Traumatic brain injury (TBI) afflicts approximately 1.4 million individuals in the U.S. each year. Following 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 rapid and sustained rise of intracellular free calcium. Although an endogenous inhibitor of calpains, calpastatin, is co- expressed, the sustained activation of these calcium-dependent proteases suggests endogenous calpastatin levels may be insufficient. The overall hypothesis of this proposal is that overexpression of calpastatin will reduce the proteolytic activity of calpains 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 calpastatin expression via lentiviral vector delivery into brain regions vulnerable to TBI (Aim 2).
Aim 1 will use a novel transgenic mouse line with human calpastatin under control of the ubiquitous prion promoter. This mouse line exhibits a 9-fold greater expression of calpastatin in the cortex and hippocampus compared to wildtype mice. hCAST transgenic and wildtype littermates will be subjected to severe controlled cortical impact (CCI) injury or sham treatment. To confirm that calpastatin overexpression decreases calpain activity, cortical and hippocampal homogenates will be evaluated for calpain-mediated cytoskeletal and membrane protein breakdown via immunoblot. Both motor and cognitive functions will be assessed after injury, after which mice will be euthanized for analysis of hippocampal neurodegeneration and cortical tissue damage to assess the neuroprotective actions of hCAST overexpression. The expectation is that hCAST transgenic mice will have reduced posttraumatic calpain proteolytic activity, offering a neuroprotective advantage.
Aim 2 establishes an alternative approach to hCAST overexpression through lentiviral vector delivery. After injection of control lentivirus or calpastatin lentivirus into the cortex or hippocampus, mice will be subjected to 1.0mm CCI brain injury or sham treatment and assessed as in Aim 1. Targeting of calpastatin to vulnerable neuronal regions prior to injury should spare affected neurons and reduce behavioral deficits.

Public Health Relevance

Given the scope of traumatic brain injury, it is imperative to understand the mechanisms of neuronal damage and death in order to develop effective treatment strategies.
My research aims will not only seek to establish a functional, neuroprotective role for calpastatin in brain trauma but also advance translational efforts for the treatment of brain injury.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31NS071804-02
Application #
8222810
Study Section
Special Emphasis Panel (ZRG1-F01-L (20))
Program Officer
Hicks, Ramona R
Project Start
2011-01-05
Project End
2013-07-04
Budget Start
2012-01-05
Budget End
2013-01-04
Support Year
2
Fiscal Year
2012
Total Cost
$30,851
Indirect Cost
Name
University of Kentucky
Department
Physiology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Schoch, Kathleen M; Madathil, Sindhu K; Saatman, Kathryn E (2012) Genetic manipulation of cell death and neuroplasticity pathways in traumatic brain injury. Neurotherapeutics 9:323-37