A novel tau mutation (N296N) in familial dementia with swollen achromatic neurons and corticobasal inclusion bodies.

Familial dementia with swollen achromatic neurons and corticobasal inclusion bodies is a neurodegenerative disease that resembles corticobasal degeneration. It is characterized by the presence of abundant neuronal and glial tau protein deposits. Here we describe a novel silent mutation in exon 10 of tau (N296N) in this familial dementia. By exon trapping, the mutation produced an increase in the splicing in of exon 10, indicating that it probably causes disease through an overproduction of four-repeat tau.

Tau gene mutation K257T causes a tauopathy similar to Pick's disease.

Exonic and intronic mutations in Tau cause neurodegenerative syndromes characterized by frontotemporal dementia and filamentous tau protein deposits. Here we describe a K257T missense mutation in exon 9 of Tau. The proband, a 47-yr-old male, presented with severe personality changes followed by semantic memory loss. A diagnosis of Pick's disease was made. The symptoms progressed until death at age 51. The proband's brain showed a marked frontotemporal atrophy that was most pronounced in the temporal lobes. Numerous tau-immunoreactive Pick bodies were present in the neocortex and the hippocampal formation, as well as in some subcortical brain regions. Their appearance and staining characteristics were indistinguishable from those of sporadic Pick's disease. Diffuse staining for hyperphosphorylated tau was also observed in some nerve cell bodies. Immunoblot analysis of sarkosyl-insoluble tau showed 2 major bands of 60 and 64 kDa and 2 very minor bands of 68 and 72 kDa. Upon dephosphorylation, these bands resolved into 6 bands consisting of 3-repeat and 4-repeat tau isoforms, with an overall preponderance of 3-repeat tau. Isolated tau filaments were narrow, irregularly twisted ribbons. Biochemically, recombinant tau proteins with the K257T mutation showed a reduced ability to promote microtubule assembly, suggesting that this may be the primary effect of the mutation. In addition, the K257T mutation was found to stimulate heparin-induced assembly of 3-repeat tau into filaments. Taken together, the present findings indicate that the K257T mutation in Tau can cause a dementing condition similar to Pick's disease.

A new case of frontotemporal dementia and parkinsonism resulting from an intron 10 +3-splice site mutation in the tau gene: clinical and pathological features.

Hereditary frontotemporal dementia and parkinsonism (FTDP) linked to chromosome 17 (FTDP-17) constitutes a new form of tauopathy, and mutations in the tau gene have recently been reported in some affected families. This report presents clinical and neuropathological data from a member of a British family (SOT 254) with a history of dementia and movement disorder. The medical history of the affected patient, a woman aged 44 years, was reviewed, and a detailed post-mortem examination of the brain was undertaken. A panel of well characterized phosphorylation-dependent and independent anti-tau antibodies was used to assess tau pathology, and inclusions were examined by electron microscopy. Neuronal loss and gliosis were widely distributed, but most severe in neocortical regions, and were associated with abundant neuronal and glial tau inclusions which consisted of a mixture of paired helical filaments (PHFs), similar to those in Alzheimer's disease, and distinct twisted ribbon-like filaments. Genomic DNA was obtained from post-mortem tissue from the index patient, and blood from two unaffected members of the same family. For the index case only, sequencing of intronic sequences flanking exon 10 of the tau gene identified a G to A transition at position +3 of the splice-donor site downstream of exon 10, identical to that reported in multiple system tauopathy with presenile dementia (MSTD). The clinical, neuropathological and genetic findings strongly suggest that SOT 254 represents a new example of FTDP-17 resulting from a mutation in the tau gene. These results are compared with those reported for other FTDP-17 families, i.e. for MSTD.

Induction of two DNA mismatch repair proteins, MSH2 and MSH6, in differentiated human neuroblastoma SH-SY5Y cells exposed to doxorubicin.

The MutS homologues MSH2 and MSH6 form a heterodimeric protein complex that is involved in the recognition of base/base mismatches and insertion/deletion loops, as well as some other types of DNA damage. We investigated the expression of these proteins in undifferentiated and retinoic acid-differentiated human neuroblastoma SH-SY5Y cells by immunocytochemistry, western blot analysis, and RT-PCR. Nuclei from undifferentiated SH-SY5Y cells were found to be immunoreactive to anti-MSH2 and anti-MSH6 antibodies. Following differentiation, the cells stop dividing and change morphology to acquire a neuron-like phenotype. Under these conditions, both anti-MSH2 and anti-MSH6 immunoreactivities were still detectable, although the signals were somewhat less intense. When these cells were exposed for 2 h to neurotoxic concentrations of doxorubicin (50 nM), they exhibited a marked and homogeneous increase of both anti-MSH2 and anti-MSH6 immunoreactivities. As revealed by western blot analysis, these effects were associated with increased protein content and were dose-dependent. Using RT-PCR technology, we also found that doxorubicin treatment did not change MSH2 or MSH6 mRNA levels. Our data indicate that human postmitotic, neuron-like cells constitutively express the molecular machinery devoted to recognition of DNA mismatches and that this system is activated by specific treatment leading to cell death. These findings might help clarify the molecular mechanisms underlying various human neurological diseases that are associated with deficiencies in DNA repair and/or a high rate of DNA damage acquisition.

Distribution and kainate-mediated induction of the DNA mismatch repair protein MSH2 in rat brain.

DNA repair is one of the most essential systems for maintaining the inherited nucleotide sequence of genomic DNA over time. Repair of DNA damage would be particularly important in neurons, because these cells are among the longest-living cells in the body. MSH2 is one of the proteins which are involved in the recognition and repair of a specific type of DNA damage that is characterized by pair mismatches. We studied the distribution of MSH2 in rat brain by immunohistochemical analysis. We found the level of MSH2 expression in rat brain to be clearly heterogeneous. The highest intensity of staining was found in the pyramidal neurons of the hippocampus and in the entorhinal and frontoparietal cortices. Positive cells were observed in the substantia nigra pars compacta, in cerebellar granular and Purkinje cells, and in the motor neurons of the spinal cord. We investigated the possible modulation of MSH2 expression after injection of kainate. Systemic administration of kainate induces various behavioural alterations and a typical pattern of neuropathology, with cell death in the hippocampal pyramidal neurons of the CA3/CA4 fields. Kainate injection also resulted in a marked, dose-dependent increase of MSH2 immunoreactivity in the hippocampal neurons of the CA3/CA4 fields. The effect was specific, since no changes in immunoreactivity were detected in the dentate gyrus nor in other brain areas. In summary, our data suggest that a mismatch DNA repair system, of which MSH2 protein is a representative component, is heterogeneously expressed in the rat brain and specifically induced by an experimental paradigm of excitotoxicity.

Characterization of tau proteins in human neuroblastoma SH-SY5Y cell line.

Here we report three experimental paradigms in which tau proteins are differentially localized and expressed in human neuroblastoma cells SH-SY5Y. We found that in undifferentiated cells, tau proteins were predominantly localized in the nucleus. Western blot analysis of nuclear extracts revealed, among the others, a high molecular weight tau isoform and evaluation of tau mRNA levels showed a single tau isoform. After differentiation, tau immunoreactivity was detected only in cytosol and along neuritic processes. The high molecular weight tau isoform disappeared and an additional tau mRNA species was detected. Treatment of differentiated cells with doxorubicin or okadaic acid resulted in an increase of tau immunoreactivity and in a subsequent cell loss. Our results indicate that both subcellular localization and pattern of expression of tau proteins vary depending on the developmental and functional state of the cells, thus suggesting different roles in cell function.

Priming of cultured neurons with sabeluzole results in long-lasting inhibition of neurotoxin-induced tau expression and cell death.

Sabeluzole was described to have antiischemic, antiepileptic, and cognitive-enhancing properties, and is currently under development for Alzheimer's disease. Recently, it was reported that repeated treatments with sabeluzole protect cultured rat hippocampal neurons against NMDA- and glutamate-induced neurotoxicity. We evaluated the possibility that sabeluzole elicits neuroprotection by acting, either directly or indirectly, on tau proteins. We found that repeated treatments during development of primary cultures of cerebellar granule cells with nanomolar concentrations of sabeluzole resulted in mature cells that were resistant to the excitotoxicity induced by glutamate. Also, sabeluzole treatment specifically prevented the glutamate-induced increase of tau expression without modifying the basal pattern of expression of tau proteins, as shown by measurement of mRNA and protein levels. In human neuroblastoma cell line SH-SY5Y, differentiated by treatment with retinoic acid, doxorubicin increased tau immunoreactivity, and later induced cell death. Both effects were prevented by sabeluzole. Our data indicate that increased tau expression is a common response to different types of cells to neurotoxic agents, and that sabeluzole-induced neuroprotection is functionally associated with the prevention of the injury-mediated increase of tau expression.