Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy

Abstract

BackgroundX-linked muscular dystrophy is a primary disease of the neuromuscular system. Primary abnormalities in the Dmd gene result in the absence of the full-length isoform of the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle wasting and cardio-respiratory complications, developmental cognitive deficits and behavioural abnormalities are clinical features of Duchenne muscular dystrophy. In order to better understand the mechanisms that underlie impaired brain functions in Duchenne patients, we have carried out a proteomic analysis of total brain extracts from the mdx-4cv mouse model of dystrophinopathy.ResultsThe comparative proteomic profiling of the mdx-4cv brain revealed a significant increase in 39 proteins and a decrease in 7 proteins. Interesting brain tissue-associated proteins with an increased concentration in the mdx-4cv animal model were represented by the glial fibrillary acidic protein GFAP, the neuronal Ca2+-binding protein calretinin, annexin AnxA5, vimentin, the neuron-specific enzyme ubiquitin carboxyl-terminal hydrolase isozyme L1, the dendritic spine protein drebrin, the cytomatrix protein bassoon of the nerve terminal active zone, and the synapse-associated protein SAP97. Decreased proteins were identified as the nervous system-specific proteins syntaxin-1B and syntaxin-binding protein 1, as well as the plasma membrane Ca2+-transporting ATPase PMCA2 that is mostly found in the brain cortex. The differential expression patterns of GFAP, vimentin, PMCA2 and AnxA5 were confirmed by immunoblotting. Increased GFAP levels were also verified by immunofluorescence microscopy.ConclusionsThe large number of mass spectrometrically identified proteins with an altered abundance suggests complex changes in the mdx-4cv brain proteome. Increased levels of the glial fibrillary acidic protein, an intermediate filament component that is uniquely associated with astrocytes in the central nervous system, imply neurodegeneration-associated astrogliosis. The up-regulation of annexin and vimentin probably represent compensatory mechanisms involved in membrane repair and cytoskeletal stabilization in the absence of brain dystrophin. Differential alterations in the Ca2+-binding protein calretinin and the Ca2+-pumping protein PMCA2 suggest altered Ca2+-handling mechanisms in the Dp427-deficient brain. In addition, the proteomic findings demonstrated metabolic adaptations and functional changes in the central nervous system from the dystrophic phenotype. Candidate proteins can now be evaluated for their suitability as proteomic biomarkers and their potential in predictive, diagnostic, prognostic and/or therapy-monitoring approaches to treat brain abnormalities in dystrophinopathies.

Mass spectrometry‐based protein analysis to unravel the tissue pathophysiology in Duchenne muscular dystrophy

“…It has been demonstrated that the dystrophin‐deficient mdx mouse displays alterations in learning, behavior, fear response, and memory . Glial fibrillary acidic protein was upregulated in the mdx‐4cv brain in an unbiased proteomic investigation, which is of considerable interest as it is a well‐established indicator of brain damage further evidencing brain involvement in DMD .…”

“…A label‐free proteomic study comparing mdx‐4cv and wild‐type brains was conducted by Murphy et al. . Overall 1.78% of 2572 proteins detected in the mdx‐4cv brain were differentially expressed when compared to wild‐type.…”

Emerging proteomic biomarkers of X-linked muscular dystrophy

“…Although dystrophinopathies are primarily classified as muscle diseases with characteristic neuromuscular symptoms [5], Duchenne muscular dystrophy is a multi-system disorder with severe complications due to cardiac failure, respiratory dysfunction, scoliosis, impaired liver metabolism, renal abnormalities and cognitive impairments [9]. Most studies into proteome-wide changes in non-skeletal muscle tissues [79][80][81][82][83][84][85][86] have focused on preparations from the dystrophin-deficient heart [38,[79][80][81]84], but muscular dystrophy-related effects on the brain [82,86], kidney [83] and liver [85] have also been studied by proteomics. Investigations using both gel-based and gel-free systems have established changed concentration levels in protein markers of energy metabolism, ion homeostasis, the extracellular matrix and cytoskeletal networks.…”

“…Investigations using both gel-based and gel-free systems have established changed concentration levels in protein markers of energy metabolism, ion homeostasis, the extracellular matrix and cytoskeletal networks. Table 3 lists proteomic biomarker candidates from non-skeletal muscle tissues that are significantly changed in X-linked muscular dystrophy [79][80][81][82][83][84][85][86]. This includes cardiac-specific changes in the core dystrophin complex, mitochondrial enzymes, the basal lamina and associated extracellular matrix [38,[79][80][81]84], which might be linked to the cardio-respiratory syndrome in X-linked muscular dystrophy [5].…”

“…A useful indicator of potential side effects of exon skipping therapy on the kidney was identified as the renal marker protein meprin MEP-1 [83]. Since a subset of children suffering from X-linked muscular dystrophy is also affected by neuropathological complications that affect emotion, language, attention and memory [5], it is of interest that the proteomic analysis of the central nervous system from the mdx-4cv mouse model has established an increased concentration of glial fibrillary acid protein GFAP [82,86]. Since GFAP is an intermediate filament component of astrocytes in the brain, its elevated concentration implies neurodegeneration-associated astrogliosis in muscular dystrophy.…”