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Elevated autophagy and mitochondrial dysfunction in the Smith – Lemli – Opitz Syndrome

View Article: PubMed Central - PubMed

ABSTRACT

Smith–Lemli–Opitz Syndrome (SLOS) is a congenital, autosomal recessive metabolic and developmental disorder caused by mutations in the enzyme which catalyzes the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. Herein we show that dermal fibroblasts obtained from SLOS children display increased basal levels of LC3B-II, the hallmark protein signifying increased autophagy. The elevated LC3B-II is accompanied by increased beclin-1 and cellular autophagosome content. We also show that the LC3B-II concentration in SLOS cells is directly proportional to the cellular concentration of 7DHC, suggesting that the increased autophagy is caused by 7DHC accumulation secondary to defective DHCR7. Further, the increased basal LC3B-II levels were decreased significantly by pretreating the cells with antioxidants implicating a role for oxidative stress in elevating autophagy in SLOS cells. Considering the possible source of oxidative stress, we examined mitochondrial function in the SLOS cells using JC-1 assay and found significant mitochondrial dysfunction compared to mitochondria in control cells. In addition, the levels of PINK1 which targets dysfunctional mitochondria for removal by the autophagic pathway are elevated in SLOS cells, consistent with mitochondrial dysfunction as a stimulant of mitophagy in SLOS. This suggests that the increase in autophagic activity may be protective, i.e., to remove dysfunctional mitochondria. Taken together, these studies are consistent with a role for mitochondrial dysfunction leading to increased autophagy in SLOS pathophysiology.

No MeSH data available.


Related in: MedlinePlus

Inhibition of DHCR7 with AY9944 increases autophagic markers and the protective effect of antioxidants. (A) MIA PaCa-2 cells were transfected with LC3-GFP followed by treatment with AY9944 for 48 h. Autophagic vacuole formation was visualized by the accumulation of LC3-GFP. (B) LC3 Western-blot for AY9944 treated control and SLOS cells showing increased LC3B-II in control fibroblasts and even greater expression in SLOS fibroblasts. (C) B27, a nutritional supplement with antioxidants, decreased LC3B-II expression in AY9944 treated fibroblasts. Lanes 1–3, AY9944 treated control fibroblasts, and lanes 4–6, effects of B27 in AY9944 treated control fibroblasts. (C) LC3B-II expression before and after B27 and (D), LC3B-II expression before and after N-acetylcysteine (NAC), a potent antioxidant.
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f0025: Inhibition of DHCR7 with AY9944 increases autophagic markers and the protective effect of antioxidants. (A) MIA PaCa-2 cells were transfected with LC3-GFP followed by treatment with AY9944 for 48 h. Autophagic vacuole formation was visualized by the accumulation of LC3-GFP. (B) LC3 Western-blot for AY9944 treated control and SLOS cells showing increased LC3B-II in control fibroblasts and even greater expression in SLOS fibroblasts. (C) B27, a nutritional supplement with antioxidants, decreased LC3B-II expression in AY9944 treated fibroblasts. Lanes 1–3, AY9944 treated control fibroblasts, and lanes 4–6, effects of B27 in AY9944 treated control fibroblasts. (C) LC3B-II expression before and after B27 and (D), LC3B-II expression before and after N-acetylcysteine (NAC), a potent antioxidant.

Mentions: The correlation between 7DHC and LC3B-II levels prompted us to use the specific DHCR7 inhibitor, AY9944 [21] to increase 7DHC levels followed by the assessment of autophagosome formation and LC3B-II levels. Fig. 5A shows MIA PaCa-2 cells transfected with LC3-GFP with and without AY9944 treatment showing an increase in autophagosomes in AY9944 treated cells. Immunoblots revealed an increased LC3B-II expression compared to cells with no AY9944 treatment (Fig. 5B). This result is consistent with the correlation between 7DHC and LC3B-II. Furthermore, the antioxidant cocktail/supplement/ B27 had a protective effect in control cells treated with AY9944 (Fig. 5, panel C), a similar effect as that seen with the single antioxidant N-acetylcysteine in Fig. 5, panel D. These results demonstrate that the antioxidant cocktail B27 and the single antioxidant N-acetylcysteine have very similar effects thereby supporting the suggestion that oxidative stress underlies the activation of autophagy in SLOS cells.


Elevated autophagy and mitochondrial dysfunction in the Smith – Lemli – Opitz Syndrome
Inhibition of DHCR7 with AY9944 increases autophagic markers and the protective effect of antioxidants. (A) MIA PaCa-2 cells were transfected with LC3-GFP followed by treatment with AY9944 for 48 h. Autophagic vacuole formation was visualized by the accumulation of LC3-GFP. (B) LC3 Western-blot for AY9944 treated control and SLOS cells showing increased LC3B-II in control fibroblasts and even greater expression in SLOS fibroblasts. (C) B27, a nutritional supplement with antioxidants, decreased LC3B-II expression in AY9944 treated fibroblasts. Lanes 1–3, AY9944 treated control fibroblasts, and lanes 4–6, effects of B27 in AY9944 treated control fibroblasts. (C) LC3B-II expression before and after B27 and (D), LC3B-II expression before and after N-acetylcysteine (NAC), a potent antioxidant.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231544&req=5

f0025: Inhibition of DHCR7 with AY9944 increases autophagic markers and the protective effect of antioxidants. (A) MIA PaCa-2 cells were transfected with LC3-GFP followed by treatment with AY9944 for 48 h. Autophagic vacuole formation was visualized by the accumulation of LC3-GFP. (B) LC3 Western-blot for AY9944 treated control and SLOS cells showing increased LC3B-II in control fibroblasts and even greater expression in SLOS fibroblasts. (C) B27, a nutritional supplement with antioxidants, decreased LC3B-II expression in AY9944 treated fibroblasts. Lanes 1–3, AY9944 treated control fibroblasts, and lanes 4–6, effects of B27 in AY9944 treated control fibroblasts. (C) LC3B-II expression before and after B27 and (D), LC3B-II expression before and after N-acetylcysteine (NAC), a potent antioxidant.
Mentions: The correlation between 7DHC and LC3B-II levels prompted us to use the specific DHCR7 inhibitor, AY9944 [21] to increase 7DHC levels followed by the assessment of autophagosome formation and LC3B-II levels. Fig. 5A shows MIA PaCa-2 cells transfected with LC3-GFP with and without AY9944 treatment showing an increase in autophagosomes in AY9944 treated cells. Immunoblots revealed an increased LC3B-II expression compared to cells with no AY9944 treatment (Fig. 5B). This result is consistent with the correlation between 7DHC and LC3B-II. Furthermore, the antioxidant cocktail/supplement/ B27 had a protective effect in control cells treated with AY9944 (Fig. 5, panel C), a similar effect as that seen with the single antioxidant N-acetylcysteine in Fig. 5, panel D. These results demonstrate that the antioxidant cocktail B27 and the single antioxidant N-acetylcysteine have very similar effects thereby supporting the suggestion that oxidative stress underlies the activation of autophagy in SLOS cells.

View Article: PubMed Central - PubMed

ABSTRACT

Smith–Lemli–Opitz Syndrome (SLOS) is a congenital, autosomal recessive metabolic and developmental disorder caused by mutations in the enzyme which catalyzes the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. Herein we show that dermal fibroblasts obtained from SLOS children display increased basal levels of LC3B-II, the hallmark protein signifying increased autophagy. The elevated LC3B-II is accompanied by increased beclin-1 and cellular autophagosome content. We also show that the LC3B-II concentration in SLOS cells is directly proportional to the cellular concentration of 7DHC, suggesting that the increased autophagy is caused by 7DHC accumulation secondary to defective DHCR7. Further, the increased basal LC3B-II levels were decreased significantly by pretreating the cells with antioxidants implicating a role for oxidative stress in elevating autophagy in SLOS cells. Considering the possible source of oxidative stress, we examined mitochondrial function in the SLOS cells using JC-1 assay and found significant mitochondrial dysfunction compared to mitochondria in control cells. In addition, the levels of PINK1 which targets dysfunctional mitochondria for removal by the autophagic pathway are elevated in SLOS cells, consistent with mitochondrial dysfunction as a stimulant of mitophagy in SLOS. This suggests that the increase in autophagic activity may be protective, i.e., to remove dysfunctional mitochondria. Taken together, these studies are consistent with a role for mitochondrial dysfunction leading to increased autophagy in SLOS pathophysiology.

No MeSH data available.


Related in: MedlinePlus