Aquino, Erika; (2018) Downregulation of glial fibrillary acidic protein (GFAP) by TNF-α - implications for the neurogenic ability of Müller glia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Müller glial cells can regenerate the retina in zebrafish throughout life. However, in the damaged adult human retina, upregulation of inflammatory cytokines, including TNF-α, leads to Müller cell gliosis, a hallmark of which is an increase in intermediate filament glial fibrillary acid protein (GFAP) production. However, a subset of these human cells has stem cell characteristics in vitro. This study investigated the role that inflammatory cytokines may play in regulating Müller cell gliosis-associated proteins and the implications this could have on the neurogenic ability of Müller glia in vitro. As determined by gene and protein analysis, culture of the human Müller glial cell line MIO-M1 in the presence of TNF-α, causes downregulation of GFAP expression. Through upregulation of TNF-receptor2 and downstream activation of the NFκB signalling pathway, a cell survival signal is initiated. MIO-M1 cells co-cultured with TNF-α and factors known to induce rod photoreceptor precursor differentiation, showed increased expression of the photoreceptor marker NR2E3. These observations suggest that TNF-α may not inhibit the neurogenic ability of these cells. A retroviral transfection method was developed to overexpress GFAP in MIO-M1 cells using molecular cloning techniques. Overexpression of GFAP resulted in no phenotypic changes as Müller cells maintained their stem cell characteristics. Culturing these transfected cells with TNF-α revealed differential transcriptional regulation of endogenous and exogenous GFAP. This indicates the importance of the GFAP promoter and transcriptional response elements in responding to TNF-α during gliosis. In conclusion, the present study has identified the downregulation of GFAP expression by TNF-α in Müller glial cells as a target that could be further explored to control scarring of the human retina. These observations pave the way for further investigations to promote endogenous regeneration of the adult human retina by Müller glia.

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