Biology subjects

Veerabhadraswamy, P.

Publications and source records attributed to Veerabhadraswamy, P..

3 recordsLinked to original sources

bioRxiv2025

p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

The interaction of amyloid-{beta} (A{beta}) peptides with the plasma membrane (PM) is a potential trigger that initiates the formation of higher-order aggregates, membrane alterations/damage, and progressive neurotoxicity in Alzheimers disease (AD). In a previous study, we showed that oligomers of A{beta}1-42 (oA{beta}1-42) induced PM damage, resulting in PM repair cascade via lysosomal exocytosis coupled with endocytosis, and facilitation of tunneling nanotubes (TNTs)-like membrane protrusions to promote direct cell-to-cell transfer of aggregates. In this study, we demonstrated that PM damage induced by oligomers of the aggregation-prone peptide A{beta}1-42 significantly facilitates PM repair by enhancing phosphorylated p21-activated kinase 1 (pPAK1)-dependent endocytosis and Rab3a-dependent exocytosis in SH-SY5Y and SK-N-SH neuronal cells compared to control and oA{beta}1-40 treated cells. We studied the kinetics of pPAK1-dependent endocytosis and the fusion of EGFP-Rab3a vesicles near the PM using total internal reflection fluorescence (TIRF) microscopy. IPA-3, a selective non-ATP competitive inhibitor of PAK1, inhibits endocytosis of oA{beta} peptides and Rab3a-dependent PM repair. Further, shRNA-mediated knockdown of the Rab3a gene inhibits pPAK1 and disrupts PM repair. Repair of damaged PM is a vital protective mechanism for non-proliferative cells like neurons, as disruption in PM repair leads to gradual neuronal cell death. However, there was no explicit understanding of PM repair in response to A{beta} oligomers. This study revealed the interconnected action of Rab3a and pPAK1 in PM repair in response to oA{beta}-mediated damage, and its potential correlation in AD pathogenesis.

cell biology↗

bioRxiv2024

Enhanced in vitro aggregation, but not phase separation, of TDP-43 and its C-terminal fragments generate deep-blue autofluorescence.

As misfolding and aggregation of the RNA/DNA-binding protein, TDP-43, are linked to devastating TDP-43 proteinopathies like amyotrophic lateral sclerosis (ALS), distinction of the nature of the aggregated TDP-43 species being liquid-like non-pathogenic or solid-like pathogenic is important for mechanistic elucidation and therapeutic targeting. Here, we examined if in vitro enhancement of the TDP-43 aggregation can generate or enhance intrinsic deep-blue autofluorescence (dbAF) previously reported for a few other protein aggregates and whether dbAF is emitted by all or only liquid-like or solid-like TDP-43 aggregates. Using thioflavin-T fluorescence, turbidimetry, atomic force microscopy and fluorescence microscopy of Alexa Fluor-labelled protein, we first tested the in vitro enhancement of the aggregation of the full-length TDP-43 and its two C-terminal fragments (CTFs), TDP-432C (aa: 193-414) and the TDP-43-low complexity domain (LCD) (aa: 274-414). We find that presence of metal ions, Zn2+ or Mn2+, that are also linked to ALS-associated metal dyshomeostasis, or addition of a kosmotropic anion, SO42-, enhance the in vitro solid-like aggregations of the full-length TDP-43 and TDP-432C that also concurrently enhance emission of dbAF. In contrast, Alexa fluor-633-labeled-TDP-43-LCD underwent a quick phase separation into globular structures in presence of Zn2+ ions and the phase-separated species failed to emit dbAF but upon further incubation when matured into solid-like irregular, but non-amyloid nature aggregates, it emitted dbAF. Strikingly, we find that the TDP-43 aggregates of both amyloid and non-amyloid nature, but not the oligomers or the phase-separated droplets of TDP-43, manifest dbAF. Overall, the observed in vitro enhancement of aggregation leading to concurrent enhancement of dbAF can enable a label-free easy detection and may facilitate distinguishing of potentially pathogenic versus non-pathogenic TDP-43 aggregates.

biochemistry↗

bioRxiv2023

Localization and function of multivesicular-bodies that release exosomes in islet cells: dysregulation during type-2 diabetes

Type-2 diabetes (T2D) is characterized by high blood glucose due to compromised insulin secretion from pancreatic {beta}-cells. {beta}-cells primarily comprise insulin-secreting large-dense-core-vesicles/insulin-secretory-granules (ISGs) and also multivesicular-bodies (MVBs). MVBs are vesicles of endosomal origin containing intraluminal vesicles, which upon fusion with the plasma membrane, secrete exosomes. These play a significant role in the physiology and pathology of T2D via intercellular communication. The role of MVBs and their influence on ISGs of {beta}-cells or their characterization is yet to be uncovered. In our study, we characterized the role of MVBs by comparing them to largely well-characterized ISGs in {beta}-cells. We compared the density, localization, and exocytosis of MVBs with ISGs in {beta}-cells. For this, we developed a novel probe where we exploit the efficiency of tetraspanins CD63 and CD151 to label the MVBs in {beta}-cells. We showed that the {beta}-cells have a significantly higher density of ISGs than MVBs. MVBs and ISGs are spatially localized apart within {beta}-cells. The proteins that localize with MVBs are different from the ones that localize with ISGs. Exocytosis of ISGs occurs at the periphery of the {beta}-cells and takes significantly lesser time when compared to exosome release, which is non-peripheral and takes a longer duration. Further, we also observed a significant reduction in the density of ISGs and MVBs in T2D patients islets compared to healthy controls. Studying the effect of MVBs on insulin secretion in physiological and T2D conditions has huge potential. This study provides a strong basis to open new avenues for such future studies.

cell biology↗