Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biomedical Sciences

First Advisor's Name

Madhavan Nair

First Advisor's Committee Title

Major Professor, Committee chair

Second Advisor's Name

Nazira El-Hage

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Hoshang Unwalla

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Ajeet Kaushik

Fourth Advisor's Committee Title

Committee member

Fifth Advisor's Name

Michal Toborek

Fifth Advisor's Committee Title

Committee member

Sixth Advisor's Name

Chenzhong Li

Sixth Advisor's Committee Title

Committee member

Keywords

Amyloid Beta, Amyloidogenesis, Amyloid precursor proteins, Withaferin A, β-secretases, Gamma-secretases, Tau phosphorylation

Date of Defense

3-4-2019

Abstract

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers.

Identifier

FIDC007683

Creative Commons License

Creative Commons Attribution 3.0 License
This work is licensed under a Creative Commons Attribution 3.0 License.

Available for download on Thursday, April 09, 2020

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