Trauma to the spinal cord promotes a complex cascade of pathophysiological events that result in greater injury than was initially sustained and which contribute to inflammation, demyelination, axon injury and an unfavorable environment for neural recovery. The factors which drive this cascade continue to be identified and critically evaluated since each may serve as a target for the rationale design of new therapies to mitigate injury and to promote repair and regeneration. Work in our laboratory during the previous funding period indicates that serine proteases of the kallikrein (KLK) family are among the complex cascade of enzymes now recognized to be deregulated with spinal cord trauma and furthermore that several KLKs are novel mediators of neurotoxicity, astrogliosis and demyelination. Importantly, we discovered that KLKs exert their cellular effects by cleaving thereby activating G-protein coupled receptors termed Protease Activated Receptors (PARs). As cell surface receptors, PARs endow the cell with the ability to respond, or to over respond, to the rapidly changing proteolytic microenvironment that occurs at sites of CNS trauma, inflammation and blood brain barrier breakdown. The CENTRAL HYPOTHESIS to be tested in the proposed studies is that proteolytic activation of select PARs regulates unique cellular responses in the traumatically injured spinal cord and that these receptors can be differentially targeted to prevent secondary injury and to promote repair. If this hypothesis is correct, PARs may serve as targets for the development of new therapies. Four complementary Aims that focus on cellular, molecular and systems outcomes are proposed to test this hypothesis.
In Aim 1, we will determine the effects of genetic targeting of PARs on neurobehavioral recovery in a murine model of traumatic spinal cord injury.
In Aim 2, we will use genetic and pharmacologic loss and gain of function approaches to establish the role of PARs in mediating the cellular effects of SCI-related PAR agonists (KLKs, thrombin and plasmin) in primary spinal cord neurons, astrocytes and oligodendroglia and their sensitivity to neurotoxic agents in vitro.
In Aim 3, we will dissect the molecular signaling and gene expression profiles that are elicited by each protease across neurons and neuroglia and the PARs responsible for mediating these effects.
In Aim 4, we will determine the effects of PAR-pharmacotherapy on neurobehavioral recovery in murine traumatic SCI. The proposed studies will identify new receptor based mechanisms regulating the SCI microenvironment that are potentially highly amenable to therapeutic intervention and given the widespread expression of PARs in the CNS, are likely to be of fundamental significance to understanding injury and repair mechanisms in a wide range of neurological conditions.
Traumatic spinal cord injury (SCI) is a devastating often paralyzing condition with an annual incidence of 15-40 cases per million in the USA and an estimated annual cost of more than $7 billion. Efforts to improve understanding of SCI pathophysiology have already resulted in the development of specific clinical strategies for management, but there remains no drug treatment that effectively improves outcome. Interventions that target the injury that occurs after the initial trauma have the chance of improving functional outcomes, including bowel and bladder control, hand grasp, limb mobility and breathing. The proposed studies will determine the role of protease activated receptors in mediating neural injury in cases of spinal cord trauma and whether these highly druggable receptors can be targeted to improve recovery at cellular, molecular and behavioral levels in a murine SCI model. The long-term goal of these studies is to develop new therapies for SCI patients.
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