Abstract
Gu F, Parada I, Yang T, Longo FM, Prince DA. PNAS. 2022;119(7):e2022726119. doi:10.1073/pnas.2022726119. Dravet syndrome (DS) is one of the most severe childhood epilepsies, characterized by intractable seizures and comorbidities including cognitive and social dysfunction and high premature mortality. DS is mainly caused by loss-of-function mutations in the Scn1a gene encoding Nav1.1 that is predominantly expressed in inhibitory parvalbumin-containing (PV) interneurons. Decreased Nav1.1 impairs PV cell function, contributing to DS phenotypes. Effective pharmacological therapy that targets defective PV interneurons is not available. The known role of brain-derived neurotrophic factor (BDNF) in the development and maintenance of interneurons, together with our previous results showing improved PV interneuronal function and antiepileptogenic effects of a TrkB receptor agonist in a posttraumatic epilepsy model, led to the hypothesis that early treatment with a TrkB receptor agonist might prevent or reduce seizure activity in DS mice. To test this hypothesis, we treated DS mice with LM22A-4 (LM), a partial agonist at the BDNF TrkB receptor, for 7 d starting at postnatal day 13 (P13), before the onset of spontaneous seizures. Results from immunohistochemistry, Western blot, whole-cell patch-clamp recording, and in vivo seizure monitoring showed that LM treatment increased the number of perisomatic PV interneuronal synapses around cortical pyramidal cells in layer V, upregulated Nav1.1 in PV neurons, increased inhibitory synaptic transmission, and decreased seizures and the mortality rate in DS mice. The results suggest that early treatment with a partial TrkB receptor agonist may be a promising therapeutic approach to enhance PV interneuron function and reduce epileptogenesis and premature death in DS.
Commentary
Dravet syndrome (DS) is a catastrophic pediatric epilepsy characterized by febrile and afebrile seizures, refractory epilepsy, and comorbid behavioral challenges. 1 Loss-of-function mutations in SCN1A, encoding the voltage-gated sodium channel Nav1.1, are the primary cause of DS. Scn1a mouse models recapitulate many of the phenotypes observed in patients with DS, including increased seizure susceptibility, spontaneous seizures, and behavioral abnormalities. Genetic mouse models of DS have significantly advanced our understanding of the pathogenesis of Scn1a-derived epilepsy. For example, previous studies have demonstrated that Scn1a mutant mice have dysfunctional fast-spiking parvalbumin (PV) interneurons, 2 and reducing Scn1a activity or expression in PV interneurons is sufficient to increase seizure susceptibility 3 and alter behavior. 4 Thus, increasing PV interneuron activity in an Scn1a mouse model of DS would be predicted to ameliorate disease phenotypes.
Previous studies have shown that modulation of brain derived neurotrophic factor (BDNF) and the receptor that mediates its biological effects, tropomyosin related kinase B (TrkB) can have conflicting roles on epileptogenesis. For example, activation and inhibition of the BDNF/TrkB signaling cascade have been shown to prevent hyperexcitability. The inconsistencies in these studies could be due to a number of factors, including acute vs. chronic treatment, activation of different downstream cascades, the different experimental epilepsy models used, or targeting different cell types. However, it is well-established that BDNF/TrkB signaling is involved in the normal development of interneurons and inhibitory transmission. Consistent with this, Gu and colleagues previously demonstrated that the administration of a partial TrkB receptor agonist, LM22A-4 (LM), in a posttraumatic epilepsy model, suppressed cortical epileptogenesis by increasing GABAergic inhibitory synaptic transmission. 5 Together, these studies suggest that increasing activity of the BDNF/TrkB signaling cascade may be therapeutic in DS.
In the current study, Gu and colleagues evaluated the effect of partial TrkB activation in a mouse model of DS. 6 Scn1a mutant mice were treated with LM (50 mg/kg intraperitoneal + 5 m/kg intranasal) once a day for 7 days, beginning at postnatal day 13 (P13). This time point was selected as it preceded the onset of spontaneous seizures. LM treatment resulted in more PV synapses onto cortical pyramidal cells in the mutants and wild-type (WT) littermates. Furthermore, in the LM-treated mutants, the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was increased by 75%, and a 59% increase in miniature IPSCs (mIPSCs) was observed, which suggests an increase in the intrinsic excitability of the PV interneurons. It was previously demonstrated that impairment in PV interneuron action potential firing in Scn1a mutant mice is transient, with normalization by P35. However, the abnormalities of PV interneuron synaptic transmission persist into adulthood. 2 Thus, in future studies, it would be important to establish whether LM treatment in adulthood could restore interneuron synaptic transmission. To determine whether LM could reduce spontaneous seizure frequency in the Scn1a mutants, EEG analysis was performed between P21-P23. The authors found that LM treatment was able to reduce the percentage of mice that had a spontaneous seizure during the 3 days of recordings. No information was provided on whether LM treatment reduced seizure severity or its ability to protect against hyperthermia-induced seizures, a prominent characteristic of Scn1a mutants and patients with DS. In addition, LM treatment was able to significantly increase the survival of the Scn1a mutants during the first month of life. Given the positive results with only 7 days of LM treatment, a logical next step would be to determine whether chronic LM treatment would confer longer-lasting benefit in the Scn1a mutants without causing adverse effects. Overall, the current study provides evidence that increasing activity of the BDNF/TrkB signaling cascade before seizure onset can improve survival and reduce spontaneous seizure frequency in an Scn1a mouse model of DS. While these results are promising, patients with DS are typically not treated before symptom onset. Thus, additional studies investigating the effect of LM intervention after the onset of spontaneous seizures could provide important, clinically-relevant information.
Surprisingly, LM treatment was able to increase Nav1.1 expression levels in the Scn1a mutants. 6 Currently, there are only a few treatments that are capable of increasing Nav1.1 expression. However, it would be important to establish whether LM treatment also alters the expression of other sodium channels, such as Nav1.2 and Nav1.6, that are localized to the axon initial segment. The underlying mechanisms by which LM increases Nav1.1 expression remain unclear. While increased Nav1.1 expression levels would be beneficial in DS, Gu et al. also demonstrated that LM treatment increased PV interneuron synapses and the intrinsic excitability of PV interneurons in WT littermates, 6 suggesting that LM could potentially have therapeutic utility in other disorders characterized by dysfunctional PV interneurons.
There are currently 3 FDA approved treatments for DS: stiripentol, cannabidiol, and fenfluramine. Like LM, these drugs affect multiple targets, which could also lead to unwanted side effects. Currently, there is significant interest in precision medicine strategies to increase Nav1.1 activity and/or expression for the treatment of DS. Richards et al., demonstrated that hm1a, a venom peptide that preferentially activates Nav1.1, was capable of restoring interneuron function and reducing spontaneous seizures in Scn1a mutant mice. 7 Genetic approaches have also been used to selectively increase Nav1.1 levels. For example, Colasante and colleagues used dCas9-mediated Scn1a gene activation to restore more normal PV interneuron excitability and protect against hyperthermia-induced seizures in Scn1a mutants. 8 Han and colleagues identified antisense oligonucleotides (ASOs) that could modulate naturally occurring nonproductive splicing events to increase expression of Scn1a . 9 The lead ASO, STK-001, was able to significantly increase Nav1.1 protein levels, reduce spontaneous seizure frequency, and restore more normal excitability of PV interneurons in Scn1a mutant mice. 10
For the potential treatment of DS, there are advantages and disadvantages to the use of pharmacological compounds and genetic approaches. Drugs like LM have the advantage of ease of delivery and effective blood-brain barrier penetrance; however, their effects may be short-lived and they often affect multiple targets. In contrast, genetic strategies are longer-lasting and have the advantage of specificity, but delivery likely will require more invasive approaches. Together, these emerging treatment strategies provide hope for improved clinical care in patients with DS and other forms of treatment-resistant epilepsy.
