Mitochondrial dysfunction in fibroblasts derived from patients with Niemann-Pick type C disease

doi:10.1016/j.abb.2016.02.012

Archives of Biochemistry and Biophysics

Volume 593, 1 March 2016, Pages 50-59

https://doi.org/10.1016/j.abb.2016.02.012 Get rights and content

Highlights

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Primary fibroblasts from NPC patients reveal alterations in mitochondrial features.
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NPC1 mutant cells show disorganization of the mitochondria network.
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NPC1 Mitochondria exhibit an increased respiration rate and lower levels of ATP and ROS.
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These dysfunctions may result from an increased rate of mitochondrial biogenesis.

Abstract

Mutations in the NPC1 or NPC2 genes lead to Niemann-Pick type C (NPC) disease, a rare lysosomal storage disorder characterized by progressive neurodegeneration. These mutations result in cholesterol and glycosphingolipid accumulation in the late endosomal/lysosomal compartment. Complications in the storage of cholesterol in NPC1 mutant cells are associated with other anomalies, such as altered distribution of intracellular organelles and properties of the plasma membrane. The pathomechanism of NPC disease is largely unknown. Interestingly, other storage diseases such as Gaucher and Farber diseases are accompanied by severe mitochondrial dysfunction. This prompted us to investigate the effect of absence or dysfunction of the NPC1 protein on mitochondrial properties to confirm or deny a putative relationship between NPC1 mutations and mitochondrial function. This study was performed on primary skin fibroblasts derived from skin biopsies of two NPC patients, carrying mutations in the NPC1 gene. We observed altered organization of mitochondria in NPC1 mutant cells, significant enrichment in mitochondrial cholesterol content, increased respiration, altered composition of the respiratory chain complex, and substantial reduction in cellular ATP level. Thus, a primary lysosomal defect in NPC1 mutant fibroblasts is accompanied by deregulation of the organization and function of the mitochondrial network.

Graphical abstract

Introduction

Niemann-Pick type C (NPC) is one of rare lysosomal storage disorders characterized by progressive neurodegeneration accompanied by liver and spleen enlargement. Mutations in the NPC1 gene are responsible for the majority of disease cases. These mutations result in a lack of or production of dysfunctional NPC1 protein, as well as cholesterol and glycolipid accumulation in the late endosomal/lysosomal (LE/LY) compartment of the cell. As a result of such accumulation the properties of the LE/LY compartment are affected [1]. Disturbances in the transport and storage of cholesterol are associated with other cell dysfunctions, such as changes in distribution of cellular organelles, altered properties of the plasma membrane and endoplasmic reticulum (ER), deregulation of intracellular localization of various proteins or changes in expression of a number of genes, including elements of important signaling pathways [2].

Changes in the lipid composition, in particular in cholesterol content, may alter the physicochemical properties of membranes with implications for membrane associated proteins [3]. At the cellular level abnormalities related to the NPC phenotype include deregulation of the entire cell vesicular traffic machinery [4], abnormalities in cytoskeleton organization [5], functioning and morphology of the Golgi apparatus [6] and endoplasmic reticulum [7]. A recent publication addresses potential consequences of increased mitochondrial cholesterol in NPC1- and/or NPC2-depleted cells due to its transport from endosomes/lysosomes [8]. However, none of these studies examined NPC1 mutant cells obtained from NPC patients.

The pathomechanism of NPC disease is still completely unknown. Therefore, in the present report we have investigated the potential influence of NPC1 protein deficiency or dysfunction on the architecture of the mitochondrial network as well as mitochondrial properties. Using human skin fibroblasts from patients carrying NPC1 mutations and from healthy volunteers, we studied morphology and function of NPC1 mutant mitochondria, e.g., their distribution in the cell, cholesterol content, oxygen consumption, potential changes in the respiratory chain complex composition and production of ATP. We tested the hypothesis that the primary lysosomal defect in NPC1 mutant fibroblasts may cause accumulation of dysfunctional mitochondria leading to impaired cellular bioenergetics. These data may potentially provide new insights into the pathogenesis of the NPC disease.

Section snippets

Cell cultures

These studies were performed on primary skin fibroblasts derived from skin biopsies of two NPC anonymous patients carrying mutations in the NPC1 gene (called throughout N466 and N521 fibroblasts or cells) and two 25 years old healthy male donors (control fibroblasts C687 and C688). The NPC1 mutant fibroblasts carried different mutations in the NPC1 gene: N466 - M1001V and P1007A, and N521 - P733fs and P1007A, within the sequence encoding the lumenal segment of the NPC1 protein. Both

Morphology of mitochondria from skin fibroblasts of patients carrying mutations in NPC1 gene

As it has been already shown by Sztolsztener et al. [9] NPC1 mutant fibroblasts from human patients are characterized by extensive accumulation of cholesterol in the LE/LY compartment [19]. This observation was confirmed in this study (Fig. 1A, right column) using different NPC donors and considered as a basis for further investigations. Human fibroblasts, carrying different mutations within the lumenal segment of the NPC1 protein, namely, M1001V and P1007A in N466 cells, and P733fs and P1007A

Discussion

It must be underlined that our studies on various properties of mitochondria in NPC1 mutant cells were the first performed on fibroblasts taken from patients suffering from NPC disease.

In this study we tested the hypothesis that the NPC disease is associated with dysfunction of mitochondria. As an experimental model we used fibroblasts from patients carrying mutations in the NPC1 gene and from healthy volunteers as controls. We decided to use NPC1 mutant cells with P1007A mutation, one of the

Author contributions

All listed authors contributed to the concept and design of the work, acquisition of data, data analysis, interpretation of data, drafting of the manuscript, revision of the work, and gave final approval to the version to be published. Furthermore, all authors agree to be accountable for all aspects of the work.

Acknowledgments

This work was supported by a grant NN401642740 from the National Science Center to JBP, and by statutory funds from the Nencki Institute of Experimental Biology. MW was a recipient of a stipend from the Mazovia scholarships for PhD students (Operational Program Human Capital 2007–2013, Priority 8.2.2 from the European Union funds by the European Social Fund).

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Mitochondrial dysfunction in fibroblasts derived from patients with Niemann-Pick type C disease

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Cell cultures

Morphology of mitochondria from skin fibroblasts of patients carrying mutations in NPC1 gene

Discussion

Author contributions

Acknowledgments

Biochim. Biophys. Acta

J. Colloid Interface Sci.

Am. J. Pathol.

J. Lipid Res.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Biochim. Biophys. Acta

Trends Cell. Biol.

Am. J. Hum. Genet.

Neurochem. Int.

J. Lipid Res.

J. Lipid Res.

J. Biol. Chem.

J. Lipid Res.

Toxicol. Appl. Pharmacol.