Biology subjects

Sivaprasadarao, A.

Publications and source records attributed to Sivaprasadarao, A..

3 recordsLinked to original sources

bioRxiv2024

A distinct signalling circuit upregulates cytotoxic ROS production in a cellular model of Parkinsons disease: key roles for TRPM2, Zn2+ and complex III

Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinsons disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP+ (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (i) MPP+ stimulates ROS production by raising cytoplasmic Ca2+ levels, rather than acting directly on mitochondria. (ii) To raise the Ca2+, MPP+ co-stimulates NADPH oxidase-2 (NOX2) and the TRPM2 (Transient Receptor Potential Melastatin2) channel that form a positive feedback loop to support each others function. (iii) Ca2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn2+. (iv) Zn2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca2+ and Zn2+, and prevention of electron escape from complexes -all abolished the ability of MPP+ to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP+, while Zn2+ ionophore replicated the effects of both MPP+ and Ca2+. Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca 2+, Zn2+, and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases.

cell biology↗

bioRxiv2020

Protein kinase Cϵ activation induces EHD-dependent degradation and downregulation of KATP channels: Implications for glucose stimulated insulin secretion

Pancreatic {beta}-cells have the unique ability to couple glucose metabolism to insulin secretion. This capacity is generally attributed to the ability of ATP to inhibit KATP channels, and the consequent {beta}-cell membrane depolarization and excitation. This notion has recently been challenged by a study which demonstrated that high glucose (HG) downregulates the cell surface KATP channels, and thereby leads to {beta}-cell depolarisation and excitation. The authors attributed the downregulation to HG-induced protein kinase C (PKC) activation and the consequent increase in channel endocytosis. This interpretation, however, is inconsistent with our previous findings that PKC activation does not affect endocytosis. To address this controversy, we revisited the problem: we have used cell biological and electrophysiological approaches combined with the pharmacological activator of PKC, PMA (phorbol 12-myristate 13-acetate). We first confirm that PKC does not play a role in KATP channel endocytosis; instead, it downregulates the channel by promoting lysosomal degradation coupled with reduced recycling. We then show that (i) mutation of the dileucine motif (355LL356) in the C-terminal domain of the Kir6.2 subunit of the KATP channel complex prevents lysosomal degradation; (ii) lysosomal targeting is mediated by the EHD (Eps15 homology domain- containing) proteins; and (iii) the PKC isoform responsible for channel degradation is PKC{varepsilon}. Taken together with the published data, we suggest that HG promotes {beta}-cell excitability via two mechanisms: ATP-dependent channel inhibition and ATP-independent, PKC{varepsilon}-dependent channel degradation. The results likely have implications for glucose induced biphasic insulin secretion.

cell biology↗

bioRxiv2018

Piezo1 channel agonist mimics high glucose as a stimulator of insulin release

ObjectiveGlucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic {beta}-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release.\n\nMethodsTwo rat {beta}-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively.\n\nResultsPiezo1 mRNA was detected in both {beta}-cell lines and mouse islets. Yoda1 evoked Ca2+ entry which was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from {beta}-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Pre-treatment with ruthenium red significantly reduced hypotonicity induced insulin release from {beta}-cells and pancreatic islets.\n\nConclusionThe data show that Piezo1 channel agonist induces insulin release from {beta}-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have a potential to be used as enhancers of insulin release.

cell biology↗