Pharmacological Inhibition of p-21 Activated Kinase (PAK) Restores Impaired Neurite Outgrowth and Remodeling in a Cellular Model of Down Syndrome.

Down syndrome (DS) is characterized by the trisomy of chromosome 21 and by cognitive deficits that have been related to neuronal morphological alterations in humans, as well as in animal models. The gene encoding for amyloid precursor protein (APP) is present in autosome 21, and its overexpression in DS has been linked to neuronal dysfunction, cognitive deficit, and Alzheimer's disease-like dementia. In particular, the neuronal ability to extend processes and branching is affected. Current evidence suggests that APP could also regulate neurite growth through its role in the actin cytoskeleton, in part by influencing p21-activated kinase (PAK) activity. The latter effect is carried out by an increased abundance of the caspase cleavage-released carboxy-terminal C31 fragment. In this work, using a neuronal cell line named CTb, which derived from the cerebral cortex of a trisomy 16 mouse, an animal model of human DS, we observed an overexpression of APP, elevated caspase activity, augmented cleavage of the C-terminal fragment of APP, and increased PAK1 phosphorylation. Morphometric analyses showed that inhibition of PAK1 activity with FRAX486 increased the average length of the neurites, the number of crossings per Sholl ring, the formation of new processes, and stimulated the loss of processes. Considering our results, we propose that PAK hyperphosphorylation impairs neurite outgrowth and remodeling in the cellular model of DS, and therefore we suggest that PAK1 may be a potential pharmacological target.

Genetic Profile of Patients with Limb-Girdle Muscle Weakness in the Chilean Population.

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.

Microencapsulation of cellular aggregates composed of differentiated insulin and glucagon-producing cells from human mesenchymal stem cells derived from adipose tissue.

BACKGROUND: In type I diabetes mellitus (T1DM) pancreatic ß 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.

Correction to: Microencapsulation of cellular aggregates composed of differentiated insulin and glucagon-producing cells from human mesenchymal stem cells derived from adipose tissue.

[This corrects the article DOI: 10.1186/s13098-020-00573-9.].

N-Acetylcysteine Reduces Skeletal Muscles Oxidative Stress and Improves Grip Strength in Dysferlin-Deficient Bla/J Mice.

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'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.

Myofibers deficient in connexins 43 and 45 expression protect mice from skeletal muscle and systemic dysfunction promoted by a dysferlin mutation.

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.

Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains.

Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.

Comparative study of the neural differentiation capacity of mesenchymal stromal cells from different tissue sources: An approach for their use in neural regeneration therapies.

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.

RCAN1 Knockdown Reverts Defects in the Number of Calcium-Induced Exocytotic Events in a Cellular Model of Down Syndrome.

In humans, Down Syndrome (DS) is a condition caused by partial or full trisomy of chromosome 21. Genes present in the DS critical region can result in excess gene dosage, which at least partially can account for DS phenotype. Although regulator of calcineurin 1 (RCAN1) belongs to this region and its ectopic overexpression in neurons impairs transmitter release, synaptic plasticity, learning and memory, the relative contribution of RCAN1 in a context of DS has yet to be clarified. In the present work, we utilized an in vitro model of DS, the CTb neuronal cell line derived from the brain cortex of a trisomy 16 (Ts16) fetal mouse, which reportedly exhibits acetylcholine release impairments compared to CNh cells (a neuronal cell line established from a normal littermate). We analyzed single exocytotic events by using total internal reflection fluorescence microscopy (TIRFM) and the vesicular acetylcholine transporter fused to the pH-sensitive green fluorescent protein (VAChT-pHluorin) as a reporter. Our analyses showed that, compared with control CNh cells, the trisomic CTb cells overexpress RCAN1, and they display a reduced number of Ca2+-induced exocytotic events. Remarkably, RCAN1 knockdown increases the extent of exocytosis at levels comparable to those of CNh cells. These results support a critical contribution of RCAN1 to the exocytosis process in the trisomic condition.

Poly(lactic acid) composites based on graphene oxide particles with antibacterial behavior enhanced by electrical stimulus and biocompatibility.

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.

Knockdown of Myo-Inositol Transporter SMIT1 Normalizes Cholinergic and Glutamatergic Function in an Immortalized Cell Line Established from the Cerebral Cortex of a Trisomy 16 Fetal Mouse, an Animal Model of Human Trisomy 21 (Down Syndrome).

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.

Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells.

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.

Microencapsulation of Parathyroid Cells for the Treatment of Hypoparathyroidism.

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.

Criopreservación de células estromales derivadas del tejido adiposo: comparación de diferentes medios de criopreservación / Cryopreservation of adipose derived stromal cells: comparison of different cryopreservation media

Objetivo Obtener 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 método Medición 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. Resultados No 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. Conclusiones No 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%.

Aim To 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 method Viability 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. Results No 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. Conclusions None 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%.

The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes.

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 P2X7 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.

Overexpressed Down Syndrome Cell Adhesion Molecule (DSCAM) Deregulates P21-Activated Kinase (PAK) Activity in an In Vitro Neuronal Model of Down Syndrome: Consequences on Cell Process Formation and Extension.

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.

Broadening the imaging phenotype of dysferlinopathy at different disease stages.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Sol-gel synthesis and in vitro bioactivity of copper and zinc-doped silicate bioactive glasses and glass-ceramics.

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.

Extracellular α-synuclein alters synaptic transmission in brain neurons by perforating the neuronal plasma membrane.

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.

Dynamin-2 in nervous system disorders.

Dynamin-2 is a pleiotropic GTPase whose best-known function is related to membrane scission during vesicle budding from the plasma or Golgi membranes. In the nervous system, dynamin-2 participates in synaptic vesicle recycling, post-synaptic receptor internalization, neurosecretion, and neuronal process extension. Some of these functions are shared with the other two dynamin isoforms. However, the involvement of dynamin-2 in neurological illnesses points to a critical function of this isoform in the nervous system. In this regard, mutations in the dynamin-2 gene results in two congenital neuromuscular disorders. One of them, Charcot-Marie-Tooth disease, affects myelination and peripheral nerve conduction, whereas the other, Centronuclear Myopathy, is characterized by a progressive and generalized atrophy of skeletal muscles, yet it is also associated with abnormalities in the nervous system. Furthermore, single nucleotide polymorphisms located in the dynamin-2 gene have been associated with sporadic Alzheimer's disease. In the present review, we discuss the pathogenic mechanisms implicated in these neurological disorders.