Biology subjects

Gupta, S. J.

Publications and source records attributed to Gupta, S. J..

3 recordsLinked to original sources

bioRxiv2025

Survival of Transplanted Retinal Ganglion Cell in Human Donor Eyes under Elevated Pressure

Glaucoma is a group of optic neuropathies characterized by visual field loss, classically due to increased intraocular pressure (IOP) and retinal ganglion cell (RGC) degeneration. Current treatment options reduce IOP, but progressive RGC degeneration persists. The ability to reprogram de novo RGCs from human corneal keratocytes provides a valuable tool to potentially restore vision in patients with late-stage disease when most RGCs are irreversibly damaged. We investigate the survival of these human induced pluripotent stem cell (hiPSC) derived RGCs after culturing them in human donor eyes under conditions of elevated and normal IOP using the pressurized ocular translaminar autonomous system (TAS) chamber. The hiPSCs were generated by reprogramming human donor keratocytes using Sendai viral vectors with Yamanaka factors. The hiPSCs were then differentiated into retinal organoids (ROs) from which RGCs were obtained. The RGCs were transduced with AAV2-CBA-EGFP (Adeno-Associated Virus serotype 2- Chicken Beta Actin- Enhanced Green Fluorescent Protein) and successfully transplanted into donor human eyes obtained from individuals having non-ocular history. They were pressurized for 5-7 days, with the left eye maintained at normal IOP and right eye at high IOP. Viability was measured by expression levels of pro-survival pathways via qRT-PCR, immunohistochemistry staining, and electroretinography (ERG) for retinal function. After RGC transplantation, increased expression of pro-survival and decreased inflammatory and apoptotic markers were identified in normal IOP conditions compared to high IOP. In conclusion, survival of RGCs was more conducive under normal IOP conditions with significantly increased degeneration observed at high IOP. Thus, suggesting that elevated IOP could potentially create a microenvironment that would significantly inhibit successful transplantation of de novo RGCs.

neuroscience↗

bioRxiv2022

Pleiotrophin signals through ALK receptor to enhance growth of neurons in the presence of inhibitory CSPGs

Chondroitin sulfate proteoglycans (CSPGs), one of the major extracellular matrix components of the glial scar that surrounds central nervous system (CNS) injuries, are known to inhibit the regeneration of neurons. This study investigated whether pleiotrophin (PTN), a growth factor upregulated during early CNS development, can overcome the inhibition mediated by CSPGs and promote the neurite outgrowth of neurons in vitro. The data showed that a CSPG matrix inhibited the outgrowth of neurites in primary cortical neuron cultures compared to a control matrix. PTN elicited a dose dependent increase in the neurite outgrowth even in the presence of the growth inhibitory CSPG matrix, with optimal growth at 15 ng mL-1 of PTN (114.8% of neuronal outgrowth relative to laminin control). The growth promoting effect of PTN was blocked by inhibition of the receptor anaplastic lymphoma kinase (ALK) by alectinib in a dose dependent manner. Neurite outgrowth in the presence of this CSPG matrix was induced by activation of the protein kinase B (AKT) pathway, a key downstream mediator of ALK activation. This study identified PTN as a dose-dependent regulator of neurite outgrowth in primary cortical neurons cultured in the presence of a CSPG matrix, and identified ALK activation as a key driver of PTN-induced growth. Summary statementFunction in the central nervous system (CNS) is attributed to the complex interactions of neurons and glia. These cells are anchored in extracellular matrix (ECM) which constitutes about 10% - 20% of brain volume. Cells in the brain produce different components of the ECM in brain including chondroitin sulfate proteoglycans (CSPGs). After a nervous system injury, glial cells produce excess CSPGs that restrict the regeneration of neurons, thus limiting functional recovery. This study examines the role of the endogenous growth factor pleiotrophin (PTN) in driving the growth of neurons even in the presence of inhibitory CSPGs, and anaplastic lymphoma kinase (ALK) receptor as a key mediator by which PTN potentiates growth.

neuroscience↗

bioRxiv2020

Inflammatory cytokine profile and plasticity of brain and spinal microglia in response to ATP and glutamate

Microglia are the primary cells in the central nervous system that identify and respond to injury or damage. Such a perturbation in the nervous system induces the release of molecules including ATP and glutamate that act as damage-associated molecular patterns (DAMPs). DAMPs are detected by microglia, which then regulate the inflammatory response in a manner sensitive to their surrounding environment. The available data indicates that ATP and glutamate can induce the release of pro inflammatory factors TNF (tumor necrosis factor), IL-1{beta} (interleukin 1 beta) and NO (nitric oxide) from microglia. However, non-physiological concentrations of ATP and glutamate were often used to derive these insights. Here, we have compared the response of spinal cord microglia (SM) relative to brain microglia (BM) using physiologically relevant concentrations of glutamate and ATP that mimic injured conditions in the central nervous system. The data show that ATP and glutamate are not significant modulators of the release of cytokines from either BM or SM. Consistent with previous studies, spinal microglia exhibited a general trend towards reduced release of inflammatory cytokines relative to brain-derived microglia. Moreover, we demonstrate that the responses of microglia to these DAMPs can be altered by modifying the biochemical milieu in their surrounding environment. Preconditioning brain derived microglia with media from spinal cord derived mixed glial cultures shifted their release of IL-{beta}, IL-6 and IL-10 to a less inflammatory phenotype consistent with a spinal microglia.

neuroscience↗