Notes

Decoding Post-Translational Modification Crosstalk With Proteomics.
Neuronal Ca2+ entry elicited by electrical activity contributes to information coding via activation of K+ and Cl- channels. While Ca2+-dependent K+ channels have been extensively studied, the molecular identity and role of Ca2+-activated Cl- channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca2+-activated Cl- conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS. Two-photon functional imaging using genetically encoded calcium indicators (GECIs) is one prominent tool to map neural activity. Under optimized experimental conditions, GECIs detect single action potentials in individual cells with high accuracy. However, using current approaches, these optimized conditions are never met when imaging large ensembles of neurons. Here, we developed a method that substantially increases the signal-to-noise ratio (SNR) of population imaging of GECIs by using galvanometric mirrors and fast smart line scan (SLS) trajectories. We validated our approach in anesthetized and awake mice on deep and dense GCaMP6 staining in the mouse barrel cortex during spontaneous and sensory-evoked activity. Compared to raster population imaging, SLS led to increased SNR, higher probability of detecting calcium events, and more precise identification of functional neuronal ensembles. SLS provides a cheap and easily implementable tool for high-accuracy population imaging of neural GCaMP6 signals by using galvanometric-based two-photon microscopes. It is widely accepted that Beta-band oscillations play a role in sensorimotor behavior. To further explore this role, we developed a hybrid platform to combine neural operant conditioning and phase-specific intracortical microstimulation (ICMS). We trained monkeys, implanted with 96 electrode arrays in the motor cortex, to volitionally enhance local field potential (LFP) Beta-band (20-30 Hz) activity at selected sites using a brain-machine interface. We find that Beta oscillations of LFP and single-unit spiking activity increase dramatically with brain-machine interface training and that pre-movement Beta power is anti-correlated with task performance. We also find that phase-specific ICMS modulates the power and phase of oscillations, shifting local networks between oscillatory and non-oscillatory states. Furthermore, ICMS induces phase-dependent effects in animal reaction times and success rates. These findings contribute to unraveling the functional role of cortical oscillations and to the future development of clinical tools for ameliorating abnormal neuronal activities in brain disease. To understand the conditions necessary to initiate and terminate seizures, we investigate optogenetically induced hippocampal seizures with LFP, fMRI, and optogenetic inhibition. During afterdischarge induction using optogenetics, LFP recordings show that stimulations with earlier ictal onset times are more likely to result in afterdischarges and are more difficult to curtail with optogenetic inhibition. These results are generalizable across two initiation sites, the dorsal and ventral hippocampus. fMRI shows that afterdischarges initiated from the dorsal or ventral hippocampus exhibit distinct networks. Short-duration seizures initiated in the dorsal and ventral hippocampus are unilateral and bilateral, respectively, while longer-duration afterdischarges recruit broader, bilateral networks. When optogenetic inhibition is ineffective at stopping seizures, the network activity spreads more extensively but largely overlaps with the network activity associated with seizures that could be curtailed. These results provide insights into how seizures can be inhibited, which has implications for targeted seizure interventions. Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties. Type I interferons (IFNs) play critical roles in anti-viral and anti-tumor immunity. However, they also suppress protective immune responses in some infectious diseases. Here, we identify type I IFNs as major upstream regulators of CD4+ T cells from visceral leishmaniasis (VL) patients. Furthermore, we report that mice deficient in type I IFN signaling have significantly improved control of Leishmania donovani, a causative agent of human VL, associated with enhanced IFNγ but reduced IL-10 production by parasite-specific CD4+ T cells. Importantly, we identify a small-molecule inhibitor that can be used to block type I IFN signaling during established infection and acts synergistically with conventional anti-parasitic drugs to improve parasite clearance and enhance anti-parasitic CD4+ T cell responses in mice and humans. Thus, manipulation of type I IFN signaling is a promising strategy for improving disease outcome in VL patients. check details
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