Proteomic profiling of the mouse diaphragm and refined mass spectrometric analysis of the dystrophic phenotype.

The diaphragm is a crucial muscle involved in active inspiration and whole body homeostasis. Previous biochemical, immunochemical and cell biological investigations have established the distribution and fibre type-specific expression of key diaphragm proteins. Building on these findings, it was of interest to establish the entire experimentally assessable diaphragm proteome and verify the presence of specific protein isoforms within this specialized subtype of skeletal muscle. A highly sensitive Orbitrap Fusion Tribrid mass spectrometer was used for the systematic identification of the mouse diaphragm-associated protein population. Proteomics established 2925 proteins by high confidence peptide identification. Bioinformatics was used to determine the distribution of the main protein classes, biological processes and subcellular localization within the diaphragm proteome. Following the establishment of the respiratory muscle proteome with special emphasis on protein isoform expression in the contractile apparatus, the extra-sarcomeric cytoskeleton, the extracellular matrix and the excitation-contraction coupling apparatus, the mass spectrometric analysis of the diaphragm was extended to the refined identification of proteome-wide changes in X-linked muscular dystrophy. The comparative mass spectrometric profiling of the dystrophin-deficient diaphragm from the mdx-4cv mouse model of Duchenne muscular dystrophy identified 289 decreased and 468 increased protein species. Bioinformatics was employed to analyse the clustering of changes in protein classes and potential alterations in interaction patterns of proteins involved in metabolism, the contractile apparatus, proteostasis and the extracellular matrix. The detailed pathoproteomic profiling of the mdx-4cv diaphragm suggests highly complex alterations in a variety of crucial cellular processes due to deficiency in the membrane cytoskeletal protein dystrophin.

Neurodegeneration Caused by S1P-Lyase Deficiency Involves Calcium-Dependent Tau Pathology and Abnormal Histone Acetylation.

We have shown that sphingosine 1-phosphate (S1P) generated by sphingosine kinase 2 (SK2) is toxic in neurons lacking S1P-lyase (SGPL1), the enzyme that catalyzes its irreversible cleavage. Interestingly, patients harboring mutations in the gene encoding this enzyme (SGPL1) often present with neurological pathologies. Studies in a mouse model with a developmental neural-specific ablation of SGPL1 (SGPL1fl/fl/Nes) confirmed the importance of S1P metabolism for the presynaptic architecture and neuronal autophagy, known to be essential for brain health. We now investigated in SGPL1-deficient murine brains two other factors involved in neurodegenerative processes, namely tau phosphorylation and histone acetylation. In hippocampal and cortical slices SGPL1 deficiency and hence S1P accumulation are accompanied by hyperphosphorylation of tau and an elevated acetylation of histone3 (H3) and histone4 (H4). Calcium chelation with BAPTA-AM rescued both tau hyperphosphorylation and histone acetylation, designating calcium as an essential mediator of these (patho)physiological functions of S1P in the brain. Studies in primary cultured neurons and astrocytes derived from SGPL1fl/fl/Nes mice revealed hyperphosphorylated tau only in SGPL1-deficient neurons and increased histone acetylation only in SGPL1-deficient astrocytes. Both could be reversed to control values with BAPTA-AM, indicating the close interdependence of S1P metabolism, calcium homeostasis, and brain health.

Dataset on the mass spectrometry-based proteomic profiling of the kidney from wild type and the dystrophic mdx-4cv mouse model of X-linked muscular dystrophy.

The proteomic data presented in this article provide supporting information to the related research article "Proteomic and cell biological profiling of the renal phenotype of the mdx-4cv mouse model of Duchenne muscular dystrophy" (Dowling et al., 2019) [1]. This article supplies additional datasets on protein species with increased versus decreased concentration in the kidney from the dystrophic mdx-4cv mouse, as well as tables with mass spectrometrically identified kidney marker proteins that exhibit characteristic tissue distributions, subcellular localizations and physiological functions. Information is provided on the underlying multi-consensus protein listings from the proteomic screening of both wild type and mdx-4cv mouse kidneys. The data article provides comprehensive information on the systematic and mass spectrometric identification of the mouse kidney proteome.

Proteomic and cell biological profiling of the renal phenotype of the mdx-4cv mouse model of Duchenne muscular dystrophy.

The X-linked inherited muscle wasting disease Duchenne muscular dystrophy, which is caused by primary abnormalities in the membrane cytoskeletal protein dystrophin, is a multi-system disorder. Highly progressive forms of dystrophinopathy are associated with a complex secondary pathophysiology, including renal dysfunction. It was therefore of interest to carry out a systematic survey of potential proteome-wide changes in the kidney of the established mdx-4cv mouse model of dystrophinopathy. Of 5878 mass spectrometrically identified kidney proteins, 82 versus 142 proteins were shown to be decreased or increased, respectively, in association with muscular dystrophy. The most decreased versus increased protein species are the ACSM3 isoform of mitochondrial acyl-coenzyme A synthetase and the FABP1 isoform of fatty acid binding protein, respectively. Both proteomic findings were verified by immunofluorescence microscopy and immunoblot analysis. Interestingly, haematoxylin/eosin staining indicated diffuse whitish deposits in the mdx-4cv kidney, and an increased intensity of Sudan Black labelling of kidney cells revealed ectopic fat deposition. Although the proteomic results and cell biological findings do not demonstrate a direct functional link between increased FABP1 and fat accumulation, the results suggest that the up-regulation of FABP1 may be related to abnormal fat metabolism. This makes FABP1 potentially a novel pathobiochemical indicator for studying kidney abnormalities in the mdx-4cv model of dystrophinopathy.

Emerging proteomic biomarkers of X-linked muscular dystrophy.

Introduction: Progressive skeletal muscle wasting is the manifesting symptom of Duchenne muscular dystrophy, an X-linked inherited disorder triggered by primary abnormalities in the DMD gene. The almost complete loss of dystrophin isoform Dp427 causes a multi-system pathology that features in addition to skeletal muscle weakness also late-onset cardio-respiratory deficiencies, impaired metabolism and abnormalities in the central nervous system. Areas covered: This review focuses on the mass spectrometry-based proteomic characterization of X-linked muscular dystrophy with special emphasis on the identification of novel biomarker candidates in skeletal muscle tissues, as well as non-muscle tissues and various biofluids. Individual sections focus on molecular and cellular aspects of the pathogenic changes in dystrophinopathy, proteomic workflows used in biomarker research, the proteomics of the dystrophin-glycoprotein complex and the potential usefulness of newly identified protein markers involved in fibre degeneration, fibrosis and inflammation. Expert opinion: The systematic application of large-scale proteomic surveys has identified a distinct cohort of both tissue- and biofluid-associated protein species with considerable potential for improving diagnostic, prognostic and therapy-monitoring procedures. Novel proteomic markers include components involved in fibre contraction, cellular signalling, ion homeostasis, cellular stress response, energy metabolism and the immune response, as well as maintenance of the cytoskeletal and extracellular matrix.

Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism.

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.