Loss of CNTNAP2 Alters Human Cortical Excitatory Neuron Differentiation and Neural Network Development.

BACKGROUND: Loss-of-function mutations in the contactin-associated protein-like 2 (CNTNAP2) gene are causal for neurodevelopmental disorders, including autism, schizophrenia, epilepsy, and intellectual disability. CNTNAP2 encodes CASPR2, a single-pass transmembrane protein that belongs to the neurexin family of cell adhesion molecules. These proteins have a variety of functions in developing neurons, including connecting presynaptic and postsynaptic neurons, and mediating signaling across the synapse. METHODS: To study the effect of loss of CNTNAP2 function on human cerebral cortex development, and how this contributes to the pathogenesis of neurodevelopmental disorders, we generated human induced pluripotent stem cells from one neurotypical control donor null for full-length CNTNAP2, modeling cortical development from neurogenesis through to neural network formation in vitro. RESULTS: CNTNAP2 is particularly highly expressed in the first two populations of early-born excitatory cortical neurons, and loss of CNTNAP2 shifted the relative proportions of these two neuronal types. Live imaging of excitatory neuronal growth showed that loss of CNTNAP2 reduced neurite branching and overall neuronal complexity. At the network level, developing cortical excitatory networks null for CNTNAP2 had complex changes in activity compared with isogenic controls: an initial period of relatively reduced activity compared with isogenic controls, followed by a lengthy period of hyperexcitability, and then a further switch to reduced activity. CONCLUSIONS: Complete loss of CNTNAP2 contributes to the pathogenesis of neurodevelopmental disorders through complex changes in several aspects of human cerebral cortex excitatory neuron development that culminate in aberrant neural network formation and function.

Loss function of SL (sekiguchi lesion) in the rice cultivar Minghui 86 leads to enhanced resistance to (hemi)biotrophic pathogens.

BACKGROUND: Serotonin, originally identified as a neurotransmitter in mammals, functions as an antioxidant to scavenge cellular ROS in plants. In rice, the conversion of tryptamine to serotonin is catalyzed by SL (sekiguchi lesion), a member of cytochrome P450 monooxygenase family. The sl mutant, originated from rice cultivar Sekiguchi-asahi, exhibits spontaneous lesions, whereas its immune responses to pathogens have not been clearly characterized. RESULTS: Here we identified three allelic mutants of SL in an indica rice restore line Minghui 86 (MH86), named as sl-MH-1, - 2 and - 3, all of which present the typical lesions under normal growth condition. Compared with those in MH86, the serotonin content in sl-MH-1 is dramatically decreased, whereas the levels of tryptamine and L-trytophan are significantly increased. The sl-MH-1 mutant accumulates high H2O2 level at its lesion sites and is more sensitive to exogenous H2O2 treatment than the wild type. When treated with the reductant vitamin C (Vc), the lesion formation on sl-MH-1 leaves could be efficiently suppressed. In addition, sl-MH-1 displayed more resistant to both the blast fungus and blight bacteria, Pyricularia oryzae (P. oryzae, teleomorph: Magnaporthe oryzae) and Xanthomonas oryzae pv. Oryzae (Xoo), respectively. The pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) responses, like reactive oxygen species (ROS) burst and callose deposition, were enhanced in sl-MH-1. Moreover, loss function of SL resulted in higher resting levels of the defense hormones, salicylic acid and jasmonic acid. The RNA-seq analysis indicated that after P. oryzae infection, transcription of the genes involved in reduction-oxidation regulation was the most markedly changed in sl-MH-1, compared with MH86. CONCLUSIONS: Our results indicate that SL, involving in the final step of serotonin biosynthesis, negatively regulates rice resistance against (hemi)biotrophic pathogens via compromising the PTI responses and defense hormones accumulation.

Physiological and metabolic features of mice with CRISPR/Cas9-mediated loss-of-function in growth hormone-releasing hormone.

Our previous study demonstrated that the loss of growth hormone releasing hormone (GHRH) results in increased lifespan and improved metabolic homeostasis in the mouse model generated by classical embryonic stem cell-based gene-targeting method. In this study, we targeted the GHRH gene using the CRISPR/Cas9 technology to avoid passenger alleles/mutations and performed in-depth physiological and metabolic characterization. In agreement with our previous observations, male and female GHRH-/- mice have significantly reduced body weight and enhanced insulin sensitivity when compared to wild type littermates. Dual-energy X-ray absorptiometry showed that there were significant decreases in lean mass, bone mineral content and density, and a dramatic increase in fat mass of GHRH-/- mice when compared to wild type littermates. Indirect calorimetry measurements showed dramatic reductions in oxygen consumption, carbon dioxide production and energy expenditure in GHRH-/- mice compared to wild type mice in both light and dark cycles. Respiratory exchange ratio was significantly lower in GHRH-/- mice during the light cycle, but not during the dark cycle, indicating a circadian related metabolic shift towards fat utilization in the growth hormone deficient mice. The novel CRISPR/Cas9 GHRH-/- mice are exhibiting the consistent and unique physiological and metabolic characteristics, which might mediate the longevity effects of growth hormone deficiency in mice.

Clarification of the dispensability of PDX1.2 for Arabidopsis viability using CRISPR/Cas9.

BACKGROUND: PDX1.2 has recently been shown to be a regulator of vitamin B6 biosynthesis in plants and is implicated in biotic and abiotic stress resistance. PDX1.2 expression is strongly and rapidly induced by heat stress. Interestingly, PDX1.2 is restricted to eudicota, wherein it behaves as a non-catalytic pseudoenzyme and is suggested to provide an adaptive advantage to this clade. A first report on an Arabidopsis insertion mutant claims that PDX1.2 is indispensable for viability, being essential for embryogenesis. However, a later study using an independent insertion allele suggests that knockout mutants of pdx1.2 are viable. Therefore, the essentiality of PDX1.2 for Arabidopsis viability is a matter of debate. Given the important implications of PDX1.2 in stress responses, it is imperative to clarify if it is essential for plant viability. RESULTS: We have studied the previously reported insertion alleles of PDX1.2, one of which is claimed to be essential for embryogenesis (pdx1.2-1), whereas the other is viable (pdx1.2-2). Our study shows that pdx1.2-1 carries multiple T-DNA insertions, but the T-DNA insertion in PDX1.2 is not responsible for the loss of embryogenesis. By contrast, the pdx1.2-2 allele is an overexpressor of PDX1.2 under standard growth conditions and not a null allele as previously reported. Nonetheless, upregulation of PDX1.2 expression under heat stress is impaired in this mutant line. In wild type Arabidopsis, studies of PDX1.2-YFP fusion proteins show that the protein is enhanced under heat stress conditions. To clarify if PDX1.2 is essential for Arabidopsis viability, we generated several independent mutant lines using the CRISPR-Cas9 gene editing technology. All of these lines are viable and behave similar to wild type under standard growth conditions. Reciprocal crosses of a subset of the CRISPR lines with pdx1.2-1 recovers viability of the latter line and demonstrates that knocking out the functionality of PDX1.2 does not impair embryogenesis. CONCLUSIONS: Gene editing reveals that PDX1.2 is dispensable for Arabidopsis viability and resolves conflicting reports in the literature on its function.

Genotypic characterisation of monepantel resistance in historical and newly derived field strains of Teladorsagia circumcincta.

Recent reports of monepantel (MPTL) resistance in UK field isolates of Teladorsagia circumcincta has highlighted the need for a better understanding of the mechanism of MPTL-resistance in order to preserve its anthelmintic efficacy in this economically important species. Nine discrete populations of T. circumcincta were genotypically characterised; three MPTL-susceptible isolates, three experimentally selected MPTL-resistant strains and three field derived populations. Full-length Tci-mptl-1 gene sequences were generated and comparisons between the MPTL-susceptible isolates, MPTL-resistant strains and one field isolate, showed that different putative MPTL-resistance conferring mutations were present in different resistant isolates. Truncated forms of the Tci-mptl-1 gene were also observed. The genetic variability of individual larvae, within and between populations, was examined using microsatellite analyses at 10 'neutral' loci (presumed to be unaffected by MPTL). Results confirmed that there was little background genetic variation between the populations, global FST <0.038. Polymorphisms present in exons 7 and 8 of Tci-mptl-1 enabled genotyping of individual larvae. A reduction in the number of genotypes was observed in all MPTL-resistant strains compared to the MPTL-susceptible strains that they were derived from, suggesting there was purifying selection at Tci-mptl-1 as a result of MPTL-treatment. The potential link between benzimidazole (BZ)-resistance and MPTL-resistance was examined by screening individual larvae for the presence of three SNPs associated with BZ-resistance in the ß-tubulin isotype-1 gene. The majority of larvae were BZ-susceptible homozygotes at positions 167 and 198. Increased heterozygosity at position 200 was observed in the MPTL-resistant strains compared to their respective MPTL-susceptible population. There was no decrease in the occurrence of BZ-resistant genotypes in larvae from each population. These differences, in light of the purifying selection at this locus in all MPTL-resistant isolates, suggests that Tci-mptl-1 confers MPTL-resistance in T. circumcincta, as in Haemonchus contortus, but that different mutations in Tci-mptl-1 can confer resistance in different populations.

Loss-of-Function Mutations in Human Regulator of G Protein Signaling RGS2 Differentially Regulate Pharmacological Reactivity of Resistance Vasculature.

Regulator of G protein signaling 2 (RGS2) plays a role in reducing vascular contraction and promoting relaxation due to its GTPase accelerating protein activity toward Gαq. Previously, we identified four human loss-of-function (LOF) mutations in RGS2 (Q2L, D40Y, R44H, and R188H). This study aimed to investigate whether those RGS2 LOF mutations disrupt the ability of RGS2 to regulate vascular reactivity. Isolated mesenteric arteries (MAs) from RGS2-/- mice showed an elevated contractile response to 5 nM angiotensin II and a loss of acetylcholine (ACh)-mediated vasodilation. Reintroduction of a wild-type (WT) RGS2-GFP plasmid into RGS2-/- MAs suppressed the vasoconstrictor response to angiotensin II. RGS2 LOF mutants failed to suppress the angiotensin II constriction response compared with RGS2 WT. In contrast, ACh-mediated vasoconstriction was restored by expression of RGS2 WT, D40Y, and R44H but not by RGS2 Q2L or R188H. Phosphorylation of RGS2 D40Y and R44H by protein kinase G (PKG) may explain their maintained function to support relaxation in MAs. This is supported by phosphomimetic mutants and suppression of vasorelaxation mediated by RGS2 D40Y by a PKG inhibitor. These results demonstrate that RGS2 attenuates vasoconstriction in MAs and that RGS2 LOF mutations cannot carry out this effect. Among them, the Q2L and R188H mutants supported less relaxation to ACh, whereas relaxation mediated by the D40Y and R44H mutant proteins was equal to that with WT protein. Phosphorylation of RGS2 by PKG appears to contribute to this vasorelaxation. These results provide insights for precision medicine targeting the rare individuals carrying these RGS2 mutations. SIGNIFICANCE STATEMENT: Regulator of G protein signaling 2 (RGS2) has been implicated in the control of blood pressure; rare mutations in the RGS2 gene have been identified in large-scale human gene sequencing studies. Four human mutations in RGS2 that cause loss of function (LOF) in cell-based assays were examined in isolated mouse arteries for effects on both vasoconstriction and vasodilation. All mutants showed the expected LOF effects in suppressing vasoconstriction. Surprisingly, the D40Y and R44H mutant RGS2 showed normal control of vasodilation. We propose that this is due to rescue of the mislocalization phenotype of these two mutants by nitric oxide-mediated/protein kinase G-dependent phosphorylation. These mechanisms may guide drug discovery or drug repurposing efforts for hypertension by enhancing RGS2 function.

Drosophila ClC-a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits.

Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. We explored the function of the Drosophila ClC-a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC-a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss-of-function ClC-a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC-a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia-mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.

Identification of candidate domestication-related genes with a systematic survey of loss-of-function mutations.

Domestication is an important key co-evolutionary process through which humans have extensively altered the genomic make-up and appearance of both plants and animals. The identification of domestication-related genes remains very arduous. In this study, we present a systematic analytical approach that harnesses two recent advances in genomics, whole-genome sequencing (WGS) and prediction of loss-of-function (LOF) mutations, to greatly facilitate the assembly of an enriched catalogue of domestication-related candidate genes. Using WGS data for 296 cultivated (Glycine max) and 64 wild soybean accessions, we identified 8699 LOF variants, and 116 genes that are uniquely fixed for one or more LOF allele(s) in domesticated soybeans. Existing soybean transcriptomic data led us to overcome analytical challenges associated with whole-genome duplications and to identify neo- or subfunctionalized genes. This systematic approach allowed us to identify 110 candidate domestication-related genes in an efficient and rapid way. This catalogue contains previously well characterized domestication genes in soybean, as well as some orthologs from other domesticated crop species. In addition, it comprises many promising candidate domestication genes. Overall, this collection of candidate domestication-related genes in soybean is almost twice as large as the sum of all previously reported candidate genes in all other crops. We believe this systematic approach could readily be used in wide range of species.

Heterogeneity in the Drosophila gustatory receptor complexes that detect aversive compounds.

Animals must detect aversive compounds to survive. Bitter taste neurons express heterogeneous combinations of bitter receptors that diversify their response profiles, but this remains poorly understood. Here we describe groups of taste neurons in Drosophila that detect the same bitter compounds using unique combinations of gustatory receptors (GRs). These distinct complexes also confer responsiveness to non-overlapping sets of additional compounds. While either GR32a/GR59c/GR66a or GR22e/GR32a/GR66a heteromultimers are sufficient for lobeline, berberine, and denatonium detection, only GR22e/GR32a/GR66a responds to strychnine. Thus, despite minimal sequence-similarity, Gr22e and Gr59c show considerable but incomplete functional overlap. Since the gain- or loss-of-function of Gr22e or Gr59c alters bitter taste response profiles, we conclude a taste neuron's specific combination of Grs determines its response profile. We suspect the heterogeneity of Gr expression in Drosophila taste neurons diversifies bitter compound detection, improving animal fitness under changing environmental conditions that present a variety of aversive compounds.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.