Tau-independent microtubule defects in Alzheimer’s disease: the potential for new drug targets

The hallmarks of Alzheimer’s disease (AD) include intracellular neurofibrillary tau tangles and extracellular beta amyloid (Aβ) plaques. The axonal response to plaques results in neurodegeneration, as evidenced by the formation of dystrophic neurites. Levels of BACE1, the enzyme responsible for the conversion of APP to the pathogenic Aβ, are elevated in the peri-plaque region, as is Aβ production. In cultured neurons, we detect Aβ-induced elevated resting Ca2+ levels and cytoskeletal defects. We propose a feed-forward mechanism by which Aβ-induced dystrophy and cytoskeletal defects lead to BACE1 elevation and increased Aβ production, contributing further to plaque and dystrophies. Our hypothesis is that Aβ-induced Ca2+ elevation in neurons causes axonal and cytoskeletal defects, increased BACE1 levels, and accelerated Aβ production in a tau-independent manner, and that stabilizing microtubules and reducing Ca2+ levels is a potential strategy for AD treatment even before tau pathophysiology is present. Aim 1: Determine the tau-independent effects of Aβ on AD pathophysiology. We will treat cultured Tau-/- and wild type neurons with oligomeric Aβ1-42. Using Fura-2 Ca2+ imaging, we will measure Ca2+ levels in neurons. Live imaging of fluorescently labeled tubulin will reveal microtubule defects. Live imaging of BACE1-YFP in these neurons will allow for the study of trafficking. Extensive analysis of fixed cultures stained with antibodies to kinesins, dynein, dynactin, and vesicular markers will allow for the identification of motor proteins and specific vesicular compartments that are affected in both wild type and Tau-/- neurons. 5XFAD/Tau-/- mice will be generated as a tau-independent AD mouse model to study plaques and dystrophies in the brain. Aim 2: Determine the therapeutic potential of microtubule stabilizing agents and CRAC channel inhibitors for treating AD. Since microtubule disruptions occur as a result of Ca2+ elevation, we will use the microtubule stabilizing agents taxol and Epothilone D to prevent and treat Aβ-induced microtubule defects. Cultured neurons will be both be pre-treated with the drugs to determine if the axonal degeneration can be prevented, and also be treated with drugs subsequent to Aβ treatment to determine if axonal disruptions can be alleviated. Similarly, the CRAC channel inhibitor BTP2 will be used to prevent Aβ-induced Ca2+ influx. We anticipate a rescue of Aβ-induced defects when microtubules are stabilized with either taxol or Epothilone D, or CRAC channels are inhibited using BTP2.