Gaia Colasante

Objective: Dravet syndrome (DS) is a developmental and epileptic encephalopathy primarily caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of Na _V 1.1 voltage‐gated sodium channel. The disease manifests with febrile and spontaneous seizures, developmental delay, cognitive impairment, and increased risk of sudden unexpected death in epilepsy. Most preclinical therapeutic strategies aim to enhance endogenous Scn1a gene expression or deliver a functional copy via perinatal interventions. Although promising, these approaches face challenges in achieving efficient brain‐wide delivery, targeting all interested brain areas. Methods: In this study, we used a conditional DS reversible mouse model ( Scn1a ^Stop/+ ) to assess the therapeutic potential of postsymptomatic restoration (postnatal day 30) of Na _V 1.1 physiological levels in all neuronal populations but restricted to minimal brain regions. Results: We found that hippocampal correction alone reduces seizure susceptibility, whereas simultaneous restoration in the hippocampus, thalamus, and overlying somatosensory and visual cortex was associated with a trend toward further reduction in seizure burden and improved survival. Significance: These findings indicate that hippocampal restoration accounts for a substantial component of the therapeutic benefit, and broader network correction may confer additional advantages.

The overexpression of MECP2 leads to severe neurological deficits in MECP2 duplication syndrome, and its dosage is considered a risk factor in gene therapy for Rett syndrome. However, in MECP2 duplication syndrome patients, MECP2 dysregulation arises at the embryonic stage while in Rett syndrome gene therapy, MECP2 is delivered into the mature brain. Here, we show that MeCP2 overexpression induces transcriptional alterations in neural progenitor cells, but has minimal effects in neurons in both mouse and human contexts. Consequently, MeCP2 overexpression in neural progenitor cells, but not mature neurons, leads to functional changes. Mechanistically, we observe that both endogenous and overexpressed Mecp2 bind to the same CpG island repertoire. In neurons, where endogenous Mecp2 is highly expressed, ectopic protein expression leads to reduced CpG island binding and accelerated protein degradation. In contrast, in neural progenitor cells, lower endogenous Mecp2 levels facilitate stronger deposition of the ectopic protein onto CpG islands, driving the transcriptional activation of many developmental bivalent genes. We show that this activation is mediated by the interaction with the SWI/SNF chromatin remodeling complex. Our findings establish that increased gene dosage-dependent effects are highly influenced by cell type, levels of proteins and their mechanisms of action.

Neurodevelopmental disorders associated with epilepsy are typically linked to postnatal dysfunction of synaptic proteins and ion channels, yet increasing evidence suggests a role for these proteins before birth. The voltage-gated sodium channel Nav1.1, encoded by SCN1A, is well studied postnatally. SCN1A mutations result in a broad range of neurological phenotypes including developmental and epileptic encephalopathies (DEEs including Dravet syndrome, DS) and fetal lethality. Here, we investigated the role of SCN1A dysfunction in early corticogenesis. By integrating data from DS patient-derived forebrain models, a DS mouse model, and DS post-mortem tissue, we report altered G2/M cell cycle transition, with a shift towards earlier neurogenic fate commitment. These changes lead to altered cortical specification at birth and throughout life. The early developmental roles of Nav1.1 complement the well-known postnatal roles in neuronal excitability. These discoveries reveal new insights into the DS pathogenesis and a new non-canonical role for Nav1.1 in early corticogenesis.

Dravet syndrome is a severe epileptic syndrome that begins during the first year of life of otherwise healthy babies. Over the years, the seizure burden changes, and pathology evolves in strong association with behavioral alterations, including cognitive delay and autistic traits. Initially, this aspect was considered a direct consequence of epilepsy severity, and DS was defined as an epileptic encephalopathy. Increasing evidence suggests that these two aspects of the disease, epilepsy and behavioral impairment, might not be so strictly connected. DS is mostly caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes for the alpha-subunit of the voltage-gated sodium channel Nav1.1, responsible for GABAergic interneuron excitability. Interneuron dysfunction is evident at symptom onset in Dravet murine models, but their activity appears to recover in the chronic phase of the disease, when a series of secondary modifications arise and likely drive the phenotype. Given that the genetic basis of the disease is clear, innovative therapies based on the restoration of sufficient expression levels of Nav1.1 to re-establish functional neuronal activity are being developed. In this work, we review such therapeutic approaches, with a specific focus on the existing evidence of their ability to address not only epilepsy but also behavioral alterations, and to recover secondary modifications.

Claudia Di Berardino, Martina Mainardi, Simone Brusco, Elena Benvenuto, Vania Broccoli, Gaia Colasante; Reply: Spatial and temporal manipulation of the Scn1a ge

Dravet syndrome is a severe epileptic encephalopathy, characterized by drug-resistant epilepsy, severe cognitive and behavioural deficits, with increased risk of sudden unexpected death (SUDEP). It is caused by haploinsufficiency of SCN1A gene encoding for the α-subunit of the voltage-gated sodium channel Na_v1.1. Therapeutic approaches aiming to upregulate the healthy copy of SCN1A gene to restore its normal expression levels are being developed. However, whether Scn1a gene function is required only during a specific developmental time-window or, alternatively, if its physiological expression is necessary in adulthood is untested up to now. We induced Scn1a gene haploinsufficiency at two ages spanning postnatal brain development (P30 and P60) and compared the phenotypes of those mice to Scn1a perinatally induced mice (P2), recapitulating all deficits of Dravet mice. Induction of heterozygous Na_v1.1 mutation at P30 and P60 elicited susceptibility to the development of both spontaneous and hyperthermia-induced seizures and SUDEP rates comparable to P2-induced mice, with symptom onset accompanied by the characteristic GABAergic interneuron dysfunction. Finally, delayed Scn1a haploinsufficiency induction provoked hyperactivity, anxiety and social attitude impairment at levels comparable to age matched P2-induced mice, while it was associated with a better cognitive performance, with P60-induced mice behaving like the control group. Our data show that maintenance of physiological levels of Na_v1.1 during brain development is not sufficient to prevent Dravet symptoms and that long-lasting restoration of Scn1a gene expression would be required to grant optimal clinical benefit in patients with Dravet syndrome.

Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the oncoprotein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that a condition in which SET is accumulated, the severe Schinzel-Giedion Syndrome (SGS), is characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of SGS and possibly other the SET-related disorders in humans.

SUMMARY: Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the onco-protein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that conditions in which SET is accumulated, including the severe Schinzel-Giedion Syndrome (SGS), are characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis -regulation as a contributing factor to the pathological basis of the SET-related disorders in humans, including SGS, neurodevelopmental disorders, myeloproliferative diseases, and cancer.

Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a ^Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Na_v1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.