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Thomas J Jentsch - One of the best experts on this subject based on the ideXlab platform.
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The FASEB Journal • Research Communication Lysosomal degradation of endocytosed proteins depends on the chloride transport protein ClC-7
2015Co-Authors: Lena Wartosch, Jens C Fuhrmann, Tobias Stauber, Michaela Schweizer, Thomas J JentschAbstract:mal/lysosomal member of the CLC family of chloride channels and transporters, or in its -subunit OSTM1 cause osteopetrosis and lysosomal storage disease in mice and humans. The severe phenotype of mice globally deleted for ClC-7 or OSTM1 and the absence of storage material in cultured cells hampered investiga-tions of the mechanism leading to lysosomal pathology in the absence of functional ClC-7/OSTM1 transporters. Tissue-specific ClC-7-knockout mice now reveal that accumulation of storage material occurs cell-autono-mously in neurons or renal proximal tubular cells lacking ClC-7. Almost all ClC-7-deficient neurons die. The activation of glia is restricted to brain regions where ClC-7 has been inactivated. The effect of ClC-7 disruption on lysosomal function was investigated in renal proximal tubular cells, which display high endo-cytotic activity. Pulse-chase endocytosis experiments in vivo with mice carrying chimeric deletion of ClC-7 in proximal tubules allowed a direct comparison of the handling of endocytosed protein between cells express-ing or lacking ClC-7. Whereas protein was endocytosed similarly in cells of either genotype, its half-life in-creased significantly in ClC-7-deficient cells. These ex-periments demonstrate that lysosomal pathology is a cell-autonomous consequence of ClC-7 disruption and that ClC-7 is important for lysosomal protein degrada
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a missense mutation accelerating the gating of the lysosomal cl h exchanger clc 7 OSTM1 causes osteopetrosis with gingival hamartomas in cattle
Disease Models & Mechanisms, 2014Co-Authors: Arnaud Sartelet, Tobias Stauber, Wouter Coppieters, Carmen F Ludwig, Corinne Fasquelle, Tom Druet, Zhiyan Zhang, Naima Ahariz, Nadine Cambisano, Thomas J JentschAbstract:Chloride-proton exchange by the lysosomal anion transporter ClC7/OSTM1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or OSTM1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/OSTM1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/OSTM1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/OSTM1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/OSTM1 in lysosomal function and bone resorption.
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A missense mutation accelerating the gating of the lysosomal Cl-/H+-exchanger ClC-7/OSTM1 causes osteopetrosis with gingival hamartomas in cattle
Disease models & mechanisms, 2013Co-Authors: Arnaud Sartelet, Tobias Stauber, Wouter Coppieters, Carmen F Ludwig, Corinne Fasquelle, Tom Druet, Zhiyan Zhang, Naima Ahariz, Nadine Cambisano, Thomas J JentschAbstract:Chloride-proton exchange by the lysosomal anion transporter ClC7/OSTM1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or OSTM1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/OSTM1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/OSTM1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/OSTM1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/OSTM1 in lysosomal function and bone resorption.
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cell biology and physiology of clc chloride channels and transporters
Comprehensive Physiology, 2012Co-Authors: Tobias Stauber, Stefanie Weinert, Thomas J JentschAbstract:Proteins of the CLC gene family assemble to homo- or sometimes heterodimers and either function as Cl(-) channels or as Cl(-)/H(+)-exchangers. CLC proteins are present in all phyla. Detailed structural information is available from crystal structures of bacterial and algal CLCs. Mammals express nine CLC genes, four of which encode Cl(-) channels and five 2Cl(-)/H(+)-exchangers. Two accessory β-subunits are known: (1) barttin and (2) OSTM1. ClC-Ka and ClC-Kb Cl(-) channels need barttin, whereas OSTM1 is required for the function of the lysosomal ClC-7 2Cl(-)/H(+)-exchanger. ClC-1, -2, -Ka and -Kb Cl(-) channels reside in the plasma membrane and function in the control of electrical excitability of muscles or neurons, in extra- and intracellular ion homeostasis, and in transepithelial transport. The mainly endosomal/lysosomal Cl(-)/H(+)-exchangers ClC-3 to ClC-7 may facilitate vesicular acidification by shunting currents of proton pumps and increase vesicular Cl(-) concentration. ClC-3 is also present on synaptic vesicles, whereas ClC-4 and -5 can reach the plasma membrane to some extent. ClC-7/OSTM1 is coinserted with the vesicular H(+)-ATPase into the acid-secreting ruffled border membrane of osteoclasts. Mice or humans lacking ClC-7 or OSTM1 display osteopetrosis and lysosomal storage disease. Disruption of the endosomal ClC-5 Cl(-)/H(+)-exchanger leads to proteinuria and Dent's disease. Mouse models in which ClC-5 or ClC-7 is converted to uncoupled Cl(-) conductors suggest an important role of vesicular Cl(-) accumulation in these pathologies. The important functions of CLC Cl(-) channels were also revealed by human diseases and mouse models, with phenotypes including myotonia, renal loss of salt and water, deafness, blindness, leukodystrophy, and male infertility.
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Comprehensive Physiology - Cell Biology and Physiology of CLC Chloride Channels and Transporters
Comprehensive Physiology, 2012Co-Authors: Tobias Stauber, Stefanie Weinert, Thomas J JentschAbstract:Proteins of the CLC gene family assemble to homo- or sometimes heterodimers and either function as Cl(-) channels or as Cl(-)/H(+)-exchangers. CLC proteins are present in all phyla. Detailed structural information is available from crystal structures of bacterial and algal CLCs. Mammals express nine CLC genes, four of which encode Cl(-) channels and five 2Cl(-)/H(+)-exchangers. Two accessory β-subunits are known: (1) barttin and (2) OSTM1. ClC-Ka and ClC-Kb Cl(-) channels need barttin, whereas OSTM1 is required for the function of the lysosomal ClC-7 2Cl(-)/H(+)-exchanger. ClC-1, -2, -Ka and -Kb Cl(-) channels reside in the plasma membrane and function in the control of electrical excitability of muscles or neurons, in extra- and intracellular ion homeostasis, and in transepithelial transport. The mainly endosomal/lysosomal Cl(-)/H(+)-exchangers ClC-3 to ClC-7 may facilitate vesicular acidification by shunting currents of proton pumps and increase vesicular Cl(-) concentration. ClC-3 is also present on synaptic vesicles, whereas ClC-4 and -5 can reach the plasma membrane to some extent. ClC-7/OSTM1 is coinserted with the vesicular H(+)-ATPase into the acid-secreting ruffled border membrane of osteoclasts. Mice or humans lacking ClC-7 or OSTM1 display osteopetrosis and lysosomal storage disease. Disruption of the endosomal ClC-5 Cl(-)/H(+)-exchanger leads to proteinuria and Dent's disease. Mouse models in which ClC-5 or ClC-7 is converted to uncoupled Cl(-) conductors suggest an important role of vesicular Cl(-) accumulation in these pathologies. The important functions of CLC Cl(-) channels were also revealed by human diseases and mouse models, with phenotypes including myotonia, renal loss of salt and water, deafness, blindness, leukodystrophy, and male infertility.
Tobias Stauber - One of the best experts on this subject based on the ideXlab platform.
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The FASEB Journal • Research Communication Lysosomal degradation of endocytosed proteins depends on the chloride transport protein ClC-7
2015Co-Authors: Lena Wartosch, Jens C Fuhrmann, Tobias Stauber, Michaela Schweizer, Thomas J JentschAbstract:mal/lysosomal member of the CLC family of chloride channels and transporters, or in its -subunit OSTM1 cause osteopetrosis and lysosomal storage disease in mice and humans. The severe phenotype of mice globally deleted for ClC-7 or OSTM1 and the absence of storage material in cultured cells hampered investiga-tions of the mechanism leading to lysosomal pathology in the absence of functional ClC-7/OSTM1 transporters. Tissue-specific ClC-7-knockout mice now reveal that accumulation of storage material occurs cell-autono-mously in neurons or renal proximal tubular cells lacking ClC-7. Almost all ClC-7-deficient neurons die. The activation of glia is restricted to brain regions where ClC-7 has been inactivated. The effect of ClC-7 disruption on lysosomal function was investigated in renal proximal tubular cells, which display high endo-cytotic activity. Pulse-chase endocytosis experiments in vivo with mice carrying chimeric deletion of ClC-7 in proximal tubules allowed a direct comparison of the handling of endocytosed protein between cells express-ing or lacking ClC-7. Whereas protein was endocytosed similarly in cells of either genotype, its half-life in-creased significantly in ClC-7-deficient cells. These ex-periments demonstrate that lysosomal pathology is a cell-autonomous consequence of ClC-7 disruption and that ClC-7 is important for lysosomal protein degrada
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a missense mutation accelerating the gating of the lysosomal cl h exchanger clc 7 OSTM1 causes osteopetrosis with gingival hamartomas in cattle
Disease Models & Mechanisms, 2014Co-Authors: Arnaud Sartelet, Tobias Stauber, Wouter Coppieters, Carmen F Ludwig, Corinne Fasquelle, Tom Druet, Zhiyan Zhang, Naima Ahariz, Nadine Cambisano, Thomas J JentschAbstract:Chloride-proton exchange by the lysosomal anion transporter ClC7/OSTM1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or OSTM1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/OSTM1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/OSTM1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/OSTM1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/OSTM1 in lysosomal function and bone resorption.
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A missense mutation accelerating the gating of the lysosomal Cl-/H+-exchanger ClC-7/OSTM1 causes osteopetrosis with gingival hamartomas in cattle
Disease models & mechanisms, 2013Co-Authors: Arnaud Sartelet, Tobias Stauber, Wouter Coppieters, Carmen F Ludwig, Corinne Fasquelle, Tom Druet, Zhiyan Zhang, Naima Ahariz, Nadine Cambisano, Thomas J JentschAbstract:Chloride-proton exchange by the lysosomal anion transporter ClC7/OSTM1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or OSTM1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/OSTM1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/OSTM1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/OSTM1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/OSTM1 in lysosomal function and bone resorption.
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Activity and fractional distribution of wild type and G213R ClC-7.
2013Co-Authors: Patrick Schulz, Johannes Werner, Tobias Stauber, Kim Henriksen, Klaus FendlerAbstract:A: Western Blot of membrane fractions against GFP (ClC-7), LAMP-1 (lysosomes) and Calnexin (ER), showing lysosomes enriched in the 31/45% fraction and ER in the 45/51% fraction. Densitometric analysis of Western blot bands (Dens.): numbers give relative intergral band densities in % left to right lane. B: Quantitative fluorescence analysis of membrane fractions. Fractions were adjusted to same total protein concentration. Fractional fluorescence was normalized to the total fluorescence in all samples. Mean and standard deviation of 2–3 individual preparations. Wt ClC-7 is significantly enriched in 31/45%, while G213R is more equally distributed between 31/45% and 45/51%. OSTM1 alters G213R localization only slightly. C: Western Blot against OSTM1. Fraction 31/45% and 45/51% of CHO control cells (CHO), wt ClC-7+OSTM1 (WT) and G213R+OSTM1 (G213R). Arrows: full length OSTM1 (∼70 kDa) and cleaved fragment (∼35 kDa), asterisk indicating an unspecific band recognized by the OSTM1 antibody [10]. D: Western Blot against ClC-3 and LAMP-1 in fraction 31/45% of control CHO, wt ClC-7+OSTM1 and G213R ClC-7+OSTM1.
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Localization of wild type and G213R ClC-7 in CHO cells.
2013Co-Authors: Patrick Schulz, Johannes Werner, Tobias Stauber, Kim Henriksen, Klaus FendlerAbstract:A: Confocal images of wt ClC-7-EGFP expressing living CHO cells stained with LysoTracker®-Red (top row). Images of EGFP fluorescence, LysoTracker®-Red fluorescence and the merge of both are shown. G213R ClC-7-EGFP stained with LysoTracker®-Red (middle row) and ER-Tracker™-Red (lower row). Individual fluorescence images and merge are shown. B: Co-expression of wt ClC-7-EGFP with its ß-subunit OSTM1. Compared images were recorded with fixed hardware settings and at constant experimental conditions. Localization of ClC-7 in OSTM1 expressing cells was not affected but a significantly increased EGFP fluorescence could be detected. C: Co-expression of G213R ClC-7-EGFP with OSTM1 partly (∼50%) restored lysosomal localization.
Craig C. Malbon - One of the best experts on this subject based on the ideXlab platform.
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OSTM1 regulates β catenin lef1 interaction and is required for wnt β catenin signaling
Cellular Signalling, 2008Co-Authors: Michael E. Feigin, Craig C. MalbonAbstract:The Wnt/beta-catenin signaling pathway controls key aspects of embryonic development and adult tissue homeostasis, including the formation and maintenance of bone. Recently, mutations in the OSTM1 gene were found to be the cause of severe autosomal recessive osteopetrosis in both the mouse and humans. This disorder is characterized by increased bone mass resulting from a defect in osteoclast maturation. The possible role of OSTM1 in signaling of the Wnt/beta-catenin "canonical" pathway was investigated in totipotent mouse F9 embryonal teratocarcinoma cells. Overexpression of OSTM1 in F9 cells increased Wnt3a-responsive beta-catenin accumulation and Lef/Tcf-sensitive transcription. Similarly, knockdown of endogenous OSTM1 attenuated the ability of Wnt3a to stimulate the canonical signaling pathway. An OSTM1 mutant (detected in humans with osteopetrosis) was expressed in F9 cells and found to inhibit Wnt-stimulated beta-catenin stabilization, gene transcription, and primitive endoderm formation. Expression of this OSTM1 C-terminal deletion mutant attenuated Lef/Tcf-sensitive gene transcription, even when transcription was activated by expression of a constitutively-active form of beta-catenin. However, expression of this OSTM1 C-terminal deletion mutant was unable to alter Lef/Tcf-sensitive gene transcription when transcription was activated by expression of a beta-catenin/Lef chimeric protein. From the standpoint of protein-protein interactions, expression of wild-type OSTM1 stimulated whereas mutant OSTM1 inhibited, the Wnt-dependent association of beta-catenin and Lef1. On the foundation of these experiments, we propose that the human mutations in OSTM1 such as the C-terminal deletion mutant studied herein provoke dysregulation of the canonical Wnt/beta-catenin signaling pathway, providing a molecular basis for severe autosomal recessive osteopetrosis.
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OSTM1 Regulates β-catenin/Lef1 Interaction and is Required for Wnt/β-catenin Signaling
Cellular signalling, 2008Co-Authors: Michael E. Feigin, Craig C. MalbonAbstract:The Wnt/beta-catenin signaling pathway controls key aspects of embryonic development and adult tissue homeostasis, including the formation and maintenance of bone. Recently, mutations in the OSTM1 gene were found to be the cause of severe autosomal recessive osteopetrosis in both the mouse and humans. This disorder is characterized by increased bone mass resulting from a defect in osteoclast maturation. The possible role of OSTM1 in signaling of the Wnt/beta-catenin "canonical" pathway was investigated in totipotent mouse F9 embryonal teratocarcinoma cells. Overexpression of OSTM1 in F9 cells increased Wnt3a-responsive beta-catenin accumulation and Lef/Tcf-sensitive transcription. Similarly, knockdown of endogenous OSTM1 attenuated the ability of Wnt3a to stimulate the canonical signaling pathway. An OSTM1 mutant (detected in humans with osteopetrosis) was expressed in F9 cells and found to inhibit Wnt-stimulated beta-catenin stabilization, gene transcription, and primitive endoderm formation. Expression of this OSTM1 C-terminal deletion mutant attenuated Lef/Tcf-sensitive gene transcription, even when transcription was activated by expression of a constitutively-active form of beta-catenin. However, expression of this OSTM1 C-terminal deletion mutant was unable to alter Lef/Tcf-sensitive gene transcription when transcription was activated by expression of a beta-catenin/Lef chimeric protein. From the standpoint of protein-protein interactions, expression of wild-type OSTM1 stimulated whereas mutant OSTM1 inhibited, the Wnt-dependent association of beta-catenin and Lef1. On the foundation of these experiments, we propose that the human mutations in OSTM1 such as the C-terminal deletion mutant studied herein provoke dysregulation of the canonical Wnt/beta-catenin signaling pathway, providing a molecular basis for severe autosomal recessive osteopetrosis.
Jens C Fuhrmann - One of the best experts on this subject based on the ideXlab platform.
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The FASEB Journal • Research Communication Lysosomal degradation of endocytosed proteins depends on the chloride transport protein ClC-7
2015Co-Authors: Lena Wartosch, Jens C Fuhrmann, Tobias Stauber, Michaela Schweizer, Thomas J JentschAbstract:mal/lysosomal member of the CLC family of chloride channels and transporters, or in its -subunit OSTM1 cause osteopetrosis and lysosomal storage disease in mice and humans. The severe phenotype of mice globally deleted for ClC-7 or OSTM1 and the absence of storage material in cultured cells hampered investiga-tions of the mechanism leading to lysosomal pathology in the absence of functional ClC-7/OSTM1 transporters. Tissue-specific ClC-7-knockout mice now reveal that accumulation of storage material occurs cell-autono-mously in neurons or renal proximal tubular cells lacking ClC-7. Almost all ClC-7-deficient neurons die. The activation of glia is restricted to brain regions where ClC-7 has been inactivated. The effect of ClC-7 disruption on lysosomal function was investigated in renal proximal tubular cells, which display high endo-cytotic activity. Pulse-chase endocytosis experiments in vivo with mice carrying chimeric deletion of ClC-7 in proximal tubules allowed a direct comparison of the handling of endocytosed protein between cells express-ing or lacking ClC-7. Whereas protein was endocytosed similarly in cells of either genotype, its half-life in-creased significantly in ClC-7-deficient cells. These ex-periments demonstrate that lysosomal pathology is a cell-autonomous consequence of ClC-7 disruption and that ClC-7 is important for lysosomal protein degrada
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Lysosomal degradation of endocytosed proteins depends on the chloride transport protein ClC-7
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2009Co-Authors: Lena Wartosch, Jens C Fuhrmann, Tobias Stauber, Michaela Schweizer, Thomas J JentschAbstract:Mutations in either ClC-7, a late endosomal/lysosomal member of the CLC family of chloride channels and transporters, or in its beta-subunit OSTM1 cause osteopetrosis and lysosomal storage disease in mice and humans. The severe phenotype of mice globally deleted for ClC-7 or OSTM1 and the absence of storage material in cultured cells hampered investigations of the mechanism leading to lysosomal pathology in the absence of functional ClC-7/OSTM1 transporters. Tissue-specific ClC-7-knockout mice now reveal that accumulation of storage material occurs cell-autonomously in neurons or renal proximal tubular cells lacking ClC-7. Almost all ClC-7-deficient neurons die. The activation of glia is restricted to brain regions where ClC-7 has been inactivated. The effect of ClC-7 disruption on lysosomal function was investigated in renal proximal tubular cells, which display high endocytotic activity. Pulse-chase endocytosis experiments in vivo with mice carrying chimeric deletion of ClC-7 in proximal tubules allowed a direct comparison of the handling of endocytosed protein between cells expressing or lacking ClC-7. Whereas protein was endocytosed similarly in cells of either genotype, its half-life increased significantly in ClC-7-deficient cells. These experiments demonstrate that lysosomal pathology is a cell-autonomous consequence of ClC-7 disruption and that ClC-7 is important for lysosomal protein degradation.
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clc 7 requires OSTM1 as a beta subunit to support bone resorption and lysosomal function
Nature, 2006Co-Authors: Philipp F Lange, Lena Wartosch, Thomas J Jentsch, Jens C FuhrmannAbstract:Mutations in ClC-7, a late endosomal/lysosomal member of the CLC family of chloride channels and transporters1,2, cause osteopetrosis3 and lysosomal storage disease4 in humans and mice. Severe osteopetrosis is also observed with mutations in the OSTM1 gene, which encodes a membrane protein of unknown function5. Here we show that both ClC-7 and OSTM1 proteins co-localize in late endosomes and lysosomes of various tissues, as well as in the ruffled border of bone-resorbing osteoclasts. Co-immunoprecipitations show that ClC-7 and OSTM1 form a molecular complex and suggest that OSTM1 is a β–subunit of ClC-7. ClC-7 is required for OSTM1 to reach lysosomes, where the highly glycosylated OSTM1 luminal domain is cleaved. Protein but not RNA levels of ClC-7 are greatly reduced in grey-lethal mice, which lack OSTM1, suggesting that the ClC-7–OSTM1 interaction is important for protein stability. As ClC-7 protein levels in OSTM1-deficient tissues and cells, including osteoclasts, are decreased below 10% of normal levels, OSTM1 mutations probably cause osteopetrosis by impairing the acidification of the osteoclast resorption lacuna, which depends on ClC-7 (ref. 3). The finding that grey-lethal mice, just like ClC-7-deficient mice4, show lysosomal storage and neurodegeneration in addition to osteopetrosis implies a more general importance for ClC-7–OSTM1 complexes.
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clc 7 requires OSTM1 as a β subunit to support bone resorption and lysosomal function
Nature, 2006Co-Authors: Philipp F Lange, Lena Wartosch, Thomas J Jentsch, Jens C FuhrmannAbstract:Mutations in ClC-7, a late endosomal/lysosomal member of the CLC family of chloride channels and transporters, cause osteopetrosis and lysosomal storage disease in humans and mice. Severe osteopetrosis is also observed with mutations in the OSTM1 gene, which encodes a membrane protein of unknown function. Here we show that both ClC-7 and OSTM1 proteins co-localize in late endosomes and lysosomes of various tissues, as well as in the ruffled border of bone-resorbing osteoclasts. Co-immunoprecipitations show that ClC-7 and OSTM1 form a molecular complex and suggest that OSTM1 is a beta-subunit of ClC-7. ClC-7 is required for OSTM1 to reach lysosomes, where the highly glycosylated OSTM1 luminal domain is cleaved. Protein but not RNA levels of ClC-7 are greatly reduced in grey-lethal mice, which lack OSTM1, suggesting that the ClC-7-OSTM1 interaction is important for protein stability. As ClC-7 protein levels in OSTM1-deficient tissues and cells, including osteoclasts, are decreased below 10% of normal levels, OSTM1 mutations probably cause osteopetrosis by impairing the acidification of the osteoclast resorption lacuna, which depends on ClC-7 (ref. 3). The finding that grey-lethal mice, just like ClC-7-deficient mice, show lysosomal storage and neurodegeneration in addition to osteopetrosis implies a more general importance for ClC-7-OSTM1 complexes.
Jean Vacher - One of the best experts on this subject based on the ideXlab platform.
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Expression pattern of the V5-OSTM1 protein in bacterial artificial chromosome transgenic mice.
Genesis (New York N.Y. : 2000), 2021Co-Authors: Monica Pata, Pardis Yousefi Behzadi, Jean VacherAbstract:Mutations in the osteopetrotic transmembrane protein 1 (OSTM1) gene are responsible for the most severe form of autosomal recessive osteopetrosis both in humans and in the gray lethal (gl/gl) mouse. This defect leads to increased bone mass with bone marrow occlusion and hematopoietic defects. To establish the expression profile of the mouse OSTM1 protein in vivo, homologous recombination in bacteria was designed to generate a V5-OSTM1 bacterial artificial chromosome (BAC) that was subsequently integrated in the mouse genome. Tissue expression of the transgene V5-OSTM1 RNA and protein in transgenic mice follow the endogenous expression profile. Immunohistochemistry analysis demonstrated expression in neuronal populations from central and peripheral nervous system and defined a unique cellular expression pattern. Importantly, together with appropriate protein post-translational modification, in vivo rescue of the osteopetrotic bone gl/gl phenotype in BAC V5-OSTM1 gl/gl mice is consistent with the expression of a fully functional and active protein. These mice represent a unique tool to unravel novel OSTM1 functions in individual tissue and neuronal cell populations and the V5-OSTM1 transgene represents an easy visual marker to monitor the expression of OSTM1 in vitro and in vivo.
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OSTM1 Bifunctional Roles in Osteoclast Maturation: Insights From a Mouse Model Mimicking a Human OSTM1 Mutation.
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2018Co-Authors: Monica Pata, Jean VacherAbstract:OSTM1 mutations are responsible for the most severe form of osteopetrosis in human and mice. To gain insight into OSTM1 cellular functions, we engineered a conditional in-frame deletion of the OSTM1 transmembrane domain and generated the first OSTM1 mouse model with a human mutation. Systemic targeting of OSTM1 loss of transmembrane domain produced osteopetrosis, as in the null OSTM1 gl/gl mouse. Significantly, conditional osteoclast targeting of OSTM1 resulted in similar osteopetrosis, thereby demonstrating that the intrinsic OSTM1 osteoclast deficiency is solely responsible for the mouse phenotype. Our analysis showed oversized osteoclasts with enhanced multinucleation associated with stimulation of intracellular calcium levels, of Nfatc1 nuclear re-localization, and of specific downstream Nfatc1 target genes, providing compelling evidence that OSTM1 is a negative regulator of preosteoclast fusion. Moreover, mature OCs with OSTM1 loss of transmembrane domain show appropriate levels of intracellular acidification but an altered distribution pattern, highlighting misregulation of endolysosome localization and dispersion. Consistently, the hydrolases tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Ctsk) normally produced are sequestered within the osteoclasts and are not extracellularly secreted. These studies defined bifunctional roles for OSTM1 as a major regulator of preosteoclast cytoskeletal rearrangements toward cell multinucleation and of mature osteoclast intracellular lysosomal trafficking and exocytosis mechanism, both of which are essential for bone resorption. Importantly, these OSTM1 molecular and regulatory functions could serve as preclinical targets in this mouse model toward osteoclastogenic pathologies as osteoporosis and inflammation-induced bone loss. © 2018 American Society for Bone and Mineral Research.
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Role of OSTM1 Cytosolic Complex with Kinesin 5B in Intracellular Dispersion and Trafficking
Molecular and cellular biology, 2015Co-Authors: Subramanya N. M. Pandruvada, Janie Beauregard, Suzanne Benjannet, Monica Pata, Claude Lazure, Nabil G. Seidah, Jean VacherAbstract:In humans and in mice, mutations in the OSTM1 gene cause the most severe form of osteopetrosis, a major bone disease, and neuronal degeneration, both of which are associated with early death. To gain insight into OSTM1 function, we first investigated by sequence and biochemical analysis an immature 34-kDa type I transmembrane OSTM1 protein with a unique cytosolic tail. Mature OSTM1 is posttranslationally processed and highly N-glycosylated and has an apparent mass of ∼60 kDa. Analysis the subcellular localization of OSTM1 showed that it is within the endoplasmic reticulum, trans-Golgi network, and endosomes/lysosomes. By a wide protein screen under physiologic conditions, several novel cytosolic OSTM1 partners were identified and validated, for which a direct interaction with the kinesin 5B heavy chains was demonstrated. These results determined that OSTM1 is part of a cytosolic scaffolding multiprotein complex, imparting an adaptor function to OSTM1. Moreover, we uncovered a role for the OSTM1/KIF5B complex in intracellular trafficking and dispersion of cargos from the endoplasmic reticulum to late endosomal/lysosomal subcellular compartments. These OSTM1 molecular and cellular functions could elucidate all of the pathophysiologic mechanisms underlying the wide phenotypic spectrum of OSTM1-deficient mice.
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severe neurodegeneration with impaired autophagy mechanism triggered by OSTM1 deficiency
Journal of Biological Chemistry, 2014Co-Authors: Subramanya N. M. Pandruvada, Monica Pata, Adam Griffiths, Céline Héraud, Manfred W Kilimann, Jean VacherAbstract:Loss of OSTM1 leads to the most severe form of osteopetrosis in mice and humans. Because functional rescue of the osteopetrotic defect in these mice extended their lifespan from ∼3 weeks to 6 weeks, this unraveled a second essential role of OSTM1. We discovered that OSTM1 is highly expressed in the mouse brain in neurons, microglia, and astrocytes. At 3–4 weeks of age, mice with OSTM1 loss showed 3–10-fold stimulation of reactive gliosis, with an increased astrocyte cell population and microglia activation. This inflammatory response was associated with marked retinal photoreceptor degeneration and massive neuronal loss in the brain. Intracellular characterization of neurons revealed abnormal storage of carbohydrates, lipids, and ubiquitinated proteins, combined with marked accumulation of autophagosomes that causes frequent axonal swelling. Stimulation of autophagy was provided by specific markers and by significant down-regulation of the mammalian target of rapamycin signaling, identifying a cellular pathologic mechanism. A series of transgenic mouse lines specifically targeted to distinct central nervous system cell subpopulations determined that OSTM1 has a primary and autonomous role in neuronal homeostasis. Complete functional complementation demonstrated that the development of severe and rapid neurodegeneration in these mice is independent of the hematopoietic lineage and has clinical implications for treatment of osteopetrosis. Importantly, this study establishes a novel neurodegenerative mouse model critical for understanding the multistep pathogenic cascade of cellular autophagy disorders toward therapeutic strategy design.
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Severe neurodegeneration with impaired autophagy mechanism triggered by OSTM1 deficiency.
The Journal of biological chemistry, 2014Co-Authors: Céline Héraud, Subramanya N. M. Pandruvada, Monica Pata, Adam Griffiths, Manfred W Kilimann, Jean VacherAbstract:Abstract Loss of OSTM1 leads to the most severe form of osteopetrosis in mice and humans. Because functional rescue of the osteopetrotic defect in these mice extended their lifespan from approximately 3 to 6 weeks, this unraveled a second essential role of OSTM1. We uncovered that OSTM1 is highly expressed in the mouse brain in neurons, microglia, and astrocytes. At 3 to 4 weeks of age, mice with OSTM1 loss showed 3- to 10-fold stimulation of reactive gliosis, with an increased astrocyte cell population and microglia activation. This inflammatory response was associated with marked retinal photoreceptor degeneration and massive neuronal loss in the brain. Intracellular characterization of neurons revealed abnormal storage of carbohydrates, lipids, and ubiquitinated-proteins, combined with marked accumulation of autophagosomes that causes frequent axonal swelling. Stimulation of autophagy was provided by specific markers and by significant downregulation of the mammalian target of rapamycin signaling identifying a cellular pathologic mechanism. Series of transgenic mouse lines specifically targeted to distinct central nervous system cell subpopulations determined that OSTM1 has a primary and autonomous role in neuronal homeostasis. Complete functional complementation demonstrated that the development of severe and rapid neurodegeneration in these mice is independent of the hematopoietic lineage and has clinical implications for treatment of osteopetrosis. Importantly, this study establishes a novel neurodegenerative mouse model critical for understanding the multi-step pathogenic cascade of cellular autophagy disorders toward therapeutic strategy design.
