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Paired helical filaments (PHFs) in the neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) brains are composed of highly phosphorylated isoforms of tau (PHFtau) that fail to bind microtubules (MTs), and the levels of MT-binding competent tau are decreased in AD brains with abundant PHFtau. Because this loss of MT binding could compromise the viability of tangle-bearing AD neurons by destabilizing MTs, we asked whether these events could be initiated by inhibiting protein phosphatase 1 (PP1) and PP2A in cultured human neurons (NT2N cells) using okadaic acid (OK) and calyculin-A (CL-A). The treatment of NT2N cells with OK and CL-A increased tau phosphorylation, decreased the binding of tau to MTs, and selectively depolymerized the more stable detyrosinated MTs but not the more labile tyrosinated MTs. Significantly, this led to the rapid degeneration of axons, which are enriched in the more stable detyrosinated MTs, and PP2A was implicated in the initiation of this cascade of events because PP2A but not PP1 was closely associated with MTs in the NT2N cells. These studies imply that inactivation of PP2A in vulnerable neurons of the AD brain may play a mechanistic role in the conversion of normal tau into PHFtau, in the depolymerization of stable MTs, and in the degeneration of axons emanating from tangle-bearing neurons.

Tau proteins isolated from paired helical filaments, the major building blocks of Alzheimer's disease neurofibrillary tangle, are abnormally phosphorylated and unable to bind microtubules. To examine the dynamics of tau phosphorylation and to identify specific tau phosphorylation sites involved in the stabilization of microtubules, we treated cultured postmitotic neuron-like cells (NT2N) derived from a human teratocarcinoma cell line (NTera2/D1) with drugs that depolymerize microtubules (i.e. colchicine or nocodazole). This led to the recovery of dephosphorylated tau from the NT2N cells as monitored by a relative increase in the electrophoretic mobility of tau and an increase in the turnover of [ Paired helical filaments (PHFs)1 are the major building blocks of neurofibrillary tangles, neuropil threads, and senile plaque neurites in Alzheimer's disease brains (reviewed in Refs. 1-3). PHFs are composed of abnormally phosphorylated central nervous system (CNS) tau proteins (PHF-tau) (4 -8) that normally promote and stabilize the assembly of microtubules (MTs) (9). Normal adult CNS tau consists of six alternatively spliced isoforms encoded by one gene (10, 11), and all are found in PHF-tau (12), which differs from normal tau in the extent and sites of phosphorylation (2,8,(13)(14)(15)(16). We recently showed that some of the putative "abnormal" phosphorylation sites in PHF-tau are normal sites of phosphorylation in adult rat (17) and adult human brain tau isolated from biopsy samples (18). These studies also showed that rat and human brain tau proteins are phosphorylated to a lesser extent at many of the same sites as in PHF-tau. Significantly, an increase in tau phosphorylation decreases MT binding (19,20), and enzymatic dephosphorylation PHF-tau restores its ability to bind MTs (20). Several phosphorylation sites in tau (including Ser 396 and Ser 262) modulate binding to MTs (20,21), and hyperphosphorylation of these sites may decrease the affinity of PHF-tau for MTs leading to the depolymerization of MTs, impaired axonal transport, neuronal degeneration, and the aggregation of tau into PHFs.The affinity of tau for MTs is regulated by the number of MT binding repeats (22, 23), proline-rich sequences adjacent to the MT binding repeats (24), linker sequences between MT binding repeats (25), and the extent and sites of phosphorylation (19 -21). The expression of different tau isoforms and their phosphorylation states are developmentally regulated (2,11,17,18,20). During human development only the smallest isoform is expressed, and this isoform contains three MT binding repeats and no N-terminal inserts (11). The six adult human brain tau isoforms differ in the number of MT binding repeats and with respect to the presence or absence of N-terminal inserts (11,22), while fetal tau is more phosphorylated than adult tau. Thus, the number of MT binding repeats and phosphorylation may facilitate reorganization of the cytoskeleton during axonal growth and synaptogenesis by modulating the affinity of fetal tau for MTs.Altho...

Lamprey axons regenerate following spinal cord transection despite the formation of a glial scar. As we were unable to detect a lamprey homologue of glial fibrillary acidic protein (GFAP), a major constituent of astrocytes, we studied the composition of intermediate filament (IF) proteins of lamprey glia. Monoclonal antibodies (mAbs) were raised to lamprey spinal cord cytoskeletal extracts and these mAbs were characterized by using Western blotting and immunocytochemistry. On two-dimensional (2-D) Western blots, five of the mAbs detected three major IF polypeptides in the molecular weight (MW) range of 45-56 kD. Further studies were conducted to determine the relationship between the lamprey glial-specific antigen and other mammalian IF proteins. Antikeratin 8 antibody recognized two of the three polypeptides. Several of the glial-specific mAbs reacted with human keratins 8 and 18 on Western blots. Keratin-like immunoreactivity was found in all parts of the central and peripheral nervous systems in both larval and adult lampreys. The immunocytochemical staining patterns of glial-specific mAbs were indistinguishable on lamprey spinal cord sections. However, on brain sections, two distinct patterns were observed. A subset of mAbs stained only a few glial fibers in the brain, whereas others stained many more brain glia, particularly the ependymal cells. The former group of mAbs recognized only the two lower MW polypeptides on 2-D Western blots, but the latter group of mAbs recognized all three major IF polypeptides. This correlation is supported by the observation that the highest MW IF polypeptide has an increased level of expression in the brain relative to the spinal cord. Thus, in the lamprey, the glial cells of both spinal cord and brain express molecules similar to simple epithelial cytokeratins, but their IFs may contain these keratins in different stoichiometric proportions. The widespread presence in the lamprey of primitive glial cells containing keratin-like intermediate filaments may have significance for the extraordinary ability of lamprey spinal axons to regenerate.

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