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Raymond Enke, Zhiyong Yang, William Hauswirth, Sanford Boye, Vince Chiodo, Donald Zack, Shannath Merbs; Conditional mutant analysis of Dnmts in murine retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):285.
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© ARVO (1962-2015); The Authors (2016-present)
We hypothesize that epigenetic mechanisms may modulate the onset and progression of disease in the retina. As a prelude to studying the role of DNA methylation in glaucoma and optic nerve degeneration, we set out to determine the expression pattern of DNA methyltransferase (Dnmt) enzymes in mature murine retinal ganglion cells (RGCs). We also use conditional Dnmt mutant lines to demonstrate RGC survival following optic nerve crush.
Eyes from adult wt mice were fixed in 4% PFA, equilibrated in a sucrose gradient, and cryopreserved. Retinal cross sections were cut and used for immunohistochemical (IHC) analysis of Dnmts. Conditional Dnmt1, Dnmt3a, and Dnmt3b mutants were created by intravitreally injecting an adeno-associated virus serotype 2 (AAV2) encoding Cre recombinase into transgenic mouse lines harboring lox P sites in each respective allele. Optic nerve crush was performed in adult mice to induce RGC degeneration. For RGC viability analysis, eyecups were fixed in 4% PFA and used for whole mount IHC quantification of Brn3 and Tuj1. Corresponding optic nerve cross sections were also stained for phosphorylated neurofilament (pNF).
IHC analysis of the adult mouse retina demonstrated that Dnmt1 and Dnmt3b are expressed in postmitotic RGCs while Dnmt3a expression is limited to amacrine cells in the ganglion cell layer (GCL) of the retina. Intravitreal delivery of AAV2-Cre results in efficient transduction of the mouse GCL cells and proves to be an effective technique for producing conditional Dnmt mutant mice. Dnmt1 mutant RGCs were partially protected from degeneration following optic nerve crush. Surviving Dnmt1 mutant RGCs also displayed more persistent expression of Brn3 following optic nerve crush compared to controls.
Expression of Dnmt1 and Dnmt3b in postmitotic RGCs suggests that DNA methylation may be involved in RGC homeostasis. Our current experiments further address this question by assaying the role of Dnmts in RGC survival following optic nerve injury. A conditional mutation in Dnmt1 imparts partial protection against RGC degeneration following optic nerve crush. This finding indicates that DNA methylation may be an important epigenetic signal in response to optic neuropathy. Ongoing analysis will determine if Dnmt3b conditional mutants also have improved RGC viability following optic nerve crush.
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