This is a proposal that uses novel, systemically deliverable, small inhibitory peptides to determine whether combined targeting of neuron-intrinsic and extracellular inhibitory factors markedly improves retinal ganglion cell (RGC) axon regeneration and survival after optic nerve injury (ONI). Severed optic axons fail to regenerate and ONI leads to life-long visual loss in patients. In addition to apoptotic RGC death following axotomy, both a reduced intrinsic growth capacity and an inhibitory molecular environment contribute to failure of mature CNS neurons to regenerate their axons. Studies using conditional knockout (KO) mice suggest that the tumor suppressor gene PTEN is one neuron-intrinsic factor that critically restricts the regenerative capacity of adult RGCs. Deletion of PTEN enhanced RGC axon growth and survival after ONI. Chondroitin sulfate proteoglycans (CSPGs) generated in glial scars are extrinsic factors that strongly suppress axon extension. Recently, we and other labs identified LAR and PTP phosphatases as receptors that mediate CSPG inhibition. Deletion of either of them partially overcomes suppression by CSPGs and stimulates growth of injured CNS axons. Some PTPs, including LAR, can also activate caspases and induce cell apoptosis. Suppressing PTEN and CSPG signaling is very promising for promoting CNS axon regeneration, but transgenic deletion of PTEN, LAR or PTP is not feasible for treating patients. We have designed small mimetic peptides to block functions of these inhibitory molecules and demonstrated their efficiency in promoting axon growth in vitro and in vivo. Because CSPG receptors appear to regulate neuronal functions via pathways different from PTEN signaling, we hypothesize that combining inhibition of PTEN and CSPG signaling promotes RGC axon regeneration and survival better than inhibiting either alone. To test this hypothesis, we will use small peptide inhibitors alone and in combinations, validating the peptide efficacy by comparison to results with KO mice available in our lab. We propose to address 3 Specific Aims by determining whether: 1) peptide blockade of PTEN promotes similar RGC axon regeneration and survival as transgenic deletion in KO mice; 2) peptide blockade of two CSPG receptors promotes greater RGC axon regeneration and survival than inhibiting one receptor; and 3) blocking both PTEN and CSPG signaling with peptides promotes greater RGC axon regeneration and survival than targeting either one alone. By simultaneously targeting neuron-intrinsic and environmental inhibitory factors with small blocking peptides, we attempt to promote axon regeneration and to reduce axotomy-induced RGC loss to greater degrees than by targeting either signal individually. Our novel strategy of administering small, systemically deliverable compounds post-injury may facilitate development of practical combinatorial therapy for optic nerve injury.