Identification of Candidate Protein Markers in Skeletal Muscle of Laminin-211-Deficient CMD Type 1A-Patients

Laminin-211 deficiency leads to the most common form of congenital muscular dystrophy in childhood, MDC1A. The clinical picture is characterized by severe muscle weakness, brain abnormalities and delayed motor milestones defining MDC1A as one of the most severe forms of congenital muscular diseases. Although the molecular genetic basis of this neurological disease is well-known and molecular studies of mouse muscle and human cultured muscle cells allowed first insights into the underlying pathophysiology, the definition of marker proteins in human vulnerable tissue such as skeletal muscle is still lacking. To systematically address this need, we analyzed the proteomic signature of laminin-211-deficient vastus muscle derived from four patients and identified 86 proteins (35 were increased and 51 decreased) as skeletal muscle markers and verified paradigmatic findings in a total of two further MDC1A muscle biopsies. Functions of proteins suggests fibrosis but also hints at altered synaptic transmission and accords with central nervous system alterations as part of the clinical spectrum of MDC1A. In addition, a profound mitochondrial vulnerability of the laminin-211-deficient muscle is indicated and also altered abundances of other proteins support the concept that metabolic alterations could be novel mechanisms that underline MDC1A and might constitute therapeutic targets. Intersection of our data with the proteomic signature of murine laminin-211-deficient gastrocnemius and diaphragm allowed the definition of nine common vulnerable proteins representing potential tissue markers

Proteomic Profiling Unravels a Key Role of Specific Macrophage Subtypes in Sporadic Inclusion Body Myositis

Unbiased proteomic profiling was performed toward the identification of biological parameters relevant in sIBM, thus giving hints about the pathophysiological processes and the existence of new reliable markers. For that purpose, skeletal muscle biopsies from 13 sIBM and 7 non-diseased control patients were analyzed with various methods, including liquid chromatography coupled to tandem mass spectrometry (four patients). Subsequent data analysis identified key molecules further studied in a larger cohort by qPCR, immunostaining, and immunofluorescence in situ. Proteomic signature of muscle biopsies derived from sIBM patients revealed the chaperone and cell surface marker CD74, the macrophage scavenger molecule CD163 and the transcription activator STAT1 to be among the highly and relevantly expressed proteins suggesting a significant contribution of immune cells among the myofibers expressing these markers. Moreover, in silico studies showed that 39% of upregulated proteins were involved in type I or mixed type I and type II interferon immunity. Indeed, further studies via immunohistochemistry clearly confirmed the prominent involvement of the key type I interferon signature-related molecules, ISG15 as well as IRF8 with MHC class II+ myofibers. Siglec1 colocalized with CD163+ macrophages and MHC class II molecules also co-localized with CD74 on macrophages. STAT1 co-localized with Siglec1+ macrophages in activemyofibremyophagocytosis while STAT6 colocalized with endomysial macrophages. These combined results show involvement of CD74, CD163, and STAT1 as key molecules of macrophage activation being crucially involved in mixed and specific type I interferon, and interferon gamma associated-pathways in sIBM. On a more general note, these results also highlight the type of immune-interaction between macrophages and myofibers in the etiopathology of sIBM

Identification of Cellular Pathogenicity Markers for SIL1 Mutations Linked to Marinesco-Sjögren Syndrome.

Background and objective: Recessive mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), a rare neuropediatric disorder. MSS-patients typically present with congenital cataracts, intellectual disability, cerebellar ataxia and progressive vacuolar myopathy. However, atypical clinical presentations associated with SIL1 mutations have been described over the last years; compound heterozygosity of SIL1 missense mutations even resulted in a phenotype not fulfilling the clinical diagnostic criteria of MSS. Thus, a read-out system to evaluate reliably the pathogenicity of amino acid changes in SIL1 is needed. Here, we aim to provide suitable cellular biomarkers enabling the robust evaluation of pathogenicity of SIL1 mutations. Methods: Five SIL1 variants including one polymorphism (p.K132Q), three known pathogenic mutations (p.V231_I232del, p.G312R, and p.L457P) and one ambiguous missense variant (p.R92W) were studied along with the wild-type proteins in Hek293 in vitro models by cell biological assays, immunoprecipitation, immunoblotting, and immunofluorescence as well as electron microscopy. Moreover, the SIL1-interactomes were interrogated by tandem-affinity-purification and subsequent mass spectrometry. Results: Our combined studies confirmed the pathogenicity of p.V231_I232del, p.G312R, and p.L457P by showing instability of the proteins as well as tendency to form aggregates. This observation is in line with altered structure of the ER-Golgi system and vacuole formation upon expression of these pathogenic SIL1-mutants as well as the presence of oxidative or ER-stress. Reduced cellular fitness along with abnormal mitochondrial architecture could also be observed. Notably, both the polymorphic p.K132Q and the ambiguous p.R92W variants did not elicit such alterations. Study of the SIL1-interactome identified POC1A as a novel binding partner of wild-type SIL1; the interaction is disrupted upon the presence of pathogenic mutants but not influenced by the presence of benign variants. Disrupted SIL1-POC1A interaction is associated with centrosome disintegration. Conclusions: We developed a combination of cellular outcome measures to evaluate the pathogenicity of SIL1 variants in suitable in vitro models and demonstrated that the p. R92W missense variant is a polymorphism rather than a pathogenic mutation leading to MSS

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3.

Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, β-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation

Proteomic Profiling Unravels a Key Role of Specific Macrophage Subtypes in Sporadic Inclusion Body Myositis

Unbiased proteomic profiling was performed toward the identification of biological parameters relevant in sIBM, thus giving hints about the pathophysiological processes and the existence of new reliable markers. For that purpose, skeletal muscle biopsies from 13 sIBM and 7 non-diseased control patients were analyzed with various methods, including liquid chromatography coupled to tandem mass spectrometry (four patients). Subsequent data analysis identified key molecules further studied in a larger cohort by qPCR, immunostaining, and immunofluorescence in situ. Proteomic signature of muscle biopsies derived from sIBM patients revealed the chaperone and cell surface marker CD74, the macrophage scavenger molecule CD163 and the transcription activator STAT1 to be among the highly and relevantly expressed proteins suggesting a significant contribution of immune cells among the myofibers expressing these markers. Moreover, in silico studies showed that 39% of upregulated proteins were involved in type I or mixed type I and type II interferon immunity. Indeed, further studies via immunohistochemistry clearly confirmed the prominent involvement of the key type I interferon signature-related molecules, ISG15 as well as IRF8 with MHC class II+ myofibers. Siglec1 colocalized with CD163+ macrophages and MHC class II molecules also co-localized with CD74 on macrophages. STAT1 co-localized with Siglec1+ macrophages in active myofibre myophagocytosis while STAT6 colocalized with endomysial macrophages. These combined results show involvement of CD74, CD163, and STAT1 as key molecules of macrophage activation being crucially involved in mixed and specific type I interferon, and interferon gamma associated-pathways in sIBM. On a more general note, these results also highlight the type of immune-interaction between macrophages and myofibers in the etiopathology of sIBM

SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology

Autosomal recessive variants in TUBGCP2 alter the γ-tubulin ring complex leading to neurodevelopmental disease.

Microtubules help building the cytoskeleton of neurons and other cells. Several components of the gamma-tubulin (γ-tubulin) complex have been previously reported in human neurodevelopmental diseases. We describe two siblings from a consanguineous Turkish family with dysmorphic features, developmental delay, brain malformation, and epilepsy carrying a homozygous mutation (p.Glu311Lys) in TUBGCP2 encoding the γ-tubulin complex 2 (GCP2) protein. This variant is predicted to disrupt the electrostatic interaction of GCP2 with GCP3. In primary fibroblasts carrying the variant, we observed a faint delocalization of γ-tubulin during the cell cycle but normal GCP2 protein levels. Through mass spectrometry, we observed dysregulation of multiple proteins involved in the assembly and organization of the cytoskeleton and the extracellular matrix, controlling cellular adhesion and of proteins crucial for neuronal homeostasis including axon guidance. In summary, our functional and proteomic studies link TUBGCP2 and the γ-tubulin complex to the development of the central nervous system in humans

Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease

Clinical presentation and proteomic signature of patients with TANGO2 mutations

Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C&gt;T/p.Arg88*; c.220A&gt;C/p.Thr74Pro; c.380+1G&gt;A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11-13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged-red/cytochrome c oxidase-negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10 ) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum-Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions.</p

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb