The control of microtubule (MT) dynamics and stability is an important regulatory process during the development of the nervous system, where MTs are involved in neurite outgrowth, and in the adult, where MTs are important for axonal transport and for maintenance of neuronal integrity. Alterations in MT stability may underlie pathological processes, such as in the formation of paired helical filaments (PHFs) in degenerating neurons of individuals afflicted with Alzheimer's disease (AD). PHFs are comprised of the MT-associated protein (MAP), tau, and the PHF tau is in a hyperphosphorylated state. Understanding the mechanism responsible for the abnormal phosphorylation of tau promises to shed light on PHF formation and AD pathology. Previously, the applicant found a critical requirement for one of the major protein phosphatases (PPase), namely PP1, in the maintenance of stable MTs in cultured cells. To extend this original study, they determined whether PPases associated directly with MTs prepared from brain tissue. They have obtained immunological and enzymological evidence that a specific form of PP1 remains tightly associated with MTs through multiple rounds of assembly/disassembly, indicating that it behaves as a MAP. This project will confirm by direct sequence analysis that this MT-associated PPase (or MAPPase) associates with MTs will be investigated, since it is likely that MAPPase is directed to MTs by a targeting subunit. The possibility that this targeting subunit is one of the known brain MAPs will be tested. If the targeting subunit and/or the catalytic subunit of MAPPase are novel, antibody probes will be made and the expression, intracellular location, etc. of the novel proteins will be characterized. Preliminary studies have suggested that MAPPase is active on phosphorylated tau protein and this will be confirmed and phosphorylated tau substrates. The possibility that MAPPase is active on sites phosphorylated in PHF tau will be tested by mapping sites dephosphorylated by MAPPase with antibodies specific for individual phosphorylation sites of PHF tau. Other MAP substrates of MAPPase will also be documented. The idea that MAPPase activity toward tau and other substrates is regulated through its targeting subunit will also be tested. The information gained and techniques developed in this study will help to understand the role of a novel ppase in the regulation of MAP-MT interactions and PHF formation in AD.
