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Ling Zhang, Zhiyong Yang, Cynthia Berlinicke, Katherine L Mitchell, Derek S Welsbie, Noriko Esumi, Donald J Zack; RIT2, A Neuron-Specific Small GTPase, Modulates Neurite Outgrowth in Primary Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2444.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) axon injury and cell death are defining features of glaucoma. Within the retina, RIT2, a member of the Ras small GTPase family that has been implicated in neurite growth, is predominantly expressed in RGCs. In this study we explored RIT2’s potential function in regulating RGC neurite outgrowth.
Primary mouse RGCs were purified by immunopanning using a Thy1.2 antibody and postnatal day 0-3 C57BL/6 mice. Loss and gain of RIT2 function was studied by treating RGCs with either Rit2 siRNA or adenoviral vectors expressing 1) Rit2 or 2) Rit2 V12 (constitutively active mutant form of Rit2). Treated RGCs were cultured and effects on survival and neurite morphology were assayed at different time points using an automated fluorescent image system (Cellomics VTI). Immunohistochemical assays in which RGC neurites were stained with Map2b, Tau and RIT2 antibodies were performed to determine whether RIT2 was expressed in axons or dendrites. Immunoblotting of RGC lysates 24, 48, and 72 hours after isolation was done using antibodies against Rit2, JNK, p-JNK, p38, p-p38, ERK, and p-ERK.
RGC RIT2 protein levels began to decrease 24 hours after RIT2 knockdown by siRNA. By 72hrs in culture, Rit2 siRNA treated RGCs had shorter neurites and the neurites that were present had fewer branches compared to RGCs treated with non- targeting siRNA (p<0.05). Rit2 over-activity was induced in the RGC cultures using Rit2 V12 adenovirus. Treated RGCs showed increased protein levels of RIT2 after 24 hours in culture, and increased neurite length and branching points after 72 hours in culture (p value<0.05). Western blot results showed that JNK, p-JNK, p38, p-p38, ERK, and p-ERK expression did not differ significantly between the RIT2 knockdown and control groups.
In this study, we explored the function of RIT2 in modulating neurite outgrowth in primary mouse RGCs. We confirmed that RIT2 is expressed in RGCs (as shown in our prior study), and demonstrated its role in modulating neurite growth in vitro. Over-expression of RIT2 promotes neurite growth, increasing both neurite length and neurite branching. Knockdown of RIT2 inhibits neurite growth, decreasing neurite length and branching. However, at the cellular level, RIT2 does not appear to affect total neurite number per cell.
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