Investigating the role of BCATc in autophagy and its implications in Alzheimer's disease

Edward, Roshita

Abstract

Background: Autophagy, crucial in maintaining cellular health, is often dysregulated in neurodegenerative conditions such as Alzheimer’s disease (AD). Recent data shows that the cytosolic branched chain aminotransferase (BCATc) protein plays a pivotal role in autophagy regulation, contrary to its canonical role as a transaminase.

Hypothesis: BCATc can regulate autophagic flux, contributing to aggregation or ineffective clearance.

Methods: SH-SY5Y cells (+/- BCATc) were nutrient-deprived and treated with autophagy inhibitors. Expression of autophagy (LC3, p-MTORC1, Vps34 and Ulk1) or AD markers (Aβ41, tau and p-tau) were analysed using Western blot analysis. Autophagic flux was monitored using the GFP-RFP-LC3B tandem sensor. Co-immunoprecipitation studies tested for interactions between Vps34 and BCATc. The functional effect of BCATc expression on Vps34 activity was established using the Vps34 Kinase ELISA kit (Echelon Biosciences). Immunocytochemistry was used to assess sub-cellular colocalisation of proteins in cells. C. elegans were used to map the changes in cognition relative to BCATc knockdown/AD aggregation.

Results: A novel role for BCATc as a regulator of autophagy, predominantly associating with Vps34 is shown. BCATc is upregulated in response to autophagy stimulation. Overexpression of BCATc resulted in an increase in autophagosome synthesis and Aβ42. Studies using a Vps34 inhibitor, co-immunoprecipitation and kinase assays, showed that BCATc directly interacts and functionally regulates Vps34, with BCATc overexpression increasing Vps34 activity (p<0.001). Interactions were confirmed using ICC, where BCATc differentially co-localised with Ulk1 and Vps34 in response to nutrient load. Importantly, co-localisation was lost when incubated with leucine, a key regulator of BCATc’s transaminase role. In C. elegans, loss of BCATc resulted in a decrease in cognition that matched AD controls.

Conclusions: These results highlight a novel role for BCATc in regulating autophagy, principally autophagosome synthesis. This moonlighting role is reversed in response to leucine. Sustained increases in BCATc expression, as seen in AD brains, results in increased autophagosome synthesis that cannot be matched by clearance. This thesis proposes that BCATc-dysregulated autophagy contributes to the accumulation of autophagosome seen in AD brains and could be targeted through leucine regulation.

• Thesis Type: Thesis
• Deposit Date: Apr 30, 2023
• Publicly Available Date: Feb 13, 2024
• Keywords: BCATc Autophagy Alzheimer's disease
• Public URL: https://uwe-repository.worktribe.com/output/10732083
• Award Date: Feb 13, 2024