Effects of Pulsed Electromagnetic Field Treatment on Skeletal Muscle Tissue Recovery in a Rat Model of Collagenase-Induced Tendinopathy: Results from a Proteome Analysis.

Tendon disorders often result in decreased muscle function and atrophy. Pulsed Electromagnetic Fields (PEMFs) have shown potential in improving tendon fiber structure and muscle recovery. However, the molecular effects of PEMF therapy on skeletal muscle, beyond conventional metrics like MRI or markers of muscle decline, remain largely unexplored. This study investigates the metabolic and structural changes in PEMF-treated muscle tissue using proteomics in a rat model of Achilles tendinopathy induced by collagenase. Sprague Dawley rats were unilaterally induced for tendinopathy with type I collagenase injection and exposed to PEMFs for 8 h/day. Gastrocnemius extracts from untreated or PEMF-treated rats were analyzed with LC-MS/MS, and proteomics differential analysis was conducted through label-free quantitation. PEMF-treated animals exhibited decreased glycolysis and increased LDHB expression, enhancing NAD signaling and ATP production, which boosted respiratory chain activity and fatty acid beta-oxidation. Antioxidant protein levels increased, controlling ROS production. PEMF therapy restored PGC1alpha and YAP levels, decreased by tendinopathy. Additionally, myosins regulating slow-twitch fibers and proteins involved in fiber alignment and force transmission increased, supporting muscle recovery and contractile function. Our findings show that PEMF treatment modulates NAD signaling and oxidative phosphorylation, aiding muscle recovery through the upregulation of YAP and PGC1alpha and increasing slow myosin isoforms, thus speeding up physiological recovery.

Characterization of Proteome Changes in Aged and Collagen VI-Deficient Human Pericyte Cultures.

Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.

Space Omics and Tissue Response in Astronaut Skeletal Muscle after Short and Long Duration Missions.

The molecular mechanisms of skeletal muscle adaptation to spaceflight are as yet not fully investigated and well understood. The MUSCLE BIOPSY study analyzed pre and postflight deep calf muscle biopsies (m. soleus) obtained from five male International Space Station (ISS) astronauts. Moderate rates of myofiber atrophy were found in long-duration mission (LDM) astronauts (~180 days in space) performing routine inflight exercise as countermeasure (CM) compared to a short-duration mission (SDM) astronaut (11 days in space, little or no inflight CM) for reference control. Conventional H&E scout histology showed enlarged intramuscular connective tissue gaps between myofiber groups in LDM post vs. preflight. Immunoexpression signals of extracellular matrix (ECM) molecules, collagen 4 and 6, COL4 and 6, and perlecan were reduced while matrix-metalloproteinase, MMP2, biomarker remained unchanged in LDM post vs. preflight suggesting connective tissue remodeling. Large scale proteomics (space omics) identified two canonical protein pathways associated to muscle weakness (necroptosis, GP6 signaling/COL6) in SDM and four key pathways (Fatty acid ß-oxidation, integrin-linked kinase ILK, Rho A GTPase RHO, dilated cardiomyopathy signaling) explicitly in LDM. The levels of structural ECM organization proteins COL6A1/A3, fibrillin 1, FBN1, and lumican, LUM, increased in postflight SDM vs. LDM. Proteins from tricarboxylic acid, TCA cycle, mitochondrial respiratory chain, and lipid metabolism mostly recovered in LDM vs. SDM. High levels of calcium signaling proteins, ryanodine receptor 1, RyR1, calsequestrin 1/2, CASQ1/2, annexin A2, ANXA2, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) pump, ATP2A, were signatures of SDM, and decreased levels of oxidative stress peroxiredoxin 1, PRDX1, thioredoxin-dependent peroxide reductase, PRDX3, or superoxide dismutase [Mn] 2, SOD2, signatures of LDM postflight. Results help to better understand the spatiotemporal molecular adaptation of skeletal muscle and provide a large scale database of skeletal muscle from human spaceflight for the better design of effective CM protocols in future human deep space exploration.

Novel Insight into the Serum Sphingolipid Fingerprint Characterizing Longevity.

Sphingolipids (SLs) are structural components of the lipid bilayer regulating cell functions. In biological fluids, their distribution is sex-specific and is at variance in aging and many disorders. The aim of this study is to identify SL species associated with the decelerated aging of centenarians. SLs, extracted from serum of adults (Ad, 35-37 years old), aged (Ag, 75-77 years old) and centenarian (C, 105-107 years old) women were analyzed by LC-MS/MS in combination with mRNA levels in peripheral blood mononuclear cells (PBMCs) of SL biosynthetic enzymes. Results indicated in Ag and C vs. Ad a comparable ceramides (Cers) increase, whereas dihydroceramide (dhCer) decreased in C vs. Ad. Hexosylceramides (HexCer) species, specifically HexCer 16:0, 22:0 and 24:1 acyl chains, increased in C vs. Ag representing a specific trait of C. Sphingosine (Sph), dihydrosphingosine (dhSph), sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), increased both in Ag and C vs. Ad, with higher levels in Ag, indicating a SL fine-tuning associated with a reduced physiological decline in C. mRNA levels of enzymes involved in ceramide de novo biosynthesis increased in Ag whereas enzymes involved in sphingomyelin (SM) degradation increased in C. Collectively, results suggest that Ag produce Cers by de novo synthesis whereas C activate a protective mechanism degrading SMs to Cers converting it into glycosphingolipids.

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin.

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Muscle Proteomic Profile before and after Enzyme Replacement Therapy in Late-Onset Pompe Disease.

Mutations in the acidic alpha-glucosidase (GAA) coding gene cause Pompe disease. Late-onset Pompe disease (LOPD) is characterized by progressive proximal and axial muscle weakness and atrophy, causing respiratory failure. Enzyme replacement therapy (ERT), based on recombinant human GAA infusions, is the only available treatment; however, the efficacy of ERT is variable. Here we address the question whether proteins at variance in LOPD muscle of patients before and after 1 year of ERT, compared withhealthy age-matched subjects (CTR), reveal a specific signature. Proteins extracted from skeletal muscle of LOPD patients and CTR were analyzed by combining gel based (two-dimensional difference gel electrophoresis) and label-free (liquid chromatography-mass spectrometry) proteomic approaches, and ingenuity pathway analysis. Upstream regulators targeting autophagy and lysosomal tethering were assessed by immunoblotting. 178 proteins were changed in abundance in LOPD patients, 47 of them recovered normal level after ERT. Defects in oxidative metabolism, muscle contractile protein regulation, cytoskeletal rearrangement, and membrane reorganization persisted. Metabolic changes, ER stress and UPR (unfolded protein response) contribute to muscle proteostasis dysregulation with active membrane remodeling (high levels of LC3BII/LC3BI) and accumulation of p62, suggesting imbalance in the autophagic process. Active lysosome biogenesis characterizes both LOPD PRE and POST, unparalleled by molecules involved in lysosome tethering (VAMP8, SNAP29, STX17, and GORASP2) and BNIP3. In conclusion this study reveals a specific signature that suggests ERT prolongation and molecular targets to ameliorate patient's outcome.

Novel Insight in Idiopathic Normal Pressure Hydrocephalus (iNPH) Biomarker Discovery in CSF.

Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological disease, causing motor and cognitive dysfunction and dementia. iNPH and Alzheimer's disease (AD) share similar molecular characteristics, including amyloid deposition, t-tau and p-tau dysregulation; however, the disease is under-diagnosed and under-treated. The aim was to identify a panel of sphingolipids and proteins in CSF to diagnose iNPH at onset compared to aged subjects with cognitive integrity (C) and AD patients by adopting multiple reaction monitoring mass spectrometry (MRM-MS) for sphingolipid quantitative assessment and advanced high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic analysis. The results indicated that iNPH are characterized by an increase in very long chains Cer C22:0, Cer C24:0 and Cer C24:1 and of acute-phase proteins, immunoglobulins and complement component fragments. Proteins involved in synaptic signaling, axogenesis, including BACE1, APP, SEZ6L and SEZ6L2; secretory proteins (CHGA, SCG3 and VGF); glycosylation proteins (POMGNT1 and DAG1); and proteins involved in lipid metabolism (APOH and LCAT) were statistically lower in iNPH. In conclusion, at the disease onset, several factors contribute to maintaining cell homeostasis, and the protective role of very long chains sphingolipids counteract overexpression of amyloidogenic and neurotoxic proteins. Monitoring specific very long chain Cers will improve the early diagnosis and can promote patient follow-up.

Skeletal Muscle Proteomic Profile Revealed Gender-Related Metabolic Responses in a Diet-Induced Obesity Animal Model.

Obesity is a chronic, complex pathology associated with a risk of developing secondary pathologies, including cardiovascular diseases, cancer, type 2 diabetes (T2DM) and musculoskeletal disorders. Since skeletal muscle accounts for more than 70% of total glucose disposal, metabolic alterations are strictly associated with the onset of insulin resistance and T2DM. The present study relies on the proteomic analysis of gastrocnemius muscle from 15 male and 15 female C56BL/J mice fed for 14 weeks with standard, 45% or 60% high-fat diets (HFD) adopting a label-free LC-MS/MS approach followed by bioinformatic pathway analysis. Results indicate changes in males due to HFD, with increased muscular stiffness (Col1a1, Col1a2, Actb), fiber-type switch from slow/oxidative to fast/glycolytic (decreased Myh7, Myl2, Myl3 and increased Myh2, Mylpf, Mybpc2, Myl1), increased oxidative stress and mitochondrial dysfunction (decreased respiratory chain complex I and V and increased complex III subunits). At variance, females show few alterations and activation of compensatory mechanisms to counteract the increase of fatty acids. Bioinformatics analysis allows identifying upstream molecules involved in regulating pathways identified at variance in our analysis (Ppargc1a, Pparg, Cpt1b, Clpp, Tp53, Kdm5a, Hif1a). These findings underline the presence of a gender-specific response to be considered when approaching obesity and related comorbidities.

Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

The Sarcolab pilot study of 2 crewmembers, investigated before and after a 6-mo International Space Station mission, has demonstrated the substantial muscle wasting and weakness, along with disruption of muscle's oxidative metabolism. The present work aimed at evaluating the pro/anti-inflammatory status in the same 2 crewmembers (A, B). Blood circulating (c-)microRNAs (miRs), c-proteasome, c-mitochondrial DNA, and cytokines were assessed by real-time quantitative PCR or ELISA tests. Time series analysis was performed ( i.e., before flight and after landing) at 1 and 15 d of recovery (R+1 and R+15, respectively). C-biomarkers were compared with an age-matched control population and with 2-dimensional proteomic analysis of the 2 crewmembers' muscle biopsies. Striking differences were observed between the 2 crewmembers at R+1, in terms of inflamma-miRs (c-miRs-21-5p, -126-3p, and -146a-5p), muscle specific (myo)-miR-206, c-proteasome, and IL-6/leptin, thus making the 2 astronauts dissimilar to each other. Final recovery levels of c-proteasome, c-inflamma-miRs, and c-myo-miR-206 were not reverted to the baseline values in crewmember A. In both crewmembers, myo-miR-206 changed significantly after recovery. Muscle biopsy of astronaut A showed an impressive 80% increase of α-1-antitrypsin, a target of miR-126-3p. These results point to a strong stress response induced by spaceflight involving muscle tissue and the proinflammatory setting, where inflamma-miRs and myo-miR-206 mediate the systemic recovery phase after landing.-Capri, M., Morsiani, C., Santoro, A., Moriggi, M., Conte, M., Martucci, M., Bellavista, E., Fabbri, C., Giampieri, E., Albracht, K., Flück, M., Ruoss, S., Brocca, L., Canepari, M., Longa, E., Di Giulio, I., Bottinelli, R., Cerretelli, P., Salvioli, S., Gelfi, C., Franceschi, C., Narici, M., Rittweger, J. Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

Urinary physiology and hypoxia: a pilot study of moderate-altitude trekking effects on urodynamic indexes.

Exposure to high altitude is one of the most widely used models to study the adaptive response to hypoxia in humans. However, little is known about the related effects on micturition. The present study addresses the adaptive urinary responses in four healthy adult lowlanders, comparing urodynamic indexes at Kathmandu [1,450 m above sea level (a.s.l.); K1450] and during a sojourn in Namche Bazar (3,500 m a.s.l.; NB3500). The urodynamic testing consisted of cistomanometry and bladder pressure/flow measurements. Anthropometrics, electrocardiographic, and peripheral capillary oxygen saturation data were also collected. The main findings consisted of significant reductions in bladder power at maximum urine flow by ~30%, bladder contractility index by 13%, and infused volume both at first (by 57%) and urgency sensation (by 14%) to urinate, indicating a reduced cystometric capacity, at NB3500. In addition to the urinary changes, we found that oxygen saturation, body mass index, body surface area, and median RR time were all significantly reduced at altitude. We submit that the hypoxia-related parasympathetic inhibition could be the underlying mechanism of both urodynamic and heart rate adaptive responses to high-altitude exposure. Moreover, increased diuresis and faster bladder filling at altitude may trigger the anticipation of being able to void, a common cause of urgency. We believe that the present pilot study represents an original approach to the study of urinary physiology at altitude.

ECM Remodeling in Breast Cancer with Different Grade: Contribution of 2D-DIGE Proteomics.

Tumor extracellular matrix (ECM) plays a pivotal role in outcome of breast cancer (BC) patients. Overexpression of 58 genes, encoding 43 structural ECM proteins, has been identified to determine a specific cluster of BC with accelerated metastatic potential only in the undifferentiated (grade III) phenotype. The scope of this study is to characterize protein repertoire able to predict patient outcome in BC according to ECM gene expression pattern and histological grade. The differential proteomic analysis is based on 2D-differential gel electrophoresis, MALDI-MS, bioinformatics, and immunoblotting. Results suggest a relationship among ECM remodeling, signal mechanotransduction, and metabolic rewiring in BCs characterized by a specific mRNA ECM signature and identified a set of dysregulated proteins characteristic of hormone receptors expression as fibrinogen-ß chain, collagen α-1(VI) chain, and α-1B-glycoprotein. Furthermore, in triple negative tumors with ECM signature, the FGG and α5ß1/αvß3 integrins increase whereas detyrosinated α-tubulin and mimecan decrease leading to unorganized integrin presentation involving focal adhesion kinase, activation of Rho GTPases associated to epithelial mesenchymal transition. In hormone receptors negative BCs characterized by a specific ECM gene cluster, the differentially regulated proteins, identified in the present study, can be potentially relevant to predict patient's outcome.

Contribution of Extracellular Matrix and Signal Mechanotransduction to Epithelial Cell Damage in Inflammatory Bowel Disease Patients: A Proteomic Study.

This study utilizes 2D-DIGE (difference gel etrophoresis), isotope-coded protein labeling and biochemical assays to characterize protein alteration in ulcerative colitis (UC) and Crohn's disease (CD) in human epithelial cell and mucosal biopsies in inflammatory bowel disease (IBD)-affected patients. The aim of this study is to identify the key molecular signatures involved in epithelial cell structure of IBDs. In non-inflamed UC (QUC) keratins, vimentin, and focal adhesion kinase (7) increased, whereas vinculin and de-tyrosinated α-tubulin decreased; inflammation (IUC) exacerbated molecular changes, being collagen type VI alpha 1 chain (COL6A1), tenascin-C and vimentin increased. In non-inflamed CD (QCD), tenascin C, de-tyrosinated α-tubulin, vinculin, FAK, and Rho-associated protein kinase 1 (ROCK1) decreased while vimentin increased. In inflamed CD (ICD), COL6A1, vimentin and integrin alpha 4 increased. In QUC, cell metabolism is characterized by a decrease of the tricarboxylic acid cycle enzymes and a decrease of short/branched chain specific acyl-CoA dehydrogenase, fatty acid synthase, proliferator-activated receptors alpha, and proliferator-activated receptors gamma. In QCD a metabolic rewiring occurs, as suggested by glycerol-3-phosphate dehydrogenase (GPD2), pyruvate dehydrogenase E1 component subunit beta, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and 4-trimethylaminobutyraldehyde dehydrogenase increment, while dihydrolipoyl dehydrogenase decreased. Macroautophagy is activated in QUC and IUC, with increased levels of p62, HSC70, major vault protein, myosin heavy chain 9, whereas it is blunted in QCD and ICD. The differing pattern of extracellular matrix, cytoskeletal derangements, cellular metabolism, and autophagy in UC and CD may contribute to the pathophysiological understanding of these disorders and serve as diagnostic markers in IBD patients.

Mapping the human skeletal muscle proteome: progress and potential.

INTRODUCTION: Human skeletal muscle represents 40% of our body mass and deciphering its proteome composition to further understand mechanisms regulating muscle function under physiological and pathological conditions has proved a challenge. The inter-individual variability, the presence of structurally and functionally different muscle types and the high protein dynamic range require carefully selected methodologies for the assessment of the muscle proteome. Furthermore, physiological studies are understandingly hampered by ethical issues related to biopsies on healthy subjects, making it difficult to recruit matched controls essential for comparative studies. Areas covered: This review critically analyses studies performed on muscle to date and identifies what still remains unknown or poorly investigated in physiological and pathological states, such as training, aging, metabolic disorders and muscular dystrophies. Expert commentary: Efforts should be made on biological fluid analyses targeting low abundant/low molecular weight fragments generated from muscle cell disruption to improve diagnosis and clinical monitoring. From a methodological point of view, particular attention should be paid to improve the characterization of intact proteins and unknown post translational modifications to better understand the molecular mechanisms of muscle disorders.

A CASQ1 founder mutation in three Italian families with protein aggregate myopathy and hyperCKaemia.

BACKGROUND: Protein aggregate myopathies are increasingly recognised conditions characterised by a surplus of endogenous proteins. The molecular and mutational background for many protein aggregate myopathies has been clarified with the discovery of several underlying mutations. Familial idiopathic hyperCKaemia is a benign genetically heterogeneous condition with autosomal dominant features in a high proportion of cases. METHODS: In 10 patients from three Italian families with autosomal dominant benign vacuolar myopathy and hyperCKaemia, we performed linkage analysis and exome sequencing as well as morphological and biochemical investigations. RESULTS AND CONCLUSIONS: We show, by Sanger and exome sequencing, that the protein aggregate myopathy with benign evolution and muscle inclusions composed of excess CASQ1, affecting three Italian families, is due to the D244G heterozygous missense mutation in the CASQ1 gene. Investigation of microsatellite markers revealed a common haplotype in the three families indicating consanguinity and a founder effect. Results from immunocytochemistry, electron microscopy, biochemistry and transfected cell line investigations contribute to our understanding of pathogenetic mechanisms underlining this defect. The mutation is common to other Italian patients and is likely to share a founder effect with them. HyperCKaemia in the CASQ1-related myopathy is common and sometimes the sole overt manifestation. It is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed, and the condition could be more common than supposed.

Changes in muscle proteomics in the course of the Caudwell Research Expedition to Mt. Everest.

This study employed differential proteomic and immunoassay techniques to elucidate the biochemical mechanisms utilized by human muscle (vastus lateralis) in response to high altitude hypoxia exposure. Two groups of subjects, participating in a medical research expedition (A, n = 5, 19 d at 5300 m altitude; B, n = 6, 66 d up to 8848 m) underwent a ≈ 30% drop of muscular creatine kinase and of glycolytic enzymes abundance. Protein abundance of most enzymes of the tricarboxylic acid cycle and oxidative phosphorylation was reduced both in A and, particularly, in B. Restriction of α-ketoglutarate toward succinyl-CoA resulted in increased prolyl hydroxylase 2 and glutamine synthetase. Both A and B were characterized by a reduction of elongation factor 2 alpha, controlling protein translation, and by an increase of heat shock cognate 71 kDa protein involved in chaperone-mediated autophagy. Increased protein levels of catalase and biliverdin reductase occurred in A alongside a decrement of voltage-dependent anion channels 1 and 2 and of myosin-binding protein C, suggesting damage to the sarcomeric structures. This study suggests that during acclimatization to hypobaric hypoxia the muscle behaves as a producer of substrates activating a metabolic reprogramming able to support anaplerotically the tricarboxylic acid cycle, to control protein translation, to prevent energy expenditure and to activate chaperone-mediated autophagy.

Disuse deterioration of human skeletal muscle challenged by resistive exercise superimposed with vibration: evidence from structural and proteomic analysis.

In the present bed rest (BR) study, 23 volunteers were randomized into 3 subgroups: 60 d BR control (Ctr); BR with resistive exercise (RE; lower-limb load); and resistive vibration exercise (RVE; RE with superimposed vibration). The aim was to analyze by confocal and electron microscopy the effects of vibration on myofibril and filament integrity in soleus (Sol) and vastus lateralis (VL) muscle; differential proteomics of contractile, cytoskeletal, and costameric proteins (TN-C, ROCK1, and FAK); and expression of PGC1α and atrophy-related master genes MuRF1 and MuRF2. RVE (but not RE) preserved myofiber size and phenotype in Sol and VL by overexpressing MYBPC1 (42%, P ≤ 0.01), WDR1 (39%, P ≤ 0.01), sarcosin (84%, P ≤ 0.01), and CKM (20%, P ≤ 0.01) and prevented myofibrillar ultrastructural damage as detectable by MuRF1 expression. In Sol, cytoskeletal and contractile proteins were normalized by RVE, and TN-C increased (59%, P ≤ 0.01); the latter also with RE (108%, P ≤ 0.01). In VL, the outcomes of both RVE (acting on sarcosin and desmin) and RE (by way of troponinT-slow and MYL2) were similar. RVE appears to be a highly efficient countermeasure protocol against muscle atrophy and ultrastructural and molecular dysregulation induced by chronic disuse.

GBA1 inactivation in oligodendrocytes affects myelination and induces neurodegenerative hallmarks and lipid dyshomeostasis in mice.

BACKGROUND: Mutations in the ß-glucocerebrosidase (GBA1) gene do cause the lysosomal storage Gaucher disease (GD) and are among the most frequent genetic risk factors for Parkinson's disease (PD). So far, studies on both neuronopathic GD and PD primarily focused on neuronal manifestations, besides the evaluation of microglial and astrocyte implication. White matter alterations were described in the central nervous system of paediatric type 1 GD patients and were suggested to sustain or even play a role in the PD process, although the contribution of oligodendrocytes has been so far scarcely investigated. METHODS: We exploited a system to study the induction of central myelination in vitro, consisting of Oli-neu cells treated with dibutyryl-cAMP, in order to evaluate the expression levels and function of ß-glucocerebrosidase during oligodendrocyte differentiation. Conduritol-B-epoxide, a ß-glucocerebrosidase irreversible inhibitor was used to dissect the impact of ß-glucocerebrosidase inactivation in the process of myelination, lysosomal degradation and α-synuclein accumulation in vitro. Moreover, to study the role of ß-glucocerebrosidase in the white matter in vivo, we developed a novel mouse transgenic line in which ß-glucocerebrosidase function is abolished in myelinating glia, by crossing the Cnp1-cre mouse line with a line bearing loxP sequences flanking Gba1 exons 9-11, encoding for ß-glucocerebrosidase catalytic domain. Immunofluorescence, western blot and lipidomic analyses were performed in brain samples from wild-type and knockout animals in order to assess the impact of genetic inactivation of ß-glucocerebrosidase on myelination and on the onset of early neurodegenerative hallmarks, together with differentiation analysis in primary oligodendrocyte cultures. RESULTS: Here we show that ß-glucocerebrosidase inactivation in oligodendrocytes induces lysosomal dysfunction and inhibits myelination in vitro. Moreover, oligodendrocyte-specific ß-glucocerebrosidase loss-of-function was sufficient to induce in vivo demyelination and early neurodegenerative hallmarks, including axonal degeneration, α-synuclein accumulation and astrogliosis, together with brain lipid dyshomeostasis and functional impairment. CONCLUSIONS: Our study sheds light on the contribution of oligodendrocytes in GBA1-related diseases and supports the need for better characterizing oligodendrocytes as actors playing a role in neurodegenerative diseases, also pointing at them as potential novel targets to set a brake to disease progression.

Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

Nitrosative Stress in Astronaut Skeletal Muscle in Spaceflight.

Long-duration mission (LDM) astronauts from the International Space Station (ISS) (>180 ISS days) revealed a close-to-normal sarcolemmal nitric oxide synthase type-1 (NOS1) immunoexpression in myofibers together with biochemical and quantitative qPCR changes in deep calf soleus muscle. Nitro-DIGE analyses identified functional proteins (structural, metabolic, mitochondrial) that were over-nitrosylated post- vs. preflight. In a short-duration mission (SDM) astronaut (9 ISS days), s-nitrosylation of a nodal protein of the glycolytic flux, specific proteins in tricarboxylic acid (TCA) cycle, respiratory chain, and over-nitrosylation of creatine kinase M-types as signs of impaired ATP production and muscle contraction proteins were seen. S-nitrosylation of serotransferrin (TF) or carbonic anhydrase 3 (CA3b and 3c) represented signs of acute response microgravity muscle maladaptation. LDM nitrosoprofiles reflected recovery of mitochondrial activity, contraction proteins, and iron transporter TF as signs of muscle adaptation to microgravity. Nitrosated antioxidant proteins, alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR), and selenoprotein thioredoxin reductase 1 (TXNRD1) levels indicated signs of altered redox homeostasis and reduced protection from nitrosative stress in spaceflight. This work presents a novel spaceflight-generated dataset on s-nitrosylated muscle protein signatures from astronauts that helps both to better understand the structural and molecular networks associated to muscular nitrosative stress and to design countermeasures to dysfunction and impaired performance control in human spaceflight missions.

Proteomic analysis by mass spectrometry of postmortem muscle protein degradation for PMI estimation: A pilot study.

The determination of the postmortem interval is a topic of great forensic interest. The possibility of using new technologies has allowed the study of postmortem decay of biomolecules in the determination of PMI. Skeletal muscle proteins are promising candidates because skeletal muscle exhibits slower postmortem decay compared to other internal organs and nervous tissues, while its degradation is faster than cartilage and bone. In this pilot study, skeletal muscle tissue from pigs was degraded at two different controlled temperatures, 21 °C and 6 °C, and analysed at predefined times points: 0, 24, 48, 72, 96, and 120 h. The obtained samples were analysed by mass spectrometry proteomics approach for qualitative and quantitative evaluation of proteins and peptides. Immunoblotting validation was performed for the candidate proteins. The results obtained appeared significant and identified several proteins useful for possible postmortem interval estimation. Of these proteins, PDLIM7, TPM1, and ATP2A2 were validated by immunoblotting at a larger number of experimental points and at different temperatures. The results obtained are in agreement with those observed in similar works. In addition, the use of a mass spectrometry approach increased the number of protein species identified, providing a larger panel of proteins for PMI assessment.