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N. J. Kunzevitzky, K. T. Willeford, J. L. Goldberg; Characterizing Developing Amacrine Cells: Neurite Extension, Survival and Gene Expression Profile. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2146.
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© ARVO (1962-2015); The Authors (2016-present)
Amacrine cells are interneurons essential for visual function, as they modulate retinal signaling onto retinal ganglion cells (RGCs). In the mammalian retina, they can be classified into more than 30 subtypes by morphology, physiology and the expression of specific markers. Interestingly, they do not degenerate after RGC death, and, although they are capable of growing long neurites, they fail to extend differentially polarized axons and dendrites. Because little is known about amacrine cell biology, here we ask: why do they fail to grow axons like RGCs? What is the molecular basis for their resistance to degeneration? What are the molecular signals necessary for amacrine cell survival?
To study how amacrine cells and RGCs extend neurites, cells were acutely purified and at 3DIV, neurites were measured and the data were processed using principal component analysis. To understand the differences in polarity between amacrine cells and RGCs, we purified amacrine cells and processed them for Genechip analysis. We classified the probes by Gene Ontology and compared the gene expression profiles of amacrines and RGCs. To study what molecular pathways might be necessary for survival, we purified amacrine cells and cultured them in a serum-free media with or without trophic factors and survival pathway inhibitors. We quantified survival at 3DIV and we performed Western Blot.
We found that, as in vivo, amacrine cells and RGCs in vitro differ widely in their intrinsic ability to extend neurites. The majority of amacrine cells (93%) had a small average neurite segment length (<30um), whereas RGCs grew longer neurites (82% had average segment length <120um). Analysis of the expression levels of "polarity genes" during development of amacrine cells and RGCs led to the identification of a subset of genes that are differentially expressed in these cell types. Finally, we found that amacrine cell survival in vitro is independent of cell density and the presence of exogenous trophic factors, but it requires Erk activation via MEK1/2 and AKT signaling.
We have begun to identify the differences in trophic requirements for survival, and in neurite extension between amacrine cells and RGCs in vitro, and we are currently trying to understand what makes amacrine cells resistant to degeneration. Switching RGCs to an "amacrine cell-like" state could help preserve their survival in neurodegenerative diseases like glaucoma. Conversely, increasing structural plasticity of amacrine cells could help alleviate the loss of RGCs.
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