Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent proteintubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.

In Alzheimer disease brain the activities of protein phosphatase (PP)-2A and PP-1 are decreased and the microtubule-associated protein tau is abnormally hyperphosphorylated at several sites at serine/threonine. Employing rat forebrain slices kept metabolically active in oxygenated artificial CSF as a model system, we investigated the role of PP-2A/PP-1 in the regulation of some of the major abnormally hyperphosphorylated sites of tau and the protein kinases involved. Treatment of the brain slices with 1.0 W WM okadaic acid inhibited V V65% of PP-2A and produced hyperphosphorylation of tau at Ser 198/199/202, Ser 396/404 and Ser 422. No significant changes in the activities of glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinases cdk5 and cdc2 were observed. Calyculin A (0.1 W WM) inhibited V V50% PP-1, V V20% PP-2A, 50% GSK-3 and V V30% cdk5 but neither inhibited the activity of cyclin AMP dependent protein kinase A (PKA) nor resulted in the hyperphosphorylation of tau at any of the above sites. Treatment of brain slices with 1 W WM okadaic acid plus 0.1 W WM calyculin A inhibited V V100% of both PP-2A and PP-1, V V80% of GSK-3, V V50% of cdk5 and V V30% of cdc2 but neither inhibited PKA nor resulted in the hyperphosphorylation of tau at any of the above sites. These studies suggest (i) that PP-1 upregulates the phosphorylation of tau at Ser 198/199/202 and Ser 396/404 indirectly by regulating the activities of GSK-3, cdk5 and cdc2 whereas PP-2A regulates the phosphorylation of tau directly by dephosphorylation at the above sites, and (ii) that a decrease in the PP-2A activity leads to abnormal hyperphosphorylation of tau at Ser 198/199/202, Ser 396/404 and Ser 422. ß

The regulation of the activity of CaMKII by PP-1 and PP-2A, as well as the role of this protein kinase in the phosphorylation of tau protein in forebrain were investigated. The treatment of metabolically active rat brain slices with 1.0 W WM okadaic acid (OA) inhibited V V65% of PP-2A and had no significant effect on PP-1 in the 16 000U Ug tissue extract. Calyculin A (CL-A), 0.1 W WM under the same conditions, inhibited V V50% of PP-1 and V V20% of PP-2A activities. In contrast, a mixture of OA and CL-A practically completely inhibited both PP-2A and PP-1 activities. The inhibition of the two phosphatase activities or PP-2A alone resulted in an V V2-fold increase in CaMKII activity and an V V8-fold increase in the phosphorylation of tau at Ser 262/356 in 60 min. Treatment of the brain slices with KN-62, an inhibitor of the autophosphorylation of CaMKII at Thr 286/287, produced V V60% inhibition in CaMKII activity and no significant effect on tau phosphorylation at Ser 262/356. The KN-62-treated brain slices when further treated with OA and CL-A did not show any change in CaMKII activity. In vitro, both PP-2A and PP-1 dephosphorylated tau at Ser 262/356 that was phosphorylated with purified CaMKII. These studies suggest (i) that in mammalian forebrain the cytosolic CaMKII activity is regulated mainly by PP-2A, (ii) that CaMKII is the major tau Ser 262/356 kinase in brain, and (iii) that a decrease in PP-2A/ PP-1 activities in the brain leads to hyperphosphorylation of tau not only by inhibition of its dephosphorylation but also by promoting the CaMKII activity. ß

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The abnormal hyperphosphorylation of tau protein is one of the hallmarks of Alzheimer disease and other tauopathies; as yet the exact role of various tau kinases in this pathology is not fully understood. Here, we show that injection of isoproterenol, an activator of cAMP-dependent kinase (PKA), into rat hippocampus bilaterally results in the activation of PKA, calcium/ calmodulin-dependent kinase II and cyclin-dependent kinase-5, inhibition of protein phosphatase-2A, hyperphosphorylation of tau at several Alzheimer-like epitopes and a disturbance of spatial memory retention 48 h after the drug injection. These findings suggest the involvement of PKA and PKA-mediated signaling pathway in the Alzheimer-like tau hyperphosphorylation and memory impairment.

The intellectual status of 28 women of over 75 years of age had been prospectively assessed by the Blessed test score. It ranged from nearly normal to deeply altered by dementia. After autopsy, the supramarginal gyrus was marked at the surface of the brain. Sections, 1 cm thick, were cut with a specially devised macrotome. The volume of the parietal lobe was measured by a point counting method, using Cavalieri principle. A sample from the supramarginal gyrus was taken from the previously marked area and the shrinkage due to the histological procedures was measured (it averaged 12%). More than 500 nucleolated neuronal profiles per case were mapped with a semi-automatic system. Density maps of the neuronal profiles were drawn and mean density was calculated using Dirichlet tessellation. The thickness of the cortical ribbon was standardized on the maps. The density of the neurons per unit volume was calculated, taking into account the section thickness measured for each sample with a length gauge fastened to the Z axis of the microscope. Statistical correlations were sought between the mean and laminar densities of the neurons on one hand, and Blessed test score, the densities of neurofibrillary tangles (NFT) and of senile plaques profiles, on the other hand. Finally, the total number of neurons present in the parietal lobe was estimated in each case. Neuronal loss appeared to be linked with the density of the NFT (r = -0.52; P < 0.004). The correlation was mainly due to a severe drop in neuronal number observed in the cases with more than 5 NFT/mm2. An average difference of 98 x 10(6) neurons per parietal lobe was found between the cases with less than 5 NFT/mm2 and those with more. The neuronal loss predominated in layers II and III (upper part). A multivariate analysis showed that the intellectual status was better correlated with the density of the tangles than with the neuronal loss.

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