LPA-induced expression of CCN2 in muscular fibro/adipogenic progenitors (FAPs): Unraveling cellular communication networks.

Cellular Communication Network Factor 2, CCN2, is a profibrotic cytokine implicated in physiological and pathological processes in mammals. The expression of CCN2 is markedly increased in dystrophic muscles. Interestingly, diminishing CCN2 genetically or inhibiting its function improves the phenotypes of chronic muscular fibrosis in rodent models. Elucidating the cell-specific mechanisms behind the induction of CCN2 is a fundamental step in understanding its relevance in muscular dystrophies. Here, we show that the small lipids LPA and 2S-OMPT induce CCN2 expression in fibro/adipogenic progenitors (FAPs) through the activation of the LPA1 receptor and, to a lower extent, by also the LPA6 receptor. These cells show a stronger induction than myoblasts or myotubes. We show that the LPA/LPARs axis requires ROCK kinase activity and organized actin cytoskeleton upstream of YAP/TAZ signaling effectors to upregulate CCN2 levels, suggesting that mechanical signals are part of the mechanism behind this process. In conclusion, we explored the role of the LPA/LPAR axis on CCN2 expression, showing a strong cytoskeletal-dependent response in muscular FAPs.

Activation of skeletal muscle FAPs by LPA requires the Hippo signaling via the FAK pathway.

Lysophosphatidic acid (LPA) is a lysophospholipid that signals through six G-protein coupled receptors (LPARs), LPA1 to LPA6. LPA has been described as a potent modulator of fibrosis in different pathologies. In skeletal muscle, LPA increases fibrosis-related proteins and the number of fibro/adipogenic progenitors (FAPs). FAPs are the primary source of ECM-secreting myofibroblasts in acute and chronic damage. However, the effect of LPA on FAPs activation in vitro has not been explored. This study aimed to investigate FAPs' response to LPA and the downstream signaling mediators involved. Here, we demonstrated that LPA mediates FAPs activation by increasing their proliferation, expression of myofibroblasts markers, and upregulation of fibrosis-related proteins. Pretreatment with the LPA1/LPA3 antagonist Ki16425 or genetic deletion of LPA1 attenuated the LPA-induced FAPs activation, resulting in decreased expression of cyclin e1, α-SMA, and fibronectin. We also evaluated the activation of the focal adhesion kinase (FAK) in response to LPA. Our results showed that LPA induces FAK phosphorylation in FAPs. Treatment with the P-FAK inhibitor PF-228 partially prevented the induction of cell responses involved in FAPs activation, suggesting that this pathway mediates LPA signaling. FAK activation controls downstream cell signaling within the cytoplasm, such as the Hippo pathway. LPA induced the dephosphorylation of the transcriptional coactivator YAP (Yes-associated protein) and promoted direct expression of target pathway genes such as Ctgf/Ccn2 and Ccn1. The blockage of YAP transcriptional activity with Super-TDU further confirmed the role of YAP in LPA-induced FAPs activation. Finally, we demonstrated that FAK is required for LPA-dependent YAP dephosphorylation and the induction of Hippo pathway target genes. In conclusion, LPA signals through LPA1 to regulate FAPs activation by activating FAK to control the Hippo pathway.

Denervation Drives YAP/TAZ Activation in Muscular Fibro/Adipogenic Progenitors.

Loss of motoneuron innervation (denervation) is a hallmark of neurodegeneration and aging of the skeletal muscle. Denervation induces fibrosis, a response attributed to the activation and expansion of resident fibro/adipogenic progenitors (FAPs), i.e., multipotent stromal cells with myofibroblast potential. Using in vivo and in silico approaches, we revealed FAPs as a novel cell population that activates the transcriptional coregulators YAP/TAZ in response to skeletal muscle denervation. Here, we found that denervation induces the expression and transcriptional activity of YAP/TAZ in whole muscle lysates. Using the PdgfraH2B:EGFP/+ transgenic reporter mice to trace FAPs, we demonstrated that denervation leads to increased YAP expression that accumulates within FAPs nuclei. Consistently, re-analysis of published single-nucleus RNA sequencing (snRNA-seq) data indicates that FAPs from denervated muscles have a higher YAP/TAZ signature level than control FAPs. Thus, our work provides the foundations to address the functional role of YAP/TAZ in FAPs in a neurogenic pathological context, which could be applied to develop novel therapeutic approaches for the treatment of muscle disorders triggered by motoneuron degeneration.

Sympathetic and electrochemical skin responses in the assessment of sudomotor function: a comparative study.

OBJECTIVES: The sympathetic skin response (SSR) is a well-established test, whereas the electrochemical skin conductance (ESC) is still under evaluation. Our aim was therefore to assess the diagnostic accuracy of ESC to detect abnormal sudomotor function, using SSR as a reference test. METHODS: A cross sectional observational study was performed of 61 neurological patients assessed for possible sudomotor dysfunction and 50 age-matched healthy controls (HC). Patients with diagnoses of vasovagal syncope (VVS, n=25), Parkinson's disease (PD, n=15), multiple system atrophy (MSA, n=11) and peripheral neuropathies (PN, n=10) were included. Sudomotor function was assessed with SSR and ESC tests in all participants. The absence of SSR in the palms or soles indicates abnormal sudomotor function. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic value of the ESC. Cardiovascular autonomic (CV-Aut) function was evaluated through the Ewing score, based on the following tests: Heart rate change with deep breathing, Valsalva ratio, 30:15 ratio, blood pressure changes on standing and during isometric exercise. A Ewing score ≥ 2 indicates the presence of CV-Aut dysfunction. RESULTS: Mean SSR amplitudes and ESC values showed differences between HC and patients with MSA or PN (p < 0.05), but not in patients with VVS or PD. Absence of SSR was associated with abnormal ESC (p < 0.05). Patients with abnormal CV-Aut dysfunction had lower ESC (p< 0.05). Palm ESC (P-ESC) and sole ESC (S-ESC) assessment had a sensitivity of 0.91 and 0.95 to predict sudomotor dysfunction, with a specificity of 0.78 and 0.85, respectively. The area under ROC curve was 0.905 and 0.98, respectively. CONCLUSIONS: ESC in palms and soles has a high diagnostic accuracy for sudomotor dysfunction as detected by absent SSR in patients with MSA and PN.

Hyperhidrosis in sleep disorders - A narrative review of mechanisms and clinical significance.

Hyperhidrosis is characterized by excessive sweating beyond thermoregulatory needs that affects patients' quality of life. It results from an excessive stimulation of eccrine sweat glands in the skin by the sympathetic nervous system. Hyperhidrosis may be primary or secondary to an underlying cause. Nocturnal hyperhidrosis is associated with different sleep disorders, such as obstructive sleep apnea, insomnia, restless legs syndrome/periodic limb movement during sleep and narcolepsy. The major cause of the hyperhidrosis is sympathetic overactivity and, in the case of narcolepsy type 1, orexin deficiency may also contribute. In this narrative review, we will provide an outline of the possible mechanisms underlying sudomotor dysfunction and the resulting nocturnal hyperhidrosis in these different sleep disorders and explore its clinical relevance.

Manejo multidisciplinario y avances terapéuticos en la esclerosis lateral amiotrófica / Multidisciplinary care and therapeutic advances in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects the motor system, resulting in progressive weakness and muscle wasting. Despite the tremendous advances in physiopathological and clinical characterization, we do not have a curative treatment yet. The progressive and fatal course of ALS makes its management particularly complex and challenging given the diversity of symptoms presenting during the disease progression. The main goal in the treatment of ALS patients is to minimize morbidity and maximize the quality of life. Currently, a series of therapeutic interventions improve the quality of life and prolong survival, including multidisciplinary care, respiratory management, and disease-modifying therapy. Within the supportive interventions, weight maintenance through nutritional and metabolic support is critical. In addition, the management of neuropsychiatric manifestations and preservation of communicative capacity before speech loss are also crucial. Lastly, early palliative care intervention is essential to optimize symptomatic management. Anticipatory guidelines to face the inevitable patient deterioration should be devised. This article updates the main therapeutic strategies used in these patients, including evolving clinical trials with promising novel therapies.

Activation of the ATX/LPA/LPARs axis induces a fibrotic response in skeletal muscle.

Several common chronic diseases, muscular dystrophies (MDs), and aging lead to progressive fibrous connective tissue (fibrosis) accumulation in skeletal muscle. Cumulative past evidence points to the role of signaling lipids such as lysophosphatidic acid (LPA) and its receptors (LPARs) in different models of fibrosis. However, the potential contribution of these molecules to the fibrotic process in skeletal muscle has not been explored. Here, we show the expression of ATX/LPA/LPARs axis components in skeletal muscle, which suggests their potential relevance for the biology of this tissue. We investigated if the skeletal muscle responds to the stimulus of intramuscular (IM) LPA injections, finding an early induction of the pro-fibrotic factor connective tissue growth factor/Cellular Communication Network factor 2 (CCN2) and extracellular matrix (ECM) proteins. Also, we found that LPA induces an increase in the number of fibro/adipogenic progenitors (FAPs), which are the primary cellular source of myofibroblasts. These effects were for the most part prevented by the inhibitor Ki16425, which inhibits the LPA receptors LPA1 and LPA3, as well as in the LPA1-KO mice.  We also evaluated the in vivo activation of extracellular signal-regulated kinases (ERK 1/2), AKT, c-Jun N-terminal kinase (JNK), and Yes-asocciated protein 1 (YAP) in response to LPA. Our results show that LPA induces ERK 1/2 phosphorylation in WT muscle, but not in LPA1-KO mice. Treatment with the ERK 1/2 inhibitor U0126 prevented the induction of fibronectin in response to LPA, suggesting that this pathway is involved in LPA-induced fibrosis. Altogether, these results demonstrate that ATX/LPA/LPARs constitute a pro-fibrotic axis and suggest a possible role in muscular diseases.

[Multidisciplinary care and therapeutic advances in amyotrophic lateral sclerosis]. / Manejo multidisciplinario y avances terapéuticos en la esclerosis lateral amiotrófica.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects the motor system, resulting in progressive weakness and muscle wasting. Despite the tremendous advances in physiopathological and clinical characterization, we do not have a curative treatment yet. The progressive and fatal course of ALS makes its management particularly complex and challenging given the diversity of symptoms presenting during the disease progression. The main goal in the treatment of ALS patients is to minimize morbidity and maximize the quality of life. Currently, a series of therapeutic interventions improve the quality of life and prolong survival, including multidisciplinary care, respiratory management, and disease-modifying therapy. Within the supportive interventions, weight maintenance through nutritional and metabolic support is critical. In addition, the management of neuropsychiatric manifestations and preservation of communicative capacity before speech loss are also crucial. Lastly, early palliative care intervention is essential to optimize symptomatic management. Anticipatory guidelines to face the inevitable patient deterioration should be devised. This article updates the main therapeutic strategies used in these patients, including evolving clinical trials with promising novel therapies.

Neurogenic Orthostatic Hypotension. Lessons From Synucleinopathies.

Maintenance of upright blood pressure critically depends on the autonomic nervous system and its failure leads to neurogenic orthostatic hypotension (NOH). The most severe cases are seen in neurodegenerative disorders caused by abnormal α-synuclein deposits: multiple system atrophy (MSA), Parkinson's disease, Lewy body dementia, and pure autonomic failure (PAF). The development of novel treatments for NOH derives from research in these disorders. We provide a brief review of their underlying pathophysiology relevant to understand the rationale behind treatment options for NOH. The goal of treatment is not to normalize blood pressure but rather to improve quality of life and prevent syncope and falls by reducing symptoms of cerebral hypoperfusion. Patients not able to recognize NOH symptoms are at a higher risk for falls. The first step in the management of NOH is to educate patients on how to avoid high-risk situations and providers to identify medications that trigger or worsen NOH. Conservative countermeasures, including diet and compression garments, should always precede pharmacologic therapies. Volume expanders (fludrocortisone and desmopressin) should be used with caution. Drugs that enhance residual sympathetic tone (pyridostigmine and atomoxetine) are more effective in patients with mild disease and in MSA patients with spared postganglionic fibers. Norepinephrine replacement therapy (midodrine and droxidopa) is more effective in patients with neurodegeneration of peripheral noradrenergic fibers like PAF. NOH is often associated with other cardiovascular diseases, most notably supine hypertension, and treatment should be adapted to their presence.

MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.

Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-ß) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy. METHODS: mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-ß signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice. RESULTS: mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-ß signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells. CONCLUSIONS: These results suggest that andrographolide could be used to improve quality of life in individuals with DMD.

Identification of a novel microRNA that regulates the proliferation and differentiation in muscle side population cells.

Muscle satellite cells are largely responsible for skeletal muscle regeneration following injury. Side population (SP) cells, which are thought to be muscle stem cells, also contribute to muscle regeneration. SP cells exhibit high mesenchymal potential, and are a possible cell source for therapy of muscular dystrophy. However, the mechanism by which muscle SP cells are committed to differentiation is poorly understood. microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of their mRNA targets. In skeletal muscle, miRNAs are known to be involved in myoblast proliferation and differentiation. To investigate mechanisms of SP cell regulation, we profiled miRNA expression in SP cells and main population (MP) cells in muscles using quantitative real-time polymerase chain reaction-based expression assays. We identified a set of miRNAs that was highly expressed in SP cells as compared with MP cells. One miRNA, miR-128a, was elevated in expression in SP cells, but decreased in expression during continued culture in vitro. Overexpression of miR-128a in SP cells resulted in inhibited cell proliferation. The differentiation potential of SP cells was also decreased when miR-128a was overexpressed. MiR-128a was found to regulate the target genes involved in the regulation of adipogenic-, osteogenic- and myogenic genes that include: PPARγ, Runx1, and Pax3. Overexpression of miR-128a suppressed the activity of a luciferase reporter fused to the 3'-untranslated region of each gene. These results demonstrate that miR-128a contributes to the maintenance of the quiescent state, and it regulates cellular differentiation by repressing individual genes in SP cells.

ß4 integrin marks interstitial myogenic progenitor cells in adult murine skeletal muscle.

Skeletal muscle growth and its regeneration following injury rely on myogenic progenitor cells, a heterogeneous population that includes the satellite cells and other interstitial progenitors. The present study demonstrates that surface expression of ß4 integrin marks a population of vessel-associated interstitial muscle progenitor cells. Muscle ß4 integrin-positive cells do not express myogenic markers upon isolation. However, they are capable of undergoing myogenic specification in vitro and in vivo: ß4 integrin cells differentiate into multinucleated myotubes in culture dishes and contribute to muscle regeneration upon delivery into diseased mice. Subfractionation of ß4 integrin-expressing cells based on CD31 expression does not further enrich for myogenic precursors. These findings support the expression of ß4 integrin in interstitial, vessel-associated cells with myogenic activity within adult skeletal muscle.

Regulation of DMD pathology by an ankyrin-encoded miRNA.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the ANKYRIN1(ANK1) gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types. RESULTS: Inhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor ß, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers in vivo, as these mice had impaired muscle regeneration. CONCLUSIONS: These studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.

Inhibition of extracellular matrix assembly induces the expression of osteogenic markers in skeletal muscle cells by a BMP-2 independent mechanism.

BACKGROUND: The conversion of one cell type into another has been suggested to be, at the molecular level, the consequence of change(s) in the expression level of key developmental genes. Myoblasts have the ability to differentiate either to skeletal muscle or osteogenic lineage depending of external stimuli. Extracellular matrix (ECM) has been shown to be essential for skeletal muscle differentiation, through its direct interaction with myoblasts' cell receptors. We attempt to address if ECM also plays a role in the osteogenic differentiation of skeletal muscle cells. RESULTS: Inhibition of proteoglycan sulfation by sodium chlorate in myoblast cultures strongly affects ECM synthesis and deposition and induces the expression of the osteogenic lineage markers alkaline phosphatase (ALP) and osteocalcin in mononuclear cells. Induction of ALP by sodium chlorate does not affect the expression of specific muscle determination transcription factors, such as MyoD and Myf-5, in the same cells. The osteogenic transcription factor Cbfa-1 expression is also unaffected. Induction of ALP is not inhibited by a soluble form of BMP receptor IA. This suggests that the deviation of the myogenic pathway of C2C12 myoblasts into the osteogenic lineage by inhibitors of proteoglycan sulfation is BMP-2 independent. The increase of osteogenic markers expression can be totally prevented by an exogenous ECM. Interestingly, a similar BMP-2-independent ALP activity induction can be observed in myoblasts cultured on an ECM previously synthesized by BMP-2 treated myoblasts. Under in vivo conditions of increased ECM turn-over and deposition, as in the mdx dystrophic muscle and during skeletal muscle regeneration, an induction and relocalization of ALP is observed in a subpopulation of skeletal muscle fibers, whereas in normal skeletal muscle, ALP expression is restricted to blood vessels and some endomysial mononuclear cells. CONCLUSION: These results suggest that signals arising from the ECM induce the expression of osteogenic markers in muscle cells by a mechanism independent of BMP-2 and without affecting the expression of key muscle or osteogenic determination genes. An induction and relocalization of ALP is also observed in mdx and regenerating skeletal muscles, in vivo conditions of increased muscle ECM deposition or turnover.

Structural and functional organization of synaptic acetylcholinesterase.

The expression of the synaptic asymmetric form of the enzyme acetylcholinesterase (AChE) depends of two different genes: the gene that encodes for the catalytic subunit and the gene that encodes for the collagenic tail, ColQ. Asymmetric AChE is specifically localized to the basal lamina at the neuromuscular junction (NMJ). This highly organized distribution pattern suggests the existence of one or more specific binding sites in ColQ required for its anchorage to the synaptic basal lamina. Recent evidence support this notion: first, the presence of two heparin-binding domains in ColQ that interact with heparan sulfate proteoglycans (HSPGs) at the synaptic basal lamina; and second, a knockout mouse for perlecan, a HSPG concentrated in nerve-muscle contact, in which absence of asymmetric AChE at the NMJ is observed. The physiological importance of collagen-tailed AChE form in skeletal muscle has been illustrated by the identification of several mutations in the ColQ gene. These mutations determine end-plate acetylcholinesterase deficiency and induce one type of synaptic functional disorders observed in Congenital Myasthenic Syndromes (CMSs).

Transient up-regulation of biglycan during skeletal muscle regeneration: delayed fiber growth along with decorin increase in biglycan-deficient mice.

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Decorin is the main proteoglycan present in the extracellular matrix (ECM) of adult muscle while biglycan expression is lower, but both are increased in mdx mice dystrophic muscle. Both of these small leucine-rich proteoglycans (SLRPs) can bind other matrix proteins and to the three TGF-beta isoforms, acting as modulators of their biological activity. We evaluated biglycan and decorin expression in skeletal muscle during barium chloride-induced skeletal muscle regeneration in mice. A transient and dramatic up-regulation of biglycan was associated with newly formed myotubes, whereas decorin presented only minor variations. Studies both in vitro and in intact developing newborn mice showed that biglycan expression is initially high and then decreases during skeletal muscle differentiation and maturation. To further evaluate the role of biglycan during the regenerative process, skeletal muscle regeneration was studied in biglycan-null mice. Skeletal muscle maintains its regenerative capacity in the absence of biglycan, but a delay in regenerated fiber growth and a decreased expression of embryonic myosin were observed despite to normal expression of MyoD and myogenin. Transient up-regulation of decorin during muscle regeneration in these mice may possibly obscure further roles of SLRPs in this process.