Biology subjects

Shyam, R.

Publications and source records attributed to Shyam, R..

5 recordsLinked to original sources

bioRxiv2025

Decreased substrate stiffness leads to mitochondrial dysfunctions and Endothelial to Mesenchymal transition through Focal Adhesion Kinase activity in corneal endothelial cells

PurposeFuchs Endothelial Corneal Dystrophy (FECD), a degenerative corneal disorder, is marked by the thickening of Descemets membrane and a progressive loss of corneal endothelial cells, ultimately leading to vision loss. A feature associated with the disease is the reduced stiffness of Descemets membrane. However, the effects of this change in Descemets membrane, on corneal endothelial cell health are not well understood. To explore this, we used in vitro, in vivo, and ex vivo studies to investigate how changes in substrate stiffness and the signaling pathways associated with these changes influence corneal endothelial functions. MethodsFor in-vitro studies, we cultured bovine corneal endothelial cells for 96 hours on stiff (32 kPa) and soft (8 kPa) substrate CytoSoft plates. By using Jess immunoassay and traditional western blotting, we evaluated changes in integrin signaling components, endothelial-to-mesenchymal transition, apoptosis, autophagy, and ubiquitin-proteasome pathway markers. Mitochondrial health and mitochondrial superoxide levels were assessed using commercial kits. We assessed the protein levels of the above-mentioned markers in the Col8a2Q455K/Q455K FECD mouse model. To evaluate whether FAK signaling contributes to the FECD pathogenesis, we injected the Col8a2Q455K/Q455K mice with an FAK inhibitor and assessed the corneal phenotypes. ResultsWe observed increased levels of phosphorylated FAK, integrins 4 and 5 in bovine corneal endothelial cells cultured on soft substrate. We also found upregulated endothelial-to-mesenchymal transition (EndMT) markers, mitochondrial dysfunction, and apoptosis in cells grown on soft substrate. In the Col8a2Q455K/Q455K mouse model of FECD, there was increased pFAK Y397 levels coincident with the onset of phenotypes. Intraperitoneal injections of a pFAK inhibitor improved antioxidant protein expression and decreased EndMT; however, it did not improve FECD-associated disease progression. ConclusionIn this study, we explored how changes in the physical characteristics of the Descemets membrane impact corneal endothelial cell health. While we discovered activation of Focal adhesion kinase as a result of stiffness changes, its inhibition alone was insufficient to improve cell health in an FECD mouse model.

molecular biology↗

bioRxiv2025

Proteasomal Dysfunction results in ER stress, Endo MT, oxidative stress, and apoptotic cell death resulting in Fuchs Corneal Endothelial Dystrophy like features in mice

Fuchs Endothelial Corneal Dystrophy (FECD) is the irreversible degeneration of the corneal endothelium. The only treatment is corneal transplantation. To develop therapies for FECD, identifying the cellular causes for the onset and progression of the disease is crucial. While cell culture studies associate elevated oxidative stress, endoplasmic reticulum stress, endothelial-to-mesenchymal transition, and apoptosis with FECD, the causes behind the disease onset remain elusive. Guttae or Descemets membrane deposits are the earliest phenotype associated with FECD and are composed of unfolded proteins. Therefore, we asked if aberrant protein clearance pathways could be responsible for disease pathogenesis. We discovered a dysfunctional ubiquitin-proteasome pathway in a FECD mouse model and end-stage FECD patient samples. Inhibiting the ubiquitin-proteasome pathway in primary corneal endothelial cells resulted in the cellular dysfunctions associated with FECD. Finally, injecting healthy wild-type mice with proteasomal inhibitors resulted in all the major phenotypes associated with FECD, including corneal edema, guttae, and corneal endothelial cell loss. Therefore, this study strongly connects proteasomal dysfunction in FECD onset and progression.

cell biology↗

bioRxiv2023

Characterization of a novel mouse model for Fuchs Endothelial Corneal Dystrophy

PurposeFuchs Endothelial Corneal Dystrophy (FECD) is a progressive blinding disorder prevalent in 4% of Americans over 40. Corneal transplantation is the standard treatment. Animal models with partial FECD features exist, but a model encompassing all the major disease characteristics is desirable to improve the understanding of the pathogenesis and to identify signaling pathways involved in the disease onset and progression. Such an animal model can be helpful to develop intervention strategies. Here, we developed a mouse model that recapitulates all the features of FECD. MethodLoss of function mutations in Slc4a11 and a knock-in mutation in Col8a2 (Q455K) are implicated in FECD. Mice with Slc4a11 and Col8a2 mutations in C57BL/6J background were crossed to generate double mutant mice at F2 generation. At five weeks of age, a subset of the animals were fed tamoxifen-enriched chow or standard chow for two weeks, followed by standard chow. Corneal thickness, endothelial cell density, and guttae were measured at 5 (baseline) and 16 weeks of age. Corneas collected from mice at 16 weeks were stained for tight and adherens junctions, and reactive oxygen species. A lactate assay was performed to evaluate the endothelial pump function. ResultsThe double mutant tamoxifen-fed mice showed increased corneal thickness, decreased endothelial cell density, presence of guttae, and elevated stromal lactate levels. The endothelial cells showed altered morphology with disrupted adherens junctions and elevated ROS. ConclusionOverall, this mouse model recapitulates all the important phenotypic features associated with FECD.

cell biology↗

bioRxiv2021

Carbon-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics.

The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters. Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.

immunology↗

bioRxiv2020

MitoROS due to loss of Slc4a11 in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

Recent studies from Slc4a11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the Slc4a11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated Slc4a11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of Slc4a11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.

cell biology↗