Deletion of ARGLU1 causes global defects in alternative splicing in vivo and mouse cortical malformations primarily via apoptosis.

Haploinsufficient mutation in arginine and glutamine-rich protein 1 (Arglu1), a newly identified pre-mRNA splicing regulator, may be linked to neural developmental disorders associated with mental retardation and epilepsy in human patients, but the underlying causes remain elusive. Here we show that ablation of Arglu1 promotes radial glial cell (RG) detachment from the ventricular zone (VZ), leading to ectopic localized RGs in the mouse embryonic cortex. Although they remain proliferative, ectopic progenitors, as well as progenitors in the VZ, exhibit prolonged mitosis, p53 upregulation and cell apoptosis, leading to reduced neuron production, neuronal loss and microcephaly. RNA seq analysis reveals widespread changes in alternative splicing in the mutant mouse embryonic cortex, preferentially affecting genes involved in neuronal functions. Mdm2 and Mdm4 are found to be alternatively spliced at the exon 3 and exon 5 respectively, leading to absence of the p53-binding domain and nonsense-mediated mRNA decay (NMD) and thus relieve inhibition of p53. Removal of p53 largely rescues the microcephaly caused by deletion of Arglu1. Our findings provide mechanistic insights into cortical malformations of human patients with Arglu1 haploinsufficient mutation.

Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits.

N-methyl-D-aspartate (NMDA) receptors are heterotetramers comprising two GluN1 and two alternate GluN2 (N2A-N2D) subunits. Here we report full-length cryo-EM structures of the human N1-N2D di-heterotetramer (di-receptor), rat N1-N2C di-receptor and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a best resolution of 3.0 Å. The bilobate N-terminal domain (NTD) in N2D intrinsically adopts a closed conformation, leading to a compact NTD tetramer in the N1-N2D receptor. Additionally, crosslinking the ligand-binding domain (LBD) of two N1 protomers significantly elevated the channel open probability (Po) in N1-N2D di-receptors. Surprisingly, the N1-N2C di-receptor adopted both symmetric (minor) and asymmetric (major) conformations, the latter further locked by an allosteric potentiator, PYD-106, binding to a pocket between the NTD and LBD in only one N2C protomer. Finally, the N2A and N2C subunits in the N1-N2A-N2C tri-receptor display a conformation close to one protomer in the N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse function in major NMDA receptor subtypes.

Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells.

Spinal motor neurons deficiency results in a series of devastating disorders such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal cord injury (SCI). These disorders are currently incurable, while human pluripotent stem cells (hPSCs)-derived spinal motor neurons are promising but suffered from inappropriate regional identity and functional immaturity for the study and treatment of posterior spinal cord related injuries. In this study, we have established human spinal cord neural progenitor cells (hSCNPCs) via hPSCs differentiated neuromesodermal progenitors (NMPs) and demonstrated the hSCNPCs can be continuously expanded up to 40 passages. hSCNPCs can be rapidly differentiated into posterior spinal motor neurons with high efficiency. The functional maturity has been examined in detail. Moreover, a co-culture scheme which is compatible for both neural and muscular differentiation is developed to mimic the neuromuscular junction (NMJ) formation in vitro. Together, these studies highlight the potential avenues for generating clinically relevant spinal motor neurons and modeling neuromuscular diseases through our defined hSCNPCs.

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

S-type Anion Channels SLAC1 and SLAH3 Function as Essential Negative Regulators of Inward K+ Channels and Stomatal Opening in Arabidopsis.

Drought stress induces stomatal closure and inhibits stomatal opening simultaneously. However, the underlying molecular mechanism is still largely unknown. Here we show that S-type anion channels SLAC1 and SLAH3 mainly inhibit inward K+ (K+in) channel KAT1 by protein-protein interaction, and consequently prevent stomatal opening in Arabidopsis. Voltage-clamp results demonstrated that SLAC1 inhibited KAT1 dramatically, but did not inhibit KAT2. SLAH3 inhibited KAT1 to a weaker degree relative to SLAC1. Both the N terminus and the C terminuses of SLAC1 inhibited KAT1, but the inhibition by the N terminus was stronger. The C terminus was essential for the inhibition of KAT1 by SLAC1. Furthermore, drought stress strongly up-regulated the expression of SLAC1 and SLAH3 in Arabidopsis guard cells, and the over-expression of wild type and truncated SLAC1 dramatically impaired K+in currents of guard cells and light-induced stomatal opening. Additionally, the inhibition of KAT1 by SLAC1 and KC1 only partially overlapped, suggesting that SLAC1 and KC1 inhibited K+in channels using different molecular mechanisms. Taken together, we discovered a novel regulatory mechanism for stomatal movement, in which singling pathways for stomatal closure and opening are directly coupled together by protein-protein interaction between SLAC1/SLAH3 and KAT1 in Arabidopsis.

Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLAC1, which is nitrate-selective in rice.

MAIN CONCLUSION: OsSAPK8 is an essential activator of OsSLAC1 by phosphorylation, and OsSLAC1 is a nitrate-selective anion channel. S-type anion channel AtSLAC1 and protein kinase AtOST1 have been well-characterized as two core components of ABA signaling cascade in Arabidopsis guard cells, and AtOST1 functions as a main upstream activator of AtSLAC1 for drought stress- and ABA-induced stomata closure. However, the identity of the ortholog of AtOST1 in rice, the main activator of OsSLAC1, is still unknown. Here, we report that protein kinase OsSAPK8 interacts with and activates OsSLAC1 mainly by phosphorylating serine 129 (S129) of OsSLAC1, and this phosphorylating site corresponds to the specific phosphorylating site serine 120 (S120) of AtSLAC1 for AtOST1. Additionally, we found that OsSLAC1 is a nitrate-selective anion channel without obvious permeability to chloride, malate, and sulfate, and the expression of OsSLAC1 in Arabidopsis slac1-3 (atslac1-3) mutant successfully rescued the hypersensitive phenotype of this mutant to drought stress. Together, this research suggests that OsSAPK8 is a counterpart of AtOST1 for the activation of OsSLAC1, which is a nitrate-selective anion channel.