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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.


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Increased autophagy in cell models on SLOS neurons. (A) Increased expression of LC3B-II in neuronal stem cells obtained from the brains of Dhcr7 knockout mice. Mutational defects of one of the SLOS genotypes (Dhcr7Δ3–05/Δ3–5) were generated in mice using genetic engineering. Whole cell protein extracts from heterozygote (+/−) and homozygote (−/−) cells were subjected to immunoblotting with LC3B-II antibody. (B) Increased LC3B-II in Dhcr7-deficient Neuro2a cells. The cellular 7-DHC was monitored in control Neuro2a (0.21 ± 0.01 ng/μg protein) and in Dhcr7-deficient cells (33.3 ± 6.2 ng/μg protein). Increased LC3B-II in neuroblastoma cells in which Dhcr7 was silenced with siRNA.
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f0040: Increased autophagy in cell models on SLOS neurons. (A) Increased expression of LC3B-II in neuronal stem cells obtained from the brains of Dhcr7 knockout mice. Mutational defects of one of the SLOS genotypes (Dhcr7Δ3–05/Δ3–5) were generated in mice using genetic engineering. Whole cell protein extracts from heterozygote (+/−) and homozygote (−/−) cells were subjected to immunoblotting with LC3B-II antibody. (B) Increased LC3B-II in Dhcr7-deficient Neuro2a cells. The cellular 7-DHC was monitored in control Neuro2a (0.21 ± 0.01 ng/μg protein) and in Dhcr7-deficient cells (33.3 ± 6.2 ng/μg protein). Increased LC3B-II in neuroblastoma cells in which Dhcr7 was silenced with siRNA.

Mentions: Lastly, since among the most common and devastating defects in SLOS children derive from CNS dysfunction including intellectual disability, we examined neural stem cells cultured from the brains of transgenic mice harboring the Dhcr7Δ3–5/Δ3–5 mutation, i.e., similar to mutations in homozygotic SLOS fetuses. As shown in Fig. 8, panel A, 1 heterozygote and 2 homozygotes have elevated LC3B-II protein supporting the suggestion that autophagy is increased in the CNS similar to that seen in dermal fibroblasts isolated from SLOS children. Further supporting a role for autophagy in the CNS, silencing Dhcr7 in neuroblastoma cells resulted in an increase in the LC3B-II/I ratio (Fig. 8B), indicative of increased autophagy in neuronal cells in which Dhcr7 is deleted. The cellular 7-DHC level was monitored in control Neuro2a (0.21 ± 0.01 ng/μg protein) and in Dhcr7-deficient cells (33.3 ± 6.2 ng/μg protein).


Elevated autophagy and mitochondrial dysfunction in the Smith – Lemli – Opitz Syndrome
Increased autophagy in cell models on SLOS neurons. (A) Increased expression of LC3B-II in neuronal stem cells obtained from the brains of Dhcr7 knockout mice. Mutational defects of one of the SLOS genotypes (Dhcr7Δ3–05/Δ3–5) were generated in mice using genetic engineering. Whole cell protein extracts from heterozygote (+/−) and homozygote (−/−) cells were subjected to immunoblotting with LC3B-II antibody. (B) Increased LC3B-II in Dhcr7-deficient Neuro2a cells. The cellular 7-DHC was monitored in control Neuro2a (0.21 ± 0.01 ng/μg protein) and in Dhcr7-deficient cells (33.3 ± 6.2 ng/μg protein). Increased LC3B-II in neuroblastoma cells in which Dhcr7 was silenced with siRNA.
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Related In: Results  -  Collection

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f0040: Increased autophagy in cell models on SLOS neurons. (A) Increased expression of LC3B-II in neuronal stem cells obtained from the brains of Dhcr7 knockout mice. Mutational defects of one of the SLOS genotypes (Dhcr7Δ3–05/Δ3–5) were generated in mice using genetic engineering. Whole cell protein extracts from heterozygote (+/−) and homozygote (−/−) cells were subjected to immunoblotting with LC3B-II antibody. (B) Increased LC3B-II in Dhcr7-deficient Neuro2a cells. The cellular 7-DHC was monitored in control Neuro2a (0.21 ± 0.01 ng/μg protein) and in Dhcr7-deficient cells (33.3 ± 6.2 ng/μg protein). Increased LC3B-II in neuroblastoma cells in which Dhcr7 was silenced with siRNA.
Mentions: Lastly, since among the most common and devastating defects in SLOS children derive from CNS dysfunction including intellectual disability, we examined neural stem cells cultured from the brains of transgenic mice harboring the Dhcr7Δ3–5/Δ3–5 mutation, i.e., similar to mutations in homozygotic SLOS fetuses. As shown in Fig. 8, panel A, 1 heterozygote and 2 homozygotes have elevated LC3B-II protein supporting the suggestion that autophagy is increased in the CNS similar to that seen in dermal fibroblasts isolated from SLOS children. Further supporting a role for autophagy in the CNS, silencing Dhcr7 in neuroblastoma cells resulted in an increase in the LC3B-II/I ratio (Fig. 8B), indicative of increased autophagy in neuronal cells in which Dhcr7 is deleted. The cellular 7-DHC level was monitored in control Neuro2a (0.21 ± 0.01 ng/μg protein) and in Dhcr7-deficient cells (33.3 ± 6.2 ng/μg protein).

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