Diabetes have been shown to cause progressive neuronal injury with pain and numbness via advanced glycation end-products(AGEs)-induced neuronal cell apoptosis;however, the valuable drug targets for diabetic neuropathy...Diabetes have been shown to cause progressive neuronal injury with pain and numbness via advanced glycation end-products(AGEs)-induced neuronal cell apoptosis;however, the valuable drug targets for diabetic neuropathy have been poorly reported so far. In this study, we discovered a natural small-molecule schisandrol A(SolA) with significant protective effect against AGEs-induced neuronal cell apoptosis. ATP6V0D1, a major subunit of vacuolar-type ATPase(V-ATPase) in lysosome was identified as a crucial cellular target of SolA. Moreover, SolA allosterically mediated ATP6V0D1 conformation via targeting a unique cysteine 335 residue to activate V-ATPase-dependent lysosomal acidification.Interestingly, SolA-induced lysosome pH downregulation resulted in a mitochondrial-lysosomal crosstalk by selectively promoting mitochondrial BH3-only protein BIM degradation, thereby preserving mitochondrial homeostasis and neuronal cells survival. Collectively, our findings reveal ATP6V0D1 is a valuable pharmacological target for diabetes-associated neuronal injury via controlling lysosomal acidification, and also provide the first small-molecule template allosterically activating V-ATPase for preventing diabetic neuropathy.展开更多
Objective: ATP6V0d2 is a subunit of the vacuolar-type H+-ATPase (V-ATPase) that pumps H+ ions into lysosomes. TRPML1 (MCOLN1/Mcoln1) transports cations out of lysosomes.Mcoln1^(-/-) mice recapitulate the lysosomal sto...Objective: ATP6V0d2 is a subunit of the vacuolar-type H+-ATPase (V-ATPase) that pumps H+ ions into lysosomes. TRPML1 (MCOLN1/Mcoln1) transports cations out of lysosomes.Mcoln1^(-/-) mice recapitulate the lysosomal storage disorder mucolipidosis type IV (MLIV) phenotype. We previously demonstrated thatMcoln1^(-/-) female mice quickly became infertile at 5 months old (5M) with degenerating corpora lutea (CL) and progesterone (P4) deficiency. We tested our hypothesis thatAtp6v0d2 deficiency could partially compensate forMcoln1 deficiency to restore CL functions inAtp6v0d2^(-/-)Mcoln1^(-/-) mice.Methods: Control andAtp6v0d2^(-/-)Mcoln1^(-/-) female mice underwent fertility test from 2M to 7M. A subset of them was dissected at 5M on day 3.5 post-coitum (D3.5). The D3.5 ovaries from 5M control,Mcoln1^(-/-), andAtp6v0d2^(-/-)Mcoln1^(-/-) mice were evaluated for CL morphology, lipid droplet staining, and markers of mitochondria and P4 steroidogenesis in the luteal cells.Results: The fertility test ofAtp6v0d2^(-/-)Mcoln1^(-/-) female mice (2M–7M) revealed normal mating activity but reduced fertility compared with the control;yet ~25% of them remained fertile at 5M to 7M but with dystocia. We analyzed a subset of 11Atp6v0d2^(-/-)Mcoln1^(-/-) mice (5M) in the fertility test on D3.5: three (27.3%) had normal P4 levels and all examined CL parameters, indicating full restoration of CL function compared withMcoln1^(-/-), whereas eight had P4 deficiency, with two (18.2%) infertile and six (54.5%) once fertile. In contrast toMcoln1^(-/-) CLs, which had extensive amorphous cellular debris, indicating cell degeneration,Atp6v0d2^(-/-)Mcoln1^(-/-) CLs had reduced amorphous cellular debris regardless of P4 levels. However, similar toMcoln1^(-/-) CLs, P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs showed impaired differentiation, enlarged lipid droplets, disorganized expression of endothelial basal lamina marker collagen IV, and reduced expression of mitochondrial marker heat shock protein 60 (HSP60) and steroidogenesis rate-limiting protein StAR, indicating that additionalAtp6v0d2 deficiency compensates forMcoln1 deficiency-induced cell degeneration, but is insufficient to restore luteal cell differentiation and P4 steroidogenesis in P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs.Conclusion: This study shows thatAtp6v0d2^(-/-)Mcoln1^(-/-) CLs had varied improvements compared withMcoln1^(-/-) CLs, and it providesin vivo genetic evidence of the coordination between different lysosomal channels in CL function.展开更多
基金supported by National Key Research and Development Program of China(Nos.2019YFC1708902 and 2019YFC1711000)National Natural Science Foundation of China(Nos.81973505,81773932 and 82104621).
文摘Diabetes have been shown to cause progressive neuronal injury with pain and numbness via advanced glycation end-products(AGEs)-induced neuronal cell apoptosis;however, the valuable drug targets for diabetic neuropathy have been poorly reported so far. In this study, we discovered a natural small-molecule schisandrol A(SolA) with significant protective effect against AGEs-induced neuronal cell apoptosis. ATP6V0D1, a major subunit of vacuolar-type ATPase(V-ATPase) in lysosome was identified as a crucial cellular target of SolA. Moreover, SolA allosterically mediated ATP6V0D1 conformation via targeting a unique cysteine 335 residue to activate V-ATPase-dependent lysosomal acidification.Interestingly, SolA-induced lysosome pH downregulation resulted in a mitochondrial-lysosomal crosstalk by selectively promoting mitochondrial BH3-only protein BIM degradation, thereby preserving mitochondrial homeostasis and neuronal cells survival. Collectively, our findings reveal ATP6V0D1 is a valuable pharmacological target for diabetes-associated neuronal injury via controlling lysosomal acidification, and also provide the first small-molecule template allosterically activating V-ATPase for preventing diabetic neuropathy.
基金funded by NIH R01HD065939(co-funded by ORWH and NICHD)NIH R03HD097384NIH R03 HD100652 to X.Y.
文摘Objective: ATP6V0d2 is a subunit of the vacuolar-type H+-ATPase (V-ATPase) that pumps H+ ions into lysosomes. TRPML1 (MCOLN1/Mcoln1) transports cations out of lysosomes.Mcoln1^(-/-) mice recapitulate the lysosomal storage disorder mucolipidosis type IV (MLIV) phenotype. We previously demonstrated thatMcoln1^(-/-) female mice quickly became infertile at 5 months old (5M) with degenerating corpora lutea (CL) and progesterone (P4) deficiency. We tested our hypothesis thatAtp6v0d2 deficiency could partially compensate forMcoln1 deficiency to restore CL functions inAtp6v0d2^(-/-)Mcoln1^(-/-) mice.Methods: Control andAtp6v0d2^(-/-)Mcoln1^(-/-) female mice underwent fertility test from 2M to 7M. A subset of them was dissected at 5M on day 3.5 post-coitum (D3.5). The D3.5 ovaries from 5M control,Mcoln1^(-/-), andAtp6v0d2^(-/-)Mcoln1^(-/-) mice were evaluated for CL morphology, lipid droplet staining, and markers of mitochondria and P4 steroidogenesis in the luteal cells.Results: The fertility test ofAtp6v0d2^(-/-)Mcoln1^(-/-) female mice (2M–7M) revealed normal mating activity but reduced fertility compared with the control;yet ~25% of them remained fertile at 5M to 7M but with dystocia. We analyzed a subset of 11Atp6v0d2^(-/-)Mcoln1^(-/-) mice (5M) in the fertility test on D3.5: three (27.3%) had normal P4 levels and all examined CL parameters, indicating full restoration of CL function compared withMcoln1^(-/-), whereas eight had P4 deficiency, with two (18.2%) infertile and six (54.5%) once fertile. In contrast toMcoln1^(-/-) CLs, which had extensive amorphous cellular debris, indicating cell degeneration,Atp6v0d2^(-/-)Mcoln1^(-/-) CLs had reduced amorphous cellular debris regardless of P4 levels. However, similar toMcoln1^(-/-) CLs, P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs showed impaired differentiation, enlarged lipid droplets, disorganized expression of endothelial basal lamina marker collagen IV, and reduced expression of mitochondrial marker heat shock protein 60 (HSP60) and steroidogenesis rate-limiting protein StAR, indicating that additionalAtp6v0d2 deficiency compensates forMcoln1 deficiency-induced cell degeneration, but is insufficient to restore luteal cell differentiation and P4 steroidogenesis in P4-deficientAtp6v0d2^(-/-)Mcoln1^(-/-) CLs.Conclusion: This study shows thatAtp6v0d2^(-/-)Mcoln1^(-/-) CLs had varied improvements compared withMcoln1^(-/-) CLs, and it providesin vivo genetic evidence of the coordination between different lysosomal channels in CL function.
