Biology subjects

Saravanan, P.

Publications and source records attributed to Saravanan, P..

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↗

bioRxiv2023

In silico and in vitro studies suggest epigallocatechin gallate (EGCG), a polyphenol in green tea, can bind and modulate the aggregation and cytotoxicity of the full-length TDP-43 protein implicated in TDP-43 proteinopathies.

Misfolding and aggregation of TDP-43 protein are implicated in several proteinopathies like ALS and FTLD. Extracellular TDP-43 is also proposed to propagate in a prion-like pathogenic manner to the neighbouring cells. Here, using turbidity and sedimentation assay, we show that a polyphenol in green tea, epigallocatechin gallate (EGCG), can inhibit the in vitro aggregation of the full-length TDP-43 protein. Furthermore, Alexa-Fluor-labelled TDP-43 protein failed to show aggregates in the presence of EGCG in fluorescence microscopy. Also, AFM imaging revealed that EGCG co-incubation with TDP-43 allows formation of only small oligomers in contrast to the larger TDP-43 aggregates formed otherwise. A physical binding of EGCG with TDP-43 was observed using triphenyl tetrazolium chloride (TTC) staining and isothermal titration calorimetry (ITC). ITC also revealed a high-affinity binding site for EGCG on TDP-43 with a Kd value of 7.8 {micro}M and a binding free energy of -6.9 kcal/mol. Furthermore, in silico molecular docking and molecular dynamic simulation (MDS) studies using different available structures of the N-terminal, RRM1-2 and C-terminal domains of TDP-43, predicted a preferable and stable binding of EGCG to the structure of the aggregation prone C-terminal domain (CTD) (PDB ID:7KWZ). Also, EGCG complexed with CTD of TDP-43 yielded a negative {Delta}G value of -20.29 kcal/mol using MM-PBSA analysis of the MDS data thereby further suggesting a stable complex formation. Also, in MDS, EGCG interacted with the amino acids Phe-313 and Ala-341 of TDP-43, which were previously projected to be important for the recruitment of monomers for the amyloid formation by CTD, thereby suggesting a possible mechanism of EGCGs inhibition of the TDP-43 aggregation. Notably, while the in vitro-made aggregates of full-length TDP-43 caused mild cytotoxicity to the HEK293 cells, the small oligomers of TDP-43 formed in presence of EGCG did not. In totality, EGCG can in vitro interact with TDP-43 and inhibit its aggregation, possibly via interaction with the amyloidogenic domain, thereby preventing it from assuming cytotoxic conformations. As EGCG is a natural molecule, it could be relevant to the therapeutic quest against the TDP-43 proteinopathies.

biochemistry↗

bioRxiv2023

MultipleXed Population Selection and Enrichment single nucleus RNA sequencing (XPoSE-seq) enables sample identity retention during transcriptional profiling of rare populations

Single nucleus RNA-sequencing is critical in deciphering tissue heterogeneity and identifying rare populations. However, current high throughput techniques are not optimized for rare target populations and require tradeoffs in design due to feasibility. We provide a novel snRNA pipeline, MulipleXed Population Selection and Enrichment snRNA-sequencing (XPoSE-seq), to enable targeted snRNA-seq experiments and in-depth transcriptomic characterization of rare target populations while retaining individual sample identity.

neuroscience↗