Background An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), referred to as C9ALS/FTD. No cure or effective treatment exist for C9ALS/FTD. Three major molecular mechanisms have emerged to explain C9ALS/FTD disease mechanisms: (1) C9ORF72 loss-of-function through haploinsufficiency, (2) dipeptide repeat (DPR) proteins mediated toxicity by the translation of the repeat RNAs, and more controversial, (3) RNA-mediated toxicity by bidirectional transcription of the repeats that form intranuclear RNA foci. Recent studies indicate a double-hit pathogenic mechanism in C9ALS/FTD, where reduced C9ORF72 protein levels lead to impaired clearance of toxic DPRs. Here we explored whether pharmacological compounds can revert these pathological hallmarks in vitro and cognitive impairment in a C9ALS/FTD mouse model (C9BAC). We specifically focused our study on small molecule inhibitors targeting chromatin-regulating proteins (epidrugs) with the goal of increasing C9ORF72 gene expression and reduce toxic DPRs. Results We generated luciferase reporter cell lines containing 10 (control) or ≥ 90 (mutant) G4C2 HRE located between exon 1a and 1b of the human C9ORF72 gene. In a screen of 14 different epidrugs targeting bromodomains, chromodomains and histone-modifying enzymes, we found that several bromodomain and extra-terminal domain (BET) inhibitors (BETi), including PFI-1 and JQ1, increased luciferase reporter activity. Using primary cortical cultures from C9BAC mice, we further found that PFI-1 treatment increased the expression of V1-V3 transcripts of the human mutant C9ORF72 gene, reduced poly(GP)-DPR inclusions but enhanced intranuclear RNA foci. We also tested whether JQ1, an BETi previously shown to reach the mouse brain by intraperitoneal (i.p.) injection, can revert behavioral abnormalities in C9BAC mice. Interestingly, it was found that JQ1 administration (daily i.p. administration for 7 days) rescued hippocampal-dependent cognitive deficits in C9BAC mice. Conclusions Our findings place BET bromodomain inhibitors as a potential therapy for C9ALS/FTD by ameliorating C9ORF72-associated pathological and behavioral abnormalities. Our finding that PFI-1 increases accumulation of intranuclear RNA foci is in agreement with recent data in flies suggesting that nuclear RNA foci can be neuroprotective by sequestering repeat transcripts that result in toxic DPRs.

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

No abstract

The masticatory system is a coordinated machine, highly adaptable to environmental demands. Increased levels of interleukin 6 (IL6) in the synovial fluid have been associated with temporomandibular disorders. Although they are mostly suggested released from inflammatory cells, it has been reported that IL6 is also a “myokine”, released by muscle cells during activity or in pathological processes. A pivotal role of IL6 in skeletal muscle and bone has been suggested, being related to both anabolic and catabolic processes. We here propose that IL6 is released by masseter muscle during activity and that a deregulated increased level of muscle‐derived IL6 correlates to the musculoskeletal impairment in animal models of masticatory muscles unloading. We induced masseter muscle hypofunction in male BalbC mice (8 weeks old) by either soft diet (SD) consumption (pellet:water=1:3) or unilateral paralysis of masseter muscles injected with Botulinum Toxin Type A (BoNTA; 0.2U/10 µL). Both masseter muscles and mandibles were dissected after 2‐14 days for morphology, histology, or molecular measurements. Masseter muscles were electrically stimulated (ES) in vitro for demonstrating activity‐evoked changes in IL6 expression and secretion. All procedures were approved by the Institutional Committee for the Care and Use of Animals of the Universidad de Chile (# 17011‐OD‐UCH). We demonstrated that IL6 is expressed and released from masseter muscle after ES, in a frequency‐dependent manner. IL6 expression after ES requires the release of ATP from muscle, and activation of P2Y/P2X receptors. In the animal models of SD or BoNTA, atrophy of masseter muscle is observed (reduction of muscle volume, mass, and fibers diameter, as well as increased expression of Atrogin‐1/MURF‐1/Myogenin) after 7‐14 days. Increased resting levels of extracellular ATP and IL6 expression were detected in masseter muscles of SD‐fed mice and BoNTA‐injected muscles. In the later, an increased expression of ATP releaser molecules (pannexin 1, connexin 43‐45) was reported, as well as increased levels of ATP receptors P2Y2 and P2X7. Interestingly, the metabolization of extracellular ATP using Apyrase restored the resting levels of IL6 expression in masseter muscles derived from SD‐fed mice. Also, changes in morphology and bone loss were observed in mandibular condyles associated with BoNTA‐injected masseter muscles. We here report that IL6 is expressed and released by masseter muscles, either at rest and after muscle activity. Moreover, masseter muscle hypofunction induces an overactivation of the extracellular ATP‐IL6 axis in muscles. We are currently addressing if this deregulated signaling is involved in the muscle atrophy and bone loss evoked by masticatory muscle unloading. Understanding the molecular pathways involved in muscle‐bone crosstalk at the masticatory system will contribute to propose strategies to avoid non‐desirable effects in pathologies or clinical interventions presenting muscle unloading.

Introduction Musculoskeletal disorders of the masticatory system impact the quality of life and have a high cost of diagnosis and treatment. Masseter muscle hyperactivity is a cause of pain in the chewing apparatus. Botulinum toxin type A (BoNTA) injection is widely used to induce paralysis of the masseter muscle, thereby decreasing impaired muscle activity. However, our laboratory has described in a preclinical model that the injection of BoNTA not only induces paralysis, but also muscle atrophy, which subsequently decreases bone quality. However, it is unknown whether apoptosis or autophagy mechanisms could contribute to muscle atrophy. Aim To evaluate the induction of apoptosis and autophagy in BoNTA‐injected masseter muscle of adult mice. Methodology Unilateral injection of BoNTA (0.2U/10µl) in the masseter muscle was performed in adult BalbC mice (approved by IACUC‐Universidad de Chile, #21446‐ODO‐UCH). Apoptosis and autophagy markers were evaluated in masseter muscles by immunoblot at 2‐7d post‐injection and immunofluorescence at 7d post‐injection. Autophagy activity was blocked by i.p injection of chloroquine. The data were evaluated with t‐test, one‐way ANOVA test, Mann‐Whitney test, or Kruskal‐Wallis test, as appropriate. The results were expressed as mean ±SEM (n=4‐8; p <0.05). Results Unilateral injection of BoNTA did not change the relative levels of apoptosis‐related proteins like cleaved Caspase 3, PARP, and AIF. There was a significant increase in protein levels related to autophagy, such as lipidated LC3 (1.86‐fold), non‐lipidated LC3 (1.66‐fold), and P62 (1.24‐fold) at 7 d post‐injection. Also, there was a strong punctuated stain for LC3 in histological sections of masseter muscle 7d post injection, suggesting autophagic vesicles. However, the injection of chloroquine for blocking the autophagy flux did not improve the accumulation of autophagy proteins in masseter muscle evoked by BoNTA in the induction. Conclusions The atrophy of masseter muscle evoked by BoNTA injection is not related to autophagy‐ or apoptosis‐induction. The increase in autophagy markers after BoNTA injection, with no further increase after autophagic flux blockade, suggests that BoNTA may be blocking autophagy in the masseter muscle, perhaps favoring a proteasomal pathway of muscle atrophy.

During exercise, there is a redox imbalance that increases reactive oxygen species, generating oxidative stress that favors muscle damage, affecting physical and sports performance. Different antioxidant supplements have been used to optimize muscle recovery after physical exercise. However, the effects of various antioxidants have been counterproductive, since the reactive oxygen species generated by exercise are necessary for muscle adaptation and their reduction prevents correct intracellular signaling and consequently the adaptation of skeletal muscle to exercise. Polyphenols are a type of antioxidant that are present in various fruits and herb extracts that have different characteristics depending on their structure and composition, some presenting a cell signaling pathway similar to exercise, generating different stimuli at the skeletal muscle level, being able to reduce the oxidative damage generated by exercise and even being able to enhance its effects. The aim of this literature review is to describe the effect of various polyphenols on muscle damage and biomarkers of oxidative stress on different forms of physical exercise and sports in adults. Durante la realización de ejercicio, existe un desbalance redox que aumenta las especies reactivas de oxígeno generando un estrés oxidativo que favorece el daño muscular incidiendo en el rendimiento físico y deportivo. Diferentes suplementos antioxidantes han sido utilizados para optimizar la recuperación muscular posterior a la realización de ejercicio físico. Sin embargo, los efectos de diversos antioxidantes han sido contraproducentes, ya que las especies reactivas de oxígeno generadas por el ejercicio son necesarias para la adaptación muscular y la disminución de éstas, impide la correcta señalización intracelular y con esto, la adaptación del músculo esquelético frente al ejercicio físico. Los polifenoles son un tipo de antioxidantes que están presentes en diversos frutos y extractos de hierbas que poseen diversas características dependiendo de su estructura y composición, presentando algunos una ruta de señalización celular similar al ejercicio físico, generando diferentes estímulos a nivel del músculo esquelético, pudiendo reducir el daño oxidativo generado por el ejercicio e incluso, pudiendo potenciar sus efectos. El objetivo de la presente revisión a la literatura es determinar y describir el efecto de diversos polifenoles en el daño muscular y marcadores de estrés oxidativo posterior a diferentes modalidades de ejercicio físico y deportes en adultos.

The masticatory system is a finely coordinated machine, where minimal deregulation induces alterations of the whole system. Botulinum toxin type A (BoNTA) injection in masseter muscle is widely used as a clinical/aesthetic procedure. However, the non‐desirable effects of masseter muscle paralysis in neighbor masticatory muscles or mandibular bone are unknown. In the current work, we aimed to evaluate musculoskeletal loss at the masticatory system after BoNTA‐injection in masseter muscle of adult mice. Unilateral paralysis of masseter muscles was induced in male BalbC mice (8 weeks‐old) by single‐injection of BoNTA (0.2U/10 µL) in the right muscle and saline solution in the left muscle. Masticatory muscles (masseter, temporalis, lateral‐ and medial‐pterygoid) and mandibles were dissected after 2‐7‐14d for molecular measurements (qPCR), histology, or morphology (microcomputed tomography). All procedures were approved by the IACUC‐Universidad de Chile (# 20381‐ODO‐UCH). T‐test or Wilcoxon‐test were used for statistical comparisons between BoNTA‐ and Control‐side (n=3‐10, p<0,05). The expression of molecular markers of atrophy (Atrogin1, Murf1) and regeneration (myogenin) significantly increased in the BoNTA‐side, as early as 2‐7d, in the intervened masseter but also in the ipsilateral temporalis and medial pterygoid muscles. There was a time‐dependent reduction in masseter fibers diameter from 2d to 14d. Masseter, temporalis, and medial pterygoid’s volumes significantly reduced in the BoNTA‐injected side at 14d. Interestingly, the volume of lateral pterygoid increased in the BoNTA side, with no change in atrophy/regeneration markers. Microstructural analysis of mandibular condyles demonstrated a progressive bone loss in the BoNTA‐injected side, starting as early as 7d and worsening 14d after the intervention. In conclusion, BoNTA injection in the masseter muscle impairs the whole masticatory system, inducing atrophy of masseter but also the ipsilateral temporalis and medial pterygoid, and even early‐bone loss of the mandibular condyle at the injected side. Then, clinical uses of BoNTA in masticatory muscles should be avoided or carried out under strict morpho‐functional monitoring.