Biology subjects

Bharathi, V.

Publications and source records attributed to Bharathi, V..

3 recordsLinked to original sources

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↗

bioRxiv2020

Role of CNC1 gene in TDP-43 aggregation-induced oxidative stress-mediated cell death in S. cerevisiae model of ALS.

TDP-43 is a multi-functional ribonucleoprotein that is also found deposited as hyper-phosphorylated and ubiquitinated TDP-43 inclusions in the brain and spinal cord of the patients of the motor neuron diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Till date, how the cell death ensues is not fully deciphered although several molecular mechanisms of the TDP-43 toxicity such as impairments of endocytosis and chromatin remodelling, mis-regulations of autophagy and proteasome function, mis-localization to the mitochondria and generation of oxidative stress etc., have been proposed. A predominantly nuclear protein, Cyclin C, can regulate the oxidative stress response by affecting the transcription of stress response genes and also by translocation to the cytoplasm for the activation of the mitochondrial fragmentation-dependent cell death pathway. Using the well-established yeast model of TDP-43 aggregation and toxicity, we examined here whether upon TDP-43 aggregation, the cell survival depends on the presence of the CNC1 gene that encodes Cyclin C protein or other genes that encode proteins that function in conjunction with Cyclin C, such as the DNM1, FIS1 and MED13 genes. We found that the TDP-43 toxicity is significantly reduced in the yeast deleted for the CNC1 or DNM1 genes. Importantly, the rescue of TDP-43 toxicity in these yeast deletion backgrounds required the presence of functional mitochondria. Also, the deletion of YBH3 gene, which encodes for a protein involved in the mitochondria-dependent apoptosis, also reduced the TDP-43 toxicity. Furthermore, Cyclin C-YFP was observed to localize from the nucleus to the cytoplasm in response to the TDP-43 co-expression. Also, this cytoplasmic localization of Cyclin C was prevented by the addition of an anti-oxidant molecule, N-acetyl-cysteine. Taken together, our data suggest that Cyclin C, Dnm1 and Ybh3 proteins are important in mediating the TDP-43-induced oxidative stress-mediated cell death in the S. cerevisiae model.

genetics↗

bioRxiv2019

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4.

TDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc. Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43s aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-432C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43s aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important molecule for further investigations on TDP-43s anti-aggregation effects with relevance to the ALS pathogenesis.

bioinformatics↗