The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury. Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic impediments to axon regrowth, we analyzed cell growth control genes using a virus-assisted in vivo conditional knockout approach. Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice, the mTOR activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the regeneration failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of the mTOR pathway, also leads to axon regeneration. Thus, our results suggest the manipulation of intrinsic growth control pathways as a therapeutic approach to promote axon regeneration after CNS injury.Axons do not regenerate after injury in the adult mammalian central nervous system (CNS), a phenomenon attributed to two properties of the adult CNS, the inhibitory extrinsic environment and a diminished intrinsic regenerative capacity of mature CNS neurons (1-4). Neutralization of the extracellular molecules identified as axon regrowth inhibitors allows only a limited degree of axon regeneration in vivo (5-7). Therefore, intrinsic mechanisms are likely to be important in controlling the process of axon regeneration. A hint about possible mechanisms of neuronal regenerative ability comes from the evolutionarily conserved molecular pathways that control cellular growth and size. For most cell types, specific mechanisms are necessary to prevent cellular overgrowth upon the completion of development (8). Because many of these molecules are often expressed in postmitotic mature neurons, we hypothesized that they may contribute to the diminished regenerative ability in adult CNS neurons.To circumvent the problem that germline knockout of individual cell growth control genes often results in compromised viability in mice, we designed a strategy based on intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) in adult mice. This procedure resulted in the expression of Cre in more than 90% of retinal ganglion cells (RGCs) and few other non-RGC cells, as indicated in two reporter lines ( fig. S1, A and B). We thus injected AAV-Cre into the vitreous body of different adult floxed mice, including Rb f/f (9), P53 f/f (9),

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Axon formation is fundamental for brain development and function. TSC1 and TSC2 are two genes, mutations in which cause tuberous sclerosis complex (TSC), a disease characterized by tumor predisposition and neurological abnormalities including epilepsy, mental retardation, and autism. Here we show that Tsc1 and Tsc2 have critical functions in mammalian axon formation and growth. Overexpression of Tsc1/Tsc2 suppresses axon formation, whereas a lack of Tsc1 or Tsc2 function induces ectopic axons in vitro and in the mouse brain. Tsc2 is phosphorylated and inhibited in the axon but not dendrites. Inactivation of Tsc1/Tsc2 promotes axonal growth, at least in part, via up-regulation of neuronal polarity SAD kinase, which is also elevated in cortical tubers of a TSC patient. Our results reveal key roles of TSC1/TSC2 in neuronal polarity, suggest a common pathway regulating polarization/growth in neurons and cell size in other tissues, and have implications for the understanding of the pathogenesis of TSC and associated neurological disorders and for axonal regeneration.[Keywords: Neuronal polarity; tuberous sclerosis complex, TSC; SAD kinase; autism] Supplemental material is available at http://www.genesdev.org.

Tuberous sclerosis complex is a disease caused by mutations in the TSC1 or TSC2 genes, which encode a protein complex that inhibits mTOR kinase signaling by inactivating the Rheb GTPase. Activation of mTOR promotes the formation of benign tumors in various organs while the mechanisms underlying the neurological symptoms of the disease remain largely unknown. Here, we report that in mice Tsc2 haploinsufficiency causes aberrant retinogeniculate projections that suggest defects in EphA receptor-dependent axon guidance. We also show that EphA receptor activation by ephrin-A ligands in neurons leads to inhibition of ERK1/2 kinase activity and decreased inhibition of Tsc2 by ERK1/2. Thus, ephrin stimulation inactivates the mTOR pathway by enhancing Tsc2 activity. Furthermore, Tsc2 deficiency and hyperactive Rheb constitutively activate mTOR and inhibit ephrin-induced growth cone collapse. Our results demonstrate that TSC2-Rheb-mTOR signaling cooperates with the ephrin-Eph receptor system to control axon guidance in the visual system.