Biomedical subjects

P Saftig

Publications and source records attributed to P Saftig.

At least 19 recordsLinked to original sources

PubMed2002

Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene.

The ADP-ribosylation factor-like protein 4 (ARL4) is a 22-kDa GTP-binding protein which is abundant in testes of pubertal and adult rodents but absent in testes from prepubertal animals. During testis development, ARL4 expression starts at day 16 when the spermatogenesis proceeds to the late pachytene. In the adult testis, the ARL4 protein was detected in pre- and postmeiotic cells, spermatocytes, and spermatides, but not in spermatogonia and mature spermatozoa. Mouse Arl4-null mutants generated by targeted disruption of the Arl4 gene were viable and grew normally; male as well as female Arl4(-/-) mice were fertile. However, inactivation of the Arl4 gene resulted in a significant reduction of testis weight and sperm count by 30 and 60%, respectively, without reduction of litter size or frequency. It is suggested that the disruption of Arl4 produces a moderate retardation of germ cell development, possibly at the stage of meiosis.

ADP-Ribosylation Factors↗

PubMed2002

The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation.

In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howship's lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howship's lacuna, in close association with an osteopontin-rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K-deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell "rescues" bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howship's lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process.

Animals↗

PubMed2001

Involvement of nitric oxide released from microglia-macrophages in pathological changes of cathepsin D-deficient mice.

Cathepsin D (CD) deficiency has been shown to induce ceroid-lipofuscin storage in lysosomes of mouse CNS neuron (Koike et al., 2000). To understand the behavior of microglial cells corresponding to these neuronal changes, CD-deficient (CD-/-) mice, which die at approximately postnatal day (P) 25 by intestinal necrosis, were examined using morphological as well as biochemical approaches. Light and electron microscopic observations revealed that microglia showing large round cell bodies with few processes appeared in the cerebral cortex and thalamus after P16. At P24, microglia often encircled neurons that were occupied with autolysosomes, indicating increased phagocytic activity. These morphologically transformed microglia markedly expressed inducible nitric oxide synthase (iNOS), which was also detected in the intestine of the mice. To assess the role of microglial nitric oxide (NO) in neuropathological changes in CD-/- mice, l-N(G)-nitro-arginine methylester (l-NAME), a competitive NOS inhibitor, or S-methylisothiourea hemisulfate (SMT), an iNOS inhibitor, was administered intraperitoneally for 13 consecutive days. The total number of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells counted in the thalamus was found to be significantly decreased by chronic treatment of l-NAME or SMT, whereas neither the neuronal accumulation of ceroid-lipofuscin nor the microglial phagocytic activity was affected by these treatments. Moreover, the chronic treatment of l-NAME or SMT completely suppressed hemorrhage-necrotic changes in the small intestine of CD-/- mice, resulting in normal growth of the body weight of the mice. These results suggest that NO production via iNOS activity in microglia and peripheral macrophages contributes to secondary tissue damages such as neuronal apoptosis and intestinal necrosis, respectively.

Animals↗

PubMed2001

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells.

Urea-based beta-amyloid (Abeta) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Abeta 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 Abeta peptides, namely, 1-40/42 and carboxyl-terminal-truncated 1-37, 1-38, and 1-39, was found. Besides Abeta1-42, we also observed a consistent elevation of amino-terminal-truncated Abeta2-42 in a detergent-soluble pool in brains of subjects with Alzheimer's disease. Abeta2-42 was also specifically elevated in cerebrospinal fluid samples of Alzheimer's disease patients. To decipher the contribution of potential different gamma-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Abeta peptides, we overexpressed beta-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-Deltact (a slow-internalizing mutant) show a decrease of all secreted Abeta peptide species, as expected, because this mutant is processed mainly by alpha-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Abeta2-42 is significantly less affected in PS1-/- neurons and in neurons transfected with the endocytosis-deficient APP-Deltact construct. Our data confirm that PS1 is closely involved in the production of Abeta1-40/42 and the carboxyl-terminal-truncated Abeta1-37, Abeta1-38, and Abeta1-39, but the amino-terminal-truncated and carboxyl-terminal-elongated Abeta2-42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Abeta peptide species could be generated by a beta(Asp/Ala)-secretase activity.

Aged↗

PubMed2001

The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein.

We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.

Amyloid Precursor Protein Secretases↗

PubMed2001

The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein.

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106-126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach that o-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrin and metalloprotease); and TACE, tumor necrosis factor alpha-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPc breakdown and thereby depleting cells of the putative 106-126 "toxic" domain of PrPc.

ADAM Proteins↗

PubMed2001

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin.

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.

Alkaline Phosphatase↗

PubMed2001

Disease model: LAMP-2 enlightens Danon disease.

Danon disease ('lysosomal glycogen storage disease with normal acid maltase') is characterized by a cardiomyopathy, myopathy and variable mental retardation. Mutations in the coding sequence of the lysosomal-associated membrane protein 2 (LAMP-2) were shown to cause a LAMP-2 deficiency in patients with Danon disease. LAMP-2 deficient mice manifest a similar vacuolar cardioskeletal myopathy. In addition to the patient reports LAMP-2 deficiency in mice causes pancreatic, hepatocytic, endothelial and leucocyte vacuolation. LAMP-2 deficient mice represent a valuable animal model of Danon disease. They will further be used to study the exact role of LAMP-2 in autophagy and to analyse the consequences of an impaired autophagic pathway in various tissues.

Animals↗

PubMed2001

Overlapping functions of lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (Acp5) revealed by doubly deficient mice.

To date, two lysosomal acid phosphatases are known to be expressed in cells of the monocyte/phagocyte lineage: the ubiquitously expressed lysosomal acid phosphatase (LAP) and the tartrate-resistant acid phosphatase-type 5 (Acp5). Deficiency of either acid phosphatase results in relatively mild phenotypes, suggesting that these enzymes may be capable of mutual complementation. This prompted us to generate LAP/Acp5 doubly deficient mice. LAP/Acp5 doubly deficient mice are viable and fertile but display marked alterations in soft and mineralised tissues. They are characterised by a progressive hepatosplenomegaly, gait disturbances and exaggerated foreshortening of long bones. Histologically, these animals are distinguished by an excessive lysosomal storage in macrophages of the liver, spleen, bone marrow, kidney and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of Acp5- and LAP/Acp5-deficient mice. In osteoclasts of phosphatase-deficient mice, vacuoles were frequently found which contained fine filamentous material. The vacuoles in Acp5- and LAP/Acp5 doubly-deficient osteoclasts also contained crystallite-like features, as well as osteopontin, suggesting that Acp5 is important for processing of this protein. This is further supported by biochemical analyses that demonstrate strongly reduced dephosphorylation of osteopontin incubated with LAP/Acp5-deficient bone extracts. Fibroblasts derived from LAP/Acp5 deficient embryos were still able to dephosphorylate mannose 6-phosphate residues of endocytosed arylsulfatase A. We conclude that for several substrates LAP and Acp5 can substitute for each other and that these acid phosphatases are essential for processing of non-collagenous proteins, including osteopontin, by osteoclasts.

Acid Phosphatase↗

PubMed2001

Implication of APP secretases in notch signaling.

Signaling 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.

ADAM Proteins↗

PubMed2000

Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.

Cathepsin D-deficient (CD-/-) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CD-/- mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CD-/- mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CD-/- neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CD-/- mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

Action Potentials↗

PubMed2000

Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice.

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danon's disease is associated with the accumulation of autophagic material in striated myocytes.

Amino Acids↗

PubMed2000

mu1A-adaptin-deficient mice: lethality, loss of AP-1 binding and rerouting of mannose 6-phosphate receptors.

The heterotetrameric AP-1 complex is involved in the formation of clathrin-coated vesicles at the trans-Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse mu1A-adaptin gene causes embryonic lethality at day 13.5. In cells deficient in micro1A-adaptin the remaining AP-1 adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of micro1A-adaptin-deficient embryos that show gamma-adaptin binding to membranes, indicating the formation of an epithelial specific AP-1B complex and demonstrating the absence of additional mu1A homologs. Mannose 6-phosphate receptors are cargo molecules that exit the TGN via AP-1-clathrin-coated vesicles. The steady-state distribution of the mannose 6-phosphate receptors MPR46 and MPR300 in mu1A-deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP-1 is required for retrograde endosome to TGN transport of the receptor.

Adaptor Protein Complex 1↗

PubMed2000

Spermatogenesis in mice is not affected by histone H1.1 deficiency.

The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S phase of the cell cycle. In adult mice the histone gene H1.1 is expressed mainly in thymus, spleen, and testis. The single-copy gene coding for the H1.1 protein was eliminated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the deficient H1.1 gene developed normally until the adult stage without H1.1 mRNA and H1.1 protein. No anatomic abnormalities could be detected. In addition, mice lacking the H1.1 gene were fertile and they showed normal spermatogenesis and testicular morphology.

Animals↗

PubMed2000

Functions of cathepsin K in bone resorption. Lessons from cathepsin K deficient mice.

Cathepsin K is a cysteine proteinase expressed predominantly in osteoclasts. Cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Pycnodysostosis, an autosomal recessive osteosclerosing skeletal disorder has recently been shown to result from mutations in the cathepsin K gene. Cathepsin K deficient mice generated by targeted disruption of this proteinase phenocopy many aspects of pycnodysostosis. They display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space accompanied by an altered ultrastructural appearance of the cathepsin K deficient osteoclasts. These cells also demonstrate an impaired resorptive activity in vitro. In contrast to other forms of osteopetrosis, which are due to disrupted osteoclastogenesis, cathepsin K deficiency is associated with an inhibition of osteoclast activity. Taken together the phenotype of cathepsin K knockout mice underlines the importance of this proteinase in bone remodelling.

Animals↗

PubMed2000

Ceramide as an activator lipid of cathepsin D.

We have identified the aspartic protease cathepsin D as a novel intracellular target protein for the lipid second messenger ceramide. Ceramide specifically binds to and induces CTSD proteolytic activity. A-SMase deficient cells derived from Niemann-Pick patients show decreased CTSD activity that was reconstituted by transfection with A-SMase cDNA. Ceramide accumulation in cells derived from A-ceramidase defective Farber patients correlates with enhanced CTSD activity. These findings suggest that A-SMase-derived ceramide targets endolysosomal CTSD.

Amidohydrolases↗

PubMed2000

Spermatogenesis proceeds normally in mice without linker histone H1t.

The histone gene H1t is expressed exclusively in pachytene spermatocytes of the testis. In this report we have eliminated the single copy H1t gene by homologous recombination from the mouse genome to analyse the function of the H1t protein during spermatogenesis. Mice homozygous for the mutated H1t gene locus developed normally and showed no anatomic abnormalities until the adult stage. In addition, H1t-deficient mice were fertile and reproduced as wild-type mice. The process of spermatogenesis and the testicular morphology remained unchanged in the absence of H1t. RNase protection analysis demonstrated that H1.1, H1.2 and H1.4 histone gene expression is enhanced during spermatogenesis in H1t-deficient mice.

Animals↗

PubMed2000

Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling.

Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain-like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL-deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes-both of ectodermal origin-are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL-deficient mice is reminiscent of the spontaneous mouse mutant furless (fs). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.

Alopecia↗