Biomedical subjects

Christian Haass

Publications and source records attributed to Christian Haass.

At least 37 records · Page 2Linked to original sources

PubMed2006

Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface.

Alzheimer's disease is characterized by brain deposition of extracellular amyloid beta-peptide (Abeta)-containing plaques. The cellular site of gamma-secretase activity, which releases Abeta and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for gamma-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by gamma-secretase, and AICD-GFP is released into the cytosol. Inhibiting gamma-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that gamma-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another gamma-secretase substrate, NotchDeltaE. Our results suggest that intracellular domains are generated by gamma-secretase at the plasma membrane and/or early endosomes.

Alzheimer Disease↗

PubMed2006

Furin-, ADAM 10-, and gamma-secretase-mediated cleavage of a receptor tyrosine phosphatase and regulation of beta-catenin's transcriptional activity.

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPkappa is cleaved by three proteases: furin, ADAM 10, and gamma-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPkappa. This is followed by gamma-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates beta-catenin, an RPTPkappa substrate. The expression of RPTPkappa suppresses beta-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of beta-catenin signaling. We also found that RPTPmu, another MAM family member, undergoes gamma-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPkappa signaling and implicate PIC in the activation of beta-catenin-mediated transcription.

ADAM Proteins↗

PubMed2006

Assembly, trafficking and function of gamma-secretase.

Gamma-secretase catalyzes the final cleavage of the beta-amyloid precursor protein to generate amyloid-beta peptide, the principal component of amyloid plaques in the brains of patients suffering from Alzheimer's disease. Here, we review the identification of gamma-secretase as a protease complex and its assembly and trafficking to its site(s) of cellular function. In reconstitution experiments, gamma-secretase was found to be composed of four integral membrane proteins, presenilin (PS), nicastrin (NCT), PEN-2 and APH-1 that are essential and sufficient for gamma-secretase activity. PS, which serves as a catalytic subunit of gamma-secretase, was identified as a prototypic member of novel aspartyl proteases of the GxGD type. In human cells, gamma-secretase could be further defined as a heterogeneous activity consisting of distinct complexes that are composed of PS1 or PS2 and APH-1a or APH-1b homologues together with NCT and PEN-2. Using green fluorescent protein as a reporter we localized PS and gamma-secretase activity at the plasma membrane and endosomes. Investigation of gamma-secretase complex assembly in knockdown and knockout cells of the individual subunits allowed us to develop a model of complex assembly in which NCT and APH-1 first stabilize PS before PEN-2 assembles as the last component. Furthermore, we could map domains in PS and PEN-2 that govern assembly and trafficking of the complex. Finally, Rer1 was identified as a PEN-2-binding protein that serves a role as an auxiliary factor for gamma-secretase complex assembly.

Alzheimer Disease↗

PubMed2006

Cellular functions of gamma-secretase-related proteins.

Amyloid-beta peptide (Abeta) is generated by gamma-secretase, a membrane protein complex with an unusual aspartyl protease activity consisting of the four components presenilin, nicastrin, APH-1 and PEN-2. Presenilin is considered the catalytic subunit of this complex since it represents the prototype of the new family of intramembrane-cleaving GxGD-type aspartyl proteases. Recently, five novel members of this family and a nicastrin-like protein were identified. Whereas one of the GxGD-type proteins was shown to be identical with signal peptide peptidase (SPP), the function of the others, now called SPP-like proteins (SPPLs), is not known. We therefore analyzed SPPL2b and SPPL3 and demonstrated that they localize to different subcellular compartments suggesting nonredundant functions. This was supported by different phenotypes obtained in knockdown studies in zebrafish embryos. In addition, these phenotypes could be phenocopied by ectopic expression of putative active site mutants, providing strong evidence for a proteolytic function of SPPL2b and SPPL3. We also identified and characterized the nicastrin-like protein nicalin which, together with the 130-kDa protein NOMO (Nodal modulator), forms a membrane protein complex different from gamma-secretase. We found that during zebrafish embryogenesis this complex is involved in the patterning of the axial mesendoderm, a process controlled by the Nodal signaling pathway.

Amyloid Precursor Protein Secretases↗

PubMed2005

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein.

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.

Amino Acid Motifs↗

PubMed2005

Stable chromosomal association of MSL2 defines a dosage-compensated nuclear compartment.

Dosage compensation in Drosophila is controlled by a complex (DCC) of proteins and noncoding RNA that binds specifically to the male X chromosome and leads to fine-tuning of transcription. Here, we employ male SL2 cells to characterize DCC function and dynamics during steady state of dosage compensation. Knocking down the key regulator of dosage compensation, male-specific-lethal 2 (MSL2), leads to loss of propagation of histone H4 lysine 16 acetylation and of the twofold elevation of transcription characteristic of the compensated male X chromosome. Surprisingly, lack of dosage compensation does not impair cell viability. Targeting of MSL2 to a reporter gene suffices to initiate dosage compensation in the cell model. Using photobleaching techniques in living cells, we found the association of MSL2 with the X chromosome to be exceptionally stable, essentially excluding dynamic redistribution of the DCC during interphase. This immobility distinguishes MSL2 from most other chromosomal proteins. Our findings have profound implications for the mechanism underlying dosage compensation and furthermore provide a new, conceptual reference of stability in an otherwise highly dynamic nuclear environment.

Acetylation↗

PubMed2005

Apolipoprotein E co-localizes with newly formed amyloid beta-protein (Abeta) deposits lacking immunoreactivity against N-terminal epitopes of Abeta in a genotype-dependent manner.

Different types of amyloid beta-protein (Abeta)-containing plaques occur in brains of Alzheimer's disease (AD) patients. Diffuse plaques seen during early stages of AD differ from neuritic plaques in later stages both with respect to the length of the Abeta peptides and the presence of other proteins, e.g., apolipoprotein-E (apoE). Since apoE is involved in Abeta transport and clearance, and the epsilon4-allele of the apolipoprotein-E gene (APOE) is a major risk factor for sporadic AD, it is plausible to speculate that apoE plays a pathophysiological role in the initiation of Abeta deposition. To address the issue of whether binding of apoE to Abeta is involved in initial Abeta deposition, we studied the human medial temporal lobe of 60 autopsy cases encompassing the full spectrum of AD-related pathology. In temporal lobe regions, which become involved for the first time at a given stage of beta-amyloidosis, all plaques represent newly formed plaques, and these were studied with immunohistochemical methods. ApoE was present in 36 cases, and was frequently co-localized with newly formed Abeta deposits detectable with anti-Abeta(42) but not with antibodies raised against N-terminal epitopes of Abeta. In 10 additional cases, immunoreactivity against apoE was completely lacking in newly formed plaques, which, at the same time, displayed immunoreactivity against N-terminal epitopes of Abeta. The failure of N-terminal epitopes of Abeta to co-localize with apoE in newly formed plaques indicates that these deposits presumably contain apoE-Abeta complexes, in which the N-terminal epitopes of Abeta are often concealed after complexing with apoE, thus preventing subsequent binding of antibodies. Moreover, apoE-positive newly formed plaques were seen more frequently in APOE epsilon4/4 cases than in non-APOE epsilon4/4 individuals, thereby underlining the potentially crucial role of apoE for the development of Abeta deposits.

Adult↗

PubMed2005

Single particle detection and characterization of synuclein co-aggregation.

Protein aggregation is the key event in a number of human diseases such as Alzheimer's and Parkinson's disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type alpha-synuclein forms co-aggregates with a mutant variant found in familial Parkinson's disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

Amyloid↗

PubMed2005

Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3.

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.

Amino Acid Motifs↗

PubMed2005

Shedding and gamma-secretase-mediated intramembrane proteolysis of the mucin-type molecule CD43.

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of gamma-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel gamma-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for gamma-secretase. However, the intracellular gamma-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without gamma-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.

Amyloid Precursor Protein Secretases↗

PubMed2005

GGA proteins regulate retrograde transport of BACE1 from endosomes to the trans-Golgi network.

Golgi-localized, gamma ear-containing, ADP ribosylation factor-binding (GGA) proteins have been shown to be implicated in the sorting of cargo proteins from the trans-Golgi network (TGN) to endosomal compartments. GGAs directly bind to DXXLL motifs in the cytoplasmic domains of cargo proteins. The Alzheimer-associated beta-secretase BACE1 also interacts with GGA proteins, but the functional relevance of this interaction was unknown. Here, we show that GGA1 regulates the retrograde transport of internalized BACE1 from endosomal compartments to the TGN by direct interaction in a phosphorylation-dependent manner. While phosphorylated BACE1 is efficiently transported from endosomes to the TGN, non-phosphorylated BACE1 enters a direct recycling route to the cell surface. Our data indicate that GGA proteins are not only involved in the sorting at the TGN but also mediate the retrograde transport of cargo proteins from endosomes to the TGN.

ADP-Ribosylation Factors↗

PubMed2005

Oxidative stress in transgenic mice with oligodendroglial alpha-synuclein overexpression replicates the characteristic neuropathology of multiple system atrophy.

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism unresponsive to dopaminergic therapy, cerebellar ataxia, and dysautonomia. Neuropathology shows a characteristic neuronal multisystem degeneration that is associated with widespread oligodendroglial alpha-synuclein (alpha-SYN) inclusions. Presently no animal model completely replicates the specific neuropathology of MSA. Here we investigated the behavioral and pathological features resulting from oligodendroglial alpha-SYN overexpression in transgenic mice exposed to mitochondrial inhibition by 3-nitropropionic acid. In transgenic mice 3-nitropropionic acid induced or augmented motor deficits that were associated with MSA-like pathology including striatonigral degeneration and olivopontocerebellar atrophy. Widespread astrogliosis and microglial activation were also observed in the presence of alpha-SYN in oligodendrocytes. Our results indicate that combined mitochondrial inhibition and overexpression of oligodendroglial alpha-SYN generates a novel model of MSA that may be useful for evaluating both pathogenesis and treatment strategies.

Animals↗

PubMed2005

Length and overall sequence of the PEN-2 C-terminal domain determines its function in the stabilization of presenilin fragments.

Gamma-secretase is an aspartyl protease complex that catalyzes the intramembrane cleavage of a subset of type I transmembrane proteins including the beta-amyloid precursor protein (APP) implicated in Alzheimer's disease. Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2) constitute the active gamma-secretase complex. PEN-2, the smallest subunit, is required for triggering PS endoproteolysis. Stabilization of the resultant N- and C-terminal fragments, which carry the catalytically active site aspartates, but not endoproteolysis itself, requires the C-terminal domain of PEN-2. To functionally dissect the C-terminal domain we created C-terminal deletion mutants and mutagenized several evolutionary highly conserved residues. The PEN-2 mutants were then probed for functional complementation of a PEN-2 knockdown, which displays deficient PS1 endoproteolysis and impaired NCT maturation. Progressive truncation of the C-terminus caused increasing loss of function. This was also observed for an internal deletion mutant as well as for C-terminally tagged PEN-2 with a twofold elongated C-terminal domain. Interestingly, only simultaneous, but not individual substitution of the highly conserved D90, F94, P97 and G99 residues with alanine interfered with PEN-2 function. All loss of function mutants identified allowed PS1 endoproteolysis, but failed to stably associate with the resultant PS1 fragments, which like the PEN-2 loss of function mutants underwent proteasomal degradation. However, complex formation of the PEN-2 mutants with PS1 fragments could be recovered when proteasomal degradation was blocked. Taken together, our data suggest that the PS-subunit stabilizing function of PEN-2 depends on length and overall sequence of its C-terminal domain.

Alzheimer Disease↗

PubMed2004

A pathogenic PrP mutation and doppel interfere with polarized sorting of the prion protein.

Several proteins linked to neurodegenerative diseases, such as the beta-amyloid precursor protein, amyloid beta-peptide, beta-secretase, and tau, undergo selective polarized sorting. We investigated polarized sorting of the mammalian prion protein (PrP(C)) and its homologue doppel (Dpl). In contrast to Dpl, which accumulates on the apical surface, PrP(C) is targeted selectively to the basolateral side in Madin-Darby canine kidney cells. An extensive deletion and domain swapping analysis revealed that the internal hydrophobic domain (HD) of PrP (amino acids 113-133) confers basolateral sorting in a dominant manner. PrP mutants lacking the HD are sorted apically, while Dpl chimeras containing the HD of PrP are directed to the basolateral membrane. Furthermore, a pathogenic PrP missense mutation within the HD leads to aberrant apical sorting of PrP as well.

Animals↗

PubMed2004

Gamma-secretase complex assembly within the early secretory pathway.

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.

Amyloid Precursor Protein Secretases↗

PubMed2004

A lipid boundary separates APP and secretases and limits amyloid beta-peptide generation.

Millions of patients suffer from Alzheimer's disease, and intensive efforts to find a cure for this devastating disorder center on the proteases, which release the deadly amyloid beta-peptide from its precursor. The cutting procedure is thought to be cholesterol dependent and strategies to lower cholesterol as therapeutic treatment are under intensive investigation. Recent findings suggest that the complete proteolytic machinery required for amyloid beta-peptide generation is located within lipid rafts. Data by Dotti and colleagues (Abad-Rodriguez et al., 2004), in this issue, suggest that rafts isolate the cutting machinery away from its deadly substrate. These findings describe a novel mechanism for controlling proteolytic activity by building a lipid boundary between proteases and their substrates.

Alzheimer Disease↗

PubMed2004

Parkin phosphorylation and modulation of its E3 ubiquitin ligase activity.

Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins. Whereas the effects of mutations on the structure and function of parkin have been intensely studied, post-translational modifications of parkin and the regulation of its enzymatic activity are poorly understood. Here we report that parkin is phosphorylated both in human embryonic kidney HEK293 cells and human neuroblastoma SH-SY5Y cells. The turnover of parkin phosphorylation was rapid, because inhibition of phosphatases with okadaic acid was necessary to stabilize phosphoparkin. Phosphoamino acid analysis revealed that phosphorylation occurred mainly on serine residues under these conditions. At least five phosphorylation sites were identified, including Ser101, Ser131, and Ser136 (located in the linker region) as well as Ser296 and Ser378 (located in the RING-IBR-RING motif). Casein kinase-1, protein kinase A, and protein kinase C phosphorylated parkin in vitro, and inhibition of casein kinase-1 caused a dramatic reduction of parkin phosphorylation in cell lysates. Induction of protein folding stress in cells reduced parkin phosphorylation, and unphosphorylated parkin had slightly but significantly elevated autoubiquitination activity. Thus, complex regulation of the phosphorylation state of parkin may contribute to the unfolded protein response in stressed cells.

Casein Kinase I↗

PubMed2004

The presenilin C-terminus is required for ER-retention, nicastrin-binding and gamma-secretase activity.

gamma-Secretase is an intramembrane cleaving protease involved in Alzheimer's disease. gamma-Secretase occurs as a high molecular weight complex composed of presenilin (PS1/2), nicastrin (NCT), anterior pharynx-defective phenotype 1 and PS enhancer 2. Little is known about the cellular mechanisms of gamma-secretase assembly. Here we demonstrate that the cytoplasmic tail of PS1 fulfills several functions required for complex formation, retention of unincorporated PS1 and gamma-secretase activity. The very C-terminus interacts with the transmembrane domain of NCT and may penetrate into the membrane. Deletion of the last amino acid is sufficient to completely block gamma-secretase assembly and release of PS1 from the endoplasmic reticulum (ER). This suggests that unincorporated PS1 is actively retained within the ER. We identified a hydrophobic stretch of amino acids within the cytoplasmic tail of PS1 distinct from the NCT-binding site, which is required to retain unincorporated PS1 within the ER. Deletion of the retention signal results in the release of PS1 from the ER and the assembly of a nonfunctional gamma-secretase complex, suggesting that at least a part of the retention motif may also be required for the function of PS1.

Amino Acid Sequence↗