Regulation of successful optic nerve regeneration by the mevalonate/cholesterol pathway

PROJECT SUMMARY Loss of vision due to optic neuropathies, like glaucoma, is a common cause of blindness in the United States. Unfortunately, these conditions are usually permanent because the central nervous system lacks the ability to regenerate damaged axons. Mammalian models of optic nerve (ON) injury recapitulate the pathology seen in patients making it difficult to understand what is needed for successful regeneration. In contrast, teleost fish, such as the zebrafish, can successfully regenerate damage to the ON and recover lost vision. We are using this organism to study the mechanisms of successful ON regeneration with the hopes of translating these findings into novel therapeutics to improve regeneration in mammalian disease models and patients. In a transcriptome- wide study of retinal ganglion cells (RGCs) during zebrafish ON regeneration we identified the mevalonate and cholesterol pathways as up regulated during this process. Our preliminary data suggests the master transcriptional regulator of these pathways, srebf2, is necessary for successful ON regeneration. We hypothesize that srebf2 mediates ON regeneration by activating the RGC intrinsic mevalonate and cholesterol synthesis pathway and/or inducing expression of receptors for extracellular sources of cholesterol and lipids. Using the powerful genetic and chemical tools available for the zebrafish system, we propose to identify the critical period of srebf2 activity and the downstream mediator(s) of its function. Aim 1 will determine when srebf2 function in RGCs is critical for ON regeneration and using novel reporter lines of srebf2 activity to delineate when transcriptional activity occurs. We will also test if activation of srebf2 activity is sufficient to accelerate ON regeneration. Lastly, we will use laser capture microdissection RNA-seq (LCM-seq) to identify differential gene expression under gain- and loss-of-srebf2 function in RGCs. Aim 2 proposes to identify which RGC intrinsic or extrinsic pathways downstream of srebf2 mediate its function. We will use combinations of drugs and gene knockdown to determine if intrinsic mevalonate/cholesterol synthesis and external supplies are independent, interdependent, and/or compensatory for successful ON regeneration. Depending upon the results of this study we will further examine the downstream intrinsic synthesis pathways for cholesterol, ubiquinone, protein prenylation, and protein N-glycosylation or low-density lipoprotein receptors and their downstream processing. Aim 3 will test the sufficiency of Srebf2 expression to provide neuroprotection and stimulate axon regeneration in a mouse model of acute ON injury. LCM-seq will be used to identify gene expression changes induced in mouse RGCs by Srebf2 and compared to those identified in zebrafish to suggest mechanisms for success or failure. These experiments will delineate the pathways downstream of srebf2 necessary for efficient ON regeneration and suggest paths forward to enhance mammalian regeneration.