Clinical-grade intranasal NGF fuels neurological and metabolic functions of Mecp2-deficient mice.

MECP2 deficiency causes a broad spectrum of neuropsychiatric disorders that can affect both genders. Rett syndrome is the most common and is characterized by an apparently normal growth period followed by a regression phase in which patients lose most of their previously acquired skills. After this dramatic period, various symptoms progressively appear, including severe intellectual disability, epilepsy, apraxia, breathing abnormalities and motor deterioration. MECP2 encodes for an epigenetic transcription factor that is particularly abundant in the brain; consequently, several transcriptional defects characterize the Rett syndrome brain. The well-known deficiency of several neurotrophins and growth factors, together with the positive effects exerted by Trofinetide, a synthetic analogue of insulin-like growth factor 1, in Rett patients and in mouse models of Mecp2 deficiency, prompted us to investigate the therapeutic potential of nerve growth factor. Initial in vitro studies demonstrated a healing effect of rhNGF on neuronal maturation and activity in cultured Mecp2-null neurons. Subsequently, we designed in vivo studies with clear translational potential using intranasally administered recombinant human GMP-grade NGF (rhNGF) already used in the clinic. Efficacy of rhNGF in vivo in Mecp2-null hemizygous male mice and heterozygous female mice was assessed. General well-being was evaluated by a conventional phenotypic score and motor performance through the Pole and Beam Walking tests, while cognitive function and interaction with the environment were measured by the Novel Object Recognition Test and the Marble Burying test, respectively. At the end of the treatment, mouse cortices were dissected and bulk RNA sequencing was performed to identify the molecular pathways involved in the protective effects of rhNGF. rhNGF exerted positive effects on cognitive and motor functions in both male and female mouse models of Rett syndrome. In male hemizygous mice, which suffer from significantly more severe and rapidly advancing symptoms, the drug's ability to slow the disease's progression was more pronounced. The unbiased research for the molecular mechanisms triggering the observed benefits revealed a strong positive effect on gene sets related to oxidative phosphorylation, mitochondrial structure and function. These results were validated by demonstrating the drug's ability to improve mitochondrial structure and respiration in Mecp2-null cerebral cortices. Furthermore, GO analyses indicated that NGF exerted the expected improvement in neuronal maturation. We conclude that intranasal administration of rhNGF is a non-invasive and effective route of administration for the treatment of Rett syndrome and possibly for other neurometabolic disorders with overt mitochondrial dysfunction.

Sickness absenteeism among health care workers in the municipal public service of Chapecó-SC, in the period 2015-2018.

Introduction: Absenteeism of health workers is important because it interferes with the quality of care provided to patients. Objectives: To characterize the absenteeism-illness of workers in the municipal public health network in Chapecó, SC, Brazil (2015-2018) and test the association of two or more absences in the year with the study variables. Methods: A crosssectional study was conducted, and the variables studied were sex; age group; professional category; acting time; International Classification of Diseases and Related Health Problems, and sick leave. Descriptive analysis were performed, the factors associated with the outcome were tested, and the prevalence ratios were calculated with their respective 95% confidence intervals using Poisson regression. Results: A total of 1,695 professionals on sick leave were identified, with a higher prevalence of women (89.40%), in the 30-39 age group (33.41%), the majority with one sick leave per year (61.24%), from 3 to 9 days (47.67%). Community health workers were the category that most frequently had sick leaves (27.15%). In the years studied, there were 2,795 sick leaves (657 employees with more than one sick leaves). Musculoskeletal disorders were the main causes (21.80%) and the highest prevalence was dorsopathies (57.60%). Working for 21 years or more had a 49% higher prevalence ratio for two or more sick leaves per year, compared to having been working for up to 5 years. Conclusions: The study allowed us to characterize absenteeism-illness among workers in the healthcare services in Chapecó, SC. The results may constitute indicators of human resource management and foster strategies to promote healthy environments, prevention of diseases and injuries, and rehabilitation.

Introdução: O absenteísmo de trabalhadores da saúde é importante por interferir na qualidade da assistência prestada aos pacientes. Objetivos: Caracterizar o absenteísmo-doença dos trabalhadores da rede pública municipal de saúde de Chapecó, no estado de Santa Catarina, no período de 2015 a 2018 e testar a associação de dois ou mais afastamentos no ano com as variáveis de estudo. Métodos: Foi realizado um estudo transversal, e as variáveis estudadas foram sexo, faixa etária, categoria profissional, tempo de atuação, Classificação Internacional de Doenças e tempo de afastamento. Foram realizadas análises descritivas, testados os fatores associados ao desfecho e calculadas as razões de prevalência com os respectivos intervalos de confiança de 95% por meio da regressão de Poisson. Resultados: Foram identificados 1.695 profissionais afastados por doença, com maior prevalência do sexo feminino (89,40%), na faixa de 30 a 39 anos (33,41%), e a maioria apresentou um afastamento ao ano (61,24%), de 3 a 9 dias (47,67%). Os agentes comunitários de saúde foram a categoria com mais afastamentos (27,15%). Nos anos estudados, houve 2.795 afastamentos (657 servidores com mais de um afastamento). As doenças osteomusculares foram as principais causas identificadas (21,80%), com maior prevalência de dorsopatias (57,60%). Atuar há 21 anos ou mais apresentou razão de prevalência 49% maior para dois ou mais afastamentos no ano em comparação a estar atuando por até 5 anos. Conclusões: O estudo permitiu caracterizar o absenteísmo-doença entre trabalhadores da rede municipal de saúde de Chapecó. Os resultados encontrados poderão constituirse como indicadores de gestão de recursos humanos e fomentar estratégias de promoção de ambientes saudáveis, prevenção de doenças e agravos e reabilitação.

Advanced genetic therapies for the treatment of Rett syndrome: state of the art and future perspectives.

Loss and gain of functions mutations in the X-linked MECP2 (methyl-CpG-binding protein 2) gene are responsible for a set of generally severe neurological disorders that can affect both genders. In particular, Mecp2 deficiency is mainly associated with Rett syndrome (RTT) in girls, while duplication of the MECP2 gene leads, mainly in boys, to the MECP2 duplication syndrome (MDS). No cure is currently available for MECP2 related disorders. However, several studies have reported that by re-expressing the wild-type gene is possible to restore defective phenotypes of Mecp2 null animals. This proof of principle endorsed many laboratories to search for novel therapeutic strategies to cure RTT. Besides pharmacological approaches aimed at modulating MeCP2-downstream pathways, genetic targeting of MECP2 or its transcript have been largely proposed. Remarkably, two studies focused on augmentative gene therapy were recently approved for clinical trials. Both use molecular strategies to well-control gene dosage. Notably, the recent development of genome editing technologies has opened an alternative way to specifically target MECP2 without altering its physiological levels. Other attractive approaches exclusively applicable for nonsense mutations are the translational read-through (TR) and t-RNA suppressor therapy. Reactivation of the MECP2 locus on the silent X chromosome represents another valid choice for the disease. In this article, we intend to review the most recent genetic interventions for the treatment of RTT, describing the current state of the art, and the related advantages and concerns. We will also discuss the possible application of other advanced therapies, based on molecular delivery through nanoparticles, already proposed for other neurological disorders but still not tested in RTT.

Ascorbic Acid Route to the Endoplasmic Reticulum: Function and Role in Disease.

Significance: Humans cannot synthesize ascorbic acid (AscH2) (vitamin C), so deficiencies in dietary AscH2 cause the life-threatening disease of scurvy and many other diseases. After oral ingestion, plasma AscH2 concentrations are strictly controlled by transporters, which are required for entry into the cell and into intracellular organelles. Recent Advances: Besides its general antioxidant function, AscH2 is a cofactor for endoplasmic reticulum (ER)-localized collagen hydroxylases. Its important role in ER homeostasis is also highlighted by the fact that AscH2 deficiency in auxotrophic species triggers ER stress. Critical Issues: Characterizations of the molecular basis of diseases suggest that intracellular AscH2 deficiency is due not only to limited dietary access but also to its limited intracellular transport and net loss under conditions of intracellular hyperoxidation in the ER. This essay will offer an overview of the different transporters of vitamin C regulating its intracellular concentration, its function inside the ER, and the phenotypes of the diseases that can be triggered by increased depletion of this vitamin in the ER. Future Directions: When considering the benefits of increasing dietary AscH2, it is important to consider pharmacokinetic differences in the bioavailability between orally and intravenously administered AscH2: the latter bypasses intestinal absorption and is, therefore, the only route that can lead to the high plasma concentrations that may provide some health effects, and it is this route that needs to be chosen in clinical trials for those diseases associated with a deficiency of AscH2. Antioxid. Redox Signal. 34, 845-855.

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

Physical and Functional Cross Talk Between Endo-Sarcoplasmic Reticulum and Mitochondria in Skeletal Muscle.

Significance: The physiological relevance of contacts between the sarcoplasmic reticulum (SR), a specialized domain of the endoplasmic reticulum (ER) in skeletal muscle, and mitochondria is still not clear. Recent Advances: An extensive close proximity of these two organelles is a late developmental event, which suggests that it does not have an essential function. Critical Issues: The intimate association of SR/mitochondria develops during murine postnatal differentiation and the recovery of denervated atrophic muscle, which suggests that this is a highly regulated process with a specific function. Analyses of mouse models for muscle diseases suggest that impaired ER/SR-mitochondrial contacts may be due to ER stress and lead to defective bioenergetics and insulin signaling. Future Directions: Future studies are necessary to identify the molecular determinants weakening insulin signaling upon impairment of ER/mitochondrial contacts in skeletal muscles as well as to analyze the distance between SR/ER and mitochondria in muscle diseases associated with ER stress.

SELENON (SEPN1) protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance.

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to a congenital myopathy associated with insulin resistance (SEPN1-related myopathy). The exact cause of the insulin resistance in patients with SELENON loss of function is not known. Skeletal muscle is the main contributor to insulin-mediated glucose uptake, and a defect in this muscle-related mechanism triggers insulin resistance and glucose intolerance. We have studied the chain of events that connect the loss of SELENON with defects in insulin-mediated glucose uptake in muscle cells and the effects of this on muscle performance. Here, we show that saturated fatty acids are more lipotoxic in SELENON-devoid cells, and blunt the insulin-mediated glucose uptake of SELENON-devoid myotubes by increasing ER stress and mounting a maladaptive ER stress response. Furthermore, the hind limb skeletal muscles of SELENON KO mice fed a high-fat diet mirrors the features of saturated fatty acid-treated myotubes, and show signs of myopathy with a compromised force production. These findings suggest that the absence of SELENON together with a high-fat dietary regimen increases susceptibility to insulin resistance by triggering a chronic ER stress in skeletal muscle and muscle weakness. Importantly, our findings suggest that environmental cues eliciting ER stress in skeletal muscle (such as a high-fat diet) affect the pathological phenotype of SEPN1-related myopathy and can therefore contribute to the assessment of prognosis beyond simple genotype-phenotype correlations.

A maladaptive ER stress response triggers dysfunction in highly active muscles of mice with SELENON loss.

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca2+ uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca2+ uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca2+ uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.

Endoplasmic Reticulum Oxidative Stress Triggers Tgf-Beta-Dependent Muscle Dysfunction by Accelerating Ascorbic Acid Turnover.

Endoplasmic reticulum (ER) and oxidative stress are two related phenomena that have important metabolic consequences. As many skeletal muscle diseases are triggered by oxidative stress, we explored the chain of events linking a hyperoxidized ER (which causes ER and oxidative stress) with skeletal muscle dysfunction. An unbiased exon expression array showed that the combined genetic modulation of the two master ER redox proteins, selenoprotein N (SEPN1) and endoplasmic oxidoreductin 1 (ERO1), led to an SEPN1-related myopathic phenotype due to excessive signalling of transforming growth factor (TGF)-beta. The increased TGF-beta activity in the genetic mutants was caused by accelerated turnover of the ER localized (anti-oxidant) ascorbic acid that affected collagen deposition in the extracellular matrix. In a mouse mutant of SEPN1, which is dependent on exogenous ascorbic acid, a limited intake of ascorbic acid revealed a myopathic phenotype as a consequence of an altered TGF-beta signalling. Indeed, systemic antagonism of TGF-beta re-established skeletal muscle function in SEPN1 mutant mice. In conclusion, this study sheds new light on the molecular mechanism of SEPN1-related myopathies and indicates that the TGF-beta/ERO1/ascorbic acid axis offers potential for their treatment.

Early modulation of pro-inflammatory microglia by minocycline loaded nanoparticles confers long lasting protection after spinal cord injury.

Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI.