188 research outputs found
Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface
No full textγ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.sponsorship: KU Leuven IDO/12/020 Hideaki Mizuno Wim Annaert KU Leuven C16/15/073 Wim Annaert Fonds Wetenschappelijk Onderzoek S006617N Wim Annaert Fonds Wetenschappelijk Onderzoek G078117N Wim Annaert Fonds Wetenschappelijk Onderzoek G056017N Wim Annaert Fonds Wetenschappelijk Onderzoek AKUL/08/58 Wim Annaert Fonds Wetenschappelijk Onderzoek AKUL/11/30 Wim Annaert Fonds Wetenschappelijk Onderzoek AKUL/13/39 Wim Annaert Agence Nationale de la Recherche ANR-10-INBS-04 Magali Mondin European Molecular Biology Organization #8358 Abril Angelica Escamilla-Ayala SAO-FRA #14017 Wim Annaert The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. (KU Leuven|IDO/12/020, KU Leuven|C16/15/073, Fonds Wetenschappelijk Onderzoek|S006617N, Fonds Wetenschappelijk Onderzoek|G078117N, Fonds Wetenschappelijk Onderzoek|G056017N, Fonds Wetenschappelijk Onderzoek|AKUL/08/58, Fonds Wetenschappelijk Onderzoek|AKUL/11/30, Fonds Wetenschappelijk Onderzoek|AKUL/13/39, Agence Nationale de la Recherche|ANR-10-INBS-04, European Molecular Biology Organization|8358, SAO-FRA|14017)status: Publishe
Morphologische en moleculaire ontleding van de gamma-secretase onafhankelijke functies van Presenilines
No full textAlzheimers disease (AD) is characterized by a gradual lossof neurons and leads consequently to cognitive decline, concomitant with theformation of amyloid plaques and hyperphosphorylated Tau. The major constituentof these plaques is the neurotoxic peptide Aß, liberated from the amyloidprecursor protein (APP) by the consecutive actions of ß- and γ-secretase.Presenilins (PSEN1&2) form the catalytic components within the γ-secretasecomplex. Interestingly, most of the mutations in PSEN1, associated with early-onsetFAD cause the increased production of Aß42, which are more prone to aggregation(De Strooper, 2010a).PSENs perform also non-catalytic functions; several of themare still poorly understood, or it is still unsure whether or not theyrepresent bona fide non-γ-secretase associated functions. Studies illustratinga role for PSENs in protein maturation/turnover (Esselens et al., 2004;Hass et al., 2009; Khandelwal et al., 2007; Wilson et al., 2004) could reflect an underlyingmodulatory role of PSENs in endosomal transport. Finally, the fact thatendosomal transport blockades are observed in non-neuronal and neuronal cellsis moreover intriguing as endosomal/lysosomal dysfunction is amongst theearliest neuropathological feature observed in sporadic Alzheimers disease.Perturbations in these endosomal pathways may be one of the initiation steps inthe ethiology of AD, preceding the harmful effects of intracellular Aßaccumulations (Cataldo et al., 2004;LaFerla et al., 2007b).In order to decipher the contributions of γ-secretase-dependent/independentfunctions to AD, we first need to bridge the gap in our knowledge regarding theγ-secretase independent roles. In my doctoral thesis, I identified a major defect in aspecific endosomal transport and redistribution route in PSEN deficient cellsand hippocampal neurons. PSEN deficiency leads to the formation ofintracellular accumulations and causes delayed turnover of degradativeorganelles and late endosomes. Failure to perform proper recycling, as occursin PSENdKO cells, leads to impaired multivesicular body maturation andlysosomal function. A first major outcome of the thesis is that I demonstratedthat lysosomal dysfunction is not at all related to lysosomal acidificationproblems, as was proposed recently. A more detailed analysis moreoverdemonstrated that proton pump function and transport is normal in PSENdeficient cells. Secondly and in contrast, I show that lysosomal defects areindirectly related to a failure in selective endosomal routes, notably therecycling routes that is majorly regulated through the small GTPase ARF6.Endosomal recycling defects of lipid raft-associated and GPI-anchored proteins,but not their internalization, resulted in a selective depletion of thesemolecules from the cell surface of PSENdKO MEFs with serious implications oncell morphology andcell migration behavior. Moreover, we were able to fullyrescue all endosomal deficits by reintroducing the proteolytically inactivePSEN1 mutant or by normalizing the levels of the small GTPase ARF6. Our dataalso support that the failure in properly recycling endosomal cargo leads to abuild-up of e.g. cholesterol which could explain the observed defects inlysosomal calcium storage and therefore the impaired capacity of lysosomes tofuse and degrade long-lived proteins. This work implies that lateendosomal-lysosomal fusion deficits originating from a defect in lysosomalcalcium storage/release, MVB maturation and cell polarity/migration all fueldown to an ARF6-mediated recycling problem in PSEN deficient cells.An expression study in aging mice/neurons and control vs. ADbrain specimens revealed that PSENs and ARF6 were collectively downregulated.This implies that a defect observed in cell culture systems might indeed betranslated into an in vivo context, ultimately affecting the early-onset of ADneuropathology.Although, in general, it remains extremely difficult todissociate the different γ-secretase dependent and independent functions ofPSENs we now provide evidence that indeed non-catalytic functions of PSENsmight contribute to disease. We postulate that in sporadic cases,ARF6-deficiency might slow down endosomal recycling, resulting in increasedendosomal volume and decreased lysosomal clearance, exacerbating the toxicityof the intraneuronal Aß pool. The physiological outcome in PSEN deficientcells/neurons is likely the result of both γ-secretase dependent andindependent functions. PSENs may therefore act as a focal point influencing adiverse array of signaling molecules and protein trafficking routes that cancontribute to AD pathogenesis. By identifying a major endosomal sorting anddegradation pathway in PSEN deficient cells and hippocampal neurons, we herebypresent ARF6 as novel therapeutic target in the initiation cascade of ADpathology.status: Publishe
LOKALISATIE EN REGULATIE VAN GAMMA-SECRETASE IN CILIOGENESE: IMPLICATIES IN GEZONDHEID EN ZIEKTE
No full textSCIENTIFIC SUMMARYCilia project from the surface of most vertebrate cells, and are important for several physiological and developmental processes. Ciliary defects are linked to a wide variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. Here we show that Rer1p, a protein quality control receptor residing in the ER-Golgi compartment, is involved in regulating the structure and function of cilia. Both in zebrafish and mammalian cells, loss of Rer1p results in the shortening of cilium and impairment of its motile and/or sensory function, which is reflected by hearing, vision and left-right asymmetry defects as well as decreased Hedgehog signaling. Importantly, cilia formation is also compromised in cell lines derived from monosomy 1p36 patients, which carry a terminal chromosomal deletion that includes the RER1 gene. A substantial part of the patients clinical picture including hearing problems, congenital heart defects and facial dysmorphism suggests ciliary dysregulation, and can be mimicked in zebrafish by knockdown of Rer1p. We further demonstrate that Rer1p controls ciliogenesis partly through its function in negatively regulating gamma-secretase activity and the signaling of at least one of its substrate, Notch, as nanomolar concentrations of a gamma-secretase inhibitor partially but significantly rescued cilium length in Rer1p-depleted cells. We therefore identify Rer1p as the first ER-Golgi localized membrane protein involved in ciliogenesis where it exerts this broader physiological function in cells and in vivo models through regulating the assembly and activity of gamma-secretase. Acting through the control of ciliogenesis, Rer1p thus may play a pivotal role in contributing to the clinical picture of monosomy 1p36, recognizing it as an underlying ciliopathy.gamma- Secretase is a multimeric protein complex responsible for the proteolytic processing of around 90 different integral membrane proteins. The diverse functions of gamma-secretase are therefore attributed to the cleavage of its various substrates and the subsequent signaling properties associated with them. Its involvement in a biologically important process such as ciliogenesis urged us to explore its effectors responsible for this process. Despite the ubiquitous existence and genuine physiological importance, the protein composition of cilia remains poorly explored. Therefore, we first used a proteomics104approach to map the protein content of isolated cilia; specifically to identify novel cilia localized gamma-secretase substrates. Here we demonstrate for the first time that Amyloid Precursor Protein (APP) and APP like protein 2 (APLP2) are localized to the primary cilium. APP plays a central role in the pathogenesis of Alzheimer's disease: APP processing generates ß-amyloid (Aß) peptides, which are deposited in amyloid plaques in brains of Alzheimers disease patients. Despite being intensively investigated, the full scope of the physiological roles of APP still remains enigmatic. Using various cell biological approaches, we demonstrate that APP is proteolytically processed in the cilium and that its sheddase, ADAM10 and gamma-secretase complex are located in the cilium as well as required to maintain proper cilia length. Knockout of APP together with APLP2 leads to short cilia which could be rescued by the stable re-expression of full length/ carboxy terminal fragment of APP (APP-CTF) but not the APP intracellular domain (AICD). We further show that APP-CTF regulates cilia length through activating the cyclic AMP/Protein Kinase A signaling pathway, thus establishing for the first time the requirement of APP processing as a novel regulator in cilia length maintenance.status: Publishe
Implication of APP secretases in notch signaling
No full textSignaling via notch receptors and their ligands is an evolutionary ancient and highly conserved mechanism governing cell-fate decisions throughout the animal kingdom. Upon ligand binding, notch receptors are subject to a two-step proteolysis essential for signal transduction. First, the ectodomain is removed by an enzyme cleaving near the outer-membrane surface ("site2"). Consecutively, the notch intracellular domain is liberated by a second protease cutting within the transmembrane sequence ("site3"). The intracellular domain is then transferred to the nucleus to act as a transcriptional coactivator. The proteases involved in notch receptor activation are shared with other proteins undergoing regulated intramembrane proteolysis, with intriguing parallels to APP. Specifically, site3 cleavage of Notch, as well as gamma-secretase processing of APP depend both critically on presenilins 1 and 2. Moreover, ADAM 10 and ADAM 17, the proteases proposed to perform site2 cleavage, are also the most probable candidate alpha-secretases to cleave APP. While the biological significance of APP processing remains to be further elucidated, interference with notch signaling has been shown to have severe consequences both in small animal models as well as in humans. Thus, a growing number of long known genetic syndromes like Alagille syndrome or Fallot's tetralogy can be caused by mutations of genes relevant for the notch signaling pathway. Likewise, the anticipated interference of gamma-secretase inhibitors with site3 cleavage may turn out to be a major obstacle for this therapeutic approach to Alzheimer's disease.status: Publishe
The predictability of bond and stock returns and the implications for portfolio allocation
No full textSuperparamagnetic nanoparticle based isolation of subcellular compartments as a method to identify spatial alterations in protein and lipid composition
No full textElevation of β-Amyloid Peptide 2–42 in Sporadic and Familial Alzheimer's Disease and Its Generation in PS1 Knockout Cells
No full textUrea-based β-amyloid (Aβ) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Aβ peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Aβ peptides, namely, 1–40/42 and carboxyl-terminal-truncated 1–37, 1–38, and 1–39, was found. Besides Aβ1–42, we also observed a consistent elevation of amino-terminal-truncated Aβ2–42 in a detergent-soluble pool in brains of subjects with Alzheimer's disease. Aβ2–42 was also specifically elevated in cerebrospinal fluid samples of Alzheimer's disease patients. To decipher the contribution of potential different γ-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Aβ peptides, we overexpressed β-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1−/− neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1−/− neurons and PS1+/+ neurons overexpressing APP-Δct (a slow-internalizing mutant) show a decrease of all secreted Aβ peptide species, as expected, because this mutant is processed mainly by α-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Aβ2–42 is significantly less affected in PS1−/− neurons and in neurons transfected with the endocytosis-deficient APP-Δct construct. Our data confirm that PS1 is closely involved in the production of Aβ1–40/42 and the carboxyl-terminal-truncated Aβ1–37, Aβ1–38, and Aβ1–39, but the amino-terminal-truncated and carboxyl-terminal-elongated Aβ2–42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Aβ peptide species could be generated by a βAsp/Ala-secretase activity
PL‐05‐01: Understanding and targeting diverse conformations of aggregated proteins that trigger neurodegeneration
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