Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons.

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

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.

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.

Molecular mechanisms in chloroquine-exposed muscle cells elucidated by combined proteomic and microscopic studies.

OBJECTIVES: Chloroquine (CQ) is an antimalarial drug with a growing number of applications as recently demonstrated in attempts to treat Covid-19. For decades, it has been well known that skeletal and cardiac muscle cells might display vulnerability against CQ exposure resulting in the clinical manifestation of a CQ-induced myopathy. In line with the known effect of CQ on inhibition of the lysosomal function and thus cellular protein clearance, the build-up of autophagic vacuoles along with protein aggregates is a histological hallmark of the disease. Given that protein targets of the perturbed proteostasis are still not fully discovered, we applied different proteomic and immunological-based studies to improve the current understanding of the biochemical nature of CQ-myopathy. METHODS: To gain a comprehensive understanding of the molecular pathogenesis of this acquired myopathy and to define proteins targets as well as pathophysiological processes beyond impaired proteolysis, utilising CQ-treated C2C12 cells and muscle biopsies derived from CQ-myopathy patients, we performed different proteomic approaches and Coherent Anti-Stokes Raman Scattering (CARS) microscopy, in addition to immunohistochemical studies. RESULTS: Our combined studies confirmed an impact of CQ-exposure on proper protein processing/folding and clearance, highlighted changes in the interactome of p62, a known aggregation marker and hereby identified the Rett syndrome protein MeCP2 as being affected. Moreover, our approach revealed-among others-a vulnerability of the extracellular matrix, cytoskeleton and lipid homeostasis. CONCLUSION: We demonstrated that CQ exposure (secondarily) impacts biological processes beyond lysosomal function and linked a variety of proteins with known roles in the manifestation of other neuromuscular diseases.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

High diagnostic rate of trio exome sequencing in consanguineous families with neurogenetic diseases.

Consanguineous marriages have a prevalence rate of 24% in Turkey. These carry an increased risk of autosomal recessive genetic conditions, leading to severe disability or premature death, with a significant health and economic burden. A definitive molecular diagnosis could not be achieved in these children previously, as infrastructures and access to sophisticated diagnostic options were limited. We studied the cause of neurogenetic disease in 246 children from 190 consanguineous families recruited in three Turkish hospitals between 2016 and 2020. All patients underwent deep phenotyping and trio whole exome sequencing, and data were integrated in advanced international bioinformatics platforms. We detected causative variants in 119 known disease genes in 72% of families. Due to overlapping phenotypes 52% of the confirmed genetic diagnoses would have been missed on targeted diagnostic gene panels. Likely pathogenic variants in 27 novel genes in 14% of the families increased the diagnostic yield to 86%. Eighty-two per cent of causative variants (141/172) were homozygous, 11 of which were detected in genes previously only associated with autosomal dominant inheritance. Eight families carried two pathogenic variants in different disease genes. De novo (9.3%), X-linked recessive (5.2%) and compound heterozygous (3.5%) variants were less frequent compared to non-consanguineous populations. This cohort provided a unique opportunity to better understand the genetic characteristics of neurogenetic diseases in a consanguineous population. Contrary to what may be expected, causative variants were often not on the longest run of homozygosity and the diagnostic yield was lower in families with the highest degree of consanguinity, due to the high number of homozygous variants in these patients. Pathway analysis highlighted that protein synthesis/degradation defects and metabolic diseases are the most common pathways underlying paediatric neurogenetic disease. In our cohort 164 families (86%) received a diagnosis, enabling prevention of transmission and targeted treatments in 24 patients (10%). We generated an important body of genomic data with lasting impacts on the health and wellbeing of consanguineous families and economic benefit for the healthcare system in Turkey and elsewhere. We demonstrate that an untargeted next generation sequencing approach is far superior to a more targeted gene panel approach, and can be performed without specialized bioinformatics knowledge by clinicians using established pipelines in populations with high rates of consanguinity.

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH.

Marinesco-Sjögren syndrome is a rare human disorder caused by biallelic mutations in SIL1 characterized by cataracts in infancy, myopathy and ataxia, symptoms which are also associated with a novel disorder caused by mutations in INPP5K. While these phenotypic similarities may suggest commonalties at a molecular level, an overlapping pathomechanism has not been established yet. In this study, we present six new INPP5K patients and expand the current mutational and phenotypical spectrum of the disease showing the clinical overlap between Marinesco-Sjögren syndrome and the INPP5K phenotype. We applied unbiased proteomic profiling on cells derived from Marinesco-Sjögren syndrome and INPP5K patients and identified alterations in d-3-PHGDH as a common molecular feature. d-3-PHGDH modulates the production of l-serine and mutations in this enzyme were previously associated with a neurological phenotype, which clinically overlaps with Marinesco-Sjögren syndrome and INPP5K disease. As l-serine administration represents a promising therapeutic strategy for d-3-PHGDH patients, we tested the effect of l-serine in generated sil1, phgdh and inpp5k a+b zebrafish models, which showed an improvement in their neuronal phenotype. Thus, our study defines a core phenotypical feature underpinning a key common molecular mechanism in three rare diseases and reveals a common and novel therapeutic target for these patients.

GFPT1 deficiency in muscle leads to myasthenia and myopathy in mice.

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterized by fatigable muscle weakness owing to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) owing to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1-/- knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggest a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity and highlight that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit α and muscle-specific kinase, which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.

Mutations in INPP5K, Encoding a Phosphoinositide 5-Phosphatase, Cause Congenital Muscular Dystrophy with Cataracts and Mild Cognitive Impairment.

Phosphoinositides are small phospholipids that control diverse cellular downstream signaling events. Their spatial and temporal availability is tightly regulated by a set of specific lipid kinases and phosphatases. Congenital muscular dystrophies are hereditary disorders characterized by hypotonia and weakness from birth with variable eye and central nervous system involvement. In individuals exhibiting congenital muscular dystrophy, early-onset cataracts, and mild intellectual disability but normal cranial magnetic resonance imaging, we identified bi-allelic mutations in INPP5K, encoding inositol polyphosphate-5-phosphatase K. Mutations impaired phosphatase activity toward the phosphoinositide phosphatidylinositol (4,5)-bisphosphate or altered the subcellular localization of INPP5K. Downregulation of INPP5K orthologs in zebrafish embryos disrupted muscle fiber morphology and resulted in abnormal eye development. These data link congenital muscular dystrophies to defective phosphoinositide 5-phosphatase activity that is becoming increasingly recognized for its role in mediating pivotal cellular mechanisms contributing to 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>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>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.

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.

JAK inhibitor improves type I interferon induced damage: proof of concept in dermatomyositis.

Dermatomyositis is an acquired auto-immune disease characterized by skin lesions and muscle-specific pathological features such as perifascicular muscle fibre atrophy and vasculopathy. Dermatomyositis patients display an upregulation of type I interferon-inducible genes in muscle fibres, endothelial cells, skin and peripheral blood. However, the effect of type I interferon on muscle tissue has not yet been determined. Our aim was to study the pathogenicity of type I interferon in vitro and to evaluate the efficacy of the type I interferon pathway blockade for therapeutic purposes. The activation of type I interferon in differentiating myoblasts abolished myotube formation with reduced myogenin expression while in differentiated myotubes, we observed a reduction in surface area and an upregulation of atrophy-associated genes. In vitro endothelial cells exposure to type I interferon disrupted vascular network organization. All the pathogenic effects observed in vitro were abolished by ruxolitinib. Finally, four refractory dermatomyositis patients were treated with ruxolitinib and improvement ensued in skin lesions, muscle weakness and a reduced serum type I interferon levels and interferon-inducbile genes scores. We propose JAK inhibition as a mechanism-based treatment for dermatomyositis, a finding that is relevant for the design of future clinical trials targeting dermatomyositis.

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.

Involvement of ferryl in the reaction between nitrite and the oxy forms of globins.

The reaction between nitrite and the oxy forms of globins has complex autocatalytic kinetics with several branching steps and evolves through chain reactions mediated by reactive species (including radicals) such as hydrogen peroxide, ferryl and nitrogen dioxide, starting with a lag phase, after which it proceeds onto an autocatalytic phase. Reported here are UV-Vis spectra collected upon stopped-flow mixing of myoglobin with a supraphysiological excess of nitrite. The best fit to the experimental data follows an A â†’ B â†’ C reaction scheme involving the formation of a short-lived intermediate identified as ferryl. This is consistent with a mechanism where nitrite binds to oxy myoglobin to generate an undetectable ferrous-peroxynitrate intermediate, whose decay leads to nitrate and ferryl. The ferryl is then reduced to met by the excess nitrite. DFT calculations reveal an essentially barrierless reaction between nitrite and the oxy heme, with a notable outer-sphere component; the resulting metastable ferrous-peroxynitrate adduct is found to feature a very low barrier towards nitrate liberation, with ferryl as a final product-in good agreement with experiment.

Oxidative protection of hemoglobin and hemerythrin by cross-linking with a nonheme iron peroxidase: potentially improved oxygen carriers for use in blood substitutes.

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

Periostin as a blood biomarker of muscle cell fibrosis, cardiomyopathy and disease severity in myotonic dystrophy type 1.

BACKGROUND AND PURPOSE: Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy and is caused by an repeat expansion [r(CUG)exp] located in the 3' untranslated region of the DMPK gene. Symptoms include skeletal and cardiac muscle dysfunction and fibrosis. In DM1, there is a lack of established biomarkers in routine clinical practice. Thus, we aimed to identify a blood biomarker with relevance for DM1-pathophysiology and clinical presentation. METHODS: We collected fibroblasts from 11, skeletal muscles from 27, and blood samples from 158 DM1 patients. Moreover, serum, cardiac, and skeletal muscle samples from DMSXL mice were included. We employed proteomics, immunostaining, qPCR and ELISA. Periostin level were correlated with CMRI-data available for some patients. RESULTS: Our studies identified Periostin, a modulator of fibrosis, as a novel biomarker candidate for DM1: proteomic profiling of human fibroblasts and murine skeletal muscles showed significant dysregulation of Periostin. Immunostaining on skeletal and cardiac muscles from DM1 patients and DMSXL mice showed an extracellular increase of Periostin, indicating fibrosis. qPCR studies indicated increased POSTN expression in fibroblasts and muscle. Quantification of Periostin in blood samples from DMSXL mice and two large validation cohorts of DM1 patients showed decreased levels in animals and diseased individuals correlating with repeat expansion and disease severity and presence of cardiac symptoms identified by MRI. Analyses of longitudinal blood samples revealed no correlation with disease progression. CONCLUSIONS: Periostin might serve as a novel stratification biomarker for DM1 correlating with disease severity, presence of cardiac malfunction and fibrosis.

Dysregulation of GSK3ß-Target Proteins in Skin Fibroblasts of Myotonic Dystrophy Type 1 (DM1) Patients.

Myotonic dystrophy type 1 (DM1) is the most common monogenetic muscular disorder of adulthood. This multisystemic disease is caused by CTG repeat expansion in the 3'-untranslated region of the DM1 protein kinase gene called DMPK. DMPK encodes a myosin kinase expressed in skeletal muscle cells and other cellular populations such as smooth muscle cells, neurons and fibroblasts. The resultant expanded (CUG)n RNA transcripts sequester RNA binding factors leading to ubiquitous and persistent splicing deregulation. The accumulation of mutant CUG repeats is linked to increased activity of glycogen synthase kinase 3 beta (GSK3ß), a highly conserved and ubiquitous serine/threonine kinase with functions in pathways regulating inflammation, metabolism, oncogenesis, neurogenesis and myogenesis. As GSK3ß-inhibition ameliorates defects in myogenesis, muscle strength and myotonia in a DM1 mouse model, this kinase represents a key player of DM1 pathobiochemistry and constitutes a promising therapeutic target. To better characterise DM1 patients, and monitor treatment responses, we aimed to define a set of robust disease and severity markers linked to GSK3ßby unbiased proteomic profiling utilizing fibroblasts derived from DM1 patients with low (80- 150) and high (2600- 3600) CTG-repeats. Apart from GSK3ß increase, we identified dysregulation of nine proteins (CAPN1, CTNNB1, CTPS1, DNMT1, HDAC2, HNRNPH3, MAP2K2, NR3C1, VDAC2) modulated by GSK3ß. In silico-based expression studies confirmed expression in neuronal and skeletal muscle cells and revealed a relatively elevated abundance in fibroblasts. The potential impact of each marker in the myopathology of DM1 is discussed based on respective function to inform potential uses as severity markers or for monitoring GSK3ß inhibitor treatment responses.

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, ß-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation.

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.

Immunofluorescence-Based Analysis of Caveolin-3 in the Diagnostic Management of Neuromuscular Diseases.

Immunohistochemistry- and/or immunofluorescence-based analysis of muscular proteins represents a standard procedure in the diagnostic management of patients suffering from neuromuscular diseases such as "Caveolinopathies" which are caused by mutations in the CAV3 gene encoding for caveolin-3. Human caveolin-3 is a 151 amino acid sized transmembrane protein localized within caveolae, predominantly expressed in cardiac and skeletal muscle cells and involved in a diversity of cellular functions crucial for muscle cell homeostasis. Loss of caveolin-3 protein abundance is indicative for the presence of pathogenic mutations within the corresponding gene and thus for the diagnosis of "Caveolinopathies." Moreover, description of abnormal immunoreactivity findings for the caveolin-3 protein is increasing in the context of other neuromuscular diseases suggesting that profound knowledge of abnormal caveolin-3-expression and/or distribution findings can be decisive also for the diagnosis of other neurological diseases as well as for a better understanding of the biology of the protein. Here, we summarize the current knowledge about the caveolin-3, report on a protocol for immunofluorescence-based analysis of the protein in the diagnostic workup of neuromuscular patients-also considering problems encountered-and confirm as well as summarize already published abnormal histological findings in muscular pathologies beyond "Caveolinopathies."