Calpastatin overexpression as a therapeutic approach to traumatic brain injury--Year 3

Following traumatic brain injury (TBI), a variety of cellular mediators contribute to neuronal death and dysfunction including the cysteine proteases, calpains. Prolonged activation of calpains occurs within neurons due to a sustained rise of intracellular free calcium. Activated calpains cleave membrane receptors, cytoskeletal elements, mitochondrial proteins, and gene regulatory elements. Calpains have been implicated in the pathophysiology of TBI through the use of pharmacological inhibitors to reduce calpain-mediated proteolysis and behavioral deficits associated with trauma; however, due to the limitations of these compounds to selectively inhibit calpain, the specific role of calpain and the efficacy of calpain inhibition following TBI remains unclear. Few studies have addressed the action of calpastatin, the endogenous and specific inhibitor of calpain, in response to TBI. Although calpastatin is co-expressed with calpains in cells, the sustained activation of these calcium-dependent proteases suggests endogenous calpastatin levels may be insufficient to effectively inhibit calpain activation and its downstream pathogenesis following injury. The central hypothesis of this proposal is that overexpression of calpastatin will reduce the proteolytic activity of calpain and associated neuronal death after trauma, thereby attenuating motor and cognitive deficits. Calpastatin overexpression will be induced in two ways—transgenic overexpression of human calpastatin (hCAST) (Aim 1) and human calpastatin expression via lentiviral vector delivery into brain regions vulnerable to TBI (Aim 2). SPECIFIC AIM 1: Test the hypothesis that calpastatin overexpression in transgenic mice reduces calpain-mediated proteolysis and neuronal death leading to improved behavioral performance following controlled cortical impact (CCI) injury. A novel transgenic mouse line with hCAST under control of the ubiquitous prion promoter will be used to address the functional role of calpain and calpastatin after injury. This mouse line exhibits a 9-fold greater expression of calpastatin in the cortex and hippocampus compared to wildtype mice as determined by immunoblot analysis. hCAST transgenic and wildtype littermates will be subjected to severe (1.0mm) CCI or sham treatment. To confirm that hCAST overexpression decreases calpain activity, cortical and hippocampal homogenates from injured mice will be evaluated for calpainmediated cytoskeletal disruption and cleavage of membrane receptor proteins via immunoblot. An additional cohort of mice will be subjected to severe CCI and euthanized for histological analysis of brain tissue to quantify cortical tissue damage and hippocampal neurodegeneration. To assess motor and cognitive function, injured mice will be tested using neuroscore and neurological severity score assessments and the Morris Water Maze. The hypothesis is that the hCAST transgenic mice will have reduced posttraumatic calpain enzymatic activity, offering a neuroprotective advantage as assessed by histological and behavioral measures. SPECIFIC AIM 2: Test the hypothesis that (a) calpastatin expression can be increased in brain regions vulnerable to TBI through lentiviral vector delivery of calpastatin using convection-enhanced diffusion (CED) and (b) lentiviral overexpression of calpastatin attenuates trauma-induced calpain activity, neuronal death, and behavioral deficits following controlled cortical impact (CCI) injury. An alternative approach to overexpress human calpastatin will be established through use of lentiviral vector delivery. (a) To characterize the lentiviral delivery method and, therefore, optimize local expression of calpastatin, naïve mice will receive injections of either control lentiviral construct or C-terminal FLAG tag human calpastatin lentiviral construct. The area of expression will then be analyzed by fluorescent imaging or anti-FLAG tag immunohistochemistry of brain tissue sections. We will evaluate variations in the rate, volume, and location of infusion using convection-enhanced diffusion in mice, with the goal of achieving increased calpastatin expression throughout regions of the cortex and hippocampus known to be affected by CCI injury. (b) Mice will be injected with control or calpastatin lentivirus and subjected to 1.0mm CCI brain injury or sham treatment once stable viral expression has been achieved. To assess the ability of lentiviral-calpastatin to inhibit calpain activity, calpain-mediated proteolysis of cytoskeletal and membrane proteins in cortical and hippocampal homogenates will be analyzed via immunoblot. Neuronal death will be assessed through measurement of cortical tissue damage and hippocampal neurodegeneration. An additional cohort of mice will undergo neuroscore, neurological severity score, and Morris Water Maze assessments to evaluate motor and cognitive behavior following viral injection and CCI injury. Targeting of calpastatin to vulnerable regions of the brain prior to injury should spare affected neurons and reduce histological and behavioral deficits.