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Impaired parkin-mediated mitochondrial targeting to autophagosomes differentially contributes to tissue pathology in lysosomal storage diseases.

de Pablo-Latorre R, Saide A, Polishhuck EV, Nusco E, Fraldi A, Ballabio A - Hum. Mol. Genet. (2012)

Bottom Line: In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction.We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death.Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine, Naples 80131, Italy.

ABSTRACT
Dysfunctional mitochondria are a well-known disease hallmark. The accumulation of aberrant mitochondria can alter cell homeostasis, thus resulting in tissue degeneration. Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by the buildup of undegraded material inside the lysosomes that leads to autophagic-lysosomal dysfunction. In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction. However, the mechanisms underlying mitochondrial aberrations and how these are involved in tissue pathogenesis remain largely unexplored. In normal conditions, mitochondrial clearance occurs by mitophagy, a selective form of autophagy, which relies on a parkin-mediated mitochondrial priming and subsequent sequestration by autophagosomes. Here, we performed a detailed analysis of key steps of mitophagy in a mouse model of multiple sulfatase deficiency (MSD), a severe type of LSD characterized by both neurological and systemic involvement. We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death. Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration. Together, these data shed new light on the mechanisms underlying mitochondrial dysfunction in LSDs and on their tissue-specific differential contribution to the pathogenesis of this group of metabolic disorders.

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Dysfunctional giant mitochondria accumulate in a time-dependent manner in MSD liver. (A) Levels of COX IV in liver total lysates of MSD (n = 3) and control (n = 3) mice at P15, 1 month and 3 months. The COX IV/actin ratio is expressed in terms of fold changes and represents an average value; **P < 0.01. (B) Analysis of mitochondrial size (diameter) using the AnalySIS software in liver ultrathin sections (70 nm) from MSD and control mice at 1 month and 3 months. Average number of mitochondria analyzed: control 1 month = 43, MSD 1 month = 56, control 3 months = 60, MSD 3 months = 127. Values are expressed in nm (scale bar = 400 nm). ANOVA P-value = 1.27 × 10−13, **P < 0.01. (C) Measurement of JC-1 fluorescence (FLU) per milligram (mg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 0.048, *P < 0.05. (D) Analysis of ATP content per microgram (μg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 4.81 × 10−3, *P < 0.05, **P < 0.01.
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DDR610F2: Dysfunctional giant mitochondria accumulate in a time-dependent manner in MSD liver. (A) Levels of COX IV in liver total lysates of MSD (n = 3) and control (n = 3) mice at P15, 1 month and 3 months. The COX IV/actin ratio is expressed in terms of fold changes and represents an average value; **P < 0.01. (B) Analysis of mitochondrial size (diameter) using the AnalySIS software in liver ultrathin sections (70 nm) from MSD and control mice at 1 month and 3 months. Average number of mitochondria analyzed: control 1 month = 43, MSD 1 month = 56, control 3 months = 60, MSD 3 months = 127. Values are expressed in nm (scale bar = 400 nm). ANOVA P-value = 1.27 × 10−13, **P < 0.01. (C) Measurement of JC-1 fluorescence (FLU) per milligram (mg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 0.048, *P < 0.05. (D) Analysis of ATP content per microgram (μg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 4.81 × 10−3, *P < 0.05, **P < 0.01.

Mentions: We then evaluated whether the mitochondrial release of cytochrome c was related to morphological and functional changes. We determined the levels of subunit IV of cytochrome oxidase (COX IV), widely used as a marker for quantifying the number of mitochondria (42). COX IV levels were measured in liver homogenates obtained from MSD (n= 3) and littermate control mice (n = 3) by western blot analysis. As shown in Figure 2A, significant changes were only observed at advanced stages, thus indicating that the accumulation of mitochondria is a progressive process in liver pathogenesis.Figure 2.


Impaired parkin-mediated mitochondrial targeting to autophagosomes differentially contributes to tissue pathology in lysosomal storage diseases.

de Pablo-Latorre R, Saide A, Polishhuck EV, Nusco E, Fraldi A, Ballabio A - Hum. Mol. Genet. (2012)

Dysfunctional giant mitochondria accumulate in a time-dependent manner in MSD liver. (A) Levels of COX IV in liver total lysates of MSD (n = 3) and control (n = 3) mice at P15, 1 month and 3 months. The COX IV/actin ratio is expressed in terms of fold changes and represents an average value; **P < 0.01. (B) Analysis of mitochondrial size (diameter) using the AnalySIS software in liver ultrathin sections (70 nm) from MSD and control mice at 1 month and 3 months. Average number of mitochondria analyzed: control 1 month = 43, MSD 1 month = 56, control 3 months = 60, MSD 3 months = 127. Values are expressed in nm (scale bar = 400 nm). ANOVA P-value = 1.27 × 10−13, **P < 0.01. (C) Measurement of JC-1 fluorescence (FLU) per milligram (mg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 0.048, *P < 0.05. (D) Analysis of ATP content per microgram (μg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 4.81 × 10−3, *P < 0.05, **P < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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DDR610F2: Dysfunctional giant mitochondria accumulate in a time-dependent manner in MSD liver. (A) Levels of COX IV in liver total lysates of MSD (n = 3) and control (n = 3) mice at P15, 1 month and 3 months. The COX IV/actin ratio is expressed in terms of fold changes and represents an average value; **P < 0.01. (B) Analysis of mitochondrial size (diameter) using the AnalySIS software in liver ultrathin sections (70 nm) from MSD and control mice at 1 month and 3 months. Average number of mitochondria analyzed: control 1 month = 43, MSD 1 month = 56, control 3 months = 60, MSD 3 months = 127. Values are expressed in nm (scale bar = 400 nm). ANOVA P-value = 1.27 × 10−13, **P < 0.01. (C) Measurement of JC-1 fluorescence (FLU) per milligram (mg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 0.048, *P < 0.05. (D) Analysis of ATP content per microgram (μg) of protein in isolated mitochondria from MSD (n = 3) and control (n = 3) mice at 1 month and 3 months. Values are expressed in terms of fold changes compared with control. ANOVA P-value = 4.81 × 10−3, *P < 0.05, **P < 0.01.
Mentions: We then evaluated whether the mitochondrial release of cytochrome c was related to morphological and functional changes. We determined the levels of subunit IV of cytochrome oxidase (COX IV), widely used as a marker for quantifying the number of mitochondria (42). COX IV levels were measured in liver homogenates obtained from MSD (n= 3) and littermate control mice (n = 3) by western blot analysis. As shown in Figure 2A, significant changes were only observed at advanced stages, thus indicating that the accumulation of mitochondria is a progressive process in liver pathogenesis.Figure 2.

Bottom Line: In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction.We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death.Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine, Naples 80131, Italy.

ABSTRACT
Dysfunctional mitochondria are a well-known disease hallmark. The accumulation of aberrant mitochondria can alter cell homeostasis, thus resulting in tissue degeneration. Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by the buildup of undegraded material inside the lysosomes that leads to autophagic-lysosomal dysfunction. In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction. However, the mechanisms underlying mitochondrial aberrations and how these are involved in tissue pathogenesis remain largely unexplored. In normal conditions, mitochondrial clearance occurs by mitophagy, a selective form of autophagy, which relies on a parkin-mediated mitochondrial priming and subsequent sequestration by autophagosomes. Here, we performed a detailed analysis of key steps of mitophagy in a mouse model of multiple sulfatase deficiency (MSD), a severe type of LSD characterized by both neurological and systemic involvement. We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death. Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration. Together, these data shed new light on the mechanisms underlying mitochondrial dysfunction in LSDs and on their tissue-specific differential contribution to the pathogenesis of this group of metabolic disorders.

Show MeSH
Related in: MedlinePlus