cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.13067048.v3
title: Metformin rescues muscle function in BAG3 myofibrillar myopathy models

description: Dominant <i>de novo</i> mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To elucidate the mechanism of disease in, and identify therapies for, BAG3 myofibrillar myopathy, we generated two zebrafish models, one conditionally expressing BAG3<sup>P209L</sup> and one with a nonsense mutation in <i>bag3</i>. While transgenic BAG3<sup>P209L</sup>-expressing fish display protein aggregation, modeling the early phase of the disease, <i>bag3</i><sup>-/-</sup> fish exhibit exercise dependent fiber disintegration, and reduced swimming activity, consistent with later stages of the disease. Detailed characterization of the <i>bag3</i><sup>-/-</sup> fish, revealed an impairment in macroautophagic/autophagic activity, a defect we confirmed in <i>BAG3</i> patient samples. Taken together, our data highlights that while BAG3<sup>P209L</sup> expression is sufficient to promote protein aggregation, it is the loss of BAG3 due to its sequestration within aggregates, which results in impaired autophagic activity, and subsequent muscle weakness. We therefore screened autophagy-promoting compounds for their effectiveness at removing protein aggregates, identifying nine including metformin. Further evaluation demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the <i>bag3</i><sup>−/-</sup> fish. Therefore, repurposing metformin provides a promising therapy for BAG3 myopathy. <b>Abbreviations:</b>ACTN: actinin, alpha; BAG3: BAG cochaperone 3; CRYAB: crystallin alpha B; DES: desmin; DMSO: dimethyl sulfoxide; DNAJB6: DnaJ heat shock protein family (Hsp40) member B6; dpf: days post fertilization; eGFP: enhanced green fluorescent protein; FDA: Food and Drug Administration; FHL1: four and a half LIM domains 1; FLNC: filamin C; hpf: hours post-fertilization; HSPB8: heat shock protein family B [small] member 8; LDB3/ZASP: LIM domain binding 3; MYOT: myotilin; TTN: titin; WT: wild-type.
DOI: 10.6084/m9.figshare.13011509
title: Inactivation of the <i>Caenorhabditis elegans</i> RNF-5 E3 ligase promotes IRE-1-independent ER functions

description: RNF5 is implicated in ERAD and in negative regulation of macroautophagy/autophagy. To better understand the function of RNF-5 under ER-stress conditions, we studied the ability of <i>Caenorhabditis elegans rnf-5(tm794)</i> mutant animals to cope with stress in the background of impaired UPR machinery. We demonstrate that downregulation of RNF-5 decreased sensitivity to tunicamycin both in wild type and in an <i>ire-1</i> mutant. Double-mutant <i>rnf-5;ire-1</i> animals showed increased starvation resistance and extended lifespan when compared to the <i>ire-1</i> mutant. This partial rescue of <i>ire-1</i> required functional autophagy. Downregulation of RNF-5 rescued ER maturation defects and protein secretion of a DAF-28::GFP intestinal reporter in the <i>ire-1</i> background. Proteomics and functional studies revealed an increase in lysosomal protease levels, in the frequency of intestinal lysosomes, and in lysosomal protease activity in <i>rnf-5(tm794)</i> animals. Together, these data suggest that RNF-5 is a negative regulator of ER stress, and that inactivation of RNF-5 promotes IRE-1-independent elevation of ER capacity.
DOI: 10.6084/m9.figshare.13011509.v4
title: Inactivation of the <i>Caenorhabditis elegans</i> RNF-5 E3 ligase promotes IRE-1-independent ER functions

description: RNF5 is implicated in ERAD and in negative regulation of macroautophagy/autophagy. To better understand the function of RNF-5 under ER-stress conditions, we studied the ability of <i>Caenorhabditis elegans rnf-5(tm794)</i> mutant animals to cope with stress in the background of impaired UPR machinery. We demonstrate that downregulation of RNF-5 decreased sensitivity to tunicamycin both in wild type and in an <i>ire-1</i> mutant. Double-mutant <i>rnf-5;ire-1</i> animals showed increased starvation resistance and extended lifespan when compared to the <i>ire-1</i> mutant. This partial rescue of <i>ire-1</i> required functional autophagy. Downregulation of RNF-5 rescued ER maturation defects and protein secretion of a DAF-28::GFP intestinal reporter in the <i>ire-1</i> background. Proteomics and functional studies revealed an increase in lysosomal protease levels, in the frequency of intestinal lysosomes, and in lysosomal protease activity in <i>rnf-5(tm794)</i> animals. Together, these data suggest that RNF-5 is a negative regulator of ER stress, and that inactivation of RNF-5 promotes IRE-1-independent elevation of ER capacity.
DOI: 10.6084/m9.figshare.13067048.v2
title: Metformin rescues muscle function in BAG3 myofibrillar myopathy models

description: Dominant <i>de novo</i> mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To elucidate the mechanism of disease in, and identify therapies for, BAG3 myofibrillar myopathy, we generated two zebrafish models, one conditionally expressing BAG3<sup>P209L</sup> and one with a nonsense mutation in <i>bag3</i>. While transgenic BAG3<sup>P209L</sup>-expressing fish display protein aggregation, modeling the early phase of the disease, <i>bag3</i><sup>-/-</sup> fish exhibit exercise dependent fiber disintegration, and reduced swimming activity, consistent with later stages of the disease. Detailed characterization of the <i>bag3</i><sup>-/-</sup> fish, revealed an impairment in macroautophagic/autophagic activity, a defect we confirmed in <i>BAG3</i> patient samples. Taken together, our data highlights that while BAG3<sup>P209L</sup> expression is sufficient to promote protein aggregation, it is the loss of BAG3 due to its sequestration within aggregates, which results in impaired autophagic activity, and subsequent muscle weakness. We therefore screened autophagy-promoting compounds for their effectiveness at removing protein aggregates, identifying nine including metformin. Further evaluation demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the <i>bag3</i><sup>−/-</sup> fish. Therefore, repurposing metformin provides a promising therapy for BAG3 myopathy. <b>Abbreviations:</b>ACTN: actinin, alpha; BAG3: BAG cochaperone 3; CRYAB: crystallin alpha B; DES: desmin; DMSO: dimethyl sulfoxide; DNAJB6: DnaJ heat shock protein family (Hsp40) member B6; dpf: days post fertilization; eGFP: enhanced green fluorescent protein; FDA: Food and Drug Administration; FHL1: four and a half LIM domains 1; FLNC: filamin C; hpf: hours post-fertilization; HSPB8: heat shock protein family B [small] member 8; LDB3/ZASP: LIM domain binding 3; MYOT: myotilin; TTN: titin; WT: wild-type.
DOI: 10.6084/m9.figshare.12998586
title: Loss of PKD1/polycystin-1 impairs lysosomal activity in a CAPN (calpain)-dependent manner

description: Mutations in the <i>PKD1</i> gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used <i>Pkd1</i>-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for <i>Pkd1</i>, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional <i>pkd1<sup>fl/fl</sup>;Pax8<sup>rtTA</sup>;TetOCre<sup>+</sup></i> knockout mice to show that the lack of <i>Pkd1</i> caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. <i>Pkd1</i>-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in <i>pkd1</i> knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux. <b>Abbreviations:</b> acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.
DOI: 10.6084/m9.figshare.13011509.v3
title: Inactivation of the <i>Caenorhabditis elegans</i> RNF-5 E3 ligase promotes IRE-1-independent ER functions

description: RNF5 is implicated in ERAD and in negative regulation of macroautophagy/autophagy. To better understand the function of RNF-5 under ER-stress conditions, we studied the ability of <i>Caenorhabditis elegans rnf-5(tm794)</i> mutant animals to cope with stress in the background of impaired UPR machinery. We demonstrate that downregulation of RNF-5 decreased sensitivity to tunicamycin both in wild type and in an <i>ire-1</i> mutant. Double-mutant <i>rnf-5;ire-1</i> animals showed increased starvation resistance and extended lifespan when compared to the <i>ire-1</i> mutant. This partial rescue of <i>ire-1</i> required functional autophagy. Downregulation of RNF-5 rescued ER maturation defects and protein secretion of a DAF-28::GFP intestinal reporter in the <i>ire-1</i> background. Proteomics and functional studies revealed an increase in lysosomal protease levels, in the frequency of intestinal lysosomes, and in lysosomal protease activity in <i>rnf-5(tm794)</i> animals. Together, these data suggest that RNF-5 is a negative regulator of ER stress, and that inactivation of RNF-5 promotes IRE-1-independent elevation of ER capacity.
DOI: 10.6084/m9.figshare.12998586.v3
title: Loss of PKD1/polycystin-1 impairs lysosomal activity in a CAPN (calpain)-dependent manner

description: Mutations in the <i>PKD1</i> gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used <i>Pkd1</i>-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for <i>Pkd1</i>, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional <i>pkd1<sup>fl/fl</sup>;Pax8<sup>rtTA</sup>;TetOCre<sup>+</sup></i> knockout mice to show that the lack of <i>Pkd1</i> caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. <i>Pkd1</i>-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in <i>pkd1</i> knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux. <b>Abbreviations:</b> acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.
DOI: 10.6084/m9.figshare.13082036.v3
title: BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation

description: Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance. <b>Abbreviations:</b> BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta
DOI: 10.6084/m9.figshare.13082036
title: BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation

description: Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance. <b>Abbreviations:</b> BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta
DOI: 10.6084/m9.figshare.12998586.v2
title: Loss of PKD1/polycystin-1 impairs lysosomal activity in a CAPN (calpain)-dependent manner

description: Mutations in the <i>PKD1</i> gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used <i>Pkd1</i>-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for <i>Pkd1</i>, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional <i>pkd1<sup>fl/fl</sup>;Pax8<sup>rtTA</sup>;TetOCre<sup>+</sup></i> knockout mice to show that the lack of <i>Pkd1</i> caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. <i>Pkd1</i>-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in <i>pkd1</i> knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux. <b>Abbreviations:</b> acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.
DOI: 10.6084/m9.figshare.13050508
title: Essential role of autophagy in restricting poliovirus infection revealed by identification of an ATG7 defect in a poliomyelitis patient

description: Paralytic poliomyelitis is a rare disease manifestation following poliovirus (PV) infection. The disease determinants remain largely unknown. We used whole exome sequencing to uncover possible contributions of host genetics to the development of disease outcome in humans with poliomyelitis. We identified a patient with a variant in <i>ATG7</i>, an important regulatory gene in the macroautophagy/autophagy pathway. PV infection did not induce a prominent type I interferon response, but rather activated autophagy in neuronal-like cells, and this was essential for viral control. Importantly, virus-induced autophagy was impaired in patient fibroblasts and associated with increased viral burden and enhanced cell death following infection. Lack of ATG7 prevented control of infection in neuronal-like cells, and reconstitution of patient cells with wild-type ATG7 reestablished autophagy-mediated control of infection. Collectively, these data suggest that ATG7 defect contributes to host susceptibility to PV infection and propose autophagy as an unappreciated antiviral effector in viral infection in humans.