ALS-associated protein FIG4 is localized in Pick and Lewy bodies, and also neuronal nuclear inclusions, in polyglutamine and intranuclear inclusion body diseases.

FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. Mutations of FIG4 lead to the development of Charcot-Marie-Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS-associated proteins (transactivation response DNA protein 43 (TDP-43), fused in sarcoma (FUS), optineurin, ubiquilin-2, charged mutivesicular body protein 2b (CHMP2B) and valosin-containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP-43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP-43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP-43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases.

Phosphorylation of soluble tau differs in Pick's disease and Alzheimer's disease brains.

Frontotemporal lobar degeneration (FTLD) is a common cause of presenile dementia characterised by behavioural and language disturbances. Pick's disease (PiD) is a subtype of FTLD, which presents with intraneuronal inclusions consisting of hyperphosphorylated tau protein aggregates. Although Alzheimer's disease (AD) is also characterised by tau lesions, these are both histologically and biochemically distinct from the tau aggregates found in PiD. What determines the distinct characteristics of these tau lesions is unknown. As phosphorylated, soluble tau has been suggested to be the precursor of tau aggregates, we compared both the level and phosphorylation profile of tau in tissue extracts of AD and PiD brains to determine whether the differences in the tau lesions are reflected by differences in soluble tau. Levels of soluble tau were decreased in AD but not PiD. In addition, soluble tau was phosphorylated to a greater extent in AD than in PiD and displayed a different phosphorylation profile in the two disorders. Consistently, tau kinases were activated to different degrees in AD compared with PiD. Such differences in solubility and phosphorylation may contribute, at least in part, to the formation of distinct tau deposits, but may also have implications for the clinical differences between AD and PiD.

Profiles of matrix metalloproteinases and their inhibitors in plasma of patients with dementia.

BACKGROUND: Matrix metalloproteinases (MMPs) are elevated in the brain tissue of patients with dementia and may play a role in the pathophysiology of dementia. MMP-9 and tissue inhibitors of MMPs (TIMPs) are elevated in postmortem brain tissue of patients with Alzheimer's disease (AD). In a previous study we showed that circulating levels of MMP-9 are elevated in AD patients. The aim of the present study was to examine circulating levels of MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in the plasma of patients with mild cognitive impairment (MCI), AD, vascular dementia (VaD), dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD), to determine, whether plasma profiles of MMPs and TIMPs differ in various types of dementia. METHODS: Gelatinolytic activity (MMP-2 and MMP-9) was measured in all plasma samples by zymography. Levels of MMP-2, MMP-9, MMP-1 as well as TIMP-1 and TIMP-2 were measured by ELISA. RESULTS: We found constitutive expression of MMP-1, -2 and -9 as well as TIMP-1 and -2 in all the samples investigated. As shown previously, MMP-9 was significantly elevated in the plasma of AD patients (p = 0.004) as compared to controls and MCI patients. Plasma levels of TIMP-1 were significantly lower in VD samples as compared to all other groups. Levels of TIMP-2 were significantly lower in patients with FTD as compared to AD, VaD and MCI patients. There were no significant changes of MMP-1 and MMP-2 levels in the samples. CONCLUSION: These findings suggest that circulating levels of MMP-9, TIMP-1 and TIMP-2 and changes in the MMP/TIMP balance in plasma differ in various types of dementia.

Identification of G-protein coupled receptor kinase 2 in paired helical filaments and neurofibrillary tangles.

G-protein coupled receptor kinases (GRKs) constitute a serine/threonine kinase family playing a major role in agonist-induced phosphorylation and desensitization of G-protein coupled receptors. Recently, GRK2 and GRK5 have been demonstrated to phosphorylate alpha-synuclein (Ser129) and other synuclein isoforms. We studied colocalization of GRK2, GRK5, alpha-synuclein, and tau in neurodegenerative disorders characterized by fibrillary tau inclusions and/or alpha-synuclein-enriched Lewy bodies. We found that Lewy bodies were negative for both GRK2 and GRK5 in Lewy body disease (LBD) and LBD mixed with Alzheimer disease (AD + LBD). Instead, GRK2 but not GRK5 colocalized with 40% to 50% of neurofibrillary tangles in AD + LBD and AD brains. In disorders with less prominent alpha-synucleinopathy, neuronal and glial fibrillary tau deposits known to contain distinct subsets of tau isoforms were also positive for GRK2. These deposits included tufted astrocytes and coiled bodies in progressive supranuclear palsy, astrocytic plaques in corticobasal degeneration, and Pick bodies in Pick disease. In addition, paired helical filaments isolated from AD and AD + LBD brains were found to immunogold-label for GRK2, suggesting that GRK2 could be a potential tau kinase associated with fibrillary tau. Our studies indicate that GRK2 is a novel component of neuronal and glial fibrillary tau deposits with no preference in tau isoform binding. GRK2 may play a role in hyperphosphorylation of tau in tauopathies.

Metalloproteinase ADAMTS-1 but not ADAMTS-5 is manifold overexpressed in neurodegenerative disorders as Down syndrome, Alzheimer's and Pick's disease.

ADAMTS-1 is a disintegrin and metalloproteinase with thrombospondin 1 (TSP1)-like motifs with ubiquitous though variable expression. Natural substrates of this protease are proteoglycans as aggrecan and versican and null mutant mice propose a role for growth, fertility, organ structure and function. As the gene for this protein is encoded on chromosome 21 and maybe overexpressed due to the gene dosage hypothesis based upon the presence of a third chromosome in trisomy 21, we decided to study expression in Down syndrome (DS) brain and used brains of patients with Alzheimer's (AD) and Pick's disease (PD) as controls. Frontal cortex of controls, DS, AD and PD were homogenized and extracted proteins were used for immunoblotting using antibodies against ADAMTS-1 and ADAMTS-5. ADAMTS-1-immunoreactivity was manifold increased in brain with DS and neurodegeneration, whereas ADAMTS-5 levels were comparable. Overexpression of this metalloproteinase maybe specifically involved in proteoglycan degradation and handling in brain of patients with neurodegenerative disease which in turn may lead to or reflect pathological lesions in DS, AD and PD brain. The manifold overexpression of ADAMTS-1 may be used as marker protein for neurodegeneration.

Active stress kinase p38 enhances and perpetuates abnormal tau phosphorylation and deposition in Pick's disease.

Abnormal tau hyperphosphorylation and deposition in Pick bodies is a major abnormality in Pick's disease (PiD). This is associated with increased expression of the stress-activated protein kinase, p38 kinase, which has the capacity to phosphorylate tau in vitro. The present study has shown increased expression of phosphorylated p38 (p38-P), which does not cross-react with phospho-tau, in sarcosyl-insoluble fractions enriched in abnormal filaments, and hyperphosphorylated tau in the brain of two PiD cases obtained and processed with very short (less than 2 h) post-mortem delay. Immunohistochemical studies have shown p38-P co-localization in 90% of neurons with Pick bodies, whereas no positive cells are encountered in control brains processed in parallel. Moreover, p38-immunoprecipitated from sarcosyl-insoluble fractions in PiD brains is functionally active as it has the capacity to phosphorylate its specific substrate ATF-2. Combined biochemical, immunohistochemical and functional studies indicate that active p38 kinase is expressed in a very high percentage of Pick bodies, thus suggesting a critical role of this kinase in enhancing and perpetuating tau hyperphosphorylation in PiD.

Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders.

An increasing body of evidence indicates that oxidative stress and damage play a role in the pathogenesis of a number of diseases associated with neurodegeneration, including Down syndrome (DS), Alzheimer's disease (AD) and Pick's disease (PD). Although oxidative stress is a common element in these diseases, specific clinico-pathological phenotypes have been described for each disorder. Development of these phenotypes might be linked, among others, to differences in antioxidant response. The present study is designed to investigate expression of peroxiredoxins (Prxs), the newly characterized family of highly conserved antioxidant enzymes, and other antioxidant enzymes in frontal cortex and cerebellum of DS, AD and PD patients using the technique of proteomics. Levels of Prx I, Mn superoxide dismutase (SOD2) and glutathione-S-transferase omega1 in DS, AD and PD were not significantly different from that of controls in both brain regions investigated. In contrast, Prx II was significantly increased (P<0.05) in frontal cortex of DS, AD and PD, whereas Prx III was decreased in frontal cortex of DS (P<0.01) and PD (P<0.001). Interestingly, Prx VI displayed a significant increase (P<0.05) only in PD frontal cortex. The present data indicate that differential regulation of antioxidant enzymes exist in DS, AD and PD, suggestive of the diversity as well as distinct functional roles of these proteins. Moreover, while up-regulation of Prx II appears to provide evidence for the existence of compensatory response in increased cell loss, up-regulation of Prx VI may be used to discriminate PD from AD as well as DS.

Human brain cytosolic histamine-N-methyltransferase is decreased in Down syndrome and increased in Pick's disease.

Histamine-N-methyltransferase (HMT) inactivates the neurotransmitter histamine. Central histaminergic deficits may contribute to the cognitive impairment of neurodegenerative disorders including Alzheimer's disease (AD) and Down syndrome (DS). However, there is no evidence for histaminergic deficits in Pick's disease (PiD). HMT levels were measured in the frontal cortex and cerebellum of brains of patients with AD, DS, and PiD, and normal aged subjects using proteomics techniques. In frontal cortex, HMT was significantly decreased in DS, but significantly increased in PiD compared with controls. HMT levels were comparable in cerebellum of all groups. Elevated HMT in PiD could lead to increased histamine degradation that in turn would be in agreement with impaired cognitive functions of PiD. Decreased HMT in DS would be compatible with findings of decreased histamine synthesis, thus reflecting a compensation mechanism to antagonize reduced synthesis by decreased degradation.

Phosphorylated mitogen-activated protein kinase (MAPK/ERK-P), protein kinase of 38 kDa (p38-P), stress-activated protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in tauopathies.

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.