Mitochondrial proteomic analysis of a cell line model of familial amyotrophic lateral sclerosis

Abstract

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.

Fig. 1. Expression levels of human SOD1 in the NSC34 cell line model of fALS

The total cell lysate of NSC34^WT-SOD1 and NSC34^G93A-SOD1 cells was resolved by 12% SDS-PAGE, transferred to a nitrocellulous membrane, and blotted with an SOD1 polyclonal antibody. The endogenous mouse SOD1 and human SOD1 are indicated by arrows. Significant levels of human WT- and G93A-SOD1 are expressed in NSC34^WT-SOD1 and NSC34^G93A-SOD1 cells, respectively.

Fig. 2. A representative 2D gel electrophoresis image of the mitochondrial fraction isolated from NSC34^WT-SOD1 cells

The gel was stained by Sypro Ruby, and the image was acquired using a Storm fluorescence scanner. PDQuest software was used to analyze the image and assign the protein spots. Two hundred forty proteins spots that have been identified are indexed as numbered, and the complete list of proteins is in the supplemental table.

Fig. 3. Summary of all proteins identified by the two independent approaches: 2D gel followed by MALDI-MS/MS and SDS-PAGE followed by LC-MS/MS

The number of proteins that have been identified by each approach is summarized here. The detailed protocols and results are described in “Experimental Procedures” and “Results.”

Fig. 4. Summary of the functions of the mitochondrial proteins that have been annotated in the EXPASY or BioKnowledge databases

One hundred seventy-one mitochondrial proteins were annotated in the databases, and the functions of other identified mitochondrial proteins need to be elucidated.

Fig. 5. Representative 2D gel protein spots that displayed differential levels between NSC34^WT-SOD1 and NSC34^G93A-SOD1 cells

A, the protein named “Similar to septin6 type II” (spot 125 in Fig. 2) has increased abundance in NSC34^G93A-SOD1 cells (NSC34^G93A-SOD1/ NSC34^WT-SOD1 = 1.97, p = 0.019). B, the protein “Heat shock 70-kDa protein 5” (spot 64 in Fig. 2) has decreased abundance in NSC34^G93A-SOD1 cells (NSC34^G93A-SOD1/NSC34^WT-SOD1 = 0.58, p = 0.038). Forty-five 2D gel spots display altered abundance between the mitochondrial fractions isolated from NSC34^WT-SOD1 and NSC34^G93A-SOD1 cells. These 45 protein spots were identified as 40 unique proteins, and they are listed in Table II.

Fig. 6. Summary of the annotated functions of the 40 differentially expressed mitochondrial proteins between NSC34^WT-SOD1 and NSC-34^G93A-SOD1 cells