Showing papers by "Bart De Strooper published in 2009"

Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function

Abstract: Mutations of the mitochondrial PTEN (phosphatase and tensin homologue)-induced kinase1 (PINK1) are important causes of recessive Parkinson disease (PD) Studies on loss of function and overexpression implicate PINK1 in apoptosis, abnormal mitochondrial morphology, impaired dopamine release and motor deficits However, the fundamental mechanism underlying these various phenotypes remains to be clarified Using fruit fly and mouse models we show that PINK1 deficiency or clinical mutations impact on the function of Complex I of the mitochondrial respiratory chain, resulting in mitochondrial depolarization and increased sensitivity to apoptotic stress in mammalian cells and tissues In Drosophila neurons, PINK1 deficiency affects synaptic function, as the reserve pool of synaptic vesicles is not mobilized during rapid stimulation The fundamental importance of PINK1 for energy maintenance under increased demand is further corroborated as this deficit can be rescued by adding ATP to the synapse The clinical relevance of our observations is demonstrated by the fact that human wild type PINK1, but not PINK1 containing clinical mutations, can rescue Complex 1 deficiency Our work suggests that Complex I deficiency underlies, at least partially, the pathogenesis of this hereditary form of PD As Complex I dysfunction is also implicated in sporadic PD, a convergence of genetic and environmental causes of PD on a similar mitochondrial molecular mechanism appears to emerge

γ-Secretase Heterogeneity in the Aph1 Subunit: Relevance for Alzheimer's Disease

Abstract: The gamma-secretase complex plays a role in Alzheimer's disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the gamma-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different gamma-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B gamma-secretase in a mouse Alzheimer's disease model led to improvements of Alzheimer's disease-relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total gamma-secretase activity in the human brain, and thus specific targeting of Aph1B-containing gamma-secretase complexes may help generate less toxic therapies for Alzheimer's disease.

The Orphan G Protein–Coupled Receptor 3 Modulates Amyloid-Beta Peptide Generation in Neurons

Abstract: Deposition of the amyloid-beta peptide is a pathological hallmark of Alzheimer's disease. A high-throughput functional genomics screen identified G protein-coupled receptor 3 (GPR3), a constitutively active orphan G protein-coupled receptor, as a modulator of amyloid-beta production. Overexpression of GPR3 stimulated amyloid-beta production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-beta peptide in vitro and in an Alzheimer's disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature gamma-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimer's disease and is elevated in the sporadic Alzheimer's disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimer's disease.

Analysis of the |[gamma]|-secretase interactome and validation of its association with tetraspanin-enriched microdomains

Abstract: Gamma-secretase, an aspartyl protease that belongs to the iCLiPs (intramembrane cleaving proteases) family, is a multiprotein complex that consists of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (ref. 1). It is responsible for generation of the beta-amyloid peptide (Abeta), the primary component of senile plaques in the brains of patients with Alzheimer's disease. Although the four components are necessary and sufficient for gamma-secretase activity, additional proteins are possibly involved in its regulation. Consequently, we purified proteins associated with the active gamma-secretase complex from reconstituted PS-deficient fibroblasts, using tandem affinity purification (TAP) and identified a series of proteins that transiently interact with the gamma-secretase complex and are probably involved in complex maturation, membrane trafficking and, importantly, the tetraspanin web. Tetraspanins form detergent-resistant microdomains in the cell membrane and regulate cell adhesion, cell signalling and proteolysis. Association of the gamma-secretase complex with tetraspanin-enriched microdomains provides an explanation for the previously documented localization of gamma-secretase to raft-like domains. Thus, these studies suggest that maintenance of the integrity of tetraspanin microdomains contributes to the refinement of proteolytic activity of the gamma-secretase complex.

Notch is a critical component of the mouse somitogenesis oscillator and is essential for the formation of the somites.

Abstract: Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation

Abstract: Alzheimer's disease neuropathology is characterized by neuronal death, amyloid β-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid β-peptide and paired helical filaments remain unknown. Here, we show that amyloid β-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase ( P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal ( n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large β-sheet aggregates in vitro and in vivo , as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid β-peptide-induced toxicity and tau pathology.

G Protein–Coupled Receptors, Cholinergic Dysfunction, and Aβ Toxicity in Alzheimer’s Disease

Abstract: The beta-amyloid (Abeta) peptide is associated with the pathogenesis of Alzheimer's disease (AD). Evidence gathered over the last two decades suggests that the gradual accumulation of soluble and insoluble Abeta peptide species triggers a cascade of events that leads to the clinical manifestation of AD. Abeta accumulation has also been associated with the cholinergic dysfunction observed in AD, which is characterized by diminished acetylcholine release and impaired coupling of the muscarinic acetylcholine receptors (mAChRs) to heterotrimeric GTP-binding proteins (G proteins). Although the mechanism of Abeta-mediated toxicity is not clearly understood, evidence shows that Abeta accumulation has an effect on the oligomerization of the angiotensin II (AngII) AT(2) (angiotensin type 2) receptor and sequestration of the Galpha(q/11) family of G proteins. Sequestration of Galpha(q/11) results in dysfunctional coupling and signaling between M(1) mAChR and Galpha(q/11) and accompanies neurodegeneration, tau phosphorylation, and neuronal loss in an AD transgenic mouse model. Collectively, these results provide a putative link among Abeta toxicity, AT(2) receptor oligomerization, and disruption of the signaling pathway through M(1) mAChR and Galpha(q/11) and potentially contribute to our understanding of the cholinergic deficit observed in AD.

Epigenetic control of aquaporin 1 expression by the amyloid precursor protein

Abstract: Cellular processing of the amyloid precursor protein (APP) has been extensively studied, but its precise function remains elusive. The intracellular domain of APP has been proposed to regulate expression of several genes by mechanisms that are largely unknown. We report that APP regulates expression of the aquaporin 1 (AQP1) gene in mouse embryonic fibroblasts and in transgenic mice. AQP1 mRNA and protein were down-regulated in fibroblasts lacking APP or presenilin 2 in which AQP1 expression was restored by stable expression of full-length APP or presenilin 2 but not by APP deleted from its carboxy-terminal domain. The transcriptional activity of the AQP1 gene promoter and the stability of AQP1 mRNA were identical in fibroblasts expressing or not expressing APP. Control of AQP1 expression by APP was sensitive to trichostatin A, an histone deacetylase inhibitor, and histone deacetylase activity coimmunoprecipitated with APP. Altogether, these data show that a presenilin-2-dependent gamma-secretase activity releases the intracellular domain of APP involved in the epigenetic control of AQP1 expression. Since AQP1 is found in astrocytes surrounding senile plaques, this epigenetic control of AQP1 expression could have important implications in Alzheimer disease.

Alzheimer's dementia by circulation disorders: when trees hide the forest.

Abstract: Deposition of amyloid β-peptide in cerebral vessel walls, termed cerebral amyloid angiopathy (CAA), enhances the cognitive deficits associated with Alzheimer's disease. The molecular details by which circulatory defects with hypoxia alter peptide clearance, contributing to brain deposition and AD, are beginning to be elucidated.

Epigenetic control of Aquaporin 1 expression by the amyloid precursor protein

Abstract: APP (sAPP) as well as an electrochemiluminescence-based multi-spot ELISA to distinguish between sAPP alpha and sAPP-beta secretion. The intraand extracelluar cleavage fragments were analyzed by Western Blot. Results: The SEAP reporter-assay showed a moderate increase of sAPP in the medium upon APP overexpression in HEK293 and N2A cells and an additional increase when overexpressed together with BACE1, indicating a sAPPbeta increase. This effect was depleted by adding lcLRP. To verify the cleavage product as sAPPbeta, we applied multi-spot ELISA. This ELISA revealed the expected increase of sAPPalpha after transfection with APP. This increase changed to sAPPbeta after BACE1 coexpression. Additional overexpression of lcLRP led to a decrease of both sAPPalpha as well as sAPPbeta in cell culture supernatant. Conclusions: We found a decrease of sAPPalpha as well as sAPPbeta upon lcLRP coexpression supporting our notion of a competition between lcLRP and APP towards the BACE1 cleavage. Our results suggest a complex role of LRP in APP processing besides influencing APP internalization.

Single domain antibodies capable of modulating BACE activity

Abstract: The present invention relates to single domain antibodies with a specificity for BACE1. More specifically, the invention provides single variable domain antibodies derived from camelids which bind to BACE1 and are capable of inhibiting the activity of BACE1. Said antibodies can be used for research and medical applications. Specific applications include the use of BACE1 specific antibodies for the treatment of Alzheimer's disease.