The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of GRIN2B: G689C and G689S. Structure simulations suggest severely impaired glutamate binding which we confirm by functional analysis. Both variants show three-orders of magnitude reductions in glutamate EC50, with G689S exhibiting the largest reductions observed in GRIN2B (~2000-fold). Moreover, variants multimerize with, and upregulate, GluN2Bwt-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the variants fail to respond due to G689's novel role in proton-sensing. Together, we describe two unique variants with extreme effects on channel function. We employ protein-stability measures to explain why current (and future) LBD mutations in GluN2B primarily instigate Loss-of-Function.

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Oxidative stress (OS) is common in inflammatory conditions and may be important in atopic dermatitis (AD) etiology. The aim of this project was to study the involvement of oxidation in FSL-1 (deacylated lipoprotein)-triggered signaling pathways leading to AD-typical cytokine expression in HaCaT keratinocytes. HaCaT keratinocytes, pretreated with the inhibitor to OS N-acetylcysteine (NAC), were exposed to FSL-1, a stimulator of AD-related cytokines. Cytokines expression was studied by real time polymerase chain reaction (PCR); nuclear factor-kappa B (NF-κB) and p38 mitogen activated protein kinase (MAPK) activities were studied by western blotting; and the oxidative state of cells was determined by the dichlorofluorescein (DCF) assay. We found that endogenous OS in keratinocytes appeared 4 h after FSL-1 administration. OS activated NF-κB, but not p38 MAPK, and the inhibition of OS reduced FSL-1 induced interleukin (IL) 33, thymic stromal lymphopoietin (TSLP) and TNFα mRNA expression. We conclude that FSL-1 triggers an OS reaction in HaCaT keratinocytes, which is probably a secondary event affecting the expression of specific AD typical cytokines, possibly through the NF-κB pathways. This role of OS in the inflammatory response in AD is worth further investigating.

The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to transduction by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is markedly lower than in rodents; likely holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing, patch clamp electrophysiology and calcium imaging. We then leveraged the cultures for the rapid screening of various viruses, only to find that all yielded poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quails led us to rationally design a custom-made AAV variant (AAV1-T593K; AAV1*) that exhibits improved transduction efficiencies in vitro and in vivo (14- and five-fold, respectively). Together, we present unique culturing method, transcriptomic profiles of quail’s brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.

The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to infection by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is scarce; markedly holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a unique protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing and patch clamp electrophysiology. We then leverage the cultures for the rapid screening of various viruses, only to find that all yielded very poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quail led us to rationally design novel AAV variants, with one variant (AAV1-T593K; AAV1*) showing improved transduction efficiency in vitro and in vivo. Together, we present a novel culturing method, unique transcriptome profiles of quail’s brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.

Purposeful induction of fever for healing, including the treatment of epilepsy, was used over 2000 years ago by Hippocrates. More recently, fever has been demonstrated to rescue behavioral abnormalities in children with autism. However, the mechanism of fever benefit has remained elusive due in large part to the lack of appropriate human disease models recapitulating the fever effect. Pathological mutations in the IQSEC2 gene are frequently seen in children presenting with intellectual disability, autism and epilepsy. We recently described a murine A350V IQSEC2 disease model, which recapitulates important aspects of the human A350V IQSEC2 disease phenotype and the favorable response to a prolonged and sustained rise in body core temperature in a child with the mutation. Our goal has been to use this system to understand the mechanism of fever benefit and then develop drugs that can mimic this effect and reduce IQSEC2-associated morbidity. In this study, we first demonstrate a reduction in seizures in the mouse model following brief periods of heat therapy, similar to what was observed in a child with the mutation. We then show that brief heat therapy is associated with the correction of synaptic dysfunction in neuronal cultures of A350V mice, likely mediated by Arf6-GTP.

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