Regenerative potential of human dental pulp stem cells in the treatment of stress urinary incontinence: In vitro and in vivo study

OBJECTIVES: To evaluate the regenerative potential of human dental pulp stem cells (hDPSCs) in an animal model of stress urinary incontinence (SUI). SUI, an involuntary leakage of urine, is due to physical stress involving an increase in bladder pressure and a damage of external urethral sphincter affecting muscles and nerves. Conventional therapies can only relieve the symptoms. Human DPSCs are characterized by peculiar stemness and immunomodulatory properties and might provide an alternative tool for SUI therapy. MATERIALS AND METHODS: In vitro phase: hDPSCs were induced towards the myogenic commitment following a 24 hours pre-conditioning with 5-aza-2'-deoxycytidine (5-Aza), then differentiation was evaluated. In vivo phase: pudendal nerve was transected in female rats to induce stress urinary incontinence; then, pre-differentiated hDPSCs were injected in the striated urethral sphincter. Four weeks later, urethral sphincter regeneration was assayed through histological, functional and immunohistochemical analyses. RESULTS: Human DPSCs were able to commit towards myogenic lineage in vitro and, four weeks after cell injection, hDPSCs engrafted in the external urethral sphincter whose thickness was almost recovered, committed towards myogenic lineage in vivo, promoted vascularization and an appreciable recovery of the continence. Moreover, hDPSCs were detected within the nerve, suggesting their participation in repair of transected nerve. CONCLUSIONS: These promising data and further investigations on immunomodulatory abilities of hDPSCs would allow to make them a potential tool for alternative therapies of SUI

The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle

Satellite cells represent myogenic stem cells that allow the homeostasis and repair of adult skeletal muscle. Here the authors report that the transcription factor NF-Y is expressed in satellite cells and is important for their maintenance and proper myogenic differentiation

Amyotrophic Lateral Sclerosis, a Multisystem Pathology: Insights into the Role of TNF\u3b1

Amyotrophic lateral sclerosis (ALS) is considered a multifactorial, multisystem disease in which inflammation and the immune system play important roles in development and progression. The pleiotropic cytokine TNF\u3b1 is one of the major players governing the inflammation in the central nervous system and peripheral districts such as the neuromuscular and immune system. Changes in TNF\u3b1 levels are reported in blood, cerebrospinal fluid, and nerve tissues of ALS patients and animal models. However, whether they play a detrimental or protective role on the disease progression is still not clear. Our group and others have recently reported opposite involvements of TNFR1 and TNFR2 in motor neuron death. TNFR2 mediates TNF\u3b1 toxic effects on these neurons presumably through the activation of MAP kinase-related pathways. On the other hand, TNFR2 regulates the function and proliferation of regulatory T cells (Treg) whose expression is inversely correlated with the disease progression rate in ALS patients. In addition, TNF\u3b1 is considered a procachectic factor with a direct catabolic effect on skeletal muscles, causing wasting. We review and discuss the role of TNF\u3b1 in ALS in the light of its multisystem nature

A 5-year clinical follow-up study from the Italian National Registry for FSHD.

BACKGROUND: The natural history of facioscapulohumeral muscular dystrophy (FSHD) is undefined. METHODS: An observational cohort study was conducted in 246 FSHD1 patients. We split the analysis between index cases and carrier relatives and we classified all patients using the Comprehensive Clinical Evaluation Form (CCEF). The disease progression was measured as a variation of the FSHD score performed at baseline and at the end of 5-year follow-up (ΔFSHD score). FINDINGS: Disease worsened in 79.4% (112/141) of index cases versus 38.1% (40/105) of carrier relatives and advanced more rapidly in index cases (ΔFSHD score 2.3 versus 1.2). The 79.1% (38/48) of asymptomatic carriers remained asymptomatic. The highest ΔFSHD score (1.7) was found in subject with facial and scapular weakness at baseline (category A), whereas in subjects with incomplete phenotype (facial or scapular weakness, category B) had lower ΔFSHD score (0.6) p < 0.0001. CONCLUSIONS: The progression of disease is different between index cases and carrier relatives and the assessment of the CCEF categories has strong prognostic effect in FSHD1 patients

Studio del meccanismo molecolare che porta alla distrofia muscolare: il ruolo della sovraespressione della proteina legante l’RNA FRG1.

La distrofia muscolare facio-scapolo-omerale (FSHD) è una miopatia genetica unica e complessa la cui patogenesi molecolare è ancora oscura. I topi che sovraesprimono FRG1, un gene situato nel locus FSHD 4q35 che codifica una proteina legante l'RNA, sono l'unico modello animale che sviluppa una distrofia muscolare con caratteristiche della malattia umana. Studiando i profili di espressione dei muscoli scheletrici di topi che sovraesprimono livelli crescenti di FRG1 a 28d (esordio della distrofia) e a 91d (distrofia completa) abbiamo osservato una profonda disregolazione trascrizionale che associa la gravità del fenotipo muscolare al livello di espressione di FRG1. Le analisi di Gene Ontology e Gene Set Enrichement mostrano un'ampia disregolazione dei geni specifici del muscolo, con il mantenimento delle isoforme embrionali e la mancanza di quelle mature. La nostra analisi ha rivelato il mantenimento eterocronico delle isoforme immature delle miosine, nonché l'espressione dell'isoforma embrionale del recettore dell'acetilcolina, della sarcolipina e dell'esochinasi 1. A partire da 14d i topi con sovraespressione di FRG1 presentano una decelerazione della curva di crescita e una ridotta area della sezione trasversale delle fibre muscolari prima della comparsa dei segni distrofici. Inoltre, il sequenziamento dell'RNA condotto a 14d ha rivelato che oltre all'espressione anomala di isoforme embrionali e neonatali legate alla contrazione muscolare, si verificano importanti cambiamenti metabolici che coinvolgono la gluconeogenesi. Inoltre, a partire dall'età di 7d nel muscolo FRG1 osserviamo l'attivazione della risposta allo stress legato all'asse IL6-CCL2-P38-AKT a causa del sovraccarico meccanico causato dall'immaturità muscolare. L'attivazione prolungata del meccanismo di risposta allo stress oltre i 14d determina l'attivazione di p53 e dei suoi effettori Cdkn1a e Gadd45a e l'insorgenza di un fenotipo secretorio associato a senescenza (SASP). Nel tempo, a partire dai 28d abbiamo rilevato una risposta infiammatoria progressiva nel muscolo FRG1 con il coinvolgimento di macrofagi, cellule T e progenitori fibroadipogenici (FAP). È interessante notare che l'analisi del profilo di espressione del muscolo transgenico FRG1 mostra una significativa sovrapposizione con i profili trascrizionali ottenuti nei muscoli di modelli animali e di pazienti affetti da distrofie muscolari e disturbi del motoneurone. Questa analisi ha rivelato un panorama trascrizionale comune che include l'alterazione delle pathway legate alla funzione muscolare, al metabolismo energetico e all'infiammazione. Questa osservazione suggerisce che a livello muscolare, indipendentemente dalla noxa originale, condizioni croniche determinano danni molecolari simili. Infine, esplorando la funzione molecolare di FRG1 abbiamo scoperto che si lega al lncRNA NEAT1 provocando la sovraespressione di NONO e HEXIM1 nei muscoli transgenici FRG1, tutti componenti dei corpi ribonucleoproteici. I nostri dati suggeriscono che NEAT1, FRG1, HEXIM1 e NONO costituiscono un complesso ribonucleoproteico che ha un ruolo nella risposta allo stress. Complessivamente i nostri studi suggeriscono il ruolo di FRG1 nella risposta allo stress e dimostrano che la sua sovraespressione cronica causa una compromissione globale della maturazione muscolare, un'alterazione del metabolismo energetico e l'attivazione del percorso di risposta allo stress che porta a senescenza e infiammazione. Tutti questi risultati hanno importanti implicazioni per la salute umana e, in particolare, per i soggetti portatori del difetto molecolare della FSHD che potrebbero essere più sensibili agli stress ambientali, aprendo una nuova prospettiva traslazionale per quanto riguarda i potenziali biomarcatori e le strategie terapeutiche.Facioscapulohumeral muscular dystrophy (FSHD) is a unique and complex genetic myopathy whose molecular pathogenesis is still obscure. Mice overexpressing FRG1, a gene located in the FSHD 4q35 locus coding a RNA binding protein, are the unique animal model developing a progressive muscular dystrophy with features of human disease. Investigating the expression profiles of skeletal muscles of mice overexpressing increasing levels of FRG1 at 28 d (dystrophy onset) and at 91 d (full dystrophy), we observed a profound transcriptional deregulation associating the severity of the muscle phenotype with the level of FRG1 expression. Gene Ontology and Gene Set Enrichment Analysis show extensive dysregulation of muscle specific genes, with maintenance of embryonic isoforms and lack of mature ones. Postnatal characterization of transgenic mice confirms the anomalous expression of embryonic/neonatal proteins fundamental for muscle structures, contraction and metabolism, revealing the heterochronic maintenance of immature isoforms of myosin light and heavy chains, as well as the expression of the embryonic isoform of the acetylcholine receptor, sarcolipin and hexokinase 1, and a global delay of muscle maturation. Starting from 14 d FRG1 overexpressing mice present deceleration of growth curve and reduced cross-sectional area of muscle fibers before the appearance of dystrophic signs. Therefore, the RNA sequencing conducted at 14 d revealed that beside the anomalous expression of embryonic and neonatal isoforms related to muscle contraction, major metabolic changes occur with the enhancement of gluconeogenesis and lipolysis. Remarkably, starting at age 7 d in FRG1 muscle we observe the activation of stress signal response pathway linked to IL6-CCL2-P38-AKT axis, due to mechanical overload caused by muscle immaturity. The prolonged activation of the stress response machinery beyond 14 d of age determines the activation of p53 and its downstream effectors Cdkn1a and Gadd45a and the onset of a secretory phenotype associated with senescence (SASP). Through time, from 28 d of age, we detected a progressive inflammatory response in FRG1 muscle with the involvement of macrophages, T-cells and fibroadipogenic progenitors (FAPs). Interestingly, expression profile analysis of FRG1 transgenic muscle shows significant overlap with transcriptional profiles obtained in muscles of animal models and patients affected by muscular dystrophies and motor neuron disorders. This analysis reveals a common transcriptional landscape including the alteration of pathways related to muscle function, energy metabolism and inflammation. This observation suggests that, regardless the original noxa, chronic conditions determine similar molecular damages in muscle. Remarkably, exploring the molecular function of FRG1 we detected its direct binding with the long non-coding RNA NEAT1, eliciting the overexpression of NONO and HEXIM1 in FRG1 transgenic muscles, all components of ribonucleoprotein bodies. Our data suggest that NEAT1, FRG1, HEXIM1 and NONO constitute a nuclear RNA-associated protein complex that is part of the cell stress response machinery. Overall our studies highlight the role of FRG1 in muscle response to contractile strain and demonstrate that the chronic overexpression of FRG1 causes a global impairment of muscle maturation, alteration of energy metabolism and activation of stress response pathways leading to senescence and inflammation. All these findings have important implications for human health. In particular, subjects carrying the FSHD molecular defect may be more sensitive to environmental stresses, opening new translational perspectives regarding potential biomarkers and therapeutic strategies for FSHD and other myopathies

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS

Phenotypic Variability Among Patients With D4Z4 Reduced Allele Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disorder, associated with the deletion of tandemly arrayed D4Z4 repetitive elements. The extensive use of molecular analysis of the D4Z4 locus for FSHD diagnosis has revealed wide clinical variability, suggesting that subgroups of patients exist among carriers of the D4Z4 reduced allele (DRA)

Additional file 3: of RNS60 exerts therapeutic effects in the SOD1 ALS mouse model through protective glia and peripheral nerve rescue

Figure S3. A-D) Representative images of LSC micrographs stained with S100β (green) and GFAP (red) at 20 weeks of age. Scale bar: 20 μm. E) Quantification of immunofluorescence showed no differences between the two transgenic groups. Bar graphs represents mean ± SEM, (n = 5 animals per group); One-way ANOVA followed by post hoc Fisher’s LSD non-parametric test (p = 0.420). (DOCX 1342 kb

Additional file 2: of RNS60 exerts therapeutic effects in the SOD1 ALS mouse model through protective glia and peripheral nerve rescue

Figure S2. Representative immunoblot for GFAP performed on ventral portion of LSC of NTG mice or transgenic mice treated with NS or RNS60, at 20 weeks of age, and relative quantification. Data are expressed as mean ± SEM, (n = 5 animals per group). Data were statistically analyzed using one way ANOVA followed by post hoc Fisher’s LSD. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, n.s. = non significant. (DOCX 162 kb