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. ...
De novo mutations in GNB1, encoding the Gβ1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy. Mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and frequent spike-wave discharges (SWD). Cultured mutant cortical neurons display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses SWD in vivo. In contrast, while valproic acid suppresses SWD, it does not restore normal network behavior, suggesting that ETX has mechanistic specificity for the effects of aberrant Gβ1 signaling. Consistent with this, we show that K78R is a gain-of-function of G protein-coupled potassium channel (GIRK) activation that is potently inhibited by ETX. This work suggests that altered Gβ1 signaling causes disease in part through effects on GIRK channels, illustrates the utility of cultured neuronal networks in pharmacological screening, and establishes effective pre-clinical models for GNB1 Encephalopathy.
The basal ganglia are a group of subcortical nuclei that contribute to action selection and reinforcement learning. The principal neurons of the striatum, spiny projection neurons of the direct (dSPN) and indirect (iSPN) pathways, maintain low intrinsic excitability, requiring convergent excitatory inputs to fire. Here, we examined the role of autophagy in mouse SPN physiology and animal behavior by generating conditional knockouts of Atg7 in either dSPNs or iSPNs. Loss of autophagy in either SPN population led to changes in motor learning but distinct effects on cellular physiology. dSPNs, but not iSPNs, required autophagy for normal dendritic structure and synaptic input. In contrast, iSPNs, but not dSPNs, were intrinsically hyperexcitable due to reduced function of the inwardly rectifying potassium channel, Kir2. These findings define a novel mechanism by which autophagy regulates neuronal activity: control of intrinsic excitability via the regulation of potassium channel function.
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