Damien D. Pearse - US grants

[unreadable] DESCRIPTION (provided by applicant): Our ultimate goal is to develop effective strategies to improve outcome after human spinal cord injury (SCI). We have demonstrated (Pearse et al., 2004) that a triple combination of a SC implant, a one-time injection of cAMP into the cord above and below the implant, and a two-week subcutaneous infusion of Rolipram induced substantial growth of serotonergic fibers into the implant and beyond into the cord caudal to the implant. A marked improvement in locomotor test scores was observed in groups with the best growth of serotonergic axons in the caudal cord. To build on these results, we propose three Specific Aims using the same injury model and SC grafting (Pearse et al., 2004). In Aim 1, we will determine the most effective delivery site (lesion, raphe somata, or both) for cAMP elevation (by measuring fibers and evaluating locomotion). BDNF is known also to promote growth of serotonergic axons into the cord caudal to a SC graft; therefore we will determine the most efficacious site for BDNF delivery using the same SCI paradigm. In Aim 2, we propose to test the possibility that the combination of cAMP elevation and BDNF administration will be more effective than either one alone. Administration of cAMP (best route) and BDNF (best route) will be combined and the serotonergic axon growth response compared to that observed with either factor alone. Behavioral test results will be correlated with axon growth in each animal. In Aim 3, we propose to determine the functionality of the serotonergic projections after our most effective treatment. First, changes in locomotion caused by pharmacologically blocking serotonergic receptors will be assessed. Second, we will determine reflex responses to somatic afferent stimulation following electrical stimulation of raphespinal pathways. Third, the release of 5-HT by descending fibers after the best treatment will be verified. Exploration of combined therapies to improve serotonergic fiber growth beyond the site of injury and determination of the role of the potentially improved growth on locomotion will lead to new clinical strategies in the future. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glial scarring following CNS injury alters the lesion environment so as to impede axonal regeneration and plasticity, thereby limiting functional restitution. Reactive astrocytes are the main cellular component of the glial scar; astrocytes undergo morphological changes and produce extracellular matrix, such as chondroitin sulfate proteoglycans (CSPGs), which physically and chemically inhibit axon growth. Strategies that inhibit astrogliosis or prevent the synthesis of, or degrade, CSPGs have been demonstrated to relieve axon growth inhibition and improve function. Intracellular mechanisms involved in the control of astrocyte reactivity and the stimulation of CSPG production remain poorly understood. Recent reports have shown that the phosphodiesterase (PDE) inhibitor Rolipram can reduce astrogliosis and Preliminary Data from our laboratory indicates that there is a chronic induction of the PDE4A isozyme in reactive astrocytes that occurs in parallel with the maturation of the glial scar. Site-specific targeting of this PDE4 isoform may then prevent astrogliosis and offer a novel therapeutic direction for scar reduction after injury to the spinal cord or brain so as to enhance axon plasticity and functional recovery. To delineate the role of PDE4A in astrocyte reactivity and CSPG production, interference RNA will be used via lentiviral vector expressed PDE4A short-hairpin RNA (shRNA) specifically within astrocytes in vitro [Specific Aim 1] and in vivo (gfap-promoter driven) after spinal cord injury (SCI) [Specific Aim 2]. In astrocyte cultures, the effectiveness of PDE4A knockdown will be refined and the role of PDE4A in mechanisms of cellular reactivity, including A) cytoskeletal rearrangements, B) enhanced cell migration, C) increased cell proliferation and, D) the production of CSPGs, will be examined. Then in vivo, these in vitro effects will be corroborated as well as the anatomical and functional benefits of PDE4A knockdown in repair assessed. A complete transection SCI model will be used to assess if PDE4A knockdown in astrocytes prevents axon dieback and/or allows axonal regeneration across the injury site, while the functional effects of molecular PDE4A inhibition in astrocytes will be examined in an incomplete contusive SCI paradigm. PUBLIC HEALTH RELEVANCE: Spinal cord injury (SCI) is a devastating condition that affects more than 1.25 million Americans (Christopher and Dana Reeve Foundation), representing a major health care cost burden to the US. The formation of a glial scar around the injury site represents a potent obstacle to axonal regeneration and functional restitution. Although experimental studies have demonstrated the promise of targeting the glial scar as a therapeutic direction for promoting SCI repair, the intracellular mechanisms responsible for maintaining the reactivity of the major cellular constituent of the glial scar, the astrocyte, as well as its inhibtory extracellular matrix production are poorly understood. The proposed work will define, through molecular manipulations, a role for the cyclic AMP- phosphodiesterase signaling cascade in astrogliosis, a pathway known to play a central role in the cellular reactivity and pathological functioning of other cell types such as immune cells, as well as provide a novel therapeutic direction for glial scar ablation.