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Hereditary myopathies are a group of genetically determined muscle disorders comprising more than 300 entities. In Chile, there are no specific registries of the distinct forms of these myopathies. We now report the genetic findings of a series of Chilean patients presenting with limb-girdle muscle weakness of unknown etiology. Eighty-two patients were explored using high-throughput sequencing approaches with neuromuscular gene panels, establishing a definite genetic diagnosis in 49 patients (59.8%) and a highly probable genetic diagnosis in eight additional cases (9.8%). The most frequent causative genes identified were DYSF and CAPN3, accounting for 22% and 8.5% of the cases, respectively, followed by DMD (4.9%) and RYR1 (4.9%). The remaining 17 causative genes were present in one or two cases only. Twelve novel variants were identified. Five patients (6.1%) carried a variant of uncertain significance in genes partially matching the clinical phenotype. Twenty patients (24.4%) did not carry a pathogenic or likely pathogenic variant in the phenotypically related genes, including five patients (6.1%) presenting an autoimmune neuromuscular disorder. The relative frequency of the different forms of myopathy in Chile is like that of other series reported from different regions of the world with perhaps a relatively higher incidence of dysferlinopathy.

In the last decade, a number of important research advances in different fields have allowed Down syndrome (DS) research to flourish, creating a time of both unparalleled opportunity and considerable challenge. Building a scientific framework that distills mechanisms involved in the developmental intellectual disability of DS as well as the early-onset component of Alzheimer disease and the several other comorbidities associated with the condition is a challenge that scientists are now tackling using novel technologies and multidisciplinary approaches. The Trisomy 21 Research Society (T21RS) was founded in 2014 to address these evolving needs and challenges. In June of 2017, the T21RS held its 2nd International Conference in Chicago, USA. With more than 200 scientists, advocates, people with DS, and family members in attendance, the meeting served as a forum for the discussion of the latest research and clinical advances as well as the most compelling needs of people with DS and their families.

Background In type I diabetes mellitus (T1DM) pancreatic β cells are destroyed. Treatment entails exogenous insulin administration and strict diet control, yet optimal glycemic control is hardly attainable. Islet transplant could be an alternative in patients with poor glycemic control, but inefficient islet purification and autoimmune response of patients is still a challenge. For these reasons, it is necessary to explore new cellular sources and immunological isolation methods oriented to develop T1DM cell-based therapies. Aims We postulate human adipose-derived stem cell (hASC) as an adequate source to generate pancreatic islet cells in vitro, and to produce islet-like structures. Furthermore, we propose microencapsulation of these aggregates as an immunological isolation strategy. Methods hASC obtained from lipoaspirated fat tissue from human donors were differentiated in vitro to insulin (Ins) and glucagon (Gcg) producing cells. Then, insulin producing cells (IPC) and glucagon producing cells (GPC) were cocultured in low adhesion conditions to form cellular aggregates, and later encapsulated in a sodium alginate polymer. Expression of pancreatic lineage markers and secretion of insulin or glucagon in vitro were analyzed. Results The results show that multipotent hASC efficiently differentiate to IPC and GPC, and express pancreatic markers, including insulin or glucagon hormones which they secrete upon stimulation (fivefold for insulin in IPC, and fourfold for glucagon, compared to undifferentiated cells). In turn, calculation of the Feret diameter and area of cellular aggregates revealed mean diameters of ~ 80 µm, and 65% of the aggregates reached 4000 µm2 at 72 h of formation. IPC/GPC aggregates were then microencapsulated in sodium-alginate polymer microgels, which were found to be more stable when stabilized with Ba2+, yielding average diameters of ~ 300 µm. Interestingly, Ba2+-microencapsulated aggregates respond to high external glucose with insulin secretion. Conclusions The IPC/GPC differentiation process from hASC, followed by the generation of cellular aggregates that are later microencapsulated, could represent a possible treatment for T1DM.

Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agent N-acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela&rsquo, s inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.

The aim of this study was to explore the preparation of porous nanocomposite scaffolds with simultaneous osteogenic – antibacterial properties by incorporating copper – doped bioactive glass nanoparticles into Poly (D,L-lactide-co-glycolide) lactide:glycolide. Bioactive glass nanoparticles were synthesized by using sol–gel technique from the SiO2 – P2O5 – CaO – Na2O – CuO system. Poly (D,L-lactide-co-glycolide) lactide:glycolide nanocomposite scaffolds with different nanoparticle contents were prepared by combined lyophilization/salt leaching. The in vitro bioactivity of the scaffolds was assessed in simulated body fluid, and cell viability and osteogenic differentiation assays were performed with stem cells. Antibacterial activity of the materials was assessed against Staphylococcus aureus. Copper – dopped bioactive glass nanoparticles particles with ∼70 nm in size and relatively crystalline structure were synthesized. Porous nanocomposite scaffolds prepared with the copper – doped nanoparticles are cytocompatible, promoted the mineralization of bone-like apatite in simulated body fluid, and stimulated the osteogenic differentiation of stem cells as judged by an increased activity the enzyme alkaline phosphatase. The antibacterial activity exhibited by the nanocomposite scaffolds was not statistically superior to that of the neat polymer scaffold. Development of greater antibacterial activity in these nanocomposites would requires further research primarily related to the synthesis of more amorphous and soluble copper – dopped bioactive glass nanoparticles.

Poly(lactic acid) (PLA) is a biodegradable and biocompatible polyester widely used in biomedical applications. Unfortunately, this biomaterial suffers from some shortcomings related with the absence of both bioactivity and antibacterial capacity. In this work, composites of PLA with either graphene oxide (GO) or thermally reduced graphene oxide (TrGO) were prepared by melt mixing to overcome these limitations. PLA composites with both GO and TrGO inhibited the attachment and proliferation of Escherichia coli and Staphylococcus aureus bacteria depending on the kind and amount of filler. Noteworthy, it is shown that by applying an electrical stimulus to the percolated PLA/TrGO, the antibacterial behavior can be dramatically increased. MTT analysis showed that while all the PLA/GO composites were more cytocompatible to osteoblast-like cells (SaOS-2) than pure PLA, only low content of TrGO was able to increase this property. These tendencies were related with changes in the surface properties of the resulting polymer composites, such as polarity and roughness. In this way, the addition of GO and TrGO into a PLA matrix allows the development of multifunctional composites for potential applications in biomedicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1051-1060, 2018.

The goal of the present work was to analyze the effect of copper nanoparticles (CNP) embedded in polymer matrices on the cell viability. Both calcium alginate (a natural hydrogel) and polypropylene (a synthetic thermoplastic) matrices were used. Cell viability was tested for cerebral cortex of normal mouse fetuses and human osteosarcoma (SAOS-2). The viability strongly depends on the polymer characteristics as hydrogels presented cytotoxicity at concentrations higher than 0.5 wt% of CNP, whereas polypropylene does not show any effect even at concentrations as high as 20 wt%. These results were explained by the different copper species released from the composites.

Down syndrome (DS) is a developmental disorder that results from the trisomy of chromosome 21. DS patients show several abnormalities including cognitive deficits. Here, we show enhanced activation of the extracellular signal-regulated kinase (ERK), a kinase that critically regulates synaptic plasticity and memory, in a hippocampal cell line derived from trisomy 16 mouse foetus. In addition, these cells show enhanced activation of p38 mitogen-activated protein kinase (p38 MAPK). The hyper-activation of ERK and p38 MAPK is significantly reduced by a small peptide, Gly-Pro-Glu (GPE), derived from insulin-like growth factor-1. In addition, the trisomic cells show reduced level of inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), which is enhanced by GPE. Furthermore, the trisomic cells do not show ERK activation in response to KCl depolarization or forskolin treatment. Importantly, ERK activation by these stimuli is observed after GPE treatment of the cells. These results suggest that GPE may help reduce aberrant signalling in the trisomic neurons by affecting MAPK and GSK-3β activation.

Mesenchymal stem cells (MSCs) can trans/differentiate to neural precursors and/or mature neurons and promote neuroprotection and neurogenesis. The above could greatly benefit neurodegenerative disorders as well as in the treatment of post-traumatic and hereditary diseases of the central nervous system (CNS). In order to attain an ideal source of adult MSCs for the treatment of CNS diseases, adipose tissue, bone marrow, skin and umbilical cord derived MSCs were isolated and studied to explore differences with regard to neural differentiation capacity. In this study, we demonstrated that MSCs from several tissues can differentiate into neuron-like cells and differentially express progenitors and mature neural markers. Adipose tissue MSCs exhibited significantly higher expression of neural markers and had a faster proliferation rate. Our results suggest that adipose tissue MSCs are the best candidates for the use in neurological diseases.

Mutations in the dysferlin gene are linked to a group of muscular dystrophies known as dysferlinopathies. These myopathies are characterized by progressive atrophy. Studies in muscle tissue from dysferlinopathy patients or dysferlin-deficient mice point out its importance in membrane repair. However, expression of dysferlin homologous proteins that restore sarcolemma repair function in dysferlinopathy animal models fail to arrest muscle wasting, therefore suggesting that dysferlin plays other critical roles in muscle function. In the present review, we discuss dysferlin functions in the skeletal muscle, as well as pathological mechanisms related to dysferlin mutations. Particular focus is presented related the effect of dysferlin on cell membrane related function, which affect its repair, vesicle trafficking, as well as Ca(2+) homeostasis. Such mechanisms could provide accessible targets for pharmacological therapies.

The fabrication of a nanoporous silica coating loaded with bioactive glass nanoparticles (nBG/NSC) on titanium dental implant surface and its in vitro and in vivo evaluation is presented. The coating was produced by a combined sol–gel and evaporation induced self-assembly process. In vitro bioactivity was assessed in simulated body fluid (SBF) and investigating the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). A rat tibial model was employed to analyze the bone response to nBG/NSC-modified titanium implant surface in vivo. The nBG/NSC coating was confirmed at nano level to be constituted by a highly ordered nanoporous silica structure. The coating nanotopography in conjunction with the bioactivity of the BG particles accelerate the in vitro apatite formation and promote the osteogenic differentiation of hBMSCs in absence of osteogenic supplements. These properties accelerate the formation of bone tissue in the periphery of the implant after 3 weeks of implantation. Backscattered scanning electron microscopy images revealed the presence of gaps and soft tissue in the unmodified implant after 6 weeks, whereas the nBG/NSC-modified implant showed mature bone in intimate contact with the implant surface. The nBG/NSC coating appears promising for accelerating the osseointegration of dental implants.

Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.

Poly(lactic acid) (PLA) is a biodegradable and biocompatible polyester widely used in biomedical applications. Unfortunately, this biomaterial suffers from some shortcomings related with the absence of both bioactivity and antibacterial capacity. In this work, composites of PLA with either graphene oxide (GO) or thermally reduced graphene oxide (TrGO) were prepared by melt mixing to overcome these limitations. PLA composites with both GO and TrGO inhibited the attachment and proliferation of Escherichia coli and Staphylococcus aureus bacteria depending on the kind and amount of filler. Noteworthy, it is shown that by applying an electrical stimulus to the percolated PLA/TrGO, the antibacterial behavior can be dramatically increased. MTT analysis showed that while all the PLA/GO composites were more cytocompatible to osteoblast-like cells (SaOS-2) than pure PLA, only low content of TrGO was able to increase this property. These tendencies were related with changes in the surface properties of the resulting polymer composites, such as polarity and roughness. In this way, the addition of GO and TrGO into a PLA matrix allows the development of multifunctional composites for potential applications in biomedicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1051-1060, 2018.

Dynamin-2 is a ubiquitously expressed GTP-ase that mediates membrane remodeling. Recent findings indicate that dynamin-2 also regulates actin dynamics. Mutations in dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital myopathy characterized by progressive weakness and atrophy of skeletal muscles. However, the muscle-specific roles of dynamin-2 affected by these mutations remain elusive. Here we show that, in muscle cells, the GTP-ase activity of dynamin-2 is involved in de novo actin polymerization as well as in actin-mediated trafficking of the glucose transporter GLUT4. Expression of dynamin-2 constructs carrying CNM-linked mutations disrupted the formation of new actin filaments as well as the stimulus-induced translocation of GLUT4 to the plasma membrane. Similarly, mature muscle fibers isolated from heterozygous knock-in mice that harbor the dynamin-2 mutation p.R465W, an animal model of CNM, exhibited altered actin organization, reduced actin polymerization and impaired insulin-induced translocation of GLUT4 to the sarcolemma. Moreover, GLUT4 displayed aberrant perinuclear accumulation in biopsies from CNM patients carrying dynamin-2 mutations, further suggesting trafficking defects. These results suggest that dynamin-2 is a key regulator of actin dynamics and GLUT4 trafficking in muscle cells. Our findings also support a model in which impairment of actin-dependent trafficking contributes to the pathological mechanism in dynamin-2-associated CNM.

The Na+/myo-inositol cotransporter (SMIT1) is overexpressed in human Down syndrome (DS) and in trisomy 16 fetal mice (Ts16), an animal model of the human condition. SMIT1 overexpression determines increased levels of intracellular myo-inositol, a precursor of phophoinositide synthesis. SMIT1 is overexpressed in CTb cells, an immortalized cell line established from the cerebral cortex of a Ts16 mouse fetus. CTb cells exhibit impaired cytosolic Ca2+ signals in response to glutamatergic and cholinergic stimuli (increased amplitude and delayed time-dependent kinetics in the decay post-stimulation), compared to our CNh cell line, derived from the cerebral cortex of a euploid animal. Considering the role of myo-inositol in intracellular signaling, we normalized SMIT1 expression in CTb cells using specific mRNA antisenses. Forty-eight hours post-transfection, SMIT1 levels in CTb cells reached values comparable to those of CNh cells. At this time, decay kinetics of Ca2+ signals induced by either glutamate, nicotine, or muscarine were accelerated in transfected CTb cells, to values similar to those of CNh cells. The amplitude of glutamate-induced cytosolic Ca2+ signals in CTb cells was also normalized. The results suggest that SMIT1 overexpression contributes to abnormal cholinergic and glutamatergic Ca2+ signals in the trisomic condition, and knockdown of DS-related genes in our Ts16-derived cell line could constitute a relevant tool to study DS-related neuronal dysfunction.

Presentado a: 24th International Annual Congress of the World-Muscle-Society (WMS)en: Copenhagen, DENMARK, OCT 01-05, 2019

Immunohistochemical detection of parafibromin in parathyroid pathology Introduction: The definitive diagnosis of parathyroid cancer is extremely difficult, from the clinical approach to the molecular diagnosis. A gene mutation was detected recently in patients with parathyroid cancer. It is a suppressor tumor gene called HRPT2, which codifies for a protein that participates in PAF1 complex, the parafibromin. It has been observed that the expression of this protein it's altered in parathyroid cancer, what would serve like method of diagnosis by immunohystochemistry, with a sensitivity and speci- ficity of 73-96% and 99-100%, respectively. Material and Method: The anti-parafibromin immunohysto - chemistry staining was made in 23 parathyroids tissue samples (5 adenomas, 6 hyperplasia, 7 normal and 5 carcinomas). Results: A positive pattern is observed in almost 100% of benign pathology and 100% in normal tissue. In the cases of carcinoma only 2 of 5 had a strong positivity. Conclusions: The pathological clinical correlation does not allow the association of the loss of parafibromin immunoreactivity in some unequivocal cases of parathyroid cancer. The parafibromin immunostaining does not allow to discriminate between benign or malign pathologies.

Cell encapsulation is an alternative to avoid rejection of grafted tissue, thus bringing an interesting alternative in cell therapy. It is particularly relevant in ailments where only the implant of small quantities of tissues is warranted. In such circumstances, the use of immunosuppressive therapy in patients implanted with tissues from donors is debatable, yet unavoidable at present in order to prevent rejection and/or sensitization of the host to the tissue, in turn jeopardizing the success of successive implants. Hence, a new line of thought, which aims to provide an immunoprivileged site for the grafted tissue, while at the same time insure its nutrition, as well as its survival and continued function, appears as a most attractive possibility. To achieve these goals, cells or tissues harvested for transplant could be encapsulated in biologically compatible matrices. Among the matrices currently in existence, sodium alginate is the most widely used polymer for tissue encapsulation.In the present chapter, we present a technique used to encapsulate parathyroid tissue, for use as cell transplant therapy in patients with secondary hypoparathyroidism. With this procedure, implanted tissue survives and remains functional for up to 18 months.

ResumenObjetivoObtener células estromales derivadas del tejido adiposo, medir y comparar las tasas de viabilidad antes e inmediatamente después un ciclo de criopreservación con diferentes combinaciones de criopreservantes de manera de obtener el mejor medio de criopreservación.Material y métodoMedición de la tasa de viabilidad poscriopreservación de células estromales derivadas del tejido adiposo obtenidas de 5 pacientes utilizando medios definidos (DMEM/Ham F12) libres de suero bovino y suplementados con una de los siguientes combinaciones de compuestos: dimetilsulfóxido (DMSO) 10%; DMSO 10% +trehalosa 7,6%; DMSO 10% +albúmina humana 10% y DMSO 10% +trehalosa 7,6% +albúmina humana 10%, mediante citometría de flujo con ioduro de propidio.ResultadosNo existen diferencias estadísticamente significativas en las tasas de viabilidad de las células estromales posterior a un ciclo de criopreservación. Sin embargo, se observa una tendencia a mejorar la tasa de recuperación de células vitales al agregar albúmina humana.ConclusionesNo se observaron diferencias significativas entre las condiciones estudiadas, sugiriendo que ninguna es superior a las demás en cuanto a rendimiento. Es así como podemos afirmar que la criopreservación de las células estromales derivadas del tejido adiposo en un medio que combine DMEM/F12 con DMSO 10%+trehalosa 7,6%+albúmina humana 10% no logra una tasa de recuperación de células vitales significativamente mayor que las congeladas solo con DMSO 10%.AbstractAimTo obtain stromal cells derived from adipose tissue, to measure and compare viability rates before and immediately after cryopreservation cycle, using different combinations of cryoprotective agents in order to identify the best cryopreservation medium.Material and methodViability rate after cryopreservation of stromal cells derived from adipose tissue were assessed by flow cytometry with propidium iodide. Samples of stromal cells obtained from 5 patients were kept defined, bovine serum-free media (DMEM/Ham-F12), supplemented with one of the following combinations of compounds: 10% dymethylsulfoxide (DMSO); Trehalose 10% DMSO +7.6%; 10% DMSO +10% human albumin and 10% DMSO +7.6% Trehalose +10% human albumin.ResultsNo statistically significant differences were observed in the viability rates of stromal cells derived from adipose tissue after a cryopreservation cycle. However, we observed a tendency towards improvement of recovery rate when human albumin was added to the medium.ConclusionsNone of the studied conditions proved superior to others in terms of cell vitality after a cryopreservation cycle. Hence, we conclude that the cryopreservation of stromal cells derived from adipose tissue in an environment that combines DMEM/F12 with 10% DMSO+7.6% Trehalose+human albumin 10% does not achieve a significantly higher recovery rate than only frozen solely with DMSO 10%.

International audience; In humans, Down syndrome (DS) is caused by the presence of an extra copy of autosome 21. The most striking finding in DS patients is intellectual disability and the onset of Alzheimer's disease (AD)-like neuropathology in adulthood. Gene overdose is most likely to underlie both developmental impairments, as well as altered neuronal function in DS. Lately, the disruption of cellular signaling and regulatory pathways has been implicated in DS pathophysiology, and many of such pathways may represent common targets for diverse DS-related genes, which could in turn represent attractive therapeutical targets. In this regard, one DS-related gene Down Syndrome Cell Adhesion Molecule (DSCAM), has important functions in neuronal proliferation, maturation, and synaptogenesis. p21-associated kinases (PAKs) appear as a most interesting possibility for study, as DSCAM is known to regulate the PAKs pathway. Hence, in DS, overexpressed DSCAM could deregulate PAKs activity and affect signaling pathways that regulate synaptic plasticity such as dendritic spine dynamics and axon guidance and growth. In the present work, we used an immortalized cell line derived from the cerebral cortex of an animal model of DS such as the trisomy 16 (Ts16) fetal mouse (named CTb), and a similar cell line established from a normal littermate (named CNh), to study the effect of DSCAM in the PAKs pathway. The present study shows that DSCAM is overexpressed in CTb cells by approximately twofold, compared to CNh cells. Congruently, PAK1, as well as its downstream effectors LIMK and cofilin, stay phosphorylated for longer periods after DSCAM activation in the CTb cells, leading to an altered actin dynamics, expressed as an increased basal F/G ratio and reduced neurite growth, in the trisomic condition. The present work presents the correlation between DSCAM gene overexpression and a dysregulation of the PAK pathway, resulting in altered morphological parameters of neuronal plasticity in the trisomic cell line, namely decreased number and length of processes.

International audience; Background: Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies. Results: Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X 7 receptor and the transient receptor potential channel, TRPV2, another Ca 2+ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca 2+ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor. Conclusions: The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca 2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.ResultsHuman deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X7 receptor and the transient receptor potential channel, TRPV2, another Ca2+ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca2+ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.ConclusionsThe data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.

Studying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders.

Parkinson’s disease is a debilitating progressive neurodegenerative disorder that results from the loss of or damage to dopaminergic cells containing neuromelanin in the substantia nigra (SN). The underlying neurodegenerative mechanism(s), however, remain elusive. Aminochrome, the precursor of neuromelanin is an endogenous substance capable of inducing selective neurotoxicity to dopaminergic neurons in SN. Nicotine, on the other hand, may offer protective effects against dopaminergic cell damage induced by various neurotoxins including MPTP and salsolinol. In this study, we sought to determine whether nicotine may also protect against aminochrome-induced toxicity in SN derived RCSN-3 cells. Exposure of RCSN-3 cells to a combination of aminochrome (50 μM) and dicoumarol (50 μM) for 48 h induced approximately 70 % cell death. Pretreatment with nicotine, dose-dependently blocked this toxicity. The effects of nicotine in turn were dose-dependently blocked by mecamylamine, a non-selective nicotinic receptor antagonist. These results suggest involvement of nicotinic receptors in protective effects of nicotine against aminochrome-induced toxicity and provide further evidence for possible therapeutic effects of nicotine or nicotinic agonists in Parkinson’s disease.

Four decades after L-dopa introduction to PD therapy, the cause of Parkinson’s disease (PD) remains unknown despite the intensive research and the discovery of a number of gene mutations and delections in the pathogenesis of familial PD. Different model neurotoxins have been used as preclinical experimental models to study the neurodegenerative process in PD, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone. The lack of success in identifying the molecular mechanism for the degenerative process in PD opens the question whether the current preclinical experimental models are suitable to understand the degeneration of neuromelanin-containing dopaminergic neurons in PD. We propose aminochrome as a model neurotoxin to study the neurodegenerative processes occurring in neuromelanin-containing dopaminergic neurons in PD. Aminochrome is an endogenous compound formed during dopamine oxidation and it is the precursor of neuromelanin, a substance whose formation is a normal process in mesencephalic dopaminergic neurons. However, aminochrome itself can induce neurotoxicity under certain aberrant conditions such as (i) one-electron reduction of aminochrome catalyzed by flavoenzymes to leukoaminochrome-o-semiquinone radical, which is a highly reactive neurotoxin; or (ii) the formation of aminochrome adducts with alpha-synuclein, enhancing and stabilizing the formation of neurotoxic protofibrils. These two neurotoxic pathways of aminochrome are prevented by DT-diaphorase, an enzyme that effectively reduces aminochrome with two-electrons, preventing both aminochrome one-electron reduction or formation alpha-synuclein protofibrils. We propose to use aminochrome as a preclinical experimental model to study the neurodegenerative process of neuromelanin-containing dopaminergic neurons in PD.

We have induced intracellular dopamine oxidation to aminochrome in RCSN-3 cells derived from rat substantia nigra by inhibiting VMAT-2 with reserpine to increase free cytosolic dopamine concentration, to study aminochrome-dependent neurotoxicity in the absence of exogenous oxidizing agents such as metals, which may potentiate an aminochrome cytotoxic effect. The expression of VMAT-2 in RCSN-3 cells was determined by reverse transcriptase-polymerase chain reaction and immunocytochemistry. We observed double membrane bodies containing melanin when RCSN-3 cells were incubated with 100 microM dopamine by using transmission electron microscopy. No significant difference in the cell death was observed when the cells were treated 100 microM dopamine and 25 microM reserpine in the absence or presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase. The lack of effect was due to the inhibitory action of 25 microM reserpine on DT-diaphorase (Ki = 24 microM). However, a significant increase in the cell death was observed when DT-diaphorase was inhibited when the cells were incubated with 1 microM reserpine and 100 microM dopamine for 12 h since at this concentration reserpine inhibits VMAT-2 but not DT-diaphorase. Under this condition, we observed (i) the formation of blebbing; (ii) chromatin condensation accompanied by the formation of massive patches in contact with the nuclear membrane; (iii) the smoothness of the cell's surface, that is, lack of surface microprojections; and (iv) mitochondrial damage characterized by disruption of cristae architecture, which remains closely packed; disorganization of the mitochondrial matrix due to separation of the outer membrane from the internal membrane and considerable enlargement of the intermembrane space; and disruption of the external mitochondrial membrane determined by transmission electron microscopy. These results support the proposed neuroprotective role of DT-diaphorase against aminochrome neurotoxicity, and it suggests that RCSN-3 cells incubated with reserpine and dopamine are an excellent and more physiological cellular experimental model to study the role of dopamine oxidation in neurotoxic effects of dopamine.

MRI characterization of dysferlinopathy has been mostly limited to the lower limbs. We aimed to broaden the MRI description of dysferlinopathy and to correlate it with objective measures of motor dysfunction.Sequential whole-body axial MRI was performed in 27 patients with genetically confirmed dysferlinopathy classified according to disease duration. Spearman correlations of fatty infiltration scores versus Motor Function Measure (MFM) were calculated.Significant fatty infiltration was symmetrically present in early stages mainly in the posterior compartments of legs and thighs, thigh adductors, pelvic girdle, and some paravertebral muscles and the subscapularis. Later, fatty infiltration involved leg and thigh anterior compartments, arms and forearms, paravertebral, and trunk muscles. MRI infiltration score correlated positively with disease duration and negatively with MFM scale.We expand MRI characterization of dysferlinopathy and provide evidence for use of MRI scoring combined with motor functional scales to assess the natural course of disease. Muscle Nerve, 2016 Muscle Nerve 54: 203-210, 2016.

Metal doping of bioactive glasses based on ternary 60SiO2?36CaO?4P2O5 (58S) and quaternary 60SiO2?25CaO?11Na2O?4P2O5 (NaBG) mol% compositions synthesized using a sol?gel process was analyzed. In particular, the effect of incorporating 1, 5 and 10?mol% of CuO and ZnO (replacing equivalent quantities of CaO) on the texture, in vitro bioactivity, and cytocompatibility of these materials was evaluated. Our results showed that the addition of metal ions can modulate the textural property of the matrix and its crystal structure. Regarding the bioactivity, after soaking in simulated body fluid (SBF) undoped 58S and NaBG glasses developed an apatite surface layer that was reduced in the doped glasses depending on the type of metal and its concentration with Zn displaying the largest inhibitions. Both the ion release from samples and the ion adsorption from the medium depended on the type of matrix with 58S glasses showing the highest values. Pure NaBG glass was more cytocompatible to osteoblast-like cells (SaOS-2) than pure 58S glass as tested by 3-(4,?5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The incorporation of metal ions decreased the cytocompatibility of the glasses depending on their concentration and on the glass matrix doped. Our results show that by changing the glass composition and by adding Cu or Zn, bioactive materials with different textures, bioactivity and cytocompatibility can be synthesized.

The effect of the nanoscale structure of bioceramics on theirin vitrobioactivity and capacity to osteogenically differentiate stem cell is studied. Nanoparticles of hydroxyapatite (nHA), bioactive glass (nBG), nanoporous bioactive glass (MBG), and nanoporous bioactive glass nanospheres (nMBG) are investigated. The nanometric particle size of bioceramics seems to be more determining in controlling the ability to induce bone-like apatite as compared to the nanoporous structure. At short incubation time, nBG also produces a bioactive extracellular medium capable of upregulating key osteogenic markers involved in the development of a mineralizing phenotype in DPSCs. The bioactive properties of nBG are promissory for accelerating the bone regeneration process in tissue engineering applications.

It has been postulated that the accumulation of extracellular α-synuclein (α-syn) might alter the neuronal membrane by formation of 'pore-like structures' that will lead to alterations in ionic homeostasis. However, this has never been demonstrated to occur in brain neuronal plasma membranes. In this study, we show that α-syn oligomers rapidly associate with hippocampal membranes in a punctate fashion, resulting in increased membrane conductance (5 fold over control) and the influx of both calcium and a fluorescent glucose analogue. The enhancement in intracellular calcium (1.7 fold over control) caused a large increase in the frequency of synaptic transmission (2.5 fold over control), calcium transients (3 fold over control), and synaptic vesicle release. Both primary hippocampal and dissociated nigral neurons showed rapid increases in membrane conductance by α-syn oligomers. In addition, we show here that α-syn caused synaptotoxic failure associated with a decrease in SV2, a membrane protein of synaptic vesicles associated with neurotransmitter release. In conclusion, extracellular α-syn oligomers facilitate the perforation of the neuronal plasma membrane, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We propose that α-synuclein (α-syn) oligomers form pore-like structures in the plasma membrane of neurons from central nervous system (CNS). We believe that extracellular α-syn oligomers facilitate the formation of α-syn membrane pore-like structures, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We think that alterations in ionic homeostasis and synaptic vesicular depletion are key steps that lead to synaptotoxicity promoted by α -syn membrane pore-like structures. C. R. Pacheco funded by Ph.D. fellowship from CONICYT and FEBS Scholarship. This work was supported by grant Anillo-PBCT ACT-04 from the Chilean Government (LGA, CO), FONDECYT grant No. 1100502 (LGA, CO), FONDECYT grant No. 1140473 (LGA) and Spanish Ministerio de Economía y Competitividad (FIS PI10/00587; ISCIII-RETIC RED HERACLES RD06/0009/002-FEDER), and La Marató de TV3 (No. 100310). The Florey Institute of Neuroscience and Mental Health acknowledges the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant.

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean ± SD uptake of 100 µm59FeCl3 in RCSN-3 cells was 25 ± 4 pmol per min per mg, which increased to 28 ± 8 pmol per min per mg when complexed with dopamine (Fe(III)–dopamine). This uptake was inhibited by 2 µm nomifensine (43%p

Leukoaminochrome o-semiquinone radical is generated during one-electron reduction of dopamine oxidation product aminochrome when DT-diaphorase is inhibited. Incubation of 100 microM aminochrome with 100 microM dicoumarol, an inhibitor of DT-diaphorase during 2 h, induces 56% cell death (P < 0.001) with concomitant formation of (i) intracellular hydroperoxides (4.2-fold increase compared to control; P < 0.001); (ii) hydroxyl radicals, detected with ESR and spin trapping agents (2.4-fold increase when cells were incubated with aminochrome in the presence of dicoumarol compared to aminochrome alone); (iii) intracellular edema, and cell membrane deterioration determined by transmission electron microscopy; (iv) absence of apoptosis, supported by using anexin-V with flow cytometry; (v) a strong decrease of mitochondrial membrane potential determined by the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanineiodide (P < 0.01); (vi) swelling and disruption of outer and inner mitochondrial membranes determined by transmission electron microscopy. These results support the proposed role of leukoaminochrome o-semiquinone radical as neurotoxin in Parkinson's disease neurodegeneration and DT-diaphorase as neuroprotective enzyme.

We have tested the idea that oxidative metabolism of dopamine may be involved in MPTP toxicity using the RCSN-3 cell line derived from the substantia nigra of an adult rat. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (10 microM), MPTP combined with 40 microM dicoumarol (an inhibitor of DT-diaphorase) and dicoumarol alone, did not induce toxicity in RCSN-3 cells after 72 h incubation. The lack of toxicity in MPTP-treated RCSN-3 cells may be explained by the fact that they are unable to metabolize MPTP to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+ as determined by HPLC. Incubation for 72 h with 100 microM MPP+ induced a 6.6 +/- 1.4% cell death of RCSN-3 cells compared to 3.5 +/- 0.4 observed in control cells. However, when the cells were treated with 100 microM MPP+ and 40 microM dicoumarol, cell death increased 4-fold compared to that of cells treated solely with MPP+ (27 +/- 2%; P

We have previously reported that restraint stress applied to the gestant mother results in long-lasting effects in the offspring that show an increase in the number of dopamine D2-type receptors in limbic areas on the adult rat brain cortex. Evidence that stress during pregnancy results in activation of the hypothalamic-pituitary-adrenal (HPA) axis has been extensively demonstrated. Therefore, high levels of corticosterone secreted in response to stress by the gestant mother might be one of the predisposing factors for the changes observed in dopamine receptors in the adult rat brain. In this study we addressed the question whether corticosterone would directly up-regulate D2-type receptors in vitro. We have investigated the effect of different concentrations of corticosterone on D4 dopamine receptor in immortalized cell lines from cerebral cortex of normal mouse fetuses, detected by immunocytochemistry employing polyclonal antibodies generated against synthetic peptides homologous to an extracellular domain of D4 receptor. The results show that corticosterone in vitro decreases the number of dopamine D4 receptors, suggesting that the increase of D2-type receptors in adult rats following prenatal stress is not related to a direct action of corticosterone on receptor expression.

We have established hippocampal cell lines from normal and trisomy 16 fetal mice, a model of human trisomy 21. Both cell lines, named H1b (derived from a normal animal) and HTk (trisomic) possess neuronal markers by immunohistochemistry (enolase, synaptophysin, microtubule associated protein-2, and choline acetyltransferase) and lack glial markers (glial fibrillary acidic protein and S-100). Also, we evaluated intracellular Ca(2+) levels ([Ca(2+)](i)) in response to neurotransmitter agonists, in cells loaded with the fluorescent Ca(2+) indicators Indo-1 and Fluo-3. Both cell lines responded to glutamatergic stimuli induced by glutamate, N-methyl-D-aspartate, I-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate. Glutamate responses were only partially prevented by addition of 5 mM EGTA and the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), increased [Ca(2+)](i) in both cell types. These results confirm the presence of glutamatergic metabotropic receptors. In glutamate-induced responses, HTk cells exhibited slower time-dependent decay kinetics than H1b cells. Cholinergic agonists (nicotine and muscarine) induced a rapid, transient increase in [Ca(2+)](i) in both cell types. Furthermore, some cells were sensitive to histamine and norepinephrine. All responses to the aforementioned agonists were prevented by addition of specific antagonists. We also studied incorporation and release of [(3)H]choline in the cells, and observed no differences in uptake parameters. However, release induced by K(+) and nicotine depolarization was greatly reduced in HTk cells. The results show that H1b and HTk cells retain neuronal characteristics and respond to specific neurotransmitter stimuli. The HTk differences could be related to neuronal pathophysiology in Down syndrome.

We report the establishment of continuously growing cell lines from spinal cords of normal and trisomy 16 fetal mice. We show that both cell lines, named M4b (derived from a normal animal) and MTh (trisomic) possess neurological markers by immunohistochemistry (neuron specific enolase, synaptophysin, microtubule associated protein-2 [MAP-2], and choline acetyltransferase) and lack glial traits (glial fibrillary acidic protein and S100). MTh cells were shown to overexpress mRNA of Cu/Zn superoxide dismutase, whose gene is present in autosome 16. We also studied intracellular Ca2+ signals ([Ca2+]i) induced by different agonists in Indo-1 loaded cells. Basal [Ca2+]i was significantly higher in MTh cells compared to M4b cells. Glutamate (200 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACDP) (100 microM) induced rapid, transient increases in [Ca2+]i in M4b and MTh cells, indicating the presence of glutamatergic metabotropic receptors. N-methyl-D-aspartate (NMDA) and kainate, but not alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), produced [Ca2+)]i rises in both cell types. MTh cells exhibited faster time-dependent decay phase kinetics in glutamate-induced responses compared to M4b cells. Nicotine induced a transient increase in [Ca2+]i in M4b and MTh cells, with significantly greater amplitudes in the latter compared to the former. Further, both cell types responded to noradrenaline. Finally, we examined cholinergic function in both cell lines and found no significant differences in the [3H]-choline uptake, but fractional acetylcholine release induced by either K+, glutamate or nicotine was significantly higher in MTh cells. These results show that M4b and MTh cells have neuronal characteristics and the MTh line shows differences which could be related to neuronal pathophysiology in Down's syndrome.

We have established two immortalized cell lines from dorsal root ganglia of normal (G4b) and trisomy 16 mice (GTI), a model for Down syndrome. By immunohistochemistry, both cell lines exhibit neuronal traits and lack glial markers. GTI cells exhibited greater [ 3 H]choline uptake than G4b cells. K + and nicotine-mediated acetylcholine release was greater in GTI cells. Basal intracellular Ca 2+ concentration ([Ca 2+ ] i ) was significantly lower in GTI cells. More GTI cells responded to neurotransmitters with a transient [Ca 2+ ] i increase compared to G4b cells, but both cell types showed similar amplitudes of [Ca 2+ ] i responses. The results show that both cell lines retain neuronal characteristics and respond to specific neurotransmitter stimuli. Altered GTI cell responses could be related to neuronal pathophysiology in Down's syndrome.