Chris De Jonghe

Flemish and Dutch Mutations in Amyloid β Precursor Protein Have Different Effects on Amyloid β Secretion

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.

Behavioral Disturbances without Amyloid Deposits in Mice Overexpressing Human Amyloid Precursor Protein with Flemish (A692G) or Dutch (E693Q) Mutation

The contribution of mutations in the amyloid precursor protein (APP) gene known as Flemish (APP/A692G) and Dutch (APP/E693Q) to the pathogenesis of Alzheimers 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.

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 PS1 and PS2 are involved in Alzheimers 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 PS1 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 PS1 to caspase‐mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase‐dependent processing.

The Glu318Gly substitution in presenilin 1 is not causally related to Alzheimer disease

We are grateful to Hubert Backhovens, Marleen Van den Broeck, and Sally Serneels for their skilled technical assistance in the mutation screening and genotyping. We thank Alewijn Ott, Frans van Harskamp, Inge de Koning, Maarten de Rijk, and Sandra Kalmijn for their contribution in the dementia diagnosis. This work was funded in part by a special research project of the University of Antwerp, the Fund for Scientific Research in Flanders, and the International Alzheimers disease Research Fund, Belgium; and by the NESTOR stimulation program for scientific research in the Netherlands (Ministry of Health and Ministry of Education), the Netherlands Organisation for Scientific research, the Netherlands Prevention fund, and the municipality of Rotterdam.

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 Alzheimers 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.

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.

Identification of a Drosophila presenilin homologue: evidence of alternatively spliced forms.

Some cases of Alzheimers 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.

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 Alzheimers 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.

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 Alzheimerss 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.

CHAPTER 5.6 Identification and functional analysis of genes and genetic risk factors in Alzheimer's disease

Publisher Summary This chapter focuses on identification and functional analysis of genes and genetic risk factors in Alzheimers disease. The chapter illustrates that although it is widely recognized that AD is a complex disorder of the CNS in which many genetic and environmental factors influence the pathogenesis, molecular genetic analysis of AD has provided with major breakthroughs in the unraveling of the molecular causes for this dementia. With the identification of genes and genetic risk factors, the knowledge about the biochemical processes underlying this disease gradually continues to grow as well. Knowledge about the biological function and dysfunction of genes involved in AD, although still fragmentarily, may lead to understanding the primary disease mechanisms. This in turn can help to develop a more efficient therapy that can either decelerate or cure this devastating disorder of human mind.