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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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Mitochondria release cytochrome c and trigger cell death in MSD liver. (A) Anti-cytochrome c immunoblots of subcellular fractions (c, cytosol; m, mitochondria) obtained from the whole brain and liver from MSD (n = 4) and control mice (n = 4) at P15, 1 month and 3 months. COX IV was used as mitochondrial loading control. In the liver, levels of cytochrome c were quantified in each cytosolic fraction and expressed as the average percentage in terms of fold changes (bottom); **P < 0.01. (B) TUNEL analysis on fixed-paraffin brain and liver sections of MSD and control mice at 3 months. Images were acquired with the 20× magnification. Positive controls were obtained by treating tissue sections with DNAase I. TUNEL-positive cells were stained in green, and nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue).
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DDR610F1: Mitochondria release cytochrome c and trigger cell death in MSD liver. (A) Anti-cytochrome c immunoblots of subcellular fractions (c, cytosol; m, mitochondria) obtained from the whole brain and liver from MSD (n = 4) and control mice (n = 4) at P15, 1 month and 3 months. COX IV was used as mitochondrial loading control. In the liver, levels of cytochrome c were quantified in each cytosolic fraction and expressed as the average percentage in terms of fold changes (bottom); **P < 0.01. (B) TUNEL analysis on fixed-paraffin brain and liver sections of MSD and control mice at 3 months. Images were acquired with the 20× magnification. Positive controls were obtained by treating tissue sections with DNAase I. TUNEL-positive cells were stained in green, and nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue).

Mentions: Mitochondria can compromise cell viability leading to tissue damage. An increase in the permeabilization of the mitochondrial membrane, also known as mitochondrial permeability transition, produces mitochondrial swelling, OMM rupture and release of pro-apoptotic factors such as cytochrome c. MSD mice display a multisystemic phenotype that involves both the central nervous system (CNS) and all main organs such as the liver. Glycosaminoglycan (GAG) accumulation, inflammation and cell death are detectable as soon as 15 days after birth and progressively increase with age. At 3 months of age, both the liver and CNS pathology are clearly manifested (30,34,41) (Supplementary Material, Figs S1 and S2). In particular, we evaluated whether mitochondria obtained from the brain and liver of MSD mice contributed to MSD pathology by releasing cytochrome c from the intermembrane space. The presence of cytochrome c was not detected in the cytosolic fractions of MSD brains at any of the time points analyzed (Fig. 1A). On the contrary, we observed high levels of cytochrome c in cytosolic fractions of MSD liver as soon as at 1 month of age.Figure 1.


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)

Mitochondria release cytochrome c and trigger cell death in MSD liver. (A) Anti-cytochrome c immunoblots of subcellular fractions (c, cytosol; m, mitochondria) obtained from the whole brain and liver from MSD (n = 4) and control mice (n = 4) at P15, 1 month and 3 months. COX IV was used as mitochondrial loading control. In the liver, levels of cytochrome c were quantified in each cytosolic fraction and expressed as the average percentage in terms of fold changes (bottom); **P < 0.01. (B) TUNEL analysis on fixed-paraffin brain and liver sections of MSD and control mice at 3 months. Images were acquired with the 20× magnification. Positive controls were obtained by treating tissue sections with DNAase I. TUNEL-positive cells were stained in green, and nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3313794&req=5

DDR610F1: Mitochondria release cytochrome c and trigger cell death in MSD liver. (A) Anti-cytochrome c immunoblots of subcellular fractions (c, cytosol; m, mitochondria) obtained from the whole brain and liver from MSD (n = 4) and control mice (n = 4) at P15, 1 month and 3 months. COX IV was used as mitochondrial loading control. In the liver, levels of cytochrome c were quantified in each cytosolic fraction and expressed as the average percentage in terms of fold changes (bottom); **P < 0.01. (B) TUNEL analysis on fixed-paraffin brain and liver sections of MSD and control mice at 3 months. Images were acquired with the 20× magnification. Positive controls were obtained by treating tissue sections with DNAase I. TUNEL-positive cells were stained in green, and nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue).
Mentions: Mitochondria can compromise cell viability leading to tissue damage. An increase in the permeabilization of the mitochondrial membrane, also known as mitochondrial permeability transition, produces mitochondrial swelling, OMM rupture and release of pro-apoptotic factors such as cytochrome c. MSD mice display a multisystemic phenotype that involves both the central nervous system (CNS) and all main organs such as the liver. Glycosaminoglycan (GAG) accumulation, inflammation and cell death are detectable as soon as 15 days after birth and progressively increase with age. At 3 months of age, both the liver and CNS pathology are clearly manifested (30,34,41) (Supplementary Material, Figs S1 and S2). In particular, we evaluated whether mitochondria obtained from the brain and liver of MSD mice contributed to MSD pathology by releasing cytochrome c from the intermembrane space. The presence of cytochrome c was not detected in the cytosolic fractions of MSD brains at any of the time points analyzed (Fig. 1A). On the contrary, we observed high levels of cytochrome c in cytosolic fractions of MSD liver as soon as at 1 month of age.Figure 1.

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