Region-Specific Phosphorylation Determines Neuroligin-3 Localization to Excitatory Versus Inhibitory Synapses.

BACKGROUND: Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. METHODS: We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. RESULTS: Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. CONCLUSIONS: These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.

Rapid modeling of an ultra-rare epilepsy variant in wild-type mice by in utero prime editing.

Generating animal models for individual patients within clinically-useful timeframes holds great potential toward enabling personalized medicine approaches for genetic epilepsies. The ability to rapidly incorporate patient-specific genomic variants into model animals recapitulating elements of the patient's clinical manifestations would enable applications ranging from validation and characterization of pathogenic variants to personalized models for tailoring pharmacotherapy to individual patients. Here, we demonstrate generation of an animal model of an individual epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A, without the need for germline transmission and breeding. Using in utero prime editing in the brain of wild-type mice, our approach yielded high in vivo editing precision and induced frequent, spontaneous seizures which mirrored specific elements of the patient's clinical presentation. Leveraging the speed and versatility of this approach, we introduce PegAssist, a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. The capability to produce individualized animal models rapidly and cost-effectively will reduce barriers to access for precision medicine, and will accelerate drug development by offering versatile in vivo platforms to identify compounds with efficacy against rare neurological conditions.

Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins.

Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[HE], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.

Brain capillary pericytes are metabolic sentinels that control blood flow through a KATP channel-dependent energy switch.

Despite the abundance of capillary thin-strand pericytes and their proximity to neurons and glia, little is known of the contributions of these cells to the control of brain hemodynamics. We demonstrate that the pharmacological activation of thin-strand pericyte KATP channels profoundly hyperpolarizes these cells, dilates upstream penetrating arterioles and arteriole-proximate capillaries, and increases capillary blood flow. Focal stimulation of pericytes with a KATP channel agonist is sufficient to evoke this response, mediated via KIR2.1 channel-dependent retrograde propagation of hyperpolarizing signals, whereas genetic inactivation of pericyte KATP channels eliminates these effects. Critically, we show that decreasing extracellular glucose to less than 1 mM or inhibiting glucose uptake by blocking GLUT1 transporters in vivo flips a mechanistic energy switch driving rapid KATP-mediated pericyte hyperpolarization to increase local blood flow. Together, our findings recast capillary pericytes as metabolic sentinels that respond to local energy deficits by increasing blood flow to neurons to prevent energetic shortfalls.

Phosphotyrosine-Mediated Regulation of Enterohemorrhagic Escherichia coli Virulence.

Enteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.