The effects of fatigue and oxidation on contractile function of intact muscle fibers and myofibrils isolated from the mouse diaphragm

Abstract The goal of this study was to investigate the effects of repetitive stimulation and the oxidant H2O2 on fatigue of diaphragm intact fibers and in myofibrils measured with different Ca2+ concentrations. Intact fibers were isolated from mice diaphragm, and twitch and tetanic contractions (500 ms duration) were performed at different frequencies of stimulation ranging from 15 Hz to 150 Hz to establish a force-frequency relation before and after a fatigue and recovery protocol, without or after a treatment with H2O2. Fatigue was induced with isometric contractions (500 ms, 40 Hz) evoked every 0.8 seconds, with a total of 625 tetani. After the fatigue, the force recovery was followed by invoking tetanic contractions (500 ms, 40 Hz) every 1 min, with a total duration of 30 min. Individual myofibrils were also isolated from the mouse diaphragm and were tested for isometric contractions before and after treatment with H2O2 and NAC. In a second series of experiments, myofibrils were activated at different pCa (pCa = −log10 [Ca2+]), before and after H2O2 treatment. After 15 minutes of H2O2 treatment, the myofibrillar force was decreased to 54 ± 12% of its control, maximal value, and a result that was reversed by NAC treatment. The force was also decreased after myofibrils were treated with H2O2 and activated in pCa ranging between 4.5 and 5.7. These results suggest that fatigue in diaphragm intact fibers and at the myofibrils level is caused partially by oxidation of the contractile proteins that may be responsible for changing the force in various levels of Ca2+ activation

Differential regulation of neurexin at glutamatergic and GABAergic synapses

Neurexins (Nrxs) have emerged as potential determinants of synaptic specificity, but little is known about their localization at central synapses. Here we show that Nrxs have a remarkably selective localization at distinct types of glutamatergic synapses and we reveal an unexpected ontogenetic regulation of Nrx expression at GABAergic synapses. Our data indicate that synapses are specified by molecular interactions that involve both Nrx-dependent and Nrx-independent mechanisms. We propose that differences in the spatio-temporal profile of Nrx expression may contribute to specify the molecular identity of synapses

Current Strategies and Future Directions to Achieve Deep Molecular Response and Treatment-Free Remission in Chronic Myeloid Leukemia

The treatment of chronic myeloid leukemia (CML) has been radically changed by the approval of tyrosine kinase inhibitors (TKIs), which target BCR-ABL1 kinase activity. CML is now managed as a chronic disease requiring long-term treatment and close molecular monitoring. It has been shown that in a substantial number of patients who have achieved a stable deep molecular response (DMR), TKI treatment can be safely discontinued without loss of response. Therefore, treatment-free remission (TFR), through the achievement of a DMR, is increasingly regarded as a feasible treatment goal in many CML patients. However, only nilotinib has approval in this setting and a number of controversial aspects remain regarding treatment choices and timings, predictive factors, patient communication, and optimal strategies to achieve successful TFR. This narrative review aims to provide a comprehensive overview on how to optimize the path to DMR and TFR in patients with CML, and discusses recent data and future directions

Neuregulin1/ErbB4-induced migration in ST14A striatal progenitors: calcium-dependent mechanisms and modulation by NMDA receptor activation

<p>Abstract</p> <p>Background</p> <p>A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence.</p> <p>Results</p> <p>RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca²⁺channels characterized by low Mg²⁺-sensitivity and low Ca²⁺-permeability, generating small, long-lasting currents. Ca²⁺-imaging experiments showed slow [Ca²⁺]_iincreases in 45% of the cells following 8 μM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca²⁺in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca²⁺]_i, in 99% of the cells. These intracellular Ca²⁺signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals.</p> <p>Conclusions</p> <p>In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca²⁺]_iincrease in ST14A neural progenitors; NRG1-induced migration is Ca²⁺-dependent and can be positively modulated by activation of the NMDA receptor.</p

Dystroglycan mediates clustering of essential GABAergic components in cerebellar Purkinje cells

Muscle dystrophin–glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular matrix. In neurons, dystroglycan and dystrophin, two major components of the DGC, localize in a subset of GABAergic synapses, where their function is unclear. Here we used mouse models to analyze the specific role of the DGC in the organization and function of inhibitory synapses. Loss of full-length dystrophin in mdx mice resulted in a selective depletion of the transmembrane β-dystroglycan isoform from inhibitory post-synaptic sites in cerebellar Purkinje cells. Remarkably, there were no differences in the synaptic distribution of the extracellular α-dystroglycan subunit, of GABAA receptors and neuroligin 2. In contrast, conditional deletion of the dystroglycan gene from Purkinje cells caused a disruption of the DGC and severely impaired post-synaptic clustering of neuroligin 2, GABAA receptors and scaffolding proteins. Accordingly, whole-cell patch-clamp analysis revealed a significant reduction in the frequency and amplitude of spontaneous IPSCs recorded from Purkinje cells. In the long-term, deletion of dystroglycan resulted in a significant decrease of GABAergic innervation of Purkinje cells and caused an impairment of motor learning functions. These results show that dystroglycan is an essential synaptic organizer at GABAergic synapses in Purkinje cells