Biomedical subjects

Christian Haass

Publications and source records attributed to Christian Haass.

At least 55 records · Page 3Linked to original sources

PubMed2004

Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme.

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Amino Acid Motifs↗

PubMed2004

Amyloid beta-induced changes in nitric oxide production and mitochondrial activity lead to apoptosis.

Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid beta (Abeta) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Abeta levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Abeta production by a functional gamma-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Abeta in these processes. Extracellular treatment of PC12 cells with comparable Abeta concentrations only leads to weak changes, demonstrating the important role of intracellular Abeta. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Abeta levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Abeta levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death.

Alzheimer Disease↗

PubMed2004

Identification of distinct gamma-secretase complexes with different APH-1 variants.

The gamma-secretase complex catalyzes the final intramembraneous cleavage of the beta-amyloid precursor protein, liberating the neurotoxic amyloid beta-peptide implicated in Alzheimer's disease. Apart from the catalytic subunit presenilin (PS), three additional subunits, nicastrin, APH-1, and PEN-2, have been identified. In mammals, two PS homologues, PS1 and PS2, which are part of distinct gamma-secretase complexes, exist. Likewise, two APH-1 homologues, APH-1a and APH-1b, have been identified. Furthermore, two APH-1a splice forms, APH-1aS and APH-1aL, have been reported. Here we show that both APH-1a splice forms and APH-1b are expressed in peripheral and neuronal cells. APH-1aS, APH-1aL, and APH-1b form separate, proteolytically active gamma-secretase complexes containing either one of the two PSs. Deficiency of APH-1a caused a decrease in nicastrin, PS, and PEN-2 levels and an increase in the levels of APH-1b, whereas deficiency of APH-1b did not affect the levels of APH-1a or the other complex components. Consistent with this finding, we found that deficiency of APH-1a was associated with reduced gamma-secretase activity, whereas deficiency of APH-1b was not. Thus, APH-1b gamma-secretase complexes may fulfill redundant functions. Taken together, our results suggest that, dependent on the tissue expression of the individual subunits, six distinct gamma-secretase complexes composed of the known subunits can exist in human cells.

Alternative Splicing↗

PubMed2004

Nicalin and its binding partner Nomo are novel Nodal signaling antagonists.

Nodals are signaling factors of the transforming growth factor-beta (TGFbeta) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated gamma-secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish.

Amyloid Precursor Protein Secretases↗

PubMed2004

Human BACE forms dimers and colocalizes with APP.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

Amyloid Precursor Protein Secretases↗

PubMed2004

Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1.

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Abeta. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity.

Alleles↗

PubMed2004

Expression of the Alzheimer protease BACE1 is suppressed via its 5'-untranslated region.

The aspartyl protease BACE1 has a pivotal role in the pathogenesis of Alzheimer's disease. Recently, it was shown that in Alzheimer's disease patients, BACE1 levels were elevated although mRNA levels were not changed compared with controls. Here, we demonstrate that the 5'-untranslated region (5'UTR) of BACE1 controls the rate of BACE1 translation. In the presence of the 5'UTR, we observed more than 90% reduction of BACE1 protein levels in HEK293, COS7 and H4 cells, and a similar reduction of BACE1 activity in vitro. mRNA levels were not affected, demonstrating that the 5'UTR repressed the translation but not the transcription of BACE1. The 3'UTR did not affect BACE1 expression. An extensive mutagenesis analysis predicts that the GC-rich region of the 5'UTR forms a constitutive translation barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA. Our data therefore demonstrate translational repression as a new mechanism controlling BACE1 expression.

5' Untranslated Regions↗

PubMed2004

Requirement of PEN-2 for stabilization of the presenilin N-/C-terminal fragment heterodimer within the gamma-secretase complex.

Gamma-secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated beta-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-DeltaC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-DeltaC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the gamma-secretase complex. In addition, expression of PEN-2-DeltaC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Deltaexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for gamma-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the gamma-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for gamma-secretase complex maturation independent of its activity in PS1 endoproteolysis.

Amyloid Precursor Protein Secretases↗

PubMed2004

Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study.

A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of alpha-synuclein (alphaS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of alphaS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of alphaS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the alphaS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of alphaS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes.

Calorimetry↗

PubMed2004

Pathological properties of the Parkinson's disease-associated protein DJ-1 in alpha-synucleinopathies and tauopathies: relevance for multiple system atrophy and Pick's disease.

Mutations in the PARK7 gene DJ-1 are associated with recessive hereditary Parkinson's disease (PD). Fibrillar inclusions of alpha-synuclein comprise the neuropathological hallmarks of PD and related Lewy body diseases as well as multiple system atrophy (MSA). Moreover, neuronal and glial inclusions containing tau have been observed in alpha-synucleinopathy patients. Using a collection of antibodies against DJ-1, we have performed a comprehensive investigation of DJ-1 in alpha-synucleinopathies and tauopathies. DJ-1 was abundantly expressed in reactive astrocytes of patients with neurodegenerative diseases. Likewise, DJ-1 antiserum immunostained reactive astrocytes that became abundant with disease progression in the brain stem of transgenic mice expressing mutant [A30P]alpha-synuclein. Human Lewy bodies as well as Lewy body-like inclusions in the alpha-synuclein transgenic mice were DJ-1 negative. Neuronal tau inclusions were DJ-1 immunopositive in Pick's disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer's disease. In addition, we found DJ-1-immunopositive glial inclusions in CBD, PSP and MSA. Biochemical extraction experiments revealed the specific presence of insoluble, modified DJ-1 in PiD and MSA. Our results suggest that DJ-1 is up-regulated in reactive astrocytes as well as in neuronal and glial cells with specific alpha-synucleinopathy and tauopathy.

Adult↗

PubMed2004

Take five--BACE and the gamma-secretase quartet conduct Alzheimer's amyloid beta-peptide generation.

In 1959, Dave Brubeck and Paul Desmond revolutionized modern jazz music by composing their unforgettable Take Five in 5/4, one of the most defiant time signatures in all music. Of similar revolutionary importance for biomedical and basic biochemical research is the identification of the minimal set of genes required to obtain a deadly time bomb ticking in all of us: Alzheimer's disease. It now appears that one needs to Take Five genes to produce a deadly peptide by a proteolytic mechanism, which paradoxically is otherwise of pivotal importance for development and cell fate decisions.

Alzheimer Disease↗

PubMed2004

beta-site amyloid precursor protein cleaving enzyme 1 increases amyloid deposition in brain parenchyma but reduces cerebrovascular amyloid angiopathy in aging BACE x APP[V717I] double-transgenic mice.

The generation of amyloid peptides (Abeta) from the amyloid precursor protein (APP) is initiated by beta-secretase (BACE), whereas subsequent gamma-secretase cleavage mediated by presenilin-1, produces Abeta peptides mainly of 40 or 42 amino acids long. In addition, alternative beta'-cleavage of APP at position 11 of the amyloid sequence results in N-truncated Abeta(11-40/42) peptides, but the functional significance or pathological impact is unknown. Here we demonstrate that in the brain of BACE x APP[V717I] double-transgenic mice, amyloidogenic processing at both Asp1 and Glu11 is increased resulting in more and different Abeta species and APP C-terminal fragments. Pathologically, BACE significantly increased the number of diffuse and senile amyloid plaques in old double-transgenic mice. Unexpectedly, vascular amyloid deposition was dramatically lower in the same BACE x APP[V717I] double-transgenic mice, relative to sex- and age-matched APP[V717I] single-transgenic mice in the same genetic background. The tight inverse relation of vascular amyloid to the levels of the less soluble N-terminally truncated Abeta peptides is consistent with the hypothesis that vascular amyloid deposition depends on drainage of excess tissue Abeta. This provides biochemical evidence in vivo for the preferential contribution of N-truncated Abeta to parenchymal amyloid deposition in contrast to vascular amyloid pathology.

Age Factors↗

PubMed2004

How does parkin ligate ubiquitin to Parkinson's disease?

Recessive mutations in the human PARKIN gene are the most common cause of hereditary parkinsonism, which arises from the degeneration of dopaminergic neurons in the substantia nigra. However, the molecular mechanisms by which the loss of parkin causes dopaminergic neurodegeneration are not well understood. Parkin is an enzyme that ubiquitinates several candidate substrate proteins and thereby targets them for proteasomal degradation. Hypothesis-driven searches have led to the discovery of aggregation-prone protein substrates of parkin. Moreover, the enzyme is upregulated when under unfolded protein stress. Thus, loss-of-function mutations of parkin might impair the removal of potentially toxic protein aggregates. However, the limited neuropathological information that is available from parkin-proven patients, as well as the recent knockout of the parkin gene in fruit flies and mice, may indicate a more complex disease mechanism, possibly involving the misfolding of parkin itself or of additional substrates. The risk factors that predispose dopaminergic neurons to degenerate on parkin failure are yet to be identified.

Animals↗

PubMed2004

Regional distribution of proteinase K-resistant alpha-synuclein correlates with Lewy body disease stage.

Lewy bodies (LBs) containing alpha-synuclein (alphaSYN) fibrils are the hallmark lesions of Parkinson disease, dementia with LBs, and related neurodegenerative diseases. Here we have investigated the susceptibility to proteolysis of alphaSYN from brain samples of patients with different subtypes of LB diseases. While soluble alphaSYN was completely degradable in all samples, the affected brain regions additionally contained insoluble, proteinase K (PK)-resistant alphaSYN. In brainstem-predominant subtype LB disease cases, PK-resistant alphaSYN was found in the medulla and substantia nigra, but not in cerebral cortex. In limbic subtype LB disease cases, PK resistance of alphaSYN spread to the cingulate and parahippocampal cortex, and further to the frontal cortex in neocortical subtype LB disease cases. Variable amounts of neuritic PK-resistant alphaSYN were found in the striatum of all cases. Thus, PK resistance of alphaSYN may be useful for the development of biomarkers of LB diseases.

Aged↗

PubMed2004

Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1.

Gamma-secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.

Alzheimer Disease↗

PubMed2004

The biochemical and genetic odyssey to the function of a nicastrin-like protein.

Gamma-secretase is a high-molecular-weight protein complex required for the proteolytic processing of various transmembrane proteins including the Alzheimer's disease-associated amyloid precursor protein and the signaling receptor Notch. One of the gamma-secretase complex components is the type I transmembrane protein nicastrin. Here we review the odyssey to a cyclopic fish, which at the end allowed the functional analysis of nicalin, a novel member of the nicastrin protein family. This 60-kDa protein is part of a previously unknown membrane protein complex unrelated to gamma-secretase and binds to Nomo (Nodal modulator, previously known as pM5), a novel 130-kDa transmembrane protein. Both proteins are highly conserved in metazoans and show almost identical tissue distribution in humans. Functional studies in zebrafish embryos and cultured human cells revealed that nicalin and Nomo collaborate to antagonize the Nodal/TGFbeta signaling pathway. Thus, nicastrin and nicalin are both associated with protein complexes involved in cell fate decisions during early embryonic development.

Amyloid Precursor Protein Secretases↗

PubMed2004

Amyloid at the cutting edge: activation of alpha-secretase prevents amyloidogenesis in an Alzheimer disease mouse model.

The amyloid beta-peptide (A beta peptide) is assumed to play a crucial and early role in the pathogenesis of Alzheimer disease. Thus, strategies for a pharmacotherapy aim at reducing A beta peptide generation, which proteolytically derives from the amyloid precursor protein (APP). The main targets so far have been beta- and gamma-secretase, the two proteases that cleave APP at the N- and C-terminus of the A beta peptide and are thus directly responsible for A beta peptide generation. A different strategy, namely the activation of alpha-secretase, has barely been investigated for its therapeutic potential. alpha-Secretase cleaves within the A beta peptide domain and thus precludes A beta peptide generation. Now, new results demonstrate that activation of alpha-secretase indeed reduces A beta peptide generation and toxicity in vivo.

Alzheimer Disease↗

PubMed2003

Differential effects of Parkinson's disease-associated mutations on stability and folding of DJ-1.

Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in beta-strand and alpha-helix conformation. Alpha-helices were more susceptible to thermal denaturation than the beta-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation.

Cell Line↗