Biomedical subjects

Stefan F Lichtenthaler

Publications and source records attributed to Stefan F Lichtenthaler.

17 recordsLinked to original sources

PubMed2026

A reproducible three-dimensional model of human brain tissue to investigate physiological and disease-associated microglia phenotypes.

Stem-cell-based in vitro models offer promising potential to elucidate human brain cell functions and interactions, but limitations in reproducibility, maturation and cell-type diversity persist. Especially, prolonged incorporation of mature microglia and studies of neuroinflammation have proven challenging. Here, we developed a human induced pluripotent stem cell-based three-dimensional cortical brain tissue model (3BTM) containing neurons, astrocytes and microglia with high reproducibility, maturity and viability. 3BTMs show morphological, functional and proteomic maturation of all cell types, leading to high similarity to their in vivo counterparts. Incorporated microglia survive for over 6 months and display mature morphology, functions and gene expression. Importantly, when engineered to model Alzheimer's disease pathology, 3BTMs recapitulate key disease hallmarks, including amyloid deposition, increased phospho-tau levels and neuroinflammation, with microglia shifting their transcriptional landscape to disease-relevant signatures. Treatment of Alzheimer's disease 3BTMs with anti-Aβ immunotherapy cleared deposits and largely reversed disease signatures in glia. Together, our microglia-containing model provides a platform for studying physiological and pathological states of human brain tissue.

Humans↗

PubMed2024

Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins.

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase, characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum. Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from endoplasmic reticulum-associated protein degradation in a catalytic-independent manner, promote autophagy, and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by the loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a deubiquitinase substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation, and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis," by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to the secretion of lysosomal proteins, including LGMN.

Humans↗

PubMed2006

GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing.

The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.

ADP-Ribosylation Factors↗

PubMed2006

Transcriptional and translational regulation of BACE1 expression--implications for Alzheimer's disease.

The proteolytical processing of the amyloid precursor protein (APP) gives rise to beta-amyloid peptides, which accumulate in brains of Alzheimer's disease (AD) patients. Different soluble or insoluble higher molecular weight forms of beta-amyloid peptides have been postulated to trigger a complex pathological cascade that may cause synaptic dysfunction, inflammatory processes, neuronal loss, cognitive impairment, and finally the onset of the disease. The generation of beta-amyloid peptides requires the proteolytical cleavage of APP by an aspartyl protease named beta-site APP-cleaving enzyme 1 (BACE1). The expression and enzymatic activity of BACE1 are increased in brains of AD patients. Here we discuss the importance of a number of recently identified transcription factors as well as post-transcriptional modifications and activation of intracellular signaling molecules for the regulation of BACE1 expression in brain. Importantly, some of these factors are known to be involved in the inflammatory and chronic stress responses of the brain, which are compromised during aging. Moreover, recent evidence indicates that beneficial effects of non-steriodal anti-inflammatory drugs on the progression of AD are mediated--at least in part--by effects on the peroxisome proliferator-activated receptor-gamma response element present in the BACE1 promoter. The identification of the cell type-specific expression and activation of NF-kappaB, Sp1 and YY1 transcription factors may provide a basis to specifically interfere with BACE1 expression and, thereby, to lower the concentrations of beta-amyloid peptides, which may prevent neuronal cell loss and cognitive decline in AD patients.

5' Untranslated Regions↗

PubMed2006

SPPL2a and SPPL2b promote intramembrane proteolysis of TNFalpha in activated dendritic cells to trigger IL-12 production.

Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.

Animals↗

PubMed2006

Expression cloning screen for modifiers of amyloid precursor protein shedding.

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the amyloid beta peptide (Abeta), which is thought to provoke the pathogenesis of Alzheimer's disease. To better understand the cellular processes that regulate ectodomain shedding of APP we used human embryonic kidney 293 cells and applied a sib-selection expression cloning approach. In addition to a known activator of APP shedding -- protein kinase A -- the following cDNAs were identified: the endocytic proteins endophilin A1 and A3, the metabotropic glutamate receptor 3 (mGluR3), palmitoyl-protein thioesterase 1 (PPT1), Numb-like and the kinase MEKK2. Endophilins A1 and A3, as well as mGluR3 activated APP shedding relatively specifically. They had little or no effect on the shedding of the unrelated membrane proteins TNF receptor 2 and P-selectin glycoprotein ligand-1. In contrast, MEKK2 and PKA also increased shedding of TNF receptor 2, suggesting that these kinases contribute to a general program regulating ectodomain shedding. The strongest activator of APP shedding, endophilin A3, reduced the rate of APP endocytosis and specifically increased APP shedding by the protease alpha-secretase, as measured in an antibody uptake assay and by immunoblot analysis. This suggests that endophilin A3 is a novel modulator of APP trafficking affecting access of APP to alpha-secretase. In summary, this study shows that expression cloning is a suitable way to identify proteins controlling ectodomain shedding of membrane proteins.

Acyltransferases↗

PubMed2006

Ectodomain shedding of the amyloid precursor protein: cellular control mechanisms and novel modifiers.

Proteolytic cleavage in the ectodomain of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer's disease amyloid-beta (Abeta) peptide and occurs through two different protease activities termed alpha- and beta-secretase. Both proteases compete for APP cleavage, but have opposite effects on Abeta generation. At present, little is known about the cellular pathways that control APP alpha- or beta-secretase cleavage and thus Abeta generation. To explore the contributory pathways in more detail we have recently employed an expression cloning screen and identified several activators of APP cleavage by alpha- or beta-secretase. Among them were known activators of APP cleavage, for example protein kinase A, and novel activators, such as endophilin and the APP homolog amyloid precursor-like protein 1 (APLP1). Mechanistic analysis revealed that both endophilin and APLP1 reduce the rate of APP endocytosis and strongly increase APP cleavage by alpha-secretase. This review summarizes the results of the expression cloning screen in the context of recent developments in our understanding of the cellular regulation of APP alpha-secretase cleavage. Moreover, it highlights the particular importance of endocytic APP trafficking as a prime modulator of APP shedding.

Amyloid Precursor Protein Secretases↗

PubMed2006

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8.

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.

ADAM Proteins↗

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↗

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

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

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

The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1.

The aspartyl protease BACE1 cleaves the amyloid precursor protein and the sialyltransferase ST6Gal I and is important in the pathogenesis of Alzheimer's disease. The normal function of BACE1 and additional physiological substrates have not been identified. Here we show that BACE1 acts on the P-selectin glycoprotein ligand 1 (PSGL-1), which mediates leukocyte adhesion in inflammatory reactions. In human monocytic U937 and human embryonic kidney 293 cells expressing endogenous or transfected BACE1, PSGL-1 was cleaved by BACE1 to generate a soluble ectodomain and a C-terminal transmembrane fragment. No evidence of the cleavage fragment was seen in primary cells derived from mice deficient in BACE1. By using deletion constructs and enzymatic deglycosylation of the C-terminal PSGL-1 fragments, the cleavage site in PSGL-1 was mapped to the juxtamembrane region within the ectodomain. In an in vitro assay BACE1 catalyzed the formation of the PSGL-1 products seen in vivo. The cleavage occurred at a Leu-Ser peptide bond as identified by mass spectrometry using a synthetic peptide. We conclude that PSGL-1 is an additional substrate for BACE1.

Alkaline Phosphatase↗

PubMed2003

gamma-Secretase cleavage site specificity differs for intracellular and secretory amyloid beta.

The final step in A beta generation is the cleavage of the C-terminal 99 amino acid residues of the amyloid precursor protein by gamma-secretase. gamma-Secretase activity is closely linked to the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of gamma-secretase leading to the formation of secreted and intracellular A beta are identical, we made use of point mutations close to the gamma-cleavage site, known to have a dramatic effect on the 42/40 ratio of secreted A beta. We found that the selected point mutations only marginally influenced the 42/40 ratio of intracellular A beta, suggesting differences in the gamma-secretase cleavage site specificity for the generation of secreted and intracellular A beta. The analysis of the subcellular compartments involved in the generation of intracellular A beta revealed that A beta is not generated in the early secretory pathway in the human SH-SY5Y neuroblastoma cell line. In this study we identified late Golgi compartments to be involved in the generation of intracellular A beta. Moreover, we demonstrate that the presence of processed PS1 is not sufficient to obtain gamma-secretase processing of the truncated amyloid precursor protein construct C99, proposing the existence of an additional factor downstream of the endoplasmic reticulum and early Golgi required for the formation of an active gamma-secretase complex.

Amino Acid Sequence↗

PubMed2003

No alterations of hippocampal neuronal number and synaptic bouton number in a transgenic mouse model expressing the beta-cleaved C-terminal APP fragment.

Previous studies in the literature have resulted in conflicting reports on the potential neurotoxicity of the beta-cleaved Alzheimer's disease C-terminal fragment (beta-CTF) of beta-amyloid precursor protein in vivo. To readdress this question by rigorous quantitative methods, we analyzed transgenic mice expressing human beta-CTF with the I45F mutation (SPA4CT) under control of the prion protein promoter by stereological techniques. The transgene was expressed in hippocampus and cortex in large pyramidal neurons and in dentate gyrus granule cells. Proteolytic processing of beta-CTF released Abeta. However, most of it remained uncleaved. Neurodegeneration was evaluated by investigating the numbers of hippocampal pyramidal and granule neurons, as well as the number of synaptophysin-immunopositive presynaptic boutons in the hippocampus of 15-month-old SPA4CT mice with design-based stereological techniques. The analyses showed that a fourfold higher expression of the transgene compared to murine APP levels had no effect on the numbers of both neurons and synaptophysin-immunopositive presynaptic boutons. These data implicate that expression of beta-CTF per se is not neurotoxic, and that other mechanisms are responsible for the neurotoxic events in Alzheimer's disease brain.

Alzheimer Disease↗

PubMed2002

The transmembrane domain of the amyloid precursor protein in microsomal membranes is on both sides shorter than predicted.

The amyloid precursor protein is cleaved within its ectodomain by beta-amyloid-converting enzyme (BACE) yielding C99, which is further cleaved by gamma-secretase within its putative transmembrane domain (TMD). Because it is difficult to envisage how a protease may cleave within the membrane, alternative mechanisms have been proposed for gamma-cleavage in which the TMD is shorter than predicted or positioned such that the gamma-cleavage site is accessible to cytosolic proteases. Here, we have biochemically determined the length of the TMD of C99 in microsomal membranes. Using a single cysteine mutagenesis scan of C99 combined with cysteine modification with a membrane-impermeable labeling reagent, we identified which residues are accessible to modification and thus located outside of the membrane. We find that in endoplasmic reticulum-derived microsomes the TMD of C99 consists of 12 residues that span from residues 37 to 48, which is N- and C-terminally shorter than predicted. Thus, the gamma-cleavage sites are positioned around the middle of the lipid bilayer and are unlikely to be accessible to cytosolic proteases. Moreover, the center of the TMD is positioned at the gamma-cleavage site at residue 42. Our data are consistent with a model in which gamma-secretase is a membrane protein that cleaves at the center of the membrane.

Amino Acid Sequence↗

PubMed2002

The intramembrane cleavage site of the amyloid precursor protein depends on the length of its transmembrane domain.

Proteolytic processing of the amyloid precursor protein by beta-secretase generates C99, which subsequently is cleaved by gamma-secretase, yielding the amyloid beta peptide (A beta). This gamma-cleavage occurs within the transmembrane domain (TMD) of C99 and is similar to the intramembrane cleavage of Notch. However, Notch and C99 differ in their site of intramembrane cleavage. The main gamma-cleavage of C99 occurs in the middle of the TMD, whereas the cleavage of Notch occurs close to the C-terminal end of the TMD, making it unclear whether both are cleaved by the same protease. To investigate whether gamma-cleavage always occurs in the middle of the TMD of C99 or may also occur at the end of the TMD, we generated C99-mutants with an altered length of the TMD and analyzed their gamma-cleavage in COS7 cells. The C terminus of A beta and thus the site of gamma-cleavage were determined by using monoclonal antibodies and mass spectrometry. Compared with C99-wild type (wt), most mutants with an altered length of the TMD changed the cleavage site of gamma-secretase, whereas control mutants with mutations outside the TMD did not. Thus, the length of the whole TMD is a major determinant for the cleavage site of gamma-secretase. Moreover, the C99-mutants were not only cleaved at one site but at two sites within their TMD. One cleavage site was located around the middle of the TMD, regardless of its actual length. An additional cleavage occurred within the N-terminal half of their TMD and thus at the opposite side of the Notch cleavage site.

Amino Acid Sequence↗