Understanding the cellular and molecular mechanism of MHV-A59 induced altered Connexin 43 (Cx43) trafficking in primary astrocytes

Bose, Abhishek (2015) Understanding the cellular and molecular mechanism of MHV-A59 induced altered Connexin 43 (Cx43) trafficking in primary astrocytes. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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MHV-A59 is a neurotrophic,demyelinating strain of mouse hepatitis virus( MHV) that causes hepatitis,meningoencephalitis in acute phase and demyelination,axonal loss in chronic phase,and serves as a viral model of human central nervous system demyelinating disease multiple schlerosis(MS).MHV is known to infect astrocytes,neuron,oligodendrocytes and microglia in brain and microglial activation in coordination with oligodendrocyte damage could lead to myelin stripping from infected neurons.But the role of astrocyte infection in MHV induced neuroinflammation is not clear.Previous studies of the lab has shown that MHV-A59 infection in astrocytes cause downregulation of Cx43 in mRNA and protein level with a significant decrease in the cell membrane. The project is focussed to understand the cellular and molecular mechanism behind the alteration of membrane Cx43 level.As a first step I have characterised and standardised the isolation and enrichment of day 0 neonatal mice brain astrocytes.Cx43 expression on the cell surface of astrocyte was observed by immunofluroscence microscopy.Primary neonatal astrocytes were consecutively infected with MHV-A59 at 2 and 5 M.O.I (Multiplicity of infection) and Cx43 was observed to be localised in the perinuclear region of the infected astrocytes most likely in the ER.Double labelling immunostaining Cx43(red) and Calnexin-ER marker(green) was performed and data demonstrated that Cx43 could be primarily retained in the ER compartment.These findings lead to the hypothesis that altered localisation of Cx43 could be due to ER stress caused by MHV replication. I am also interested to explore whether altered trafficking of Cx43 could be due to disruption of microtubule as Cx43 is known to traffick along microtubule of cell.On the other hand MHV uses microtubule as a conduit to spread from one cell to another.We have studied the effect of disruption of microtubule by colchicine,(a potent microtubule disruptor) in a dose dependant manner.Cx43 internalisation increased with increased dose of colchicine which signifies that Cx43 in astrocytes could follow the trajectory of microtubule to go to the cell surface from ER.Further studies are ongoing to understand the ER stress mediated altered trafficking of Cx43 in astrocytes and its implication in MHV induced neurobiology of diseases.Simultaneously we are looking for microtubule based cellular trafficking of Cx43 in astrocytes.

Item Type: Thesis (Masters)
Additional Information: Supervisor: Dr. Jayasri Das Sarma
Uncontrolled Keywords: Altered Connexin 43 (Cx43) ; Altered Connexin 43 (Cx43) Trafficking; Cellular Mechanism; MHV-A59; Molecular Mechanism; Primary Astrocytes
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Department of Biological Sciences
Depositing User: IISER Kolkata Librarian
Date Deposited: 15 Jun 2016 07:57
Last Modified: 15 Jun 2016 07:57
URI: http://eprints.iiserkol.ac.in/id/eprint/290

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