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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

Activation of Pink-1 in SLOS cells. Immunostaining of Pink-1 and Parkin in SLOS and control cells. Panel A: representative images from immunostaining of Pink-1 in SLOS and control cells, red fluorescence for Pink-1 and blue fluorescence for nuclei, top row for control cells and the lower row for SLOS cells. Panel B: representative images from immunostaining of Parkin in SLOS and control cells, red fluorescence for Parkin and blue fluorescence for nuclei, top row for control cells and lower row for SLOS cells. Panel C: Western-blot of Pink-1 and Parkin for SLOS and control cells; β-actin was used as an internal control to normalize protein loading. Panel D: Image analysis of Pink-1 and Parkin immunostaining. The relative intensity of Pink-1 or Parkin was normalized by nuclei staining. Pink-1 was significantly increased in SLOS cells (n = 3, p < 0.05), while there was no significant change of the ratio of red/blue fluorescence in Parkin immunostaining.
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f0035: Activation of Pink-1 in SLOS cells. Immunostaining of Pink-1 and Parkin in SLOS and control cells. Panel A: representative images from immunostaining of Pink-1 in SLOS and control cells, red fluorescence for Pink-1 and blue fluorescence for nuclei, top row for control cells and the lower row for SLOS cells. Panel B: representative images from immunostaining of Parkin in SLOS and control cells, red fluorescence for Parkin and blue fluorescence for nuclei, top row for control cells and lower row for SLOS cells. Panel C: Western-blot of Pink-1 and Parkin for SLOS and control cells; β-actin was used as an internal control to normalize protein loading. Panel D: Image analysis of Pink-1 and Parkin immunostaining. The relative intensity of Pink-1 or Parkin was normalized by nuclei staining. Pink-1 was significantly increased in SLOS cells (n = 3, p < 0.05), while there was no significant change of the ratio of red/blue fluorescence in Parkin immunostaining.

Mentions: The activation of mitophagy by dysfunctional mitochondria is regulated by the accumulation of Pink-1 on the mitochondrial outer membrane [23]. To determine whether the presence of dysfunctional mitochondria in SLOS cells serves to stimulate mitophagy, we assessed the level of Pink-1 in SLOS cells. Confocal images revealed an obvious increase in Pink-1 staining in SLOS cells compared with control (Fig. 7A), while a difference in Parkin between SLOS and control was not observed (Fig. 7B). Using nuclei staining as the internal control, the relative intensity of Pink1 was remarkably increased by 40% in SLOS vs control cells, and there was no clear change of Parkin as shown in Fig. 7D and confirmed in immunoblots (Fig. 7C) from control and SLOS cell protein isolates (n = 3). These data support the conclusion that defects in mitochondrial health contribute to the stimuli elevating autophagy in SLOS cells.


Elevated autophagy and mitochondrial dysfunction in the Smith – Lemli – Opitz Syndrome
Activation of Pink-1 in SLOS cells. Immunostaining of Pink-1 and Parkin in SLOS and control cells. Panel A: representative images from immunostaining of Pink-1 in SLOS and control cells, red fluorescence for Pink-1 and blue fluorescence for nuclei, top row for control cells and the lower row for SLOS cells. Panel B: representative images from immunostaining of Parkin in SLOS and control cells, red fluorescence for Parkin and blue fluorescence for nuclei, top row for control cells and lower row for SLOS cells. Panel C: Western-blot of Pink-1 and Parkin for SLOS and control cells; β-actin was used as an internal control to normalize protein loading. Panel D: Image analysis of Pink-1 and Parkin immunostaining. The relative intensity of Pink-1 or Parkin was normalized by nuclei staining. Pink-1 was significantly increased in SLOS cells (n = 3, p < 0.05), while there was no significant change of the ratio of red/blue fluorescence in Parkin immunostaining.
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Related In: Results  -  Collection

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f0035: Activation of Pink-1 in SLOS cells. Immunostaining of Pink-1 and Parkin in SLOS and control cells. Panel A: representative images from immunostaining of Pink-1 in SLOS and control cells, red fluorescence for Pink-1 and blue fluorescence for nuclei, top row for control cells and the lower row for SLOS cells. Panel B: representative images from immunostaining of Parkin in SLOS and control cells, red fluorescence for Parkin and blue fluorescence for nuclei, top row for control cells and lower row for SLOS cells. Panel C: Western-blot of Pink-1 and Parkin for SLOS and control cells; β-actin was used as an internal control to normalize protein loading. Panel D: Image analysis of Pink-1 and Parkin immunostaining. The relative intensity of Pink-1 or Parkin was normalized by nuclei staining. Pink-1 was significantly increased in SLOS cells (n = 3, p < 0.05), while there was no significant change of the ratio of red/blue fluorescence in Parkin immunostaining.
Mentions: The activation of mitophagy by dysfunctional mitochondria is regulated by the accumulation of Pink-1 on the mitochondrial outer membrane [23]. To determine whether the presence of dysfunctional mitochondria in SLOS cells serves to stimulate mitophagy, we assessed the level of Pink-1 in SLOS cells. Confocal images revealed an obvious increase in Pink-1 staining in SLOS cells compared with control (Fig. 7A), while a difference in Parkin between SLOS and control was not observed (Fig. 7B). Using nuclei staining as the internal control, the relative intensity of Pink1 was remarkably increased by 40% in SLOS vs control cells, and there was no clear change of Parkin as shown in Fig. 7D and confirmed in immunoblots (Fig. 7C) from control and SLOS cell protein isolates (n = 3). These data support the conclusion that defects in mitochondrial health contribute to the stimuli elevating autophagy in SLOS cells.

View Article: PubMed Central - PubMed

ABSTRACT

Smith&ndash;Lemli&ndash;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