Alpha 1-antichymotrypsin (ACT) is an acute phase serum glycoprotein that belongs to a class of serine protease inhibitors named serpins and is an integral component of the amyloid plaques in Alzheimer's disease (AD) patients. It is greatly overexpressed in the astrocytes surrounding the affected area in brain and has been shown to enhance A-beta fibrillization and plaque formation. The localization and fibrillogenic activity prompted us to ask whether ACT also played a role in tangle formation in AD. Studies conducted in brain samples from tauopathy patients, brains from transgenic mice overexpressing human ACT, and in vitro cultured cortical neurons exposed to purified ACT showed a correlation between higher levels of ACT and tau hyperphosphorylation, suggesting that ACT overexpression in AD contributes to tangle formation. ACT treated neurons showed hyperphosphorylation of tau at PHF-1 (P-Ser396/404), P-Ser202 and P-Thr231, the sites that showed hyperphosphorylation in Alzheimer's disease brain, and the neurons underwent apoptosis suggesting that ACT plays a significant role in both tau hyperphosphorylation and neurodegeneration. Here we propose to unravel the pathological significance of and the mechanisms involved in ACT-induced tau phosphorylation and apoptosis using transgenic mice overexpressing ACT and human tau. Our preliminary studies in cultured neurons suggest that glycogen synthase kinase 3 (GSK-3 alpha and beta), extracellular signal regulated kinase ERK, and one or more roscovitine (a cyclin dependent kinase inhibitor of cdk2, cdk5 and cdc2)-sensitive kinases may be involved in ACT-induced tau hyperphosphorylation. Utilizing inhibitors of different kinases, we propose to identify the mechanisms involved in ACT-induced tau hyperphosphorylation and neuronal apoptosis in vivo. In order to determine the effect of kinase inhibitors on ACT-induced tau hyperphosphorylation in vivo we will infuse ACT alone or with kinase inhibitors into the brains of htau mice using an Alzet pump. At the end of the infusion period the brains will be analyzed using P-Thr231, PHF-1, AT100, and P-Ser202 specific tau antibodies to determine the efficacy of the inhibitors on ACT-induced tau phosphorylation. Apoptosis in the brains of these mice will be analyzed by TUNEL assay and/or FLIVOTM in vivo detection method. We will examine the brains of the ACT/htau mice for glial activation using astrocyte-specific GFAP and microglia-specific cd11b antibodies. Any alterations in cytokine levels will be detected by multi-plex bead-based assay using LUMINEX and ELISA techniques. ACT has been shown to induce behavioral deficits in APP mice. Whether ACT induces similar deficits in tau mice will be analyzed using a set of different sensorimotor and cognitive tasks. Although earlier studies were conducted in ACT/APP mice, the ACT/hTau mice will be more appropriate for the proposed studies as human tau mice show a significant level of tau pathology and neuronal apoptosis. These studies will provide insights into the mechanisms involved in ACT's role in development of tau pathologies that are associated with AD or tauopathies, and will identify novel targets for development of therapies towards AD.
Along with neuritic plaques and neurofibrillary tangles Alzheimer's disease is characterized by neuroinflammation and neuronal loss. Our in vivo experiments with alpha 1- antichymotrypsin (ACT), an inflammatory protein overexpressed in Alzheimer's disease brain, are designed to help identify the regulatory mechanisms involved in inflammation-induced tau hyperphosphorylation and neuronal apoptosis leading to possible behavioral deficits. An understanding of the specific molecules involved in inflammation-induced neurodegeneration will allow us to develop novel drugs that will interfere with the disease development at an early stage.
