Robert McKeon - US grants

DESCRIPTION: The astrocytic response to CNS injury has long been implicated in the failure of damaged axons to regenerate beyond the injured area. Recent experiments have demonstrated that reactive astrocytes, originally considered physical barriers to regenerating axons produce an extracellular matrix (ECM) that inhibits axon outgrowth. In vitro assays suggest that the inhibitory component of this ECM is associated with the expression of chondroitin sulfate proteoglycans (CS-PGs) and that decreasing CS-PG expression after injury would enhance axonal regeneration through areas of reactive astrogliosis. CS-PGs are a group of complex molecules whose synthesis in a variety of peripheral cells is upregulated by transforming growth factor-beta (TGF-beta). The role of TGF-beta on CS-PG expression in the CNS is less clear. TGF-beta and CS-PG levels are highest in embryonic animals and both are downregulated shortly after birth. TGF-beta levels increase following CNS injury and this increase is correlated temporally and spatially with the re-expression of CS-PGs. Preliminary data demonstrate that in response to TGF-beta, cultured astrocytes synthesize a neurite growth inhibitory CS-PG and that this response is blocked by the addition of TGF-beta neutralizing antibodies. Data demonstrating that CS-PG expression is blocked after CNS injury by the in vivo administration of these TGF-beta neutralizing antibodies is now included. Based on these significant findings, it is hypothesized that TGF-beta stimulates CS-PG production after CNS injury and that neutralizing TGF-beta bioactivity will enhance axonal regeneration in vivo. These hypotheses will be tested by: ( 1 ) determining whether TGF-beta regulates the production of CS-PG after CNS injury by continuous intraventricular delivery of TGF-beta neutralizing antibodies and assessing CS-PG levels in the injured area; (2) examining the expression of specific CNS CS-PGs in response to TGF-beta, and; (3) examining whether neutralizing the function of TGF-beta enhances axonal regeneration following spinal cord injury. These studies will help identify signals that stimulate the production of axon growth inhibitory CS-PGs by reactive astrocytes. Decreasing the expression of these inhibitory molecules, combined with the use of factors known to promote axon outgrowth, may lead to the development of comprehensive strategies designed to optimize axonal regeneration after CNS injury.

DESCRIPTION: The astrocytic response to CNS injury has long been implicated in the failure of damaged axons to regenerate beyond the injured area. Recent experiments have demonstrated that reactive astrocytes, originally considered physical barriers to regenerating axons produce an extracellular matrix (ECM) that inhibits axon outgrowth. In vitro assays suggest that the inhibitory component of this ECM is associated with the expression of chondroitin sulfate proteoglycans (CS-PGs) and that decreasing CS-PG expression after injury would enhance axonal regeneration through areas of reactive astrogliosis. CS-PGs are a group of complex molecules whose synthesis in a variety of peripheral cells is upregulated by transforming growth factor-beta (TGF-beta). The role of TGF-beta on CS-PG expression in the CNS is less clear. TGF-beta and CS-PG levels are highest in embryonic animals and both are downregulated shortly after birth. TGF-beta levels increase following CNS injury and this increase is correlated temporally and spatially with the re-expression of CS-PGs. Preliminary data demonstrate that in response to TGF-beta, cultured astrocytes synthesize a neurite growth inhibitory CS-PG and that this response is blocked by the addition of TGF-beta neutralizing antibodies. Data demonstrating that CS-PG expression is blocked after CNS injury by the in vivo administration of these TGF-beta neutralizing antibodies is now included. Based on these significant findings, it is hypothesized that TGF-beta stimulates CS-PG production after CNS injury and that neutralizing TGF-beta bioactivity will enhance axonal regeneration in vivo. These hypotheses will be tested by: ( 1 ) determining whether TGF-beta regulates the production of CS-PG after CNS injury by continuous intraventricular delivery of TGF-beta neutralizing antibodies and assessing CS-PG levels in the injured area; (2) examining the expression of specific CNS CS-PGs in response to TGF-beta, and; (3) examining whether neutralizing the function of TGF-beta enhances axonal regeneration following spinal cord injury. These studies will help identify signals that stimulate the production of axon growth inhibitory CS-PGs by reactive astrocytes. Decreasing the expression of these inhibitory molecules, combined with the use of factors known to promote axon outgrowth, may lead to the development of comprehensive strategies designed to optimize axonal regeneration after CNS injury.

DESCRIPTION (provided by applicant): The astrocytic response to CNS injury is complex and multifaceted, with important implications for protecting vulnerable neurons from cell death. Although specific neuroprotective functions of astrocytes, e.g., glutamate transport, are known to be energy dependent, little is known about how, or if, astrocytes alter their bioenergetic state in response to injury. We have discovered that expression of the Adenine Nucleotide Translocator-1 (Ant1) by reactive astrocytes increases after cortical injury and that this increase is mediated by the injury-induced cytokine transforming growth factor beta1(TGF-beta1). Ant1 is an inner mitochondrial membrane protein that exchanges mitochondrial ATP for cytosolic ADP, thereby regulating the supply of the substrate required for continued mitochondrial energy production (ADP) as well as the delivery of ATP to the cytosol for energy dependent functions. Importantly, the expression of the closely related isoform, Ant2, does not appear to change. Additional preliminary data demonstrate that glutamate uptake by Ant1 null astrocytes is significantly impaired and that Ant1 null mutant animals are prone to cortical damage following combined hypoxic/ischemic injury. These data suggest that reactive astrocytes experience a bioenergetic demand and are consistent with the hypothesis that Ant1 is a critical component of the astrocytic response to injury by mobilizing the energy required during reactive astrogliosis. This hypothesis will be tested in vitro and in an in vivo model of cerebral hypoxic/ischemic injury in Ant1 null mutant, Ant2 neural null transgenic, and genetically matched control mice. To test this hypothesis, we will: (1) examine whether astrocytic Ant1 is required to protect neurons from excitotoxic injury or death in vitro, by focusing on the critical astrocytic function of glutamate uptake; and (2) determine if astrocytic Ant1 expression is required for neuronal survival in vivo after hypoxic/ischemic injury. These studies will provide important mechanistic insight into the biological response of reactive astrocytes by examining the requirement for a molecule that facilitates mitochondrial ATP production and mobilization.