Shuxin Li, M.D. P.h.D - US grants

DESCRIPTION (provided by applicant): After CNS axonal injuries, medical treatments to enhance recovery from neurological deficits are extremely limited. Non-permissive environments for axonal growth at least partially contribute to growth failure in the adult CNS. Specifically, several groups of inhibitory molecules strongly suppress axonal extension following CNS lesions, including chondroitin sulfate proteoglycans (CSPGs) generated by glial scars. CSPGs are the principal inhibitory components of glial scars and form a major barrier to regenerating axons. Although several strategies have been reported, digestion of CSPGs with local application of bacterial chondroitinase ABC is the major in vivo approach to surmount growth inhibition of CSPGs after CNS injuries. Important disadvantages, however, preclude the use of this enzyme as a therapeutic option for axonal injury patients, including incomplete removal of inhibitory components from CSPGs, short-period of enzymatic activity at body temperature and inability to cross the blood-brain barrier. In this proposal, we aim to develop novel strategies for treating CNS axonal injury based on inhibition of CSPGs alone or in combination with our previously identified approaches. We hypothesize that peptide antagonists of CSPGs will augment both morphological and functional recovery in a mouse model of CNS injury. Using a bioinformatics approach to define the conserved elements of several CSPGs, we have identified two selective peptide antagonists for CSPGs. Our preliminary studies suggest that these peptides at low nanomolar concentrations principally overcome neurite growth restrictions of CSPGs in neuronal cultures. Systemic application of a CSPG-blocking peptide significantly improves behavioral recovery in CNS axon-injured mice in vivo. In this study, we will characterize the therapeutic potential of these CSPG antagonistic peptides in mouse spinal cord injury (SCI) model. In addition to CSPGs, a number of inhibitory molecules contribute to axonal growth suppression intracellularly mediated via activation of convergent RhoA or glycogen synthase kinase 32 (GSK-32). Recently, we have demonstrated that inactivation of RhoA with ibuprofen or GSK-32 with lithium overcomes growth inhibition of different molecules and significantly promotes axonal growth of descending motor neurons and locomotor recovery in SCI rodents. Thus, we also aim to stimulate a more dramatic axonal regeneration in SCI mice by combining a CSPG-blocking peptide with RhoA-inhibiting ibuprofen or GSK-32-inactivating lithium, two drugs widely used in humans. The use of our novel antagonists for CSPGs, alone or in combination with ibuprofen or lithium, may significantly advance our ability to treat CNS axonal injuries in adult mammals by promoting axonal regeneration and functional recovery. PUBLIC HEALTH RELEVANCE: We aim to develop novel therapies for CNS axonal injuries based on strong inhibitory properties of chondroitin sulfate proteoglycans, a group of extracellular matrix molecules generated by reactive glial scars. Development of novel peptide antagonists for these axonal growth inhibitors may advance our ability to treat CNS axonal injuries in the adult mammals. We hope that the translation of our novel therapeutic strategies from neuronal cultures in vitro to mouse model in vivo will ultimately lead to key strategies in patients with spinal cord injury and other CNS lesions.

DESCRIPTION (provided by applicant): By using novel, systemically deliverable, small inhibitory peptides developed in the PI's lab, we aim to determine whether targeting both extracellular inhibitory and neuron-intrinsic factors can markedly improve axon regeneration and functional recovery after spinal cord injury (SCI). Severed CNS axons fail to regenerate due to the extrinsic inhibitory environment and the reduced intrinsic growth capacity of mature neurons. Chondroitin sulfate proteoglycans (CSPGs) generated by glial scars strongly suppress axon extension into and beyond the lesion area and are the major molecular targets for treating SCI. Recently, we and other labs identified the LAR and PTP¿ phosphatases as receptors that mediate CSPG inhibition. Deleting either of them stimulated axon growth after SCI. Recent studies using conditional knockout mice suggested that PTEN critically restricts the intrinsic regenerative capacity of injured CNS axons. Thus, suppressing CSPG receptors and PTEN is promising for promoting axon regeneration after CNS injury. We have designed small peptides to block functions of these inhibitory molecules by targeting their specific domains and demonstrated the high efficacy of our peptides for promoting axon growth in vitro and in vivo. Since CSPGs and PTEN appear to limit growth by different signaling pathways, inhibition of both may act synergistically to promote axon regeneration by reducing environmental inhibitory influence at the lesion site and enhancing intrinsic growth capacity of mature neurons. We hypothesize that CSPGs and PTEN are critical contributors to regenerative failure of CNS neurons and that combined inhibition of both promotes axon regeneration better than inhibition of either one alone. We propose to address the following 3 Specific Aims: 1) determine whether transgenic deletion or peptide blockade of two CSPG receptors yields better axon growth in vitro and in vivo and functional recovery in adult mice with SCI than suppressing either receptor alone; 2) determine whether PTEN blockade with peptides stimulates similar degrees of axon growth and functional recovery as transgenic PTEN deletion in vitro and in vivo; 3) determine whether blocking both CSPG signaling and PTEN with peptides promotes greater axon regeneration and behavioral recovery after SCI than blocking either one alone. The results of peptide treatments will be compared with those of transgenic mouse experiments. Our novel strategy to administer small compounds systemically alone or in combination may facilitate development of a practical therapy for CNS injury.

DESCRIPTION (provided by applicant): 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.

Project Summary Severed CNS axons fail to regenerate largely because of the reduced intrinsic growth capacity of adult neurons and poor environment for axon extension. The Co-I's Lab finds that the Wnt family molecules and their receptors are upregulated after spinal cord injury (SCI) and mediates regrowth Among adult failure of injured fiber tracts. several Wnt receptors, Ryk is crucial for mediating repulsive axon growth during development and in CNS after injury.Chondroitin sulfate proteoglycans (CSPGs) generated by glial scars strongly suppress axon extension and are major extrinsic molecular targets for treating CNS injury. The PI's group designed small peptides to block functions of CSPG receptors LAR and PTP? by targeting their critical activity domains and demonstrated their high efficiency for promoting axon growth. Blocking each of the two receptors with 3 combined peptides promotes robust regeneration of injured CNS axons. We hypothesize that inhibiting both Wnt and CSPG signals represents a dual approach of enhancing neuronal growth capacity and reducing environmental inhibitory influence at the lesion site. We propose to stimulate robust axon regrowth in adult rodents with transection or contusion SCI by inhibiting Ryk and LAR/PTP? with genetic and pharmacological approaches available in our labs. In Aim 1, we will study synergistic actions of transgenically deleting Ryk plus each of LAR/PTP? receptors on promoting axon regeneration and recovery in mice with SCI. We will determine whether deleting Ryk plus LAR or Ryk plus PTP? receptors acts synergistically to stimulate axon growth and enhance neuronal plasticity in double knockout mice after SCI. In Aim 2, we will determine whether blocking each of Ryk, LAR and PTP? receptors with antibody or selective antagonists pharmacologically promotes axon regeneration and recovery in adult rats with SCI. We will compare effectiveness of the treatments that target individual receptors in promoting regrowth of multiple descending tracts and recovery of locomotor functions after SCI. In Aim 3, we will study whether combination therapies that block two or three receptors yield better axon regrowth and functional recovery in rats with transection or contusion SCI, aiming to identify the optimal therapy for mammals with SCI. Based on the promising results from our pilot studies, we predict that our combined strategies will promote dramatic regeneration of injured axon tracts and recovery of locomotion function in vivo. Our novel strategy of administering deliverable compounds post-injury may facilitate development of a practical combinatorial therapy for CNS lesions.

Abstract We propose to promote long-distance axon regeneration of injured optic nerve or tract and recovery of visual function in adult mammals by enhancing intrinsic growth capacity and growth cone dynamics of mature neurons. We will study whether inhibiting let-7 and/or upregulating its suppressors lin28 and lin41 in retinal cells promotes robust axon regeneration and functional recovery in adult mammals with optic axon injury. We will also study whether upregulating cytoskeletal TACC3 protein stimulates dramatic axon regeneration by targeting growth cones directly. CNS neurons lose the ability to regenerate axons with age, and this limits functional recovery after injury. Many genes have been identified to control the growth ability of mature neurons, but none have been translated to clinical use. The best targets are probably those with the potential to impact multiple genes simultaneously. Among them, let-7 miRNA seems important for regulating age- dependent decline in axon regeneration. We propose to enhance the growth capacity of mature neurons by targeting the lin28/let-7/lin41 pathway. Because dystrophic growth cones in axotomized CNS contribute to axon regeneration failure, we also propose to enhance cytoskeletal growth dynamics by upregulating the TACC3 gene. We hypothesize that the let-7 pathway regulates axon regeneration in mammalians and that targeting this pathway plus the cytoskeletal TACC3 gene stimulates robust axon regeneration and functional recovery of visual pathways. Using the novel AAV vectors developed in the PI?s lab, we will determine whether inhibiting let-7 and/or upregulating lin28, lin41, or TACC3 in retinal cells promotes robust axon regeneration and functional recovery in adult rodents with optic nerve or tract injury. Aim 1 proposes to study whether intravitreal injections of the individual or combined viral vectors for let-7 inhibitor, lin28, or lin41 enhance optic axon regeneration, retinal ganglion cells survival, and functional recovery in adult mice. In Aim 2, we will use our AAV vectors to study whether upregulating TACC3 stimulates dramatic axon regeneration and whether combination therapies targeting both let-7 and TACC3 signals yield better axon regeneration and functional recovery in adult rodents with optic axon injury than either individual approach. Use of our unique viral vectors has the potential not only to provide important new insights into the molecular control of growth in mature CNS neurons, but also to develop practical and effective strategies to promote axon regeneration and functional recovery in mammals. Our experiments with combined strategies to target both somatic neuronal program and growth cone cytoskeletal dynamics should stimulate further axon regeneration and functional recovery. We thus anticipate identifying extremely promising regenerative strategies in adult mammals. Our viral vectors, which are administered post-injury, can be applied to multiple axon tracts and readily translated into clinical trials. The success of this project may reverse the visual functional deficits, improve the quality of life in many patients, and reduce the financial burdens to patients, families, and the public.