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Examines the significance of phosphatidylinositol 3-kinase (PI 3-K) activity for the expression of glial fibrillary acidic protein (GFAP) upon cAMP-dependent induction in rat C6 glioma. Use of specific PI 3-k inhibitors wortmannin and LY294002; Decrease of the enzymatic activity of PI 3-K; Factors affecting the expression and cellular distribution of GFAP.

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is a rare autosomal dominant disorder caused by an amyloid-beta precursor protein (AbetaPP) 693 mutation that clinically leads to recurrent hemorrhagic strokes and dementia. The disease is pathologically characterised by the deposition of Abeta in cerebral blood vessels and as plaques in the brain parenchyma. This study measured the Abeta40 and Abeta42 concentration in plasma of Dutch AbetaPP693 mutation carriers and controls. We found that the Abeta40 concentration was not different between AbetaPP693 mutation carriers and controls. However, the Abeta42 concentration was significantly decreased in the mutation carriers. No correlation exists between the APOE(epsilon)4 allele and the plasma of Abeta40 and Abeta42 levels in HCHWA-D patients. This finding contrasted with the increased concentrations found in Alzheimer's disease. Therefore it is suggested that the Dutch AbetaPP693 mutation located within the Abeta coding region of the AbetaPP gene has a different effect not only on clinical and pathological expression but also on Abeta processing.

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event., (Copyright 2001 Academic Press.)

Most cases of familial presenile Alzheimer's disease are caused by mutations in the presenilin-1 (PSEN-1) gene, most of these mutations being missense mutations. A mutation in the splice donor site of intron 4 of PSEN-1 has been described recently which results in aberrant splicing of PSEN-1 mRNA, causing insertion of an additional amino acid, Thr113-114ins, into the protein. We studied the neuropathology of four cases bearing this mutation in an attempt to clarify the pathology of this hereditary form of Alzheimer's disease and to determine whether it differs from other familial forms of the disease. The disease presented as a progressive cognitive decline, myoclonus and seizures developing later in the disease, a feature common to PSEN-1-linked Alzheimer's disease. The course of the disease was relatively rapid, death occurring approximately 6 years after onset. Pathology in the intron 4 cases demonstrated a severe Alzheimer's disease pathology with abundant deposition of ss-amyloid (Ass) 1-42 senile plaques and the formation of neurofibrillary tangles. Amyloid angiopathy was present in these cases and was readily demonstrated by Ass 1-40 staining, particularly in the cerebellum. Cases with the intron 4 mutation appear clinically and pathologically similar to other cases of early-onset Alzheimer's disease bearing PSEN-1 mutations.

Amyloidogenic processing of the amyloid precursor protein (APP) with deposition in brain of the 42 amino acid long amyloid beta-peptide (A beta(42)) is considered central to Alzheimer's disease (AD) pathology. However, it is generally believed that nonfibrillar pre-amyloid A beta(42) deposits have to mature in the presence of A beta(40) into fibrillar amyloid plaques to cause neurodegeneration. Here, we describe an aggressive form of AD caused by a novel missense mutation in APP (T714I) directly involving gamma-secretase cleavages of APP. The mutation had the most drastic effect on A beta(42)/A beta(40) ratio in vitro of approximately 11-fold, simultaneously increasing A beta(42) and decreasing A beta(40) secretion, as measured by matrix-assisted laser disorption ionization time-of-flight mass spectrometry. This coincided in brain with deposition of abundant and predominant nonfibrillar pre-amyloid plaques composed primarily of N-truncated A beta(42) in complete absence of A beta(40). These data indicate that N-truncated A beta(42) as diffuse nonfibrillar plaques has an essential but undermined role in AD pathology. Importantly, inhibiting secretion of full-length A beta(42 )by therapeutic targeting of APP processing should not result in secretion of an equally toxic N-truncated A beta(42).

Several mutations in the amyloid precursor protein (APP) gene may lead to either Alzheimer's disease or cerebral haemorrhage due to congophilic amyloid angiopathy (CAA). A single family is known in which both types of pathology are expressed because of a missense mutation at codon 692 of the APP gene (APP692). Here we describe the clinical and pathological expression of APP692 in eight patients with the mutation. Furthermore, 21 first-degree relatives with an a priori risk of 50% of being a carrier were tested for the APP692 mutation and studied for presymptomatic signs by neurological examination, neuropsychological testing and brain MRI. Patients with APP692 presented with haemorrhage, dementia or both. The dementia in patients with the APP692 mutation was compatible with Alzheimer's disease both clinically and neuropathologically. Of the 21 healthy relatives at 50% risk, five carried the APP692 mutation. The presymptomatic carriers showed a subtle, non-significant impairment of cognitive function compared with relatives without APP692. A significant increase in the number of periventricular and subcortical white matter lesions at young age was seen in presymptomatic carriers (mean age 26.4 years). The findings of this study suggest that a single (genetic) mechanism may underlie the pathology of Alzheimer's disease and CAA. These diseases are manifested subclinically by white matter pathology. Further insight into the relationship between CAA and Alzheimer's disease may provide clues about the aetiology of Alzheimer's disease.

Background: Mutations in the presenilin (PSEN) genes are responsible for the majority of early-onset Alzheimer disease (AD) cases. PSEN1 is a component of a high molecular weight, endoplasmic reticulum, membrane-bound protein complex, including beta-catenin. Pathogenic PSEN1 mutations were demonstrated to have an effect on beta-catenin and glycogen synthase kinase-3beta(GSK-3beta), two members of the wingless Wnt pathway. The nuclear translocation and the stability of beta-catenin, and the interaction between GSK3beta and PSEN1 were influenced., Materials and Methods: Stably transfected human embryonic kidney (HEK) 293 cells overexpressing wild-type (wt) and mutant (mt) PSEN1, treated with and without LiCl, were used to isolate cytoplasmic and nuclear fractions. By Western blot analysis, endogenous beta-catenin levels were examined. By analyzing cytosolic fractions of PSEN1, transfected and nontransfected HEK 293 cells, and total brain extracts of AD patients and controls, we evaluated the effect of PSEN1 overexpression on beta-catenin stability. Finally, we analyzed the effect of pathogenic PSEN1 mutations on the interaction between PSEN1 and GSK3beta by co-immunoprecipitation experiments., Results: We report reduced nuclear translocation of beta-catenin in cells stably expressing I143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutation showed a similar pronounced effect on nuclear translocation of beta-catenin, as reported for processing of amyloid precursor protein (APP) into amyloid beta(Abeta). Overexpression of PSEN1 and the presence of pathogenic mutations in PSEN1 had no significant effect on the stability of beta-catenin. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3beta was observed when GSK3beta was immunoprecipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3beta., Conclusion: Our results further establish the nuclear translocation assay of beta-catenin as an adequate alternative for traditional Abeta measurement to evaluate the effect of PSEN1 mutations on biochemical processes. We detected no significant effect of overexpressed wt or mt PSEN1 on the stability of beta-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3beta was not observed in our experimental setup.

The contribution of mutations in the amyloid precursor protein (APP) gene known as Flemish (APP/A692G) and Dutch (APP/E693Q) to the pathogenesis of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type, respectively, was studied in transgenic mice that overexpress the mutant APP in brain. These transgenic mice showed the same early behavioral disturbances and defects and increased premature death as the APP/London (APP V717I), APP/Swedish (K670N, M671L), and other APP transgenic mice described previously. Pathological changes included intense glial reaction, extensive microspongiosis in the white matter, and apoptotic neurons in select areas of the brain, while amyloid deposits were absent, even in mice over 18 months of age. This contrasts with extensive amyloid deposition in APP/London transgenic mice and less pronounced amyloid deposition in APP/Swedish transgenic mice generated identically. It demonstrated, however, that the behavioral deficiencies and the pathological changes in brain resulting from an impaired neuronal function are caused directly by APP or its proteolytic derivative(s). These accelerate or impinge on the normal process of aging and amyloid deposits per se are not essential for this phenotype., (Copyright 2000 Academic Press.)

Presenilin-1 (PSEN1) mutations I143T and G384A give rise to severe early onset Alzheimers's disease in two extensively studied Belgian families, AD/A and AD/B. In this study we investigated the influence of the I143T and G384A mutations on PSEN1 proteolytic processing. Hereto we analyzed PSEN1 processing in lymphoblasts by immunodetection with PSEN1-specific antibodies and densitometric analysis of the immunoreactive banding pattern. No differences were observed between presymptomatic mutation carriers, patients or escapees, demonstrating that the PSEN1 mutations I143T and G384A do not alter PSEN1 proteolytic processing in lymphoblasts.

Proteolytic processing and degradation tightly regulate the amount of stable, functional presenilin 1 (PSEN1) in the cell. The approximately 46-kDa PSEN1 holoprotein is cleaved into a approximately 30-kDa N-terminal fragment (NTF) and a approximately 20-kDa C-terminal fragment (CTF) by an unknown protease. The fragments are stabilized in a high molecular weight complex and nonincorporated fragments and excess holoprotein are degraded by the 26S proteasome. The tight balance between, on the one hand, processing and incorporation into the stable complex and, on the other hand, proteolytic degradation of excess PSEN1, indicates that minor changes in one of these two processes could be pathologically relevant. Here we demonstrate the direct physical interaction between PSEN1 and two subunits, HC5 and ZETA, of the 20S proteasome. These interactions were identified using an interaction trap screening and were further established in an in vitro binding assay. Furthermore, we were able to coimmunoprecipitate the transfected binding partners, as well as the endogenous PSEN1 and ZETA proteins from HEK 293T cells. Finally, degradation of ubiquitinated wild-type and mutant PSEN1 by the 26S proteasome was demonstrated. In conclusion, we report a direct interaction between PSEN1 and subunits of the 20S catalytic particle of the 26S proteasome, further establishing the involvement of proteasomal degradation in the regulation of PSEN1 turnover., (Copyright 1999 Academic Press.)

We previously described a splice donor site mutation in intron 4 of presenilin-1 (PSEN1) in two patients with autopsy-confirmed early-onset Alzheimer's disease (AD). Here we provide evidence that the intron 4 mutation is present in four additional unrelated early-onset AD cases, that the mutation segregates in an autosomal dominant manner and that all cases have one common ancestor. We demonstrate that the intron 4 mutation produces three different transcripts, two deletion transcripts (Delta4 and Delta4cryptic) and one insertion transcript (insTAC), by aberrant splicing. The deletion transcripts result in the formation of C-truncated (approximately 7 kDa) PSEN1 proteins while the insertion transcript produces a full-length PSEN1 with one extra amino acid (Thr) inserted between codons 113 and 114 (PSEN1 T113-114ins). The truncated proteins were not detectable in vivo in brain homogenates or lymphoblast lysates of mutation carriers. In vitro HEK-293 cells overexpressing Delta4, Delta4cryptic or insTACPSEN1 cDNAs showed increased Abeta42 secretion (approximately 3.4 times) only for the insertion cDNA construct. Increased Abeta42 production was also observed in brain homogenates. Our data indicate that in the case of intron 4 mutation, the AD pathophysiology results from the presence of the PSEN1 T113-114ins protein comparable with cases carrying dominant PSEN1 missense mutations.

Mutations in the presenilin 1 (PSEN1) gene are an important cause of autosomal dominant Alzheimer's disease (AD). Both in vitro and in vivo experiments showed that PSEN1 mutations increase secretion of amyloid beta42 (Abeta42), the longer and more fibrillogenic isoform of Abeta. We measured secreted Abeta42 in plasma of patients, presymptomatic mutation carriers, and escapees of two extended Belgian early-onset AD families, AD/A and AD/B, with a similar severe phenotype in terms of onset age (mean 35 years), duration of the disease (mean 6.5 years), and pathology. Both families segregate a different missense mutation in PSEN1 located in different parts of the protein: I143T in family AD/A and G384A in family AD/B. A significant increase in Abeta42 concentrations was observed in plasma of mutation carriers in family AD/B, but not in family AD/A. A differential effect of the two PSEN1 mutations on Abeta42 secretion was also detected in conditioned medium of stably transfected HEK293 cells. Both mutations increased Abeta42 secretion significantly; however, the increase was highest for G384A (5.5-fold over wild-type PSEN1), the largest effect observed for missense PSEN1 mutations to date. Although the Abeta42 concentrations measured in vivo and in vitro did not correlate with onset age, a positive correlation was obtained with age in the presymptomatic mutation carriers and a negative correlation with duration of disease in the patients. Our data obtained for PSEN1 mutation carriers suggest that measuring Abeta42 concentrations in plasma will be informative as a diagnostic marker in a limited number of cases., (Copyright 1999 Academic Press.)

Presenilin1 (PSEN1) 1143T and G384A mutations give rise to severe early-onset Alzheimer's disease in two extensively studied Belgian families. In the present study, we examined the effect of PSEN1 1143T and G384A mutations on tau phosphorylation in human SH-SY5Y and mouse Neuro-2a neuroblastoma cell lines that were transiently transfected with wild type (WT) or mutant PSEN1. With a phosphorylation independent antibody, no alteration in the electrophoretic mobility of tau was observed between wild type and mutant PSEN1 transfectants. Also, densitometric analysis of Tau1 immunoreactivity, characteristic of unphosphorylated tau, demonstrated no significant differences between WT and mutant PSEN1 transfectants. Our data suggest that in the cellular models we used, transient overexpression of 1143T and G384A mutant PSEN1 does not lead to increased tau phosphorylation.

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.

Mutations in the amyloid beta precursor protein (APP) gene cosegregate with autosomal dominant Alzheimer disease (AD). Brain pathology of AD is characterized by amyloid deposition in senile plaques and by neurofibrillary tangles. Amyloid deposits in AD brains consist of amyloid beta (A beta), a 4-kDa proteolytic product of APP. In contrast, two other mutations in APP, the Flemish APP692 and Dutch APP693 mutations, are associated with autosomal dominant cerebral hemorrhages due to congophilic amyloid angiopathy (CAA) in the presence or absence of AD pathology, respectively. Both mutations are located within A beta near the constitutive cleavage site. While a common effect of AD-linked mutations is to elevate A beta 42 extracellular concentrations, not much is known about the effect of APP692 and APP693. Here we provide evidence that APP692 and APP693 have a different effect on A beta secretion as determined by cDNA transfection experiments. While APP692 upregulates both A beta 40 and A beta 42 secretion, APP693 does not. These data corroborate with previous findings that increased A beta secretion and particularly of A beta 42, is specific for AD pathology.

Mutations in the presenilin-1 gene (PS-1) on chromosome 14 are causative for early-onset familial Alzheimer's disease (AD). In order to study the localization of PS-1 in human brain, a polyclonal antibody, SB63, against a N-terminal epitope of PS-1 (25VRSQNDNRERQEHND40), was raised in rabbits and characterized. Immunolabeling with SB63 of formalin-fixed sections of hippocampus from cases of PS-1-linked AD (PS-1 I143T (AD/A), G384A (AD/B)), sporadic AD, and controls showed a predominant neuronal staining pattern with a stronger immunoreactivity in pyramidal neurons. Staining was mainly granular and localized in the neuronal cell body as well as in neuronal processes. In AD some dystrophic neurites surrounding the amyloid plaques were stained, but no immunoreactivity was observed in the amyloid core. Although PS-1 was present in tangle bearing neurons, colocalization of PS-1 and tau could not be detected using immunofluorescence double labeling. Our data indicate that the pattern of PS-1 immunoreactivity in the hippocampus does not substantially differ between PS-1-linked AD, sporadic AD, and controls.

Some cases of Alzheimer's disease are inherited as a dominant trait in humans. To date, mutations in three genes account for some of them: the amyloid precursor protein (APP) and presenilins 1 and 2 (PS-1 and PS-2, respectively). The function of the presenilins is still unclear, although they belong to a transmembrane protein-gene family, probably involved in some signaling pathway. We report here the isolation of the Drosophila presenilin homologue using the human PS-1 and PS-2 cDNAs as probes. Only one single gene has been detected in the Drosophila genome and evidence for alternatively spliced forms is presented and compared to the isoforms reported in humans. Temporal and spatial expression has been assessed by Northern blot and in situ hybridization on embryos of different developmental stages.

Mutations in the presenilin genes are involved in the aetiology of the majority of familial early-onset Alzheimer disease (AD) cases. Presenilin 1 (PS1) is proteolytically processed in vivo, resulting in the accumulation of N-terminal approximately 27-28 kDa and C-terminal approximately 18 kDa derivatives. To examine the metabolism of PS1 in brains of patients with AD harbouring PS1 mutations I143T and G384A, we performed immunoblot analyses of brain homogenates using well characterized antibodies. We document that approximately 27-28 kDa N-terminal and approximately 18 kDa C-terminal PS1 proteolytic fragments accumulate in brain of these individuals, and that in large part the accumulation pattern is indistinguishable from that observed in brains from individuals with sporadic AD or controls. Notably, little, if any, full-length PS1 was detected in brain tissue of patients carrying PS1 mutations or in those with sporadic AD, indicating that failed proteolysis of PS1 is not a central feature of pathogenesis in these patients.

Genetic linkage studies have indicated that chromosome 14q24.3 harbours a major locus for early-onset (onset age <65 years) Alzheimer's disease (AD3). Positional cloning efforts have identified a novel gene S182 or presenilin 1 as the AD3 gene. We have mapped S182 in the AD3 candidate region between D14S277 and D14S284 defined by genetic linkage studies in the two chromosome 14 linked, early-onset AD families AD/A and AD/B. We have shown that S182 is expressed in lymphoblasts and have determined the complete cDNA in both brain and lymphoblasts by RT-PCR sequencing. S182 is alternatively spliced in both brain and lymphoblasts within a putative phosphorylation site located 5' in the coding region. We identified two novel mutations, Ile143Thr and Gly384la located in, respectively, the second transmembrane domain and in the sixth hydrophilic loop of the putative transmembrane structure of S182. As families AD/A and AD/B have very similar AD phenotype our observation of two mutations in functionally different domains suggest that onset age and severity of AD may not be very helpful predictors of the location of putative S182 mutations.