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Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+and Ca2+entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENTNeuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.

Graft copolymers between conducting PEDOT and biodegradable PCL were synthesized and investigated for 3D printing scaffolds for patterning of muscle cells.

Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material. We thank Anna Wojtalewicz and Leire Escudero for technical help, and Dr. Javier Ruiz-Ederra for helpful discussions on HRM assay. This work was supported by funding from Health Institute Carlos III (ISCIII) and the European Regional Development Fund (ERDF/ESF, "Investing in your future"), Grant PI17/00676; from Duchenne Parent Project Spain (DPPE); from the Basque Government (2016111091); and from the University of the Basque Country UPV/EHU (PPGA19/58). H.L.-F. and L.M.-M. hold predoctoral fellowships from the University of the Basque Country, J.L.-E. holds a predoctoral fellowship from the Basque Government, and A.V.-I. holds a Ramon y Cajal contract funded by the Spanish Ministry of Economy and Competitiveness

[EN] LGMDR1 is caused by mutations in the CAPN3 gene that encodes calpain 3 (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Our previous findings show that CAPN3 deficiency leads to reduced SERCA levels through increased protein degradation. This work investigates the potential contribution of the ubiquitin-proteasome pathway to increased SERCA degradation in LGMDR1. Consistent with our previous results, we observed that CAPN3-deficient human myotubes exhibit reduced SERCA protein levels and high cytosolic calcium concentration. Treatment with the proteasome inhibitor bortezomib (Velcade) increased SERCA2 protein levels and normalized intracellular calcium levels in CAPN3-deficient myotubes. Moreover, bortezomib was able to recover mutated CAPN3 protein in a patient carrying R289W and R546L missense mutations. We found that CAPN3 knockout mice (C3KO) presented SERCA deficits in skeletal muscle in the early stages of the disease, prior to the manifestation of muscle deficits. However, treatment with bortezomib (0.8 mg/kg every 72 h) for 3 weeks did not rescue SERCA levels. No change in muscle proteasome activity was observed in bortezomib-treated animals, suggesting that higher bortezomib doses are needed to rescue SERCA levels in this model. Overall, our results lay the foundation for exploring inhibition of the ubiquitin-proteasome as a new therapeutic target to treat LGMDR1 patients. Moreover, patients carrying missense mutations in CAPN3 and presumably other genes may benefit from proteasome inhibition by rescuing mutant protein levels. Further studies in suitable models will be necessary to demonstrate the therapeutic efficacy of proteasome inhibition for different missense mutations. This research was funded by Diputación Foral de Gipuzkoa (AV-I, 2018-000117-01-B, 2019-00362-01-B); Fundación Gangoiti Barrera, Ministerio de Ciencia e Innovación (AV-I,PID 2020-119780RB-I00), the Basque Government (AV-I, ETORTEK-KK-2019/00093); the University of the Basque Country (AV-I, GIU20/057), and Instituto de Salud Carlos III, co-funded by European Regional Development Fund/ European Social Fund, “Investing in your future” (AV-I, PI17/00676; AL, PI17/01841). JL-E held a PhD fellowship from the Basque Government, LM-M holds a PhD fellowship from the UPV/EHU and GG holds a Juan de la Cierva- Incorporación 2019 contract funded by the Spanish Ministry of Science and Innovation.

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin-deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co-localise in human foetal muscle, in the dystrophin-competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies. This work was supported by funding from Health Institute Carlos III (ISCIII, Spain) and the European Regional Development Fund, (ERDF/FEDER), `A way of making Europe': Grant PI15/00333; Basque Government (grants 2016111029, 2018222035 and 2020333012) and Duchenne Parent Project Spain (grant 05/2016). P. S--M holds a Rio Hortega Fellowship from ISCIII (CM19/00104). V.A--G holds a Miguel Servet Fellowship from the ISCIII (CPII17/00004), part-funded by ERDF/FEDER. A. L--M acknowledges funding by Biocruces Bizkaia Health Research Institute (BC/I/DIV/19/001). V. A--G also acknowledges funding from Ikerbasque (Basque Foundation for Science). None of this funding represents a conflict of interest with the content of this review.

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na(+) and Ca(2+) entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca(2+)-dependent way. We find that Ca(2+) upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca(2+) binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca(2+) uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca(2+) flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP(3)R, are responsible for Ca(2+) release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca(2+)-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation. SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca(2+) is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca(2+)-dependent way, independently of effects of Ca(2+) as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca(2+) stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca(2+) stimulation is blunt in the absence of Aralar, a Ca(2+)-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca(2+)-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD(+)/NADH ratio.

Tailor-made polymers are needed to fully exploit the possibilities of additive manufacturing, constructing complex, and functional devices in areas such as bioelectronics. In this paper, the synthesis of a conducting and biocompatible graft copolymer which can be 3D printed using direct melting extrusion methods is shown. For this purpose, graft copolymers composed by conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and a biocompatible polymer polylactide (PLA) are designed. The PEDOT-g-PLA copolymers are synthesized by chemical oxidative polymerization between 3,4-ethylenedioxythiophene and PLA macromonomers. PEDOT-g-PLA copolymers with different compositions are obtained and fully characterized. The rheological characterization indicates that copolymers containing below 20 wt% of PEDOT show the right complex viscosity values suitable for direct ink writing (DIW). The 3D printing tests using the DIW methodology allows printing different parts with different shapes with high resolution (200 µm). The conductive and biocompatible printed patterns of PEDOT-g-PLA show excellent cell growth and maturation of neonatal cardiac myocytes cocultured with fibroblasts.

The hypothesis of rescuing FKBP12/RyR1 interaction and intracellular calcium homeostasis through molecular “reshaping” of FKBP12 was investigated. To this end, novel 4-arylthioalkyl-1-carboxyalkyl-1,2,3-triazoles were designed and synthesized, and their efficacy was tested in human myotubes. A library of 17 compounds (10a-n) designed to dock the FKBP12/RyR1 hot-spot interface contact residues, was readily prepared from free α-amino acids and arylthioalkynes using CuAAC “click” protocols amenable to one-pot transformations in high overall yields and total configurational integrity. To model nitro-oxidative stress, human myotubes were treated with the peroxynitrite donor SIN1, and evidence was found that some triazoles 10 were able to normalize calcium levels, as well as FKBP12/RyR1 interaction. For example, compound 10 b at 150 nM rescued 46% of FKBP12/RyR1 interaction and up to 70% of resting cytosolic calcium levels in human myotubes under nitro-oxidative stress. All compounds 10 analyzed showed target engagement to FKBP12 and low levels of cytotoxicity in vitro. Compounds 10b, 10c, 10h, and 10iR were identified as potential therapeutic candidates to protect myotubes in muscle disorders with underlying nitro-oxidative stress, FKBP12/RyR1 dysfunction and calcium dysregulation.

Summary Resident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR+ precursors of human foreskin can be ascribed to the Schwann (CD56+) and perivascular (CD56−) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR+CD56+ Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread., Graphical Abstract, Highlights • Human dermis-derived cultures show two types of SOX2+ cells: Schwann and perivascular • p75NTR+CD56+ Schwann cells are responsible for neural progeny • SOX2 expression levels regulate the neural competence of dermal precursors • p75NTR+CD56+ neural precursor cells similarly arise from human cardiospheres, In this article, Izeta and colleagues show that neural precursor cell (NPC) cultures from adult human dermis include two SOX2+ cell subpopulations (Schwann and perivascular), and neural progeny is derived only from Schwann cells in a process regulated by their SOX2 expression levels. NPCs similarly arise from human cardiospheres, potentially elucidating the origin of seemingly unrelated NPCs observed in multiple organs.

Amyotrophic lateral sclerosis (ALS) is a severe neuromuscular disease characterised by a progressive loss of motor neurons that usually results in paralysis and death within 2 to 5 years after disease onset. The pathophysiological mechanisms involved in ALS remain largely unknown and to date there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of copper homeostasis in the central nervous system is a crucial underlying event in motor neuron degeneration and ALS pathophysiology. We also review and discuss novel approaches seeking to target copper delivery to treat ALS. These novel approaches may be clinically relevant not only for ALS but also for other neurological disorders with abnormal copper homeostasis, such as Parkinson's, Huntington's and Prion diseases.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a rare disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness of shoulder, pelvic, and proximal limb muscles that usually appears in children and young adults and results in loss of ambulation within 20 years after disease onset in most patients. The pathophysiological mechanisms involved in LGMDR1 remain mostly unknown, and to date, there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of Ca2+ homeostasis in the skeletal muscle is a significant underlying event in this muscular dystrophy. We also review and discuss specific clinical features of LGMDR1, CAPN3 functions, novel putative targets for therapeutic strategies, and current approaches aiming to treat LGMDR1. These novel approaches may be clinically relevant not only for LGMDR1 but also for other muscular dystrophies with secondary calpainopathy or with abnormal Ca2+ homeostasis, such as LGMD2B/LGMDR2 or sporadic inclusion body myositis. This research was funded by Instituto de Salud Carlos III, co-funded by European Regional Development Fund/European Social Fund, "Investing in your future" (A.V.-I., PI17/00676; A.L.d.M., PI17/01841); the Basque Government (A.V.-I., 2016111091) and Diputacion Foral de Gipuzkoa (A.V.-I., 2018-000117-01-B and 2019-00362-01-B). A.V.-I. holds a Ramon y Cajal contract funded by the Spanish Ministry of Economy and Competitiveness, and J.L.-E. holds a PhD fellowship from the Basque Government.

Summary Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Integrin-linked kinase (ILK) is a serine/threonine kinase and a major regulator of integrin signaling. It links integrins to the actin cytoskeleton and recruits other adaptor molecules into a large complex to regulate actin dynamics and integrin function. Using the Cre-lox system, we deleted Ilk from NCCs of mice to investigate its role in NCC morphogenesis. The resulting mutants developed a severe aneurismal arterial trunk that resulted in embryonic lethality during late gestation. Ilk mutants showed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and the outflow tract. Within the conotruncal cushions, Ilk-deficient NCCs exhibited disorganization of F-actin stress fibers and a significantly rounder morphology with shorter cellular projections. Additionally, absence of ILK resulted in reduced in vivo phosphorylation of Smad3 in NCCs, which correlated with reduced αSMA levels. Our findings resemble those seen in Pinch1 and β1 integrin conditional mutant mice, and therefore support that, in neural crest-derived cells, ILK and Pinch1 act as cytoplasmic effectors of β1 integrin in a pathway that protects against aneurysms. In addition, our conditional Ilk mutant mice might prove useful as a model to study aortic aneurysms caused by reduced Smad3 signaling, as occurs in the newly described aneurysms-osteoarthritis syndrome, for example.

In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Focal adhesion kinase (FAK) mediates signal transduction by integrin and growth factor receptors, each of which is important for normal cardiovascular development. To investigate the role of FAK in NCC morphogenesis, we deleted it in murine NCCs using Wnt1cre, yielding craniofacial and cardiovascular malformations resembling those observed in individuals with DiGeorge syndrome. In these mice, we observed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and impaired cardiac outflow tract rotation, which resulted in a dextroposed aortic root. Moreover, within the conotruncal cushions, Fak-deficient NCCs formed a less organized mesenchyme, with reduced expression of perlecan and semaphorin 3C, and exhibited disorganized F-actin stress fibers within the aorticopulmonary septum. Additionally, absence of Fak resulted in reduced in vivo phosphorylation of Crkl and Erk1/2, components of a signaling pathway essential for NCC development. Consistent with this, both TGF-beta and FGF induced FAK and Crkl phosphorylation in control but not Fak-deficient NCCs in vitro. Our results indicate that FAK plays an essential role in cardiac outflow tract development by promoting the activation of molecules such as Crkl and Erk1/2.

Integrin-linked kinase (Ilk) is a serine/threonine kinase and an adaptor protein that links integrins to the actin cytoskeleton and to a number of signaling pathways involved in integrin action. We hypothesized that Ilk may act as an important effector of integrins in skeletal muscle, where these receptors provide a critical link between the sarcolemma and the extracellular matrix. Using the cre/lox system, we deleted Ilk from skeletal muscles of mice. The resulting mutants developed a progressive muscular dystrophy with multiple degenerating and regenerating muscle fibers, increased central nuclei, and endomysial fibrosis. These defects were widespread but were most severe near myofascial junctions where Ilk mutants showed displacement of focal adhesion-related proteins, including vinculin, paxillin, focal adhesion kinase, dystrophin, and the alpha 7 beta 1D-integrin subunits. Distal ends of mutant muscle fibers appeared irregular, and there was restructuring of the actin cytoskeleton. These findings resemble those seen in humans and mice lacking the alpha 7-integrin subunit and suggest that Ilk may act as a cytoplasmic effector of alpha 7 beta1-integrin in the pathogenesis of these deficiencies.

Recent studies have shown that glutamate excitotoxicity may be a component in the etiology of multiple sclerosis (MS). Glutamate transporters determine the levels of extracellular glutamate and are essential to prevent excitotoxicity. We have analyzed here the expression of the glutamate transporters EAAT1, EAAT2 and EAAT3 in control and in MS optic nerve samples. We observed an overall increase in the level of the glutamate transporters EAAT1 and EAAT2 mRNA and protein. In turn, functional assays showed that glutamate uptake was also increased in MS samples. Furthermore, glutamate transporter increases were mimicked in rat optic nerves treated with excitotoxic levels of glutamate. Together, these results indicate that enhanced expression of glutamate transporters in MS constitutes a regulatory response of glial cells to toxic levels of glutamate in the CNS during inflammation and neurodegeneration.

We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available.

Muscular dystrophies are a group of diseases characterised by the primary wasting of skeletal muscle, which compromises patient mobility and in the most severe cases originate a complete paralysis and premature death. Existing evidence implicates calcium dysregulation as an underlying crucial event in the pathophysiology of several muscular dystrophies, such as dystrophinopathies, calpainopathies or myotonic dystrophy among others. Duchenne muscular dystrophy is the most frequent myopathy in childhood, and calpainopathy or LGMD2A is the most common form of limb-girdle muscular dystrophy, whereas myotonic dystrophy is the most frequent inherited muscle disease worldwide. In this review, we summarise recent advances in our understanding of calcium ion cycling through the sarcolemma, the sarcoplasmic reticulum and mitochondria, and its involvement in the pathogenesis of these dystrophies. We also discuss some of the clinical implications of recent findings regarding Ca2+handling as well as novel approaches to treat muscular dystrophies targeting Ca2+regulatory proteins.

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25--35 mg kg(-1) day(-1) orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 +/- 4.5%) than in the posterior (53.2 +/- 15.4%) cortex. L-[(3)H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 +/- 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 +/- 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 +/- 0.7 microM) and the KCl-evoked (28.7 +/- 7.7 microM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

Background: Cardiac neural crest cells (NCCs) participate in the remodeling of the outflow tract and pharyngeal arch arteries during cardiovascular development. Abnormal growth factor and integrin signaling in cardiac NCCs leads to cardiovascular malformations. Focal adhesion kinase (FAK) is a ubiquitous protein kinase that transduces integrin and growth factor signaling pathways. However, a direct role for FAK in cardiac NCC morphogenesis has not been demonstrated. Here we have used a Cre/loxP method for NCC-specific deletion of FAK in mouse embryos. Results: We found that targeted deletion of FAK in NCC results in craniofacial and cardiovascular malformations, leading to early postnatal lethality. Mutant mice display cleft palate together with several cardiovascular defects, including persistent truncus arteriosus, transposition of the great arteries, overriding aorta, ventricular septal defect and type B interruption of the aortic arch, all of which resemble common forms of human congenital heart disease. We found that NCC counts in the outflow tract of FAK mutant and control littermates are similar at E11, suggesting that FAK is not required for cardiac NCC migration to the outflow tract. Furthermore, NCCs appear to differentiate normally into smooth muscle cells in the outflow tract of FAK conditional mutant embryos at E11 and E12.5. In contrast, NCCs in the aortic arch arteries of FAK conditional mutants show impaired differentiation into smooth muscle cells in these embryonic stages. Additionally, we observed that rotation of the outflow tract fails to occur by E11 in FAK mutants, resulting in a dextroposed aortic root. Moreover, FAK-deficient NCCs form a less compact mesenchyme in the conotruncal cushions and show disorganized F-actin stress fibers in the aorticopulmonary septum. Conclusions: Our results indicate that FAK plays an essential role in the development of the cardiac outflow tract and in the remodeling of aortic arch arteries. However, it is dispensable for migration and differentiation of cardiac NCC in the outflow tract. Further studies with this novel model of congenital heart disease may help elucidate the NCC signaling pathways that orchestrate septation of the outflow tract.

In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.

EAAT1 is a major glutamate transporter in the CNS and is required for normal neurotransmission and neuroprotection from excitotoxicity. In the present study, we have identified a novel form of the human EAAT1, named here as EAAT1ex9skip, which lacks the entire exon 9. Quantitative PCR analysis indicates that this variant is expressed throughout the CNS, both in grey matter and axonal tracts, at levels ranging between 10% and 20% of the full-length EAAT1 form. When expressed in HEK293 cells, EAAT1ex9skip mRNA is translated into a truncated protein localized in the endoplasmic reticulum. EAAT1ex9skip has no functional glutamate uptake activity but instead, exerts a dominant negative effect over full-length EAAT1 function. In turn, co-expression of full-length EAAT1 and EAAT1ex9skip variants reduces the insertion of the former into the plasma membrane. Together, these results indicate that the EAAT1ex9skip splice variant is a negative regulator of full-length EAAT1 function in the human brain.

To test the hypothesis that clozapine-induced reduction of glutamate transporter-1 (GLT-1) expression is mediated by astrocytes, we studied the effects of clozapine on Glu transporters and Glu uptake in primary astrocyte cultures of the cerebral cortex. Astrocyte cultures treated for 48 h with clozapine exhibited a reduction in GLT-1 levels of about 50%, whereas glutamate-aspartate transporter (GLAST) levels remained unchanged. Glu uptake was also lowered, and this reduction was dose-dependent. Our findings indicate that clozapine reduces GLT-1 expression and function by a mechanism that directly involves astrocytes. A better understanding of the molecular events by which antipsychotics regulate Glu uptake can contribute to identify new targets for the treatment of schizophrenia.

Excitation–contraction (E-C) coupling in adult skeletal muscle is permitted through interaction between dihydropyridine receptors (DHPRs) located in the membrane T-tubular system and closely apposed ryanodine receptors (RyRs), which are Ca2+ release channels in the sarcoplasmic reticulum (SR). Both receptors, along with other interacting proteins, form part of a triad, a highly specialized anatomical structure that is the basis of E-C coupling. Muscle contraction is initiated by depolarization of the sarcolemma that activates DHPRs, which in turn evokes SR Ca2+ release through RyRs. This cytoplasmatic Ca2+ rise leads to muscle contraction through Ca2+ binding to troponin C, which triggers a contractile response of the myofibrils. However, abnormally sustained Ca2+ levels may elicit other cell processes such as proteolysis and apoptosis, and therefore Ca2+ influxes within the cell must be tightly regulated. In particular, increased cytoplasmic Ca2+ levels can cause disruption of communication between DHPRs and RyRs, and therefore E-C uncoupling, through a mechanism involving endogenous Ca2+-dependent proteolysis, although the complete pathway remains elusive. Several lines of evidence suggest involvement of μ-calpain in E-C uncoupling. First, this protease is localized in close proximity to the SR; second, exogenous application of μ-calpain results in cleavage of triad junctions; and finally, both μ-calpain activation and E-C uncoupling have been found to occur at similar [Ca2+] range (Murphy et al. 2013). Nevertheless, until now none of the triad proteins has been identified as undergoing Ca2+-dependent proteolysis in tandem with E-C coupling disruption, and specifically DHPR, RyR and triadin seem to be unaffected by μ-calpain proteolysis. Within the triad junctions, junctophilins, a group of proteins anchored to both the T-tubule system and SR, have been suggested as mediators that enable apposition of DHPRs and RyRs in both skeletal and cardiac muscle. In Corona et al. (2010), a significant reduction of junctophilins (JP1 and JP2) as well as E-C coupling failure was observed after eccentric contractions performed in mice skeletal muscle. However, these authors did not find any evidence for μ-calpain or extracellular Ca2+ involvement in junctophilin reduction. In contrast, Zhang et al. (2012) have shown activation of μ-calpain with eccentric contractions using a similar model, and thus the matter still remains to be clarified. In a recent issue of The Journal of Physiology, Murphy et al. (2013) focus on the role of junctophilins in muscle E-C coupling and provide the first evidence of an association between junctophilins, Ca2+-dependent proteolysis and E-C uncoupling. In a first set of experiments they used different models, such as human muscle cryosections and rat muscle homogenates, to simulate E-C coupling disruption by increasing extracellular Ca2+, which resulted in a significant reduction of JP1 and JP2 accompanied by increased levels of activated μ-calpain. These results were further confirmed in skinned extensor digitorum longus (EDL) fibres in rat, where they reported reduced levels of JP1 after disruption of E-C coupling following 1 min incubation with 40 μm extracellular [Ca2+]. Using a more physiological model, in vitro overstimulation of whole EDL rat muscles also revealed higher proteolytic levels of JP1 with no evidence of RyR1 cleavage compared to non-stimulated ones. The authors showed that Ca2+-dependent proteolysis of JP1 (90 kDa) resulted in a diffusible 75 kDa N-terminal fragment plus a 15 kDa fixed C-terminal fragment. Therefore, they propose a model in which DHPR and RyR uncoupling results from JP1 proteolysis, where a small C-terminal fragment of JP1 remains anchored to the SR. In another set of experiments using a mouse model of Duchenne muscular dystrophy, Murphy et al. (2013) analyse proteolysis of junctophilins and levels of active μ-calpain in mdx mice skeletal muscle. In tibialis anterior muscles, the authors found by Western blot increased proteolysis of JP1 and JP2 in 28-day-old mdx compared with wild-type mice. In agreement with mdx dystrophic phenotype progress, in tibialis anterior muscles from 70-day-old mdx mice proteolysis of junctophilins was not detected. By contrast, using mdx diaphragms, which develop a more progressive degeneration than tibialis anterior muscles, Murphy et al. (2013) found increased proteolysis of JP1 in 7-month-old mdx mice compared with control mice. In this model, increased proteolysis of junctophilins also seems to be concomitant with increased μ-calpain activity, although data from tibialis anterior are not clearly shown in the paper. The fact that in control mice both JP1 and JP2 protein levels increase with age suggests their involvement in muscle development and regeneration. This is in line with previous results by Ito et al. (2001) from JP1 knock-out mice that showed a reduced number of triad junctions, less muscle contractile force and perinatal lethality. Finally, Murphy et al. (2013) test their E-C uncoupling model in cardiac muscles. To this end, they used rat cardiac ventricular homogenates that were exposed to 500 μm extracellular Ca2+ levels for 1 h to induce disruption of E-C coupling, and they found a significant reduction of JP2 as a result. Furthermore, using a cardiac model of ischaemia-reperfusion in which increased calpain activity has been reported (Singh et al. 2012), the authors found decreased JP2 levels but no associated RyR2 cleavage. Together, these results suggest that junctophilin proteolysis and μ-calpain activation could be used as potential markers of skeletal and cardiac muscle damage induced by Ca2+, which is characteristic of several muscular and cardiac disorders. The work carried out by Murphy et al. (2013) is presented with a well-designed structure and its relevance lies in the display of new evidence regarding disruption of triad proteins in skeletal and cardiac muscle fibres when Ca2+ homeostasis is misregulated. The study provides confirmation of the key role that junctophilins play in the maintenance of triad structure, which allows an appropriate Ca2+ release from SR by RyRs under low basal cytoplasmic Ca2+ levels. In addition, it is the first study indicating an association between μ-calpain activation and proteolysis of junctophilins in different situations where increased intracellular Ca2+ levels lead to disruption of E-C coupling, such as in muscular dystrophy or heart failure. With regard with muscular dystrophy, the results found by Murphy and colleagues are in line with previous studies reporting increased extracellular Ca2+ influx and abnormal SR Ca2+ release (for citations see Murphy et al. 2013). Similarly, in rat cardiac muscle, previously reported impaired SR Ca2+ release and increased calpain activity after ischaemia-reperfusion (Singh et al. 2012) are in accordance with reduced JP2 protein found in this study. However, further experiments are needed to clarify and complete some aspects of the proposed model, as the body of evidence is not strong enough to establish μ-calpain as the sole factor responsible for cleavage of junctophilins. Therefore, future experiments should focus on demonstrating a direct association between high intracellular Ca2+ levels, activation of μ-calpain and proteolysis of junctophilins, and on testing involvement of other players such as proteases or triad proteins in this process. To do so, it would be interesting to analyse, for example, proteolysis of junctophilins in μ-calpain-deficient skeletal myotubes after calcium disruption of E-C coupling. Furthermore, knocked down myotubes for each junctophilin isoform would help to differentiate their specific role in the E-C uncoupling process. Another possibility would be to follow their previous work analyse proteolysis of junctophilins in muscle fibres after exogenous application of preactivated μ-calpain. Also, this model needs to be verified in a more physiological human model, such as skeletal muscle fibres from control and dystrophic patients. Additionally, it would be helpful to test whether other proteins interacting with junctophilins and/or triads are involved in E-C disruption and Ca2+ misregulation. Interesting candidates may be TRPC3, Caveolin 3 and Orai1, which have been associated with junctophilins in a previous study (Golini et al. 2011). The work presented by Murphy et al. (2013) has potential clinical implications regarding involvement of μ-calpain activation and junctophilin proteolysis in muscular dystrophy and heart failure. Thus, future research could focus on exploring a mechanism to avoid proteolysis of junctophilins, or activation of μ-calpain to prevent disruption of E-C coupling. This would be useful as a treatment of disorders comprising skeletal and cardiac muscular damage. In summary, the authors have provided the novel finding of μ-calpain activation associated with proteolysis of junctophilins that, in turn, may trigger processes involved in muscle damage such as E-C uncopling and impaired Ca2+ homeostasis. Therefore, these results open new potential therapeutic targets for future research concerning muscular dystrophies and cardiac dysfunction.

To verify whether altered glial glutamate uptake contributes to the reduced efficacy of glutamatergic transmission reported in the prefrontal cortex of schizophrenics, we studied the expression of GLT-1, the transporter responsible for most glutamate transport, in autoptic samples of prefrontal cortex using real time quantitative RT-PCR, immunocytochemistry, and functional assays. GLT-1 mRNA levels in medication-free patients were 2.5-fold higher than in controls, whereas they were normal or reduced in patients treated with antipsychotics. We also observed a 4-fold increase in L-[(3)H]-Glu uptake in Xenopus oocytes injected with mRNA from the prefrontal cortex of a medication-free schizophrenic and a 2-fold increase in GLT-1 protein in the same cortical area of another medication-free patient. Results suggest that GLT-1 mRNA, protein and function are increased in prefrontal cortex of schizophrenics.