A homozygous hypomorphicBNIP1variant causes an increase in autophagosomes and reduced autophagic flux and results in a spondylo‐epiphyseal dysplasia
Tess HollingGandham SriLakshmi BhavaniLeonie von ElsnerHitesh ShahNeethukrishna KausthubhamShaila BhattacharyyaAnju ShuklaGeert MortierThorsten SchinkeTatyana DanyukovaSandra PohlKerstin KutscheKatta M. Girisha
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BNIP1 (BCL2 interacting protein 1) is a soluble N-ethylmaleimide-sensitive factor-attachment protein receptor involved in ER membrane fusion. We identified the homozygous BNIP1 intronic variant c.84+3A>T in the apparently unrelated patients 1 and 2 with disproportionate short stature. Radiographs showed abnormalities affecting both the axial and appendicular skeleton and spondylo-epiphyseal dysplasia. We detected ~80% aberrantly spliced BNIP1 pre-mRNAs, reduced BNIP1 mRNA level to ~80%, and BNIP1 protein level reduction by ~50% in patient 1 compared to control fibroblasts. The BNIP1 ortholog in Drosophila, Sec20, regulates autophagy and lysosomal degradation. We assessed lysosome positioning and identified a decrease in lysosomes in the perinuclear region and an increase in the cell periphery in patient 1 cells. Immunofluorescence microscopy and immunoblotting demonstrated an increase in LC3B-positive structures and LC3B-II levels, respectively, in patient 1 fibroblasts under steady-state condition. Treatment of serum-starved fibroblasts with or without bafilomycin A1 identified significantly decreased autophagic flux in patient 1 cells. Our data suggest a block at the terminal stage of autolysosome formation and/or clearance in patient fibroblasts. BNIP1 together with RAB33B and VPS16, disease genes for Smith-McCort dysplasia 2 and a multisystem disorder with short stature, respectively, highlight the importance of autophagy in skeletal development.Keywords:
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Abstract Background Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. Results This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome–lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome–lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. Conclusions Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome–lysosome fusion.
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Endo-lysosomal system through the process of autophagy is involved in the pathogenesis of many diseases. Acidification of these organelles is carried out by V-type H+-ATPases, which is inhibited by bafilomycin A1. Endosomes and lysosomes are also important Ca2+-storage in a cell. Nіcotіnіc acіd adenіne dіnucleotіde phosphate (NAADP) releases Cа2+ from endo-lysosomes. The main purpose of the study was to found out the effect of bafilomycin A1 and NAADP on stored Ca2+ and on the ATPase activity of rat hepatocytes. The stored Ca2+ was estimated using chlorotetracycline in permeabilized hepatocytes of rats. ATPase activity was determined by level of orthophosphate spectrophotometrically. It was found that bafilomycin A1 reduces stored Ca2+ in permeabilized hepatocytes of rats in the micromolar range of concentration (20 and 0.04 mkM) and averted the effect of NAADP on calcium content. Lower concentrations of bafilomycin A1 (0.001 mkM) did not alter the content of stored calcium, but prevented the influence of NAADP in permeabilized hepatocytes of rats. In the subcellular fraction of rat liver bafilomycin A1 (0.001 mkM) increased Ca2+-ATPase and basal Mg2+-ATPase activities and reduced Na+/K+-ATPase activity. Preincubation of the subcellular fraction with bafilomycin A1 completely averts any changes in the activity of estimated ATPases by means of NAADP. It was concluded that the bafilomycin-sensitive store in hepatocytes of rats is NAADP-sensitive endo-lysosomal Ca2+-store. Using of bafilomycin A1 may be useful in treating autophagy-depended diseases.
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Abstract Bafilomycin A 1 , known as an inhibitor of vacuolar type H + ‐ATPase, was used to study involvement of the vacuolar ATP‐dependent H + ‐pump in the vacuolar pH regulation in a fresh water charophyte, Chara corallina . When bafilomycin A 1 (100 nM) was externally given to intact cells, the vacuolar pH (about 5) was not affected. Internodal cells were then pretreated with 100 nM bafilomycin for 1 − 2 h and the vacuolar sap was replaced with a weakly buffered solution of pH 7.4. The readjustment of the modified vacuolar pH in bafilomycin‐treated cells was significantly retarded compared with that in untreated cells. Next, bafilomycin A 1 was directly introduced into the vacuole by vacuolar perfusion with the artificial cell sap of pH 7.4. At 100 nM bafilomycin A 1 , the decrease in the vacuolar pH was significantly inhibited. When cell sap was replaced with the artificial cell sap containing no buffer (pH 5.2 − 5.5), the vacuolar pH increased in the presence of vacuolar bafilomycin, suggesting that the PP 1 ‐ dependent H + pumping alone was not sufficient for the pH regulation of Chara vacuoles. Intracellular bafilomycin A 1 had no effect on the plasma membrane potential of tonoplast‐free cells, which is evidence that it does not affect the electrogenic H + ‐pump in the plasma membrane. Bafilomycin A 1 inhibited the ATP‐dependent H + transport of tonoplast vesicles but not the PP 1 ‐dependent H + transport. The ATPase activity of tonoplast vesicles was also inhibited by bafilomycin A 1 .
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Mammalian target of rapamycin (mTOR) is activated by numerous stimuli, including amino acids and growth factors. This kinase is part of the mTOR complex 1 (mTORC1) which regulates cell proliferation, differentiation, and autophagy. Active mTORC1 is located on lysosomes and has been reported to disassociate from the lysosomal surface in the absence of amino acids. Furthermore, mTORC1 activity has been linked to the vacuolar H+-ATPases (V-ATPases), the proton pumps responsible for lysosomal acidification; however, the exact role of the V-ATPases in mTORC1 signaling is not known. To elucidate the mechanisms involved in mTORC1 regulation by the V-ATPases, we used primary osteoclasts derived from mice carrying a point (R740S) mutation in the a3 subunit of the V-ATPase. In these cells, the mutant protein is expressed but the pump is not functional, resulting in higher lysosomal pH. By analyzing mTOR activation, mTOR/lysosome co-localization, and lysosomal positioning using confocal microscopy, fractionation, and ultrapure lysosomal purification methods, we demonstrate that in primary osteoclasts, mTOR is localized on the lysosomal surface even when mTOR activity is inhibited. Our findings reveal that mTOR targeting to the lysosome in osteoclasts is activity-independent, and that its disassociation from the lysosome during starvation is not universal.
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Macroautophagy (autophagy) is a process wherein bulk cytosolic proteins and damaged organelles are sequestered and degraded via the lysosome. Alterations in autophagy-associated proteins have been shown to cause neural tube closure defects, neurodegeneration, and tumor formation. Normal lysosome function is critical for autophagy completion and when altered may lead to an accumulation of autophagic vacuoles (AVs) and caspase activation. The tumor suppressor p53 is highly expressed in neural precursor cells (NPCs) and has an important role in the regulation of both autophagy and apoptosis. We hypothesized that altered lysosome function would lead to NPC death via an interaction between autophagy- and apoptosis-associated proteins. To test our hypothesis, we utilized FGF2-expanded NPCs and the neural stem cell line, C17.2, in combination with the lysosomotropic agent chloroquine (CQ) and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1). Both CQ and Baf A1 caused concentration- and time-dependent AV accumulation, p53 phosphorylation, increased damage regulator autophagy modulator levels, caspase-3 activation, and cell death. Short hairpin RNA knockdown of Atg7, but not Beclin1, expression significantly inhibited CQ- and Baf A1-induced cell death, indicating that Atg7 is an upstream mediator of lysosome dysfunction-induced cell death. Cell death and/or caspase-3 activation was also attenuated by protein synthesis inhibition, p53 deficiency, or Bax deficiency, indicating involvement of the intrinsic apoptotic death pathway. In contrast to lysosome dysfunction, starvation-induced AV accumulation was inhibited by either Atg7 or Beclin1 knockdown, and Atg7 knockdown had no effect on starvation-induced death. These findings indicate that Atg7- and Beclin1-induced autophagy plays a cytoprotective role during starvation but that Atg7 has a unique pro-apoptotic function in response to lysosome dysfunction.
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SUMMARY An earlier report indicated that acid secretion in turtle urinary bladder is driven by an unusual vacuolar H+-ATPase and that the ATPase accounts for essentially all acid secreted. These results, however, are difficult to reconcile with the acid transporters currently ascribed to the renal collecting duct. Here, we re-examine the effect of bafilomycin A1, an inhibitor of vacuolar (V-type) H+-ATPases, on acid secretion by intact isolated bladders from Pseudemys scriptaturtles. Serosal-side bafilomycin had no effect on the transepithelial acidification current (AC). In the mucosal solution, bafilomycin inhibited the AC, with inhibition developing over the range 0.1-10 nmol l-1, with a sigmoidal dose—response curve, and an IC50 of 0.47 nmol l-1. At saturation, approximately 70 % of H+ secretion was inhibited. The remaining 30 % could be abolished by 30 μmol l-1 Sch-28080, which is a level that in other systems is known to inhibit H+/K+-ATPase transport activity specifically and essentially completely. When the order of addition was reversed (Sch-28080 first), there was no change in the magnitude of the effect produced by either inhibitor, and the two together again eliminated the AC. The data indicate that baseline acid secretion in intact bladders is due (i) in part to a highly bafilomycin-sensitive process, with sensitivity typical of vacuolar H+ ATPases; and (ii) in part to a more bafilomycin-resistant process that is sensitive to Sch-28080.
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In vivo treatment with external bafilomycin A(1), a selective inhibitor of V-ATPase H(+) pumps, reduced whole-body Na(+) influx by up to 90 % in young tilapia and 70 % in young carp. The inhibition was rapidly reversible, with whole-body Na(+) influx rebounding to 280 % of pre-treatment values within 20 min of removal from the bafilomycin. This rebound effect is consistent with the prior accumulation of protons during the period when the cells were exposed to bafilomycin. Bafilomycin also inhibited Cl(-) uptake, an effect that was still apparent 30 min after the removal of bafilomycin. These data provide circumstantial evidence for previous suggestions that Na(+) uptake in freshwater fish is associated with a proton-motive force created by a proton pump and indirect evidence for the major significance of this mechanism in the branchial uptake of Na(+) by freshwater fish.
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Abstract Resident alveolar macrophages (mφ) possess plasmalemmal vacuolar-type H+-ATPase (V-ATPase) that plays a crucial role in regulation of intracellular pH (pHi). To assess the importance of this V-ATPase to mφ effector functions, resident alveolar mφ from rabbits were activated with E. coli-derived lipopolysaccharide (LPS) and exposed to bafilomycin A1 a specific inhibitor of V-ATPase. Bafilomycin caused a significant cytosolic acidification in both the absence and presence of CO2-HCO3-, and in both unstimulated and activated mφ. Superoxide production and Fc receptor-mediated phagocytosis also were reduced in bafilomycin-treated mφ. Similar effects were elicited by acidifying the cytoplasm in the absence of bafilomycin, by lowering extracellular pH (pHo) from 7.4 to 6.5–6.6. Thus, the effects of bafilomycin on phagocytosis and superoxide production probably were related to cytosolic acidification, secondary to blockade of V-ATPase-mediated H+ extrusion across the plasma membrane. Conversely, bafilomycin significantly increased TNF-α release. This effect cannot be explained by a bafilomycin-induced acidosis because acidic pHo significantly reduced TNF-α release. The results demonstrate that V-ATPase activity is an important determinant of the effector functions of LPS-activated mφ. J. Leukoc. Biol. 57: 275–281; 1995.
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