Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome.

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 Nav1.1 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.

CRISPR/dCas9-based Scn1a gene activation in inhibitory neurons ameliorates epileptic and behavioral phenotypes of Dravet syndrome model mice.

Dravet syndrome is a severe infantile-onset epileptic encephalopathy which begins with febrile seizures and is caused by heterozygous loss-of-function mutations of the voltage-gated sodium channel gene SCN1A. We designed a CRISPR-based gene therapy for Scn1a-haplodeficient mice using multiple guide RNAs (gRNAs) in the promoter regions together with the nuclease-deficient Cas9 fused to transcription activators (dCas9-VPR) to trigger the transcription of SCN1A or Scn1a in vitro. We tested the effect of this strategy in vivo using an adeno-associated virus (AAV) mediated system targeting inhibitory neurons and investigating febrile seizures and behavioral parameters. In both the human and mouse genes multiple guide RNAs (gRNAs) in the upstream, rather than downstream, promoter region showed high and synergistic activities to increase the transcription of SCN1A or Scn1a in cultured cells. Intravenous injections of AAV particles containing the optimal combination of 4 gRNAs into transgenic mice with Scn1a-haplodeficiency and inhibitory neuron-specific expression of dCas9-VPR at four weeks of age increased Nav1.1 expression in parvalbumin-positive GABAergic neurons, ameliorated their febrile seizures and improved their behavioral impairments. Although the usage of transgenic mice and rather modest improvements in seizures and abnormal behaviors hamper direct clinical application, our results indicate that the upregulation of Scn1a expression in the inhibitory neurons can significantly improve the phenotypes, even when applied after the juvenile stages. Our findings also suggest that the decrease in Nav1.1 is directly involved in the symptoms seen in adults with Dravet syndrome and open a way to improve this condition.

Anticonvulsive effect of vitamin C on pentylenetetrazol-induced seizures in immature rats.

Vitamin C helps to prevent brain oxidative stress and participate in the synthesis of progesterone. It also possesses a progesterone-like effect and acts synergistically with progesterone on the brain. Progesterone and its metabolites, but also vitamin C have been associated with anticonvulsant effects. We evaluated the progesterone concentration 30min and 24h after the last administration of vitamin C (500mg/kg, i.p. for five days). We also evaluated how vitamin C altered pentylenetetrazol (PTZ)-induced seizures by measuring the onset latency of seizures, percentage of incidence and mortality as well as amino acid levels after seizures. Vitamin C treatment alone increased basal progesterone concentrations to 531% after 30min compared to 253% after 24h. Furthermore, vitamin C significantly increased the latency to the first myoclonic, clonic and tonic seizure induced by PTZ (80mg/kg, i.p.) and decreased the percentage of incidence of clonic and tonic seizures as well as the mortality rate. Changes in tissue concentration of amino acids were primarily observed at 24h after vitamin C treatment. Our results suggest that vitamin C together with progesterone and/or its metabolites are involved in the protection against PTZ-induced seizures in immature rats.

Clonazepam prophylaxis and busulfan-related myoclonic epilepsy in autografted acute leukemia patients.

A prospective neurological and electroencephalographic (EEG) study was performed in sixteen leukemia patients receiving busulfan (BU) and cyclophosphamide before autologous bone marrow transplantation. All patients were given anticonvulsant prophylaxis with a combination of phenobarbital (PB) and clonazepam (CLZ). Neurological examination and EEG were performed prior to and soon after completion of BU treatment and were repeated two months later. No tonic-clonic and/or myoclonic convulsions were observed. In two patients, comparison of EEG recorded before and upon completion of BU administration revealed modification of features. EEG re-evaluated two months after BU showed normalization in one of the two patients. BU may trigger both generalized and myoclonic seizures together with EEG abnormalities; PB combined with CLZ may be useful prophylactic treatment.