NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.
Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated 5 neuroanatomical abnormalities, global developmental delay and impaired ultrasonic vocalizations, and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the 10 subiculum-a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlighted the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPUmediated disease mechanisms, and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease. chromatin organization 17,18 . We and others have reported de novo loss-of-function variants [19][20][21][22][23] and microdeletions 24,25 encompassing HNRNPU in pediatric patients with a severe, and often treatment refractory, developmental and epileptic encephalopathy (DEE) characterized by earlyonset epilepsy, moderate to severe developmental delay, autistic features, structural brain abnormalities, hypotonia, short stature and variable renal and cardiac abnormalities. HnRNP U is 35 essential for mammalian development as lethality results by embryonic day 11.5 in mice carrying homozygous hypomorphic mutations 26 . Furthermore, homozygous pathogenic mutations have yet to be reported in humans 27 . Conditional loss of Hnrnpu in mouse cardiomyocytes was also associated with a lethal dilated cardiomyopathy and widespread transcriptional and splicing 3 dysregulation including known cardiomyopathy disease genes 16 . However, the transcriptomic and 40 physiologic effects of Hnrnpu haploinsufficiency in the brain have yet to be characterized.Here we assess the neurophysiological consequences and face validity of an Hnrnpu mouse disease model using in vivo developmental, morphological, electrophysiological, and behavioral studies. We also perform a comprehensive cell-type-and brain region-specific characterization of reduced Hnrnpu levels on gene expression using scRNAseq. Using these 45 data, we generate cell-type-specific disease expression signatures and identify vulnerable cell types in the mutant mouse brain. We then compare these signatures to publicly-available gene expression signatures of cells treated with small molecules in order to identify compounds that correct disease-associated transcriptomic changes towards a normal state. ...
While several studies have attributed the development of tumor-associated seizures to an excitatory-inhibitory imbalance, we have yet to resolve the spatiotemporal interplay between different types of neurons in glioma-infiltrated cortex. Herein, we combined methods for single unit analysis of microelectrode array recordings with wide-field optical mapping of Thy1-GCaMP pyramidal cells in an ex vivo acute slice model of diffusely infiltrating glioma. This enabled simultaneous tracking of individual neurons from both excitatory and inhibitory populations throughout seizure-like events. Moreover, our approach allowed for observation of how the crosstalk between these neurons varied spatially, as we recorded across an extended region of glioma-infiltrated cortex. In tumor-bearing slices, we observed marked alterations in single units classified as putative fast-spiking interneurons, including reduced firing, activity concentrated within excitatory bursts, and deficits in local inhibition. These results were correlated with increases in overall excitability. Mechanistic perturbation of this system with the mTOR inhibitor AZD8055 revealed increased firing of putative fast-spiking interneurons and restoration of local inhibition, with concomitant decreases in overall excitability. Altogether, our findings suggest that diffusely infiltrating glioma affect the interplay between excitatory and inhibitory neuronal populations in a reversible manner, highlighting a prominent role for functional mechanisms linked to mTOR activation.
Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 ( DNM1 ) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1 -targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product.
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