Synapses are the basic functional units in Central Nervous System (CNS). The synapses enable neuron-to-neuron communication via releasing and uptake of neurotransmitters. The synapse formation is modulated by specialized CNS cell type, glia. The glia modulates neuronal function via synapse formation, modification and elimination and, the glia even affects reorganization of neuronal circuit. Understanding cellular and molecular interactions of these glial cells is of great importance because they offer insight into CNS recovery and regenerative mechanisms.
This research proposal aims to study the role of retinal glia, Muller glia. Muller glia is a specialized glia in retina that is a part of CNS. Previous studies in brain have shown that astrocytes, …show more content…
The experimental procedures of this research will be carried out in two stages; the first stage explores genetic methodologies, and the second stage studies biochemical and computational analysis of the outcomes. The genetic methodologies include designing gene targeting vector using CRISPR-Cas9 system to knock-out MERTK. Customized small guide RNA (sgRNA) will be cloned into the CRISPR vector, and its activity will be tested in rat astrocytes. The biochemical methodologies involve comparing synaptogenic activity of rat astrocytes with MERTK knockout. As a control, pharmacological agent gabapentin that completely blocks TSP binding to its synaptogenic receptor will be utilized. Expression and secretion of synaptogenic proteins TSPs and Hevin will be monitored by biochemical and immunohistochemical methods. Since gabapentin strongly blocks TSP-induced synapse formation within its therapeutic concentration, it is possible that inhibition of excitatory synapse formation is an important mode of its therapeutic action in epilepsy and pain. We expect that astrocytes will have reduced excitory synapse formation with MERTK knockout, similar to astrocyte cultured media in presence of gabapentin. Understanding the correlation between the knockout MERTK-expressing gene and gabapentin in astrocytes can also help us
This research proposal aims to study the role of retinal glia, Muller glia. Muller glia is a specialized glia in retina that is a part of CNS. Previous studies in brain have shown that astrocytes, …show more content…
The experimental procedures of this research will be carried out in two stages; the first stage explores genetic methodologies, and the second stage studies biochemical and computational analysis of the outcomes. The genetic methodologies include designing gene targeting vector using CRISPR-Cas9 system to knock-out MERTK. Customized small guide RNA (sgRNA) will be cloned into the CRISPR vector, and its activity will be tested in rat astrocytes. The biochemical methodologies involve comparing synaptogenic activity of rat astrocytes with MERTK knockout. As a control, pharmacological agent gabapentin that completely blocks TSP binding to its synaptogenic receptor will be utilized. Expression and secretion of synaptogenic proteins TSPs and Hevin will be monitored by biochemical and immunohistochemical methods. Since gabapentin strongly blocks TSP-induced synapse formation within its therapeutic concentration, it is possible that inhibition of excitatory synapse formation is an important mode of its therapeutic action in epilepsy and pain. We expect that astrocytes will have reduced excitory synapse formation with MERTK knockout, similar to astrocyte cultured media in presence of gabapentin. Understanding the correlation between the knockout MERTK-expressing gene and gabapentin in astrocytes can also help us