Lipid rafts are membrane microdomains intimately associated with cell signaling. These biochemical microstructures are characterized by their high contents of sphingolipids, cholesterol and saturated fatty acids and a reduced content of polyunsaturated fatty acids (PUFA). Here, we have purified lipid rafts of human frontal brain cortex from normal and Alzheimer's disease (AD) and characterized their biochemical lipid composition. The results revealed that lipid rafts from AD brains exhibit aberrant lipid profiles compared to healthy brains. In particular, lipid rafts from AD brains displayed abnormally low levels of n-3 long chain polyunsaturated fatty acids (LCPUFA, mainly 22:6n-3, docosahexaenoic acid) and monoenes (mainly 18:1n-9, oleic acid), as well as reduced unsaturation and peroxidability indexes. Also, multiple relationships between phospholipids and fatty acids were altered in AD lipid rafts. Importantly, no changes were observed in the mole percentage of lipid classes and fatty acids in rafts from normal brains throughout the lifespan (24-85 years). These indications point to the existence of homeostatic mechanisms preserving lipid raft status in normal frontal cortex. The disruption of such mechanisms in AD brains leads to a considerable increase in lipid raft order and viscosity, which may explain the alterations in lipid raft signaling observed in AD.

Hyperphosphorylation and accumulation of tau in neurons (and glial cells) is one the main pathologic hallmarks in Alzheimer's disease (AD) and other tauopathies, including Pick's disease (PiD), progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17 due to mutations in the tau gene (FTDP-17-tau). Hyperphosphorylation of tau is regulated by several kinases that phosphorylate specific sites of tau in vitro. GSK-3-immunoprecipitated sarcosyl-insoluble fractions in AD have the capacity to phosphorylate recombinant tau. In addition, GSK-3 phosphorylated at Ser9, that inactivates GSK-3, is found in the majority of neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in AD, and in Pick bodies and other phospho-tau-containing neurons and glial cells in other tauopathies. Increased expression of active kinases, including stress-activated kinase, c-Jun N-terminal kinase (SAPK/JNK) and kinase p38 has been found in brain homogenates in all the tauopathies. Strong active SAPK/JNK and p38 immunoreactivity has been observed restricted to neurons and glial cells containing hyperphosphorylated tau, as well as in dystrophic neurites of senile plaques in AD. Moreover, SAPK/JNK- and p38-immunoprecipitated sub-cellular fractions enriched in abnormal hyperphosphorylated tau have the capacity to phosphorylate recombinant tau and c-Jun and ATF-2 which are specific substrates of SAPK/JNK and p38 in AD and PiD. Interestingly, increased expression of phosphorylated (active) SAPK/JNK and p38 and hyperphosphorylated tau containing neurites have been observed around betaA4 amyloid deposits in the brain of transgenic mice (Tg 2576) carrying the double APP Swedish mutation. These findings suggest that betaA4 amyloid has the capacity to trigger the activation of stress kinases which, in turn, phosphorylate tau in neurites surrounding amyloid deposits. Complementary findings have been reported from the autopsy of two AD patients who participated in an amyloid-beta immunization trial and died during the course of immunization-induced encephalitis. The neuropathological examination of the brain showed massive focal reduction of amyloid plaques but not of neurofibrillary degeneration. Activation of SAPK/JNK and p38 were reduced together with decreased tau hyperphosphorylation of aberrant neurites in association with decreased amyloid plaques in both Tg2576 mice and human brains. These findings support the amyloid cascade hypothesis of tau phosphorylation mediated by stress kinases in dystrophic neurites of senile plaques but not that of neurofibrillary tangles and neuropil threads in AD.

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Abnormal tau phosphorylation and deposition in neurones and glial cells is one of the major features in taupathies. The present study examines the involvement of the Ras/MEK/ERK pathway of tau phosphorylation in Alzheimer disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), by Western blotting, single and double-labelling immunohistochemistry, and p21Ras activation assay. Since this pathway is also activated in several paradigms of cell death and cell survival, activated ERK expression is also analysed with double-labelling immunohistochemistry and in situ end-labelling of nuclear DNA fragmentation to visualise activated ERK in cells with increased nuclear DNA vulnerability. The MEK1 antibody recognises one band of 45 kD that identifies phosphorylation-independent MEK1, whose expression levels are not modified in diseased brains. The ERK antibody recognises one band of 42 kD corresponding to the molecular weight of phosphorylation-independent ERK2; the expression levels, as well as the immunoreactivity of ERK in individual cells, is not changed in AD, PiD, PSP and CBD. The antibody MAPK-P distinguishes two bands of 44 kD and 42 kD that detect phosphorylated ERK1 and ERK2. MAPK-P expression levels, as seen with Western blotting, are markedly increased in AD, PiD, PSP and CBD. Moreover, immunohistochemistry discloses granular precipitates in the cytoplasm of neurones in AD, mainly in a subpopulation of neurones exhibiting early tau deposition, whereas neurones with developed neurofibrillary tangles are less commonly immunostained. MAPK-P also decorates neurones with Pick bodies in PiD, early tau deposition in neurones in PSP and CBD, and cortical achromatic neurones in CBD. In addition, strong MAPK-P immunoreactivity is found in large numbers of tau-positive glial cells in PSP and CBD, as seen with double-labelling immunohistochemistry. Yet no co-localisation of enhanced phosphorylated ERK immunoreactivity and nuclear DNA fragmentation is found in AD, PiD, PSP and CBD. Finally, activated Ras expression levels are increased in AD cases when compared with controls. These results demonstrate increased phosphorylated (active) ERK expression in association with early tau deposition in neurones and glial cells in taupathies, and suggest activated Ras as the upstream activator of the MEK/ERK pathway of tau phosphorylation in AD.

Calcium/calmodulin-dependent kinase II (alpha- and beta-CaM kinase II), and phosphorylated mitogen-activated extracellular signal-regulated protein kinase (MAPK/ERK-P), phosphorylated protein kinase of 38 kDa (p38-P) and phosphorylated stress-activated protein kinase (SAPK/JNK-P) expression have been examined in Alzheimer disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The study was carried out to increase understanding of the signals that may regulate tau phosphorylation in tauopathies. MAPK/ERK-P was found in a subset of neurons and glial cells bearing abnormal tau deposition, but rarely in neurofibrillary tangles. Strong p38-P immunoreactivity was observed in about 50-70% of neurons with neurofibrillary tangles and in dystrophic neurites of senile plaques in AD. Strong p38-P immunoreactivity was seen in practically all Pick bodies in PiD, and in most neurons with neurofibrillary degeneration or with tau deposits (pre-tangle neurons) in PSP and CBD, as revealed with single and double-labeling immunohistochemistry to p38-P and tau. In addition, strong p38-P immunoreactivity was present in tau-positive astrocytes and in coiled bodies in PSP and CBD. Single and double-labeling immunohistochemistry to MAPK/ERK-P and p38-P disclosed that MAPK/ERK-P appeared at early stages of tau phosphorylation in neurons and glial cells in tauopathies, and that MAPK/ERK-P and p38-P co-localize only in a subset of neurons and glial cells with phosphorylated tau deposits. SAPK/JNK-P immunoreactivity was seen in a subset of neurons, including many neurons with neurofibrillary degeneration, and in glial cells accumulating abnormal tau, in AD, PiD, PSP and CBD. Double-labeling immunohistochemistry disclosed partial co-localization of SAPK/JNK-P and either MAPK/ERK-P or p-38-P immunoreactivity. These findings indicate that MAPK/ERK-P, SAPK/JNK-P and p-38-P are differentially expressed in association with tau deposits in tauopathies. Finally, CaM kinase II is present in neurons but not in glial cells, thus suggesting no role of CaM kinase II in tau phosphorylation of glial cells. These observations, together with previous results of in vitro studies, support the idea that several MAPK/ERK, SAPK/JNK, p38 and CaM kinase II may participate in tau phosphorylation in tauopathies. Lack of co-localization between MAPK/ERK-P, SAPK/JNK-P and p-38-P over-expression, and staining with the method of in situ end-labeling of nuclear DNA fragmentation in individual cells indicate that over-expression of these kinases is not linked with increased nuclear DNA vulnerability in AD, PiD, PSP and CBD.

Even though the idea that amyloid B peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of B-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimer's disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between B-amyloid and tau alterations may take place in vivo. D

The expression of mitogen-activated protein kinases, extracellular signal-regulated kinases (MAPK/ERK), stress-activated protein kinases, c-Jun N-terminal kinases (SAPK/JNK), and p38 kinases is examined in Parkinson disease (PD), in Dementia with Lewy bodies (DLB), covering common and pure forms, and in age-matched controls. The study is geared to gaining understanding about the involvement of these kinases in the pathogenesis of Lewy bodies (LBs) and associated tau deposits in Alzheimer changes in the common form of DLB. Active, phosphorylation dependent MAPK (MAPK-P) is found as granular cytoplasmic inclusions in a subset of cortical neurons bearing abnormal tau deposits in common forms of DLB. Phosphorylated p-38 (p-38-P) decorates neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in common forms of DLB. Phosphorylated SAPK/JNK (SAPK/JNK-P) expression occurs in cortical neurons with neurofibrillary tangles in the common form of DLB. Lewy bodies (LBs) in the brain stem of PD and DLB are stained with anti-ERK-2 antibodies, but they are not recognized by MAPK-P, SAPK/JNK-P and p-38-P. Yet MAPK-P, p-38-P and SAPK/JNK-P immunoreactivity is found in cytoplasmic granules in the vicinity of LBs or in association with irregular-shaped or diffuse alpha-synuclein deposits in a small percentage of neurons, not containing phosphorylated tau, of the brain stem in PD and DLB. MAPK-P, p-38-P and SAPK-P are not expressed in cortical LBs or in cortical neurons with alpha-synuclein-only inclusions in DLB. MAPK-P, p-38-P and SAPK/JNK-P are not expressed in alpha-synuclein-positive neurites (Lewy neurites) in PD and DLB as revealed by double-labeling immunohistochemistry. These results show that MAPKs are differentially regulated in neurons with alpha-synuclein-related inclusions and in neurons with abnormal tau deposits in DLB. Moreover, different kinase expression in brain stem and cortical LBs suggest a pathogenesis of brain stem and cortical LBs in LB diseases. Finally, no relationship has been observed between MAPK-P, p-38-P and SAPK/JNK-P expression and increased nuclear DNA vulnerability, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation, and active, cleaved caspase-3 expression in neurons and glial cells in the substantia nigra in PD and DLB.

BackgroundThe cellular prion protein (PrPC) fulfils several yet not completely understood physiological functions. Apart from these functions, it has the ability to misfold into a pathogenic scrapie form (PrPSc) leading to fatal transmissible spongiform encephalopathies. Proteolytic processing of PrPC generates N- and C-terminal fragments which play crucial roles both in the pathophysiology of prion diseases and in transducing physiological functions of PrPC. A-disintegrin-and-metalloproteinase 10 (ADAM10) has been proposed by cell culture experiments to be responsible for both shedding of PrPC and its α-cleavage. Here, we analyzed the role of ADAM10 in the proteolytic processing of PrPC in vivo.ResultsUsing neuron-specific Adam10 knockout mice, we show that ADAM10 is the sheddase of PrPC and that its absence in vivo leads to increased amounts and accumulation of PrPC in the early secretory pathway by affecting its posttranslational processing. Elevated PrPC levels do not induce apoptotic signalling via p53. Furthermore, we show that ADAM10 is not responsible for the α-cleavage of PrPC.ConclusionOur study elucidates the proteolytic processing of PrPC and proves a role of ADAM10 in shedding of PrPC in vivo. We suggest that ADAM10 is a mediator of PrPC homeostasis at the plasma membrane and, thus, might be a regulator of the multiple functions discussed for PrPC. Furthermore, identification of ADAM10 as the sheddase of PrPC opens the avenue to devising novel approaches for therapeutic interventions against prion diseases.