Pilot Project: Role of specific calpain isoforms in mild cognitive impairment and Alzheimer's disease

In the United States there has been a major increase in life expectancy over the past century and with this there are approximately 35 million people 65 or older. Of these, roughly 5 million have Alzheimer's Disease (AD). One contributing factor is age-related calcium dysregulation in neurons, causing activation of calpains--a family of calcium-activated proteases. Excessive calpain activation is implicated in numerous components of AD pathology including Amyloid ? protein (A?) deposition, tau cleavage and phosphorylation, somatodendritic dystrophy, and neuronal degeneration. The calpain family consists of fifteen isoforms, seven of which (calpains 1, 2, 5, 7, 10, 12, and 15) are expressed in the CNS. Although calpain activation is implicated in the pathogenesis of AD, it is unknown which calpain isoforms are involved and previous studies have not examined calpains in mild cognitive impairment (MCI), an intermediate stage between intact cognition and AD. In preliminary studies using brain tissue obtained from the Sanders-Brown ADC repository, we examined mRNA and protein levels of calpains 1, 2, 5, 7 and 10 in posterior cingulate cortex, frontal cortex (area 9), and cerebellum obtained postmortem from six individuals with AD, six with MCI, and six age-matched individuals without cognitive impairment. Our preliminary results indicate that elevated calpain 7 expression is associated with MCI, with increased calpain 2 levels occurring later in the disease progression to AD. This proposal examines the hypothesis that calpain 2 and calpain 7 contribute to neuropathology associated with MCI and AD. Specifically, Aim 1 will examine the hypothesis that calpain 2 and 7 expression is elevated in vulnerable areas (posterior cingulate cortex and prefrontal cortex) compared to less affected areas (cerebellum and motor cortex) in postmortem brains from MCI and AD patients. It is proposed that by increasing the current sample size from n=6 in each group to n=18 (based on power analysis) will yield a more accurate analysis of the expression of individual calpain isoforms in MCI and AD. AD pathology in these samples will be assessed by examination of the neuropathological slides including tau and A? immunocytochemistry, as well as measurement of ?-amyloid precursor protein (APP), A? and APP C-terminal fragments (CTFs); and tau protein levels and phosphorylation in brain homogenates along with analysis of protein and mRNA levels of individual calpain isoforms. Aim 2 will evaluate the hypothesis that reduced levels or activity of calpain 2 or calpain 7 decreases AD-like pathology in AD mouse models. Knockdown will be achieved using lentiviral shRNA vectors injected unilaterally into the hippocampal formation of mice prior to accumulation of AD-like neuropathology. The mouse models to be utilized include 5xFAD mice, which develop senile plaques by six weeks of age, and rTg4510 inducible tau model. AD neuropathology will be assessed in mice at six months of age using immunohistochemical analysis of A?1-42 and tau protein/phosphorylation; western blot analysis of P35/p25, CDK5 activation and APP CTFs; and enzyme-linked immunosorbent assays (ELISAs) for tau phosphorylation and A?1-42. As cognitive impairment in mouse models of AD-like pathology is unclear, our analysis will focus on neuropathology. Because calpains serve important physiological roles, targeting specific calpain isoforms is a preferred potential therapeutic strategy as compared to a broad spectrum calpain inhibitor. Our research program will provide insight into role of calpains, particularly calpains 2 and 7, in the pathogenesis of AD-related neuropathology.