Harold David Shine, Ph.D. - US grants

We propose to identify and characterize the cellular element(s) of PNS responsible for the miliue that supports CNS and PNS axonal regeneration. Evidence from experiments in which PNS nerve was used to bridge regions of CNS shows that CNS and PNS axons are not demonstrably different in their regeneration potential but that their local environments play a decisive role in the success or failure of axonal regeneration after injury. In the PNS, transected sciatic nerve axons will regenerate through an initially-empty tube if the proximal and distal stumps are inserted into the ends. Regeneration into the tube fails if the distal stump is omitted from the tube and the end is closed off. However, we found that a heterogeneous population of cultured PNS cells from embryonic dorsal root ganglia placed inside the tube will support axonal regeneration in the absence of the distal stump -- an indication that these cells can mimic the effect of the distal stump in providing the proper miliue for axonal regeneration. We propose to extend this work by implanting pure populations and defined mixtures of cultured PNS cells (endoneurial fibroblasts, Schwann cells and neurons) to identify the cellular source of the influence. The spatial/temporal events in axonal regeneration through tubes containing cultured cells and the fate of cultured cells in implants will be studied. Culture medium, conditioned by PNS cells, will be tested for an influence on regrowing axons through the tube implants. The mechanism of the influence will be characterized by using specially-designed tubes to provide regenerating axons with alternate environments in which to grow. We will test a mixture of CNS and PNS glial cells to determine if CNS cells actively inhibit axon regrowth.

DESCRIPTION (Adapted from Applicant's Abstract): Patients with malignant brain tumors have poor prognoses even when treated with the best conventional therapy of surgical resection, radiation therapy, and chemotherapy. Hence, investigation of new therapeutic approaches for these neurological diseases is needed. One novel approach has been the introduction of genes into tumor cells that render them sensitive to cytotoxic drugs. Previous experiments demonstrated a robust tumoricidal activity against experimental brain using the herpes simplex virus thymidine kinase (HSVtk) gene that converts ganciclovir (GCV) to a form that is cytotoxic to rapidly dividing cells. The treatment was not as effective when tested with tumors generated from cell lines with lesser immunogenicity. These observations suggest that the tumoricidal activity results from two processes: (1) direct tumoricidal activity of HSV-tk conversion of GCV and (2) cellular immune responses. The applicant will test the hypothesis that cell death from HSV-tk/GCV treatment causes antigen presenting cells (APCS) to activate cytotoxic T lymphocyte (CTL) precursors which then destroy residual tumor and prohibit further cell growth. The presence of an immunological component can explain the weaker tumoricidal activity observed in models in which the hosts were immunoincompetent or in which the cell lines were less immunogenic. If this hypothesis is correct then (1) depending on their immunogenicity, different tumor cell lines will elicit different immune responses to HSV-tk/GCV-mediated cell death in vivo as measured by immunological analyses, and (2) neuroimmune modulation by cytokine gene transfer will elicit a heightened CTL response to the tumor cells. The objective of this project is to delineate the neuroimmunological mechanisms of cell death in experimental brain tumors following initial killing by HSV-tk/GCV treatment. The Specific Aims are to: (1) characterize the immunological response to HSV-tk/GCV-mediated death of syngeneic experimental tumors generated by 3 glial tumor cell lines with different degrees of immunogenic potential, (2) measure the tumoricidal effects of in situ immune modulation by adenovirus-mediated transduction of tumors with the genes for the cytokines IL-2, GM-CSF, TNFA. and IL-12 with and without HSV-tk/GCV treatment. and (3) characterize the immunological responses elicited by adenoviral-mediated cytokine immune modulation with and without HSV-tk/GCV treatment.

DESCRIPTION: (adapted from applicant's abstract) Treatment of spinal cord injury (SCI) encompasses the rescue of injured neurons, the promotion of axonal regeneration and the formation of functional synapses. This project focuses on one aspect of an envisioned multi-modai therapy - the promotion of axonal regeneration. The proposed experiments are designed to test the hypothesis that local release of induced neurotrophic factors (NFs) in the spinal cord will enhance axonal regeneration after trauma. When adenoviral vectors carrying NF genes (Adv-nf) are injected into peripheral muscles they are transported to motoneurons where the genes are expressed and NFs are released. The NFs protect spinal cord motoneurons from trauma-induced death without evoking an inflammatory response. To test the hypothesis that local expression of NFs will support axonal regeneration adenoviral vectors will be delivered to the spinal cord in which the corticospinal tract (CST) has been unilaterally lesioned at the level of the pyramids. In this lesion model there is no trauma in the spinal cord so that tissue destruction, ischemia, and paralysis are not factors in determining the outcome and may allow better understanding of the effects of NFs on sprouting. The specific aims of this proposal are to: Test whether expression of NFs by genetically modified cells in the spinal cord will induce and sustain axonal sprouting after injury (Specific aim 1). Genes for neurotrophin 3 (NT-3), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived growth factor (GDNF) will be delivered singly and in combination to the spinal cord by: (1) direct injection of Adv-nfs into spinal cord tissue, (2) transduction of ependymal cells so that they release NFs into the cerebrospinal fluid, and (3) retrograde delivery of Adv-nfs to dorsal root ganglion and motoneurons. A panel of adenoviral vectors will be generated that contain a FLAG sequence to aid in immunological detection and the Elongation Factor (EF) promoter that is more efficient in mammalian cells. Specific aim 2 is designed to characterize the extent of expression and the pattern of sprouting. Specific aim 3 is designed to characterize the effects of Adv-nf-induced sprouting on motor behavior. If the investigators demonstrate that this strategy is effective in inducing axonal growth it would be a basis of future studies leading to a treatment of SCI.

DESCRIPTION (provided by applicant): Over-expression of Neurotrophin-3 (NT-3) in lumbar motoneurons induces axons to grow from the contralateral corticospinal tract (CST) towards the source of NT-3 in rats but only if the CST ipsilateral to the transduced motoneurons is lesioned and if the NT-3 over-expression is coincident with the time of lesion. If NT-3 over-expression is delayed 4m after the lesion there is no axonal growth suggesting that at least one factor associated with the acute phase of the CST injury is involved. If the rats with acute CST lesions were immunosuppressed axonal growth in response to NT-3 is blocked. When the experiment was repeated in athymic nude (AN) rats there was no axonal growth in response to NT-3. If the immune response is re-activated in chronically lesioned rats with systemic lipopolysaccharide (LPS) NT-3 will induce axonal growth. This is the first demonstration of inducing axonal growth with neurotrophins in achronically lesioned CNS. These observations suggest that NT-3-induced axonal growth requires processes associated with immune-mediated wound healing; specifically activated T- cells. The goals of this project are to identify the factor or factors associated with immune-mediated wound healing and test them in conjunction with NT-3 in our model of chronic spinal cord injury. The specific aims are to: (1) verify the role of T-cells in NT-3-induced axonal growth, identify the T-cell subtype responsible, (2) determine the role of microglial activation in this process, and (3) identify genes and proteins differentially expressed in conditions that support NT-3 induced axonal growth compared to those that do not using qPCR and protein expression using multiplex array immunoassays in AN rats. Our results show that neuroplasticity can be induced in the chronically injured spinal cord distal to the lesion site. This neuroplasticity is likely induced by the presence of NT-3 and a factor, or factors, associated with immune-mediated wound healing. If the identity of the factor(s) associated with the acute injury can be identified then it may be possible to recapitulate the acute environment in patients with chronic spinal cord injuries to induce neuroplasticity in spared axonal tracts. Two-thirds of spinal cord injury patients have incomplete cord transections so strategies to enhance the function of the remaining connections by enabling the inherent plasticity of the CNS may provide improvement in function of patients with chronic spinal cord injury. PUBLIC HEALTH RELEVANCE: The Armed Forces' activities in both peace- and war-time present situations for catastrophic neural injuries. It is improbable with today's biomedical technologies that the injured CNS can be repaired by inducing the regeneration of severed axons. However, it is not improbable that some function may be gained by the CNS's innate ability to mount a compensatory reorganization through collateral sprouting of spared axons. This proposal is based upon our recent discoveries that immune-related processes are involved in axonal growth after injuryand that reactivating these immune processes will enable axonal growth in the chronic injured CNS. This work addresses the basic biology of recovery from SCI with the belief that this knowledge may be used to develop new, or refine existing, strategies to help repair these neural injuries. It seeks to determine the underlying mechanism of neurotrophin-induced axonal regeneration after SCI in order to develop means to treat chronic CNS injuries.