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The hippocampus is vulnerable to deterioration in Alzheimer's disease (AD). It is, however, a heterogeneous structure, which may contribute to the differential volumetric changes along its septotemporal axis during AD progression. Here, we investigated amyloid plaque deposition along the dorsoventral axis in two strains of transgenic AD (ADTg) mouse models. We also used patch-clamp physiology in these mice to probe for functional consequences of AD pathogenesis in ventral hippocampus, which we found bears significantly higher plaque burden in the aged ADTg group compared to corresponding dorsal regions. Despite dorsoventral differences in amyloid load, ventral CA1 pyramidal neurons of aged ADTg mice exhibited subthreshold physiological changes similar to those previously reported in dorsal neurons, indicative of an HCN channelopathy, but lacked exacerbated suprathreshold accommodation. Additionally, HCN channel function could be rescued by pharmacological manipulation of the endoplasmic reticulum. These observations suggest that an AD-linked HCN channelopathy emerges in both dorsal and ventral CA1 pyramidal neurons, but that the former encounter an additional integrative obstacle in the form of reduced intrinsic excitability.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Solute carrier family 6 member 1 (SLC6A1) gene encodes GAT-1, a GABA transporter expressed on glia and presynaptic terminals of inhibitory neurons. Mutations in SLC6A1 are associated with myoclonic atonic epilepsy, absence epilepsy, autism, and intellectual disability. However, the mechanisms leading to these defects are unknown. Here, we used a novel mouse model harboring a point mutation (S295L) recently identified in the human SLC6A1 gene that results in impaired membrane trafficking of the GAT-1 protein. We performed chronic wireless telemetry recordings of heterozygous (GAT-1S295L/+) mice, and of mice lacking one or both copies of the gene (GAT-1+/− and GAT-1−/−). We assessed their behaviors and pharmacosensitivity, and investigated the relationship between seizure burden and behavioral performance. GAT-1S295L/+ mice exhibited frequent spikewave discharges (SWDs) associated with behavioral arrest, and there was a dose-effect relationship between GAT-1 gene copy number and the severity of electrocorticogram (ECoG) abnormalities. Seizure burden was inversely correlated with behavioral performance. Forelimb grip strength was reduced in female mice. Acute administration of GAT-1 antagonist NO-711 induced SWDs in wildtype mice, exacerbated the phenotype in GAT-1S295L/+ and GAT-1+/− mice, and had no effect on GAT-1−/− mice lacking the drug target. By contrast, ethosuximide normalized the ECoG in GAT-1S295L/+ and GAT-1+/− mice. In conclusion, GAT-1S295L/+ mice show haploinsufficiency with evidence of GAT-1 hypofunction. This mouse model reconstitutes major aspects of human disease and thus provides a useful preclinical model for drug screening and gene therapy.Significance StatementThe SLC6A1 gene encodes for GAT-1, a major GABA transporter. Mutations in SLC6A1 lead to a spectrum of neurodevelopmental disorders and epilepsies collectively referred to as SLC6A1-related disorders (SRD). A critical contributor to disability in SRD patients is the burden of seizures and their sequelae. There is an urgent need to understand the mechanisms of SRD as they will inform therapeutic interventions. Mouse models can provide critical information allowing both the assessment of candidate therapies and the design of next generations therapies. Here we used behavioral assessments and wireless electrophysiology in a new mouse model of SRD to understand the disease pathogenesis and the association between seizure burden and behavioral deficits.

Genome-wide association studies identified the BIN1 locus as a leading modulator of genetic risk in Alzheimer’s disease (AD). One limitation in understanding BIN1’s contribution to AD is its unknown function in the brain. AD-associated BIN1 variants are generally noncoding and likely change expression. Here, we determined the effects of increasing expression of the major neuronal isoform of human BIN1 in cultured hippocampal neurons. Higher BIN1 induced network hyperexcitability on multielectrode arrays, increased frequency of synaptic transmission, and elevated calcium transients, indicating that increasing BIN1 drives greater neuronal activity. In exploring the mechanism of these effects on neuronal physiology, we found that BIN1 interacted with L-type voltage-gated calcium channels (LVGCCs) and that BIN1–LVGCCs interactions were modulated by Tau in vitro and in vivo. Finally, Tau reduction prevented BIN1-induced network hyperexcitability. These data shed light on BIN1’s neuronal function and suggest that it may contribute to Tau-dependent hyperexcitability in AD.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved “poison” exon, 20N, whose inclusion is predicted to lead to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A expression or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy., Author summary Dravet syndrome (DS) is a neurological disorder affecting approximately 1:15,700 Americans that causes generalized epilepsy and associated complications. While most patients have a mutation in the SCN1A gene that encodes the Nav1.1 voltage-gated sodium channel, about 20% do not have a mutation identified by exome or targeted sequencing. Recently, we identified variants in intron 20, a noncoding region of SCN1A, in some DS patients. We hypothesized that these variants alter SCN1A transcript processing, decrease Nav1.1 function, and lead to DS pathophysiology via inclusion of exon 20N, a “poison” exon that leads to a premature stop codon. In this study, we generated a knock-in mouse model, Scn1a+/KI, of one of these variants, which resides in a genomic region that is extremely conserved across vertebrate species. We found that Scn1a+/KI mice have reduced levels of Scn1a transcript and Nav1.1 protein and develop DS-related phenotypes. We find that transcripts from brains of Scn1a+/KI mice show elevated rates of Scn1a exon 20N inclusion. We also explored the relationship between exon 20N inclusion and Scn1a expression during development, and found that, during brain development when Scn1a expression is low, exon 20N inclusion is high; postnatally, as Scn1a expression increases, there is a corresponding decrease in exon 20N usage. Expression of another voltage-gated sodium channel transcript, Scn8a (Nav1.6), was similarly regulated. Together, these data demonstrate that poison exon inclusion is a conserved mechanism to control sodium channel expression in the brain, and that an intronic mutation that disrupts the normal developmental regulation of poison exon inclusion leads to reduced Nav1.1 and generalized epilepsy.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved “poison” exon, 20N, whose inclusion leads to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A transcript processing or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.Author SummaryDravet syndrome (DS) is a neurological disorder affecting approximately 1:15,700 Americans[1]. While most patients have a mutation in the SCN1A gene encoding Nav1.1 sodium channels, about 20% do not have a mutation identified by exome sequencing. Recently, we identified variants in intron 20N, a noncoding region of SCN1A, in some DS patients [2]. We predicted that these variants alter SCN1A transcript processing, decrease Nav1.1 function, and lead to DS pathophysiology via inclusion of exon 20N, a “poison” exon that leads to a premature stop codon. In this study, we generated a knock-in mouse model, Scn1a+/KI, of one of these variants, NM_006920.4(SCN1A):c.3969+2451G>C, which resides in a genomic region that is extremely conserved across vertebrate species. We found that Scn1a+/KI mice have reduced levels of Scn1a transcript and Nav1.1 protein and develop DS-related phenotypes. Consistent with the poison exon hypothesis, transcripts from brains of Scn1a+/KI mice showed elevated rates of Scn1a exon 20N inclusion. Since Scn1a expression in the brain is regulated developmentally, we next explored the developmental relationship between exon 20N inclusion and Scn1a expression. During normal embryogenesis, when Scn1a expression was low, exon 20N inclusion was high; postnatally, as Scn1a expression increased, there was a corresponding decrease in exon 20N usage. Expression of another voltage-gated sodium channel transcript, Scn8a (Nav1.6), was similarly regulated, with inclusion of a poison exon termed as 18N early in development when Scn8a expression was low, followed by a postnatal decrease in exon 18N inclusion and corresponding increase in Scn8a expression. Together, these data demonstrate that poison exon inclusion is a conserved mechanism to control sodium channel expression in the brain, and that an intronic mutation that disrupts the normal developmental regulation of poison exon inclusion leads to reduced Nav1.1 and DS pathophysiology.

Voltage-gated ion channels are critical for neuronal integration. Some of these channels, however, are mis-regulated in several neurological disorders, causing both gain- and loss-of-function channelopathies in neurons. Using several transgenic mouse models of Alzheimer’s disease (AD), we find that sub-threshold voltage signals strongly influenced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels progressively deteriorate over chronological aging in hippocampal CA1 pyramidal neurons. The degraded signaling via HCN channels in the transgenic mice is accompanied by an age-related global loss of their non-uniform dendritic expression. Both the aberrant signaling via HCN channels and their mislocalization could be restored using a variety of pharmacological agents that target the endoplasmic reticulum (ER). Our rescue of the HCN channelopathy helps provide molecular details into the favorable outcomes of ER-targeting drugs on the pathogenesis and synaptic/cognitive deficits in AD mouse models, and implies that they might have beneficial effects on neurological disorders linked to HCN channelopathies.