxicity by suppressing the expression of tau and its signalling pathway. The attenuation of Aβ by NE-4C cells when NE-4C cells co-cultured with BV2 cells were unlikely to be due to the attenuation of APP and BACE1 expression alone. Further studies to determine the mechanisms involved in the attenuation of Aβ by NE4C through regulation of BV2 cells is required. Collectively, these results suggested that the neuroprotective effect of NE4C against microglia-mediated toxicity involved the attenuation of tau phosphorylation and amyloidogenesis, adding to the inherent benefits of NSCs in AD treatment.

General overview: Neuroinflammation plays an important role in Alzheimer's disease (AD) pathogenesis. Prolonged activation of microglia can contribute to excessive production of pro-inflammatory cytokines. Neuronal stem cells (NSCs), is a potential AD therapy due to its regenerative abilities. Therefore, this study aims to investigate the regulatory effect of NSCs in lipopolysaccharides (LPS)-stimulated BV2 microglia cells. Methods: The effect of NE4C neuronal stem cell on LPS-stimulated BV2 cells were examined using transwell co-culture system. Real time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting were used to study the regulation of inflammasome receptors expression that regulates the pro-inflammatory protein productions. The tested samples were further investigated for the inhibition of caspase-1 and pro-inflammatory cytokine, interleukin 1β (IL-1β) by testing the supernatants in the co-culture using caspase-1 colorimetric assay and enzyme-linked immunosorbent assay (ELISA) respectively. Results: Transwell co-culture of LPS-stimulated BV2 cells with NE4C significantly downregulated (p<0.01) mRNA expression of inflammasome receptors. Further studies on inflammasome dependent caspase-1 expression and IL-1β secretion showed NE4C inhibited inflammasome-mediated inflammation by regulating pro-inflammatory cytokines involved in activation of inflammation. Conclusion: The NE4C inhibits LPS induced neuroinflammation in BV2 cells by suppressing caspase-1 expression and IL-1β secretion through regulation of inflammasome activation.