Tiwari, Anuj Satish (2022) Study on Membrane Parameters Involved in ZnONP Penetration and Amyloid Beta Interaction in Neurodegeneration. PhD thesis.
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This thesis attempts to investigate the membrane parameters associated with neurodegeneration caused by zinc oxide nanoparticles (ZnONPs) and Amyloid Beta-40 (Aβ- 40) leading to neuronal cell deformation. Intrinsically disordered peptides (IDPs) like Aβ- 40 are known to cause neurodegeneration, although the exact mechanism through which they elicit this toxicity is still elusive. And lately, the extensive use of nanoparticles, specifically ZnONP, in cosmetics, coating, and pigments has increased tremendously. This leads to the possibility of unintentional exposure of NPs as a hazardous pollutant for humans. Recent studies have shown the implication of ZnONP exposure that induces neuronal damage. But the mechanism of entry of these NPs into the cells is poorly understood. The current understanding of nanoparticle–membrane interaction is drawn mostly from computational studies and lacks sufficient experimental evidence. We firstly explored the ZnONP-membrane interactions. Here, we try to investigate the lipid specificity and the role of the membrane biochemical and physical forces at play in modulating the penetration of ZnONPs. Using confocal fluorescence imaging and potentiometric dye-based fluorimetry, we first investigated the interaction of ZnONP in both multi-component and individual lipid membranes using cell-like giant unilamellar vesicles to dissect the lipid specificity; also, we measured the changes in membrane order, anisotropy and hydrophobicity. Amongst the single lipid membranes, ZnONP interacted strongly with phosphatidylinositol followed by phosphatidylcholine head-group containing lipids. We further compared the interaction of ZnONP with three physiologically relevant membrane conditions varying in composition and dipole potential. We found that ZnONP interaction leads to a photoinduced enhancement of phase separation that ranges from partial to complete depending upon the membrane composition and cholesterol content. Interestingly, while the lipid order of a partially-phase-separated membrane remained unchanged upon ZnONP crowding, a fully-phase-separated membrane showed an increase in the lipid order. Strikingly, ZnONP crowding induced a contrasting effect on the rigidity of the membrane upon binding to the two membrane conditions, which was inferred through fluorescence anisotropy, in line with the measured diffusion coefficient. ZnONP seems to preferentially penetrate through the liquid disordered areas of the membrane and the boundaries of the phase-separated regions driven by the interplay between the electrostatic forces and phase boundary conditions, which are collectively dictated by the composition and ZnONP induced lipid reorganization. The results may lead to a greater understanding of the interplay of membrane parameters and ZnONP interaction in driving passive penetration. Coming to Amyloid-beta (Aβ-40) aggregation mediated neuronal membrane deformation, although poorly understood, is implicated in Alzheimer's Disease (AD). The peptide aggregates, forming amyloid plaques which were long suspected to be toxic to the neurons. But lately, the leitmotif in the field has changed, where now the more soluble forms of the peptide are considered toxic for the neurons. Hence, we set out with a motive to find synthetic molecules, specifically the arylamines that could compete with the aggregating peptide for the binding pockets leading to retardation or complete arrest of the fibrillation of the peptide. Further, we also looked into whether these molecules had the potential to dissolve pre-formed aggregates. The screening of these molecules led to two potential molecules that retarded the Aβ-40 aggregation and one of which could also dissolve the preformed aggregates. Furthermore, We then wanted to emulate the Aβ-40 mediated myelin membrane deformation observed physiologically. For which myelin membrane mimic was incubated with Aβ-40 in vitro and temporally mapped for 24 hours using confocalmicroscopy, initial strong binding and extensive tubulated structures at longer timescales were observed. To dissect the lipid specificity of Aβ-40 in the myelin membrane, we used the single lipid models. ThT assay & fluorescence microscopy were used to check for potential modulation of the Aβ-40 aggregation and binding. Binding was observed in PI, PG, PC/BSM, and DOPC/PIP2. As observed, negatively charged lipids bound strongly, and hence electrostatic forces seem to play a role in binding. Interestingly zwitterionic PC/BSM too showed strong binding leading us to suspect that something else apart from electrostatics is also at play. Using the differences in the shapes of the individual lipids, we formulated three different conditions along with myelin membrane and estimated the packing defect densities through MD simulation and then further experimentally confirming The binding of Aβ-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin membrane in the early time scales. Furthermore, elongation of Aβ-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the myelin membrane followed by extensive membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aβ-40 - myelin membrane interaction and demonstrate how short timescale, localphenomena of binding, and fibril mediated load generation manifests into long timescale, global phenomena of myelin tubulation and demonstrates the ability of Aβ-40 to demyelinate.
|Item Type:||Thesis (PhD)|
|Uncontrolled Keywords:||Neurodegeneration; Alzheimer's disease; Biomimetic membranes; ZnONP; Amyloid-Beta; Compressibility modulus; Membrane diffusion; Membrane fluidity|
|Subjects:||Life Science > Molecular Meidicine|
Life Science > Biochemistry
Life Science > Microbiology
|Divisions:||Sciences > Department of Life Science|
|Deposited By:||IR Staff BPCL|
|Deposited On:||06 Dec 2022 12:09|
|Last Modified:||06 Dec 2022 12:09|
|Supervisor(s):||Saleem, Mohammed and Jha, Suman|
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