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BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

View Article: PubMed Central - PubMed

ABSTRACT

Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.

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Related in: MedlinePlus

Activation of BK by NS1619 reduces lipofuscin and cholesterol accumulation in NPC1 cells in a TRPML1-dependent manner.(A,B) Abnormal lipofuscin accumulation (detected by autofluorescence) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 16 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, dextran staining was employed to indicate the presence of cells. More than 42 cells were analyzed for each condition. (C,D) Abnormal cholesterol accumulation (filipin staining) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 18 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, bright field images were included to indicate the presence of cells. More than 41 cells were analyzed for each condition. (E,F) NS1619 treatment (15 μM, 16 hrs) reduced the abnormal lipofuscin accumulation in NPC1 human fibroblasts. This NS1619 effect was inhibited by overexpressing Syt VII DN that suppresses lysosomal exocytosis. (G) NS1619 (15 μM, 16 hrs) increased lysosomal exocytosis (indicated by the elevation of lysosomal enzyme β-hexosaminidase in cell culture supernatant), which was inhibited by applying ML-SI1 or expressing Syt VII DN. (H) Comparable LDH in culture medium under conditions indicated, suggesting the elevation of β-hexosaminidase in culture media was not attributed to cell death which releases lysosomal enzymes. The data represents mean ± SEM, and experiments were repeated independently 3 times in triplicate.
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f1: Activation of BK by NS1619 reduces lipofuscin and cholesterol accumulation in NPC1 cells in a TRPML1-dependent manner.(A,B) Abnormal lipofuscin accumulation (detected by autofluorescence) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 16 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, dextran staining was employed to indicate the presence of cells. More than 42 cells were analyzed for each condition. (C,D) Abnormal cholesterol accumulation (filipin staining) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 18 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, bright field images were included to indicate the presence of cells. More than 41 cells were analyzed for each condition. (E,F) NS1619 treatment (15 μM, 16 hrs) reduced the abnormal lipofuscin accumulation in NPC1 human fibroblasts. This NS1619 effect was inhibited by overexpressing Syt VII DN that suppresses lysosomal exocytosis. (G) NS1619 (15 μM, 16 hrs) increased lysosomal exocytosis (indicated by the elevation of lysosomal enzyme β-hexosaminidase in cell culture supernatant), which was inhibited by applying ML-SI1 or expressing Syt VII DN. (H) Comparable LDH in culture medium under conditions indicated, suggesting the elevation of β-hexosaminidase in culture media was not attributed to cell death which releases lysosomal enzymes. The data represents mean ± SEM, and experiments were repeated independently 3 times in triplicate.

Mentions: NPC1 disease is caused by defects in the gene NPC1 that transports cholesterol across lysosomal membrane. NPC1 mutations result in impaired lysosomal membrane trafficking, leading to abnormal lipofuscin, cholesterol and sphingomyelin accumulation in lysosomes415. Recent studies have suggested that TRPML1-mediated Ca2+ release is compromised in NPC1 human fibroblasts47. We have shown that BK overexpression facilitates TRPML1 function, thereby rescuing abnormal lysosomal storage in NPC1 human fibroblasts7. To help develop a potential therapeutic strategy to cure NPC1 by potentiating BK, we aim to study whether NS1619, a chemical reported to activate BK in the plasma membrane (PM)16, eliminates NPC1 cellular phenotypes. As shown in Fig. 1A, human skin fibroblasts from NPC1 patients showed significantly higher signal of lipofuscin, a non-degradable auto-fluorescent polymeric substance often accumulated with age or seen in LSDs and aging diseases781718. This was inhibited by NS1619 treatment in a time and does-dependent manner (Fig. S1A,B). Additionally, the abnormal lipofuscin in NPC1 human fibroblasts could be rescued by NS11021 (Fig. S1C), another BK agonist with high potency and selectivity19. Interestingly, the inhibitory effect of NS1619 (15 μM, 16 h) on lipofuscin signal in NPC1 fibroblasts was eliminated by TRPML1-DD/KK, a dominant negative mutant of TRPML12021, but not Lamp1 (Fig. 1A,B). These data suggest that NS1619 corrects lipofuscin accumulation in NPC1 cells through promoting TRPML1 activity. Similarly, NS1619 (15 μM, 18 h) rescued cholesterol accumulation in NPC1 cells using a TRPML1-dependent mechanism (Fig. 1C,D).


BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases
Activation of BK by NS1619 reduces lipofuscin and cholesterol accumulation in NPC1 cells in a TRPML1-dependent manner.(A,B) Abnormal lipofuscin accumulation (detected by autofluorescence) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 16 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, dextran staining was employed to indicate the presence of cells. More than 42 cells were analyzed for each condition. (C,D) Abnormal cholesterol accumulation (filipin staining) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 18 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, bright field images were included to indicate the presence of cells. More than 41 cells were analyzed for each condition. (E,F) NS1619 treatment (15 μM, 16 hrs) reduced the abnormal lipofuscin accumulation in NPC1 human fibroblasts. This NS1619 effect was inhibited by overexpressing Syt VII DN that suppresses lysosomal exocytosis. (G) NS1619 (15 μM, 16 hrs) increased lysosomal exocytosis (indicated by the elevation of lysosomal enzyme β-hexosaminidase in cell culture supernatant), which was inhibited by applying ML-SI1 or expressing Syt VII DN. (H) Comparable LDH in culture medium under conditions indicated, suggesting the elevation of β-hexosaminidase in culture media was not attributed to cell death which releases lysosomal enzymes. The data represents mean ± SEM, and experiments were repeated independently 3 times in triplicate.
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f1: Activation of BK by NS1619 reduces lipofuscin and cholesterol accumulation in NPC1 cells in a TRPML1-dependent manner.(A,B) Abnormal lipofuscin accumulation (detected by autofluorescence) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 16 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, dextran staining was employed to indicate the presence of cells. More than 42 cells were analyzed for each condition. (C,D) Abnormal cholesterol accumulation (filipin staining) in NPC1 human fibroblasts and its rescue by activation of BK with NS1619 (15 μM, 18 hrs). Expression of TRPML1-DD/KK reversed the rescue effect of BK activation. In some groups, bright field images were included to indicate the presence of cells. More than 41 cells were analyzed for each condition. (E,F) NS1619 treatment (15 μM, 16 hrs) reduced the abnormal lipofuscin accumulation in NPC1 human fibroblasts. This NS1619 effect was inhibited by overexpressing Syt VII DN that suppresses lysosomal exocytosis. (G) NS1619 (15 μM, 16 hrs) increased lysosomal exocytosis (indicated by the elevation of lysosomal enzyme β-hexosaminidase in cell culture supernatant), which was inhibited by applying ML-SI1 or expressing Syt VII DN. (H) Comparable LDH in culture medium under conditions indicated, suggesting the elevation of β-hexosaminidase in culture media was not attributed to cell death which releases lysosomal enzymes. The data represents mean ± SEM, and experiments were repeated independently 3 times in triplicate.
Mentions: NPC1 disease is caused by defects in the gene NPC1 that transports cholesterol across lysosomal membrane. NPC1 mutations result in impaired lysosomal membrane trafficking, leading to abnormal lipofuscin, cholesterol and sphingomyelin accumulation in lysosomes415. Recent studies have suggested that TRPML1-mediated Ca2+ release is compromised in NPC1 human fibroblasts47. We have shown that BK overexpression facilitates TRPML1 function, thereby rescuing abnormal lysosomal storage in NPC1 human fibroblasts7. To help develop a potential therapeutic strategy to cure NPC1 by potentiating BK, we aim to study whether NS1619, a chemical reported to activate BK in the plasma membrane (PM)16, eliminates NPC1 cellular phenotypes. As shown in Fig. 1A, human skin fibroblasts from NPC1 patients showed significantly higher signal of lipofuscin, a non-degradable auto-fluorescent polymeric substance often accumulated with age or seen in LSDs and aging diseases781718. This was inhibited by NS1619 treatment in a time and does-dependent manner (Fig. S1A,B). Additionally, the abnormal lipofuscin in NPC1 human fibroblasts could be rescued by NS11021 (Fig. S1C), another BK agonist with high potency and selectivity19. Interestingly, the inhibitory effect of NS1619 (15 μM, 16 h) on lipofuscin signal in NPC1 fibroblasts was eliminated by TRPML1-DD/KK, a dominant negative mutant of TRPML12021, but not Lamp1 (Fig. 1A,B). These data suggest that NS1619 corrects lipofuscin accumulation in NPC1 cells through promoting TRPML1 activity. Similarly, NS1619 (15 μM, 18 h) rescued cholesterol accumulation in NPC1 cells using a TRPML1-dependent mechanism (Fig. 1C,D).

View Article: PubMed Central - PubMed

ABSTRACT

Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.

No MeSH data available.


Related in: MedlinePlus