Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord

Abstract

Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain‐of‐function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild‐type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane‐anchored cytoskeleton.

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

“…Recently, proteomic analysis demonstrated that membrane SOD (SOD1) is present in LR fractions (441), a fact consistent with previous reports that SOD1 is detectable in LRs (351). Reported SOD1 levels, for example, in LRs fractions were much higher than that in other areas of the plasma membrane.…”

“…These results support the view that in aggregation the LRs may play an important role for the SOD1 actions (6,201). It is assumed that localization and subsequent aggregation of SOD1 in LRs could affect cellular functions as well as the interplay between different cell types, as LRs are rich in receptors and the signaling molecules necessary for cell-cell communications (441). Indeed, a more recent study has reported that H 2 O 2 , generated extracellularly by extracellular SOD, anchored to ECs surface via the heparin-binding domain (HBD), enhances VEGF-induced VEGF receptor 2 (VEGFR2) autophosphorylation in caveolin-enriched LRs, but not in noncaveolar LRs.…”

Proteomic analysis of muscle affected by motor neuron degeneration: The wobbler mouse model of amyotrophic lateral sclerosis

“…Immobilization, disuse or denervation triggers muscular atrophy, which clearly shows that neuromuscular innervation patterns are directly linked to gene expression, protein abundance and isoform patterns in skeletal muscles [29]. In analogy to the recent proteomic profiling of cerebrospinal fluids from patients suffering from ALS and the spinal cord from the wobbler mouse [19][20][21][22][23], this study describes the findings of the MS-based proteomic survey of skeletal muscle from the WR mouse. From the detailed analysis of the isoform patterns of the sarcomeric MyHCs it became clear that the response of gene expression in skeletal muscle to denervation is characteristically different from that to hyper-excitability and muscular dystrophy [28].…”

“…Expression levels were reduced in the case of 3 protein species and increased for 21 proteins. The mass spectrometric hit with the highest increase of 6.5 relative concentration is not listed in Table 1 proteins spots which both represent fast myosin binding protein C (spots 23,24). In order to verify key proteomic findings and to put this study into perspective with previous analyses of WR muscle tissues, representative immunoblots are shown in Fig.…”

“…The current international effort to discover reliable biomarkers of ALS [16] includes the verification of both suitable metabolites [17] and protein factors [18]. Recent proteomic profiling studies of ALS patient samples and the WR animal model have focused on the analysis of cerebrospinal fluid [19][20][21] and cervical and lumbar spinal cord [22,23], respectively. The studies on the WR mouse showed that the majority of differentially expressed proteins were related to the glutamate-glutamine cycle, energy transduction, redox functions and astrogliosis [22], thus confirming previous studies [24].…”