Nonfibrillar diffuse amyloid deposition due to a gamma(42)-secretase site mutation points to an essential role for N-truncated A beta(42) in Alzheimer's disease.

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).

Proteolytic processing of presenilin-1 in human lymphoblasts is not affected by the presence of the I143T and G384A mutations.

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.

Alzheimer's disease associated presenilin 1 interacts with HC5 and ZETA, subunits of the catalytic 20S proteasome.

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.

Evidence that Abeta42 plasma levels in presenilin-1 mutation carriers do not allow for prediction of their clinical phenotype.

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.

No influence of presenilin1 I143T and G384A mutations on endogenous tau phosphorylation in human and mouse neuroblastoma cells.

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.

Aberrant splicing in the presenilin-1 intron 4 mutation causes presenile Alzheimer's disease by increased Abeta42 secretion.

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.

Identification of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases.

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.

Immunoreactivity of presenilin-1 and tau in Alzheimer's disease brain.

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.