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Helen Erickson, Pooja Teotia, Iqbal Ahmad, Matthew J Van Hook, Nam Nguyen; Unlocking the neurogenic potential of mammalian Müller glia by small molecules. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2495.
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© ARVO (1962-2015); The Authors (2016-present)
Müller glia (MG) are the primary support cells in the vertebrate retina, regulating homeostasis in one of the most metabolically active tissues. In lower vertebrates such as fish, they respond to injury by proliferating and reprogramming to regenerate retinal neurons. In mammals, MG may also react to injury by proliferating, but they fail to initiate regeneration efficiently. Several genes and pathways have emerged as candidates for unlocking the neurogenic potential of mammalian MG. For example, we have observed that molecular axes defined by Lin28, Ascl1, REST, and miRNAs such as miR-124, miR-29, and miR-9 may regulate the transition of MG along the neuronal lineage. These observations are based on perturbation of expression by exogenous genes, precluding the approach from clinical application. Here, we have examined the influence of small molecules on neurogenic conversion of MG.
MG were enriched (eMG) from rat retina with a hypoxia-based method and maintained as a primary culture. The enrichment of the culture was ensured by complete absence of microglia, astrocytes, and retinal neurons. eMG were exposed to various small molecules (for example I-BET 151, ISX-9, Forskolin, Y-27632) in different combinations for 12 days and culture was examined temporally for changes in morphology, immunoreactivities, gene expression, and physiology. Temporal RNA seq analysis was carried out to examine the neurogenic effects of small molecules on global gene expression.
eMG acquired neuronal morphology by 2 days in vitro (DIV) displaying long processes and small refractile cell bodies. By 6DIV these cells were coexpressing immunoreactivities corresponding to β-TUBULIN and MAP2/TAU. Their processes became longer with time. Temporal examination of select gene expression revealed a decrease in levels of transcripts corresponding to GLAST and increase in those of ASCL1/ MAP2/β-TUBULIN/KV1.5. Temporal RNAseq analysis demonstrated a global shift in the expression of glial-specific to neural-specific genes. These cells displayed outwardly rectifying sodium currents, albeit small, one of the functional attributes of neurons.
Our results demonstrate that the neurogenic properties of MG are labile to culture conditions and therefore can be exposed by altering the niche through specific small molecules.
This is a 2020 ARVO Annual Meeting abstract.
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