Influence of glial cells on postnatal differentiation of rat retinal ganglion cells
GÖRITZ, Christian (2005) Influence of glial cells on postnatal differentiation of rat retinal ganglion cells. Thèses de doctorat, Université Louis Pasteur.
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The interplay of neurons and glia is highly relevant for many aspects of nervous system development. One important phase is the formation of synapses. Previous studies had shown that soluble glia-derived factors enhance the formation and efficacy of synapses in cultured retinal ganglion cells (RGCs) (Nägler et al., 2001), and identified one of these factors as cholesterol (Mauch et al., 2001). The aim of my project was to investigate how cholesterol supports synapse formation and function and whether other glia derived factors are involved in synaptogenesis in cultured RGCs. In the second part, I was searching for neuronal target genes involved in glia-dependent postnatal differentiation of rat RGCs using GeneChip expression analysis. Searching for the mechanism of glia- and cholesterol-induced synapse formation, I could show by immunostaining with pre- and postsynaptic markers that glia conditioned medium (GCM) and cholesterol enhanced the number of synapses slowly within several days of treatment. Cholesterol induced a smaller but significant increase in synapse number than GCM. Filipin staining, an indicator of the cholesterol content, revealed a tenfold enhancement in neuritic cholesterol content within 72 hours of GCM and cholesterol treatment but with a strikingly different time course. This indicated that the increase in cholesterol content does not determine the rate of synaptogenesis. Immunostaining of dendrites using a MAP2 antibody revealed that GCM and cholesterol induced dendrite differentiation with remarkably similar time course as the increase in synapse number. This indicated that dendrite differentiation, which involved a redistribution of MAP2 and GluR2/3 from the somata to dendrites, was the rate limiting step in glia-induced synapto-genesis. Interestingly, I also found that neighboring cells impede dendrite differentiation. Cholesterol was indispensable for dendrite differentiation but less efficient than GCM, which may explain the lower number of synapses in cholesterol- compared to GCM-treated RGCs. I found that glia-derived laminin containing the γ1 chain together with cholesterol promoted dendrite differentiation to the same extent then GCM, suggesting that laminin act as dendrite promoting signal for RGCs. Furthermore, using electrophysiological recordings, I showed by removal of GCM and replacement with cholesterol, that cholesterol is essential for continuous synatogenesis and for the stability of evoked transmitter release. To detect neuronal genes whoses expression is regulated by glia, I performed GeneChip analysis analysis of GCM- and cholesterol-treated RGCs. My experiments revealed several genes whose level of expression changed under the influence of GCM and cholesterol. Interestingly, there was very little overlap between the two groups except for genes involved in cholesterol biosynthesis and homeostasis. To test if the neuronal cholesterol synthesis was influnced by these changes I labeled new synthesized lipids, using a radioactive precursor. I observed that GCM and cholesterol treatment clearly down regulated the cholesterol synthesis in neurons, confirming this expression result. GCM highly up regulated the genes of matrix Gla protein and heme oxygenase 1, which could be confirmed on protein level. Immunostaining revealed that both proteins were located in the somatodendritic compartment. Together, these results revealed new roles of cholesterol and laminin in neuronal differentiation and underline the importance of neuron-glia interactions during brain development.
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