Jean Pierre Brion

Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau : generation of paired helical filament epitopes and neuronal localisation of the kinase

Glycogen synthase kinase-3 (GSK-3) reduced the mobility of human tau on SDS-PAGE, prevented binding of the monoclonal antibody (mAb), Tau.1, and induced binding of the mAb 8D8. Recombinant tau phosphorylated by GSK-3 aligned on SDS-PAGE with the abnormally phosphorylated tau (PHF-tau) associated with the paired helical filaments in Alzheimers disease brain. Phosphorylated serine396 (numbering of the largest human brain tau isoform) was identified as a binding site on tau for mAb 8D8. The localisation of GSK-3 within granular structures in pyramidal cells indicates that GSK-3 alpha and GSK-3 beta may have a role in the production of PHF-tau in Alzheimers disease.

Increased level of active GSK-3β in Alzheimer’s disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration

The somatodendritic accumulation of hyperphosphorylated tau proteins is an early event preceding the appearance of neurofibrillary tangles (NFT) in Alzheimer’s disease (AD) and might be necessary for their formation. Glycogen synthase kinase‐3β (GSK‐3β) is a physiological kinase for tau that generates many tau phosphorylation sites identified in NFT and in other tau‐positive inclusions. We have studied the cellular distribution and the expression of the active form of GSK‐3β (GSK‐3 pTyr216) in AD patients, in argyrophilic grain disease and in diffuse Lewy body disease. By Western blotting analysis, a significant increase in the level of GSK‐3 (pTyr216) was observed in the frontal cortex of AD patients. A population of neurones showed a somatodendritic accumulation of GSK‐3 (pTyr216) but not of the inactive form of GSK‐3β (GSK‐3 pSer9). Most of these GSK‐3 (pTyr216)‐positive cells were positive for six different phosphotau epitopes known to be generated by GSK‐3β. By using a quadruple labelling method using GSK‐3 (pTyr216) and phosphotau immunolabelling combined with Gallyas and DAPI staining, we examined neurones containing a somatodendritic GSK‐3 (pTyr216) immunoreactivity at different stages of neurodegeneration. A majority of neurones at the pretangle stage without Gallyas‐positive inclusions were GSK‐3 (pTyr216) positive and this GSK‐3 (pTyr216) immunoreactivity remained in most cells containing Gallyas and phosphotau‐positive inclusions excepted in extracellular NFT. A GSK‐3 (pTyr216) immunoreactivity was present in argyrophilic grains but not in cortical Lewy bodies. These results directly suggest that the activity of GSK‐3β is increased in AD and that somatodendritic accumulation and activation of GSK‐3β is an early event preceding and accompanying the formation of NFT and of other tau‐positive inclusions.

Transgenic expression of the shortest human tau affects its compartmentalization and its phosphorylation as in the pretangle stage of Alzheimer's disease.

We have generated transgenic mice expressing the shortest human tau protein, the microtubule-associated protein that composes paired helical filaments in Alzheimers disease. Transgenic tau transcripts and proteins were strongly expressed in neurons in the developing and adult brain. In contrast to the endogenous tau that progressively disappeared from neuronal cell bodies during development, the human transgenic tau remained abundant in cell bodies and dendrites of a subset of neurons in the adult. This somatodendritic transgenic tau was immunoreactive with antibodies to tau phosphorylated on Thr181 and Thr231 and with the conformation-dependent Alz50 antibody. A few astrocytes expressing the transgenic tau were strongly immunoreactive with antibodies to additional tau phosphorylation sites, ie, at Ser262/ 356 and Ser396/404. All of these phosphorylation sites have been identified in paired helical filaments-tau proteins. In electron microscopy, the transgenic tau was detected into microtubules in axons and in dendrites but not in cell bodies. Neurofibrillary tangles were not detected in transgenic animals examined up to the age of 19 months. These results indicate that transgenic manipulation of tau expression and intracellular targeting is sufficient per se to affect tau compartmentalization, phosphorylation, and conformation partly as it is observed at the pretangle stage in Alzheimers disease.

Developmental expression and localization of glycogen synthase kinase-3β in rat brain

Glycogen synthase kinase (GSK)-3beta is a protein kinase in the wingless/wnt pathway and as such is involved in the regulation of growth and development of the neural tissue in Drosophila and in vertebrates. This enzyme is also abundantly expressed in the mammal adult brain, where it might play a role in the regulation of several substrates. The expression and the neuroanatomical distribution of GSK-3beta immunoreactivity in the rat brain from embryonic up to adult stages has been studied. GSK-3beta was expressed in the developing brain with the highest expression observed from 18 days of embryonic life up to 10 days of postnatal life. Its expression decreased thereafter and was lowest in the adult. GSK-3beta was strongly expressed in developing neurons but only weakly expressed in layers containing neuroblasts. In the adult and during development, GSK-3beta was detected in the pericarya and proximal part of dendrites. In the embryo, an intense GSK-3beta immunoreactivity was also observed in axonal tracts. This axonal immunoreactivity had markedly decreased by 10 days of postnatal life and was absent at 20 days of postnatal life and in the adult. No GSK-3beta immunoreactivity was detected in astrocytes. The GSK-3beta immunoreactivity was found in most brain regions, although significant local variations of GSK-3beta expression were observed. The developmental evolution of GSK-3beta compartmentalization in neurons parallels that of phosphorylated tau, a protein considered to be a physiological substrate for the kinase.

Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation.

In vitro evidence has suggested a change in the ability of tau bearing mutations associated with fronto‐temporal dementia to promote microtubule assembly. We have used a cellular assay to quantitate the effect of both isoform differences and mutations on the physiological function of tau. Whilst all variants of tau bind to microtubules, microtubule extension is reduced in cells transfected with 3‐relative to 4‐repeat tau. Mutations reduce microtubule extension with the P301L mutation having a greater effect than the V337M mutation. The R406W mutation had a small effect on microtubule extension but, surprisingly, tau with this mutation was less phosphorylated in intact cells than the other variants.

The active form of glycogen synthase kinase-3beta is associated with granulovacuolar degeneration in neurons in Alzheimer's disease.

Abstract. Glycogen synthase kinase-3β (GSK-3β) is a physiological kinase for tau and is a candidate protein kinase involved in the hyperphosphorylation of tau present in paired helical filament (PHF)-tau of neurofibrillary tangles (NFT) in Alzheimers disease (AD). GSK-3β is also a key element of several signaling cascades (including cell death cascades). We have investigated the immunocytochemical localization of GSK-3 immunoreactivity in AD. Neurons exhibiting strongly GSK-3-immunoreactive granules were observed in AD, with a much higher frequency than in control subjects. This immunoreactivity was found to co-localize with the granulovacuolar degeneration (GVD) and to be associated with the granules of the granulovacuolar bodies. The GVD granules showed a strong GSK-3α and GSK-3β immunoreactivity, and this immunoreactivity was abolished by preabsorption with recombinant GSK-3. In addition, the GVD immunoreactivity was observed with an antibody against the tyrosine-phosphorylated and active form of GSK-3. Some granules of the granulovacuolar degeneration were also intensely labeled with an antibody specific for tau isoforms containing insert 1 (exon 2) and with antibodies specific for tau phosphorylated on Ser262 and for tau phosphorylated on Thr212/Ser214, two phosphorylation sites generated in vitro by GSK-3α and β. GSK-3β was expressed in neurons containing NFT but only a small proportion of intracellular NFT were observed to be GSK-3β immunoreactive. Immunoblotting analysis of fractions enriched in PHF-tau did not reveal any GSK-3β immunoreactivity in these fractions, indicating that GSK-3β was only loosely associated to NFT. These results suggest that neurons developing GVD sequester an active, potentially deleterious, form of GSK-3 in this compartment and that increased GSK-3 immunoreactivity in a subset of neurons quantitatively differentiates normal aging from AD.

Abnormalities of Wnt signalling in schizophrenia - evidence for neurodevelopmental abnormality

THE Wnt signalling pathway is central to normal brain development in vertebrates and invertebrates and mediates cell fate determination, cell adhesion and cell proliferation. However, its relevance to disorders of cerebral development in man is untested. We evaluated the potential involvement of the Wnt signalling pathway in schizophrenia, a disorder of neurodevelopment origin in which alterations in neuronal lamination and orientation have been described. Using immunohistochemistry and semi-quantitative rating scales, we examined the distribution of two components of the Wnt signalling pathway, β-catenin and γ-catenin in the hippocampus and subiculum of 12 schizophrenic (DSMIIR criteria) and 14 control subjects. Both catenins were distributed as intraneuronal diffuse and/or ring shaped forms. The diffuse staining of both forms catenin were reduced in the CA3 and β-catenin was also reduced in the CA4 hippocampal subregion among schizophrenic subjects. These alternations may represent the basis of the developmental brain abnormalities found in schizophrenia and would have functionally important consequences in the adult.

Tanycytes: morphology and functions. A review

Publisher Summary The chapter presents a review of the morphology and functions of tanycytes. The name “tanycyte,” derived from the Greek word “tanus,” which means “elongated,” was chosen to stress the shape of these bipolar cells, in relation at their apical pole with the infundibular recess of the third ventricle and, at their distal pole, with the portal vessels of the median eminence and the floor of the brain. The morphology of tanycytes has been thoroughly explored by means of photonic observations as well as by transmission (TEM) and scanning (SEM) electron microscopy. The review of the chapter will be restricted to the tanycytes lining the ventral region of the third ventricle in mammals, thus, excluding the study of similar cells lining the circumventricular organs (subfornical organ, subcommissural organ, organum vasculosum of the lamina terminalis), the aqueduct, and the floor of the fourth ventricle. The possible functions of the tanycytes will also be reviewed. The Golgi impregnation has been used by several authors to study the morphology of the tanycytes in mammals. Because of the thickness of the sections impregnated by this method, it is possible in favorable incidences to follow the entire length of these elongated cells, allowing a dissection in situ. This method demonstrates two main types of tanycytes according to their location and their morphology. The ventral tanycytes are located on the floor and lower third of the infundibular recess. Dorsal tanycytes have a longer tail process, starting from the base of the tanycyte and arching ventrolaterally in the neuropil of the arcuate nucleus. Glycerophosphate dehydrogenase (GPDH), lactic dehydrogenase (LDH), and glucosed-phosphate dehydrogenase (G6PD) are more active in the perikaryons of β tanycytes as compared to α.

Tau, paired helical filaments and amyloid in the neocortex: a morphometric study of 15 cases with graded intellectual status in aging and senile dementia of Alzheimer type.

SummaryTau immunoreactivity was studied in temporal neocortex, area 22, in 15 cases with graded intellectual status and compared with the immunoreactivity observed with an antiserum against paired helical filaments (PHF) and with the density of amyloid revealed by thioflavin S. Samples came from women over 75 years either intellectually normal or affected by senile dementia of the alzheimer type at various degrees of severity. Mental status had been prospectively assessed by the Blesseds test score. Antitau labelled a neuropil meshwork, the density of which increased with the severity of the disease. This meshwork was denser in layers II, III and V in the most affected cases. The number and the size of the taupositive fibers within the senile plaques increased with the intellectual deficit. Senile plaques were more numerous in layers II and III and neurofibrillary tangles in layers III and V whatever the staining technique: tau or PHF immunocytochemistry, and thioflavin S. The densities of senile plaques and of neurofibrillary tangles (NFT) were correlated with the severity of the disease whatever the staining method. The three methods revealed a systematically different number of changes. This systematic difference could greatly influence the neuropathological diagnosis. It could be the consequence of various factors: different sensitivities of the staining methods or changes in the antigenic and amyloid composition of the lesion according to the stage of the disease. In line with the last hypothesis, a higher proportion of amyloid-rich plaques was noted in the less affected cases, suggesting that tau and PHF epitopes appeared secondarily. Tau epitopes seemed to be present at least as early as PHF epitopes in the NFT. The pathological changes best linked to dementia were NFT revealed by tau antiserum.

Lithium reduces tau phosphorylation: effects in living cells and in neurons at therapeutic concentrations.

BACKGROUND The mechanism of action of lithium remains to be determined satisfactorily. Recent studies suggested a possible role in inhibiting glycogen synthase kinase-3 (GSK-3), previously shown to phosphorylate the protein tau. Tau is expressed mainly in neurons, where it functions to stabilize microtubules in a phosphorylation-dependent manner. METHODS Neurons and transfected non-neuronal cells were treated with lithium and the phosphorylation of tau at multiple epitopes examined by western blotting and by immunocytochemistry. Using green fluorescent protein as a tag we examined the effects of lithium on phosphorylated tau in living cells. RESULTS Lithium reversibly reduced tau phosphorylation at therapeutic concentrations, and even at high concentrations did not alter neuronal morphology. Green fluorescent protein tagged-tau when phosphorylated by GSK-3 was diffusely distributed; treatment with lithium resulted in association with microtubules and then bundle formation. Removing lithium allowed observation of the dissolution of bundles and gradual dissociation of tau from microtubules in living cells. CONCLUSIONS Lithium may have multiple effects in brain, but at least one action is demonstrated to be a relative inhibition of GSK-3-induced tau phosphorylation. These results carry implications for future studies of the actions of mood-stabilizing drugs and indeed of the molecular mechanisms of affective disorders.