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.
Voltage-gated ion channels are critical for neuronal integration. Some of these channels, however, are misregulated 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.
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 rat 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–LVGCC interactions were modulated by Tau in rat hippocampal neurons and mouse brain. 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.
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.Genetic discoveries have provided critical insights into potential mechanisms of Alzheimer's disease (AD), the most common neurodegenerative disease. Mutations in APP, PSEN1, or PSEN2 cause early-onset, autosomal dominantly inherited AD, but are quite rare. Several more common genetic variants that increase AD risk to differing degrees have been identified. Among these, variants near BIN1 have particularly high population attributable risk, because the risk allele is highly prevalent (~40% allele frequency for the index SNP, rs6733839) and has a relatively large effect size (odds ratio: 1.20; 95% confidence interval: 1.17-1.23) (Kunkle et al., 2019).BIN1 was first linked to AD in early genome-wide associated studies (GWAS) (Harold et al., 2009;Seshadri et al., 2010) and remains second only to APOE in genomewide significance in the recent meta-analysis of 94,437 individuals by the International Genomics of Alzheimer's Disease Project (Kunkle et al., 2019). This association has been replicated in datasets with subjects from diverse genetic backgrounds (Carrasquillo
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.
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