Emerging Therapy Opportunities for Progressive Myoclonus in Epilepsy

Abstract

Original Article Abstract:

Original Article Citation: Feng H, Clatot J, Kaneko K, Flores-Mendez M, Wengert ER, Koutcher C, Hoddeson E, Lopez E, Lee D, Arias L, Liang Q, Zhang X, Somarowthu A, Covarrubias M, Gunthorpe MJ, Large CH, Akizu N, Goldberg EM. Targeted therapy improves cellular dysfunction, ataxia, and seizure susceptibility in a model of a progressive myoclonus epilepsy. Cell Reports Medicine, Volume 5, Issue 2, 2024, 101389.

The recurrent variant KCNC1-p.Arg320His causes progressive myoclonus epilepsy type 7 (EPM7), defined by progressive myoclonus, epilepsy, and ataxia, and is without effective treatment. KCNC1 encodes the voltage-gated potassium channel subunit Kv3.1, specifically expressed in high-frequency-firing neurons. Variant subunits act via loss of function; hence, EPM7 pathogenesis may involve impaired excitability of Kv3.1-expressing neurons, while enhancing Kv3 activity could represent a viable therapeutic strategy. We generate a mouse model, Kcnc1-p.Arg320His/+, which recapitulates the core features of EPM7, including progressive ataxia and seizure susceptibility. Kv3.1-expressing cerebellar granule cells and neocortical parvalbumin-positive GABAergic interneurons exhibit abnormalities consistent with Kv3 channel dysfunction. AKv3-specific positive modulator (AUT00206) selectively enhances the firing frequency of Kv3.1-expressing neurons and improves motor function and seizure susceptibility in Kcnc1-Arg320His/+ mice. This work identifies a cellular and circuit basis of dysfunction in EPM7 and demonstrates that Kv3 positive modulators such as AUT00206 have therapeutic potential for the treatment of EPM7.

Commentary

Channelopathies contribute to multiple neurological conditions that include epilepsy and developmental epileptic encephalopathies. Kv3 potassium channels play a critical role in action potential repolarization, where their repolarizing drive minimizes action potential duration and maximizes firing frequency. ¹ Mutations in this ion channel have been linked to movement and other neurological disorders. ² For instance, myoclonic epilepsy and ataxia due to potassium channel mutation (MEAK) arise during childhood or early adolescence and result from mutations in the KCNC1 gene, ³ which encodes the Kv3.1 subunit of voltage-gated potassium channels. Furthermore, recent work has tied progressive myoclonus epilepsy type 7 (EPM7) to KCNC1 mutation and MEAK.^3,4 The key elements of EPM7 that make it particularly devastating are its progressive nature and lack of effective therapies. Patients lose limb use and experience severe neurologic decline. Importantly, EPM7 is caused by a heterozygous mutation in KCNC1 (KCNC1-p.Arg320His; R320H), and, as described by Feng et al, ⁵ this mutation modeled in mice recapitulates critical elements of EPM7 and serves as a tool to test targeted therapies.

Mice were generated using CRISPR-Cas9 for selective editing to produce the R320H mutation. Behavioral characterization of heterozygous (H/+) mice revealed normal development through post-natal day 15, consistent with normal development and childhood–adolescence onset of EPM7. However, at later ages (i.e., 4–6 months of age), the mice developed gait disruption and other motor impairments, along with seizure susceptibility and incidence. This model therefore demonstrates sufficient construct and face validity to warrant additional analysis.

Kv3.1 (Kv3 voltage-gated potassium channel subfamily) is expressed synaptically in high-frequency-firing neurons, including interneuron populations of the hippocampus and cerebellum. In particular, Kv3.1 plays a critical role in the ability of parvalbumin-positive GABAergic interneurons in the hippocampus and cortex to fire with high frequency, thus contributing significantly to overall excitatory-inhibitory balance in these and other brain regions. Mutations in Kv3.1 likely hinder firing frequency and action potential dynamics that alter neurotransmitter release. Thus, in GABAergic interneurons, the authors observed increased action potential halfwidth and time, with a concomitant decrease in firing frequency.

To link ataxia and gait dysfunction with cerebellar pathology, the authors studied cerebellar granule cells and parallel fiber synapses, which play a key role in fine motor control through their signaling to Purkinje cells. Thus dysfunction within this neurocircuitry in mice with Kv3.1 mutations may demonstrate the importance of this subunit for motor dysfunction in EPM7. The R320H mutation produced spike broadening in whole-cell patch clamp recordings in cerebellar granule cells, along with increased after hyperpolarization time, decreased firing frequency, and decreased short-term plasticity. It is noteworthy, however, that other factors may contribute to changes in motor control in mice with Kv3.1 mutations. While it appears that knockout of Kv3.1 may not be associated with compensatory upregulation of other potassium channel subunits, ¹ it is also important to point out that Kv3.3 has parallel and potentially redundant impact on Kv3 channels in other brain regions. ¹ Thus, it may be beneficial to better understand changes in Kv3.3 and other subunits in this model and in patients with EPM7.

The Kv3 voltage-gated potassium channel subfamily, which includes Kv3.1, requires tetrametric assemblies to form functional channels. The R320H mutation is loss-of-function and dominant negative, thus contributing to diminished Kv3 channel activity. However, as the authors state, the R320H mutation only produces a partial loss of function, and, as described above, other Kv3 channel subunits may play an important role in the presence of diminished Kv3.1 function. Another possible explanation for Kv3 dysfunction may be that mutated Kv3.1 proteins have altered interactions with wild-type subunits, resulting in reduced Kv3 performance. ⁶

The authors demonstrate the potential predictive validity for the R320H model by evaluating a positive modulator of Kv3 channels, AUT00206 (AUT6). They hypothesized that increasing Kv3 activity may reduce cellular, circuit, and behavioral abnormalities observed in R320H mutants. While the molecular mechanism for AUT6 is not fully understood, previous work with another Kv3 positive modulator demonstrated that the compound stabilizes the open state by binding to Kv3.1 at a unique site in the interface between voltage-sensing and pore-forming domains. ⁷ This turret-like region serves important gating functions for potassium channels ⁸ and binding to this region confers positive modulation properties and differential activity on different Kv3 subunits. ⁷ Using cellular recordings in mammalian cells, neocortex, and cerebellum, the authors demonstrate the potential for Kv3 positive modulation, and AUT6 partially reversed the loss of conductance and reduced firing frequency resulting from R320H. Importantly, the in vivo efficacy of AUT6 was also demonstrated, as treatment led to enhanced rotarod performance and survival following chemoconvulsant administration. An underpowered experiment also suggested the potential of this compound in reducing seizure frequency, but a significant effect was not observed. To further demonstrate the therapeutic potential for Kv3.1 modulation on seizure outcomes, further studies in R320H mice could use sub-chronic administration of this compound to reduce spontaneous seizures. Such a study could also be beneficial in determining whether the improvements in ataxia persist following repeat administration.

This study demonstrates the utility and translational importance of the R320H animal model for EPM7. Furthermore, the benefits of AUT6 were demonstrated in different cell types and in vivo following systemic administration, which adds enthusiasm to this approach for an eventual therapy for EPM7. To this point, a positive Kv3.1 modulator (AUT00201) is currently under clinical investigation for treatment in EPM7 (www.autifony.com). However, there are important additional questions warranting consideration. EPM7 is a progressive disease, and it is unclear from the present studies whether prolonged Kv3.1 modulation would fully reverse ataxia and seizures. While AUT6 treatment acutely and sub-chronically demonstrates the potential for reversing or perhaps limiting the progression of ataxia, such a treatment would need to be evaluated at additional time points and following prolonged administration to see if Kv3.1 positive modulation in R320H mutants could reverse the phenotype.

Beyond EPM7, it is important that the findings described by Feng et al also point to the potential of Kv3.1 modulation for the treatment of other epilepsies. KCNC1 mutations are associated with a variety of neurologic outcomes ⁹ and it is yet to be seen whether underlying Kv3.1 dysfunction is a key cause of other epilepsy types. Given the importance, for example, of fast-spiking interneurons in network excitability, Kv3.1 modulation provides a therapeutic opportunity, even in the absence of loss-of-function mutations. For instance, in Dravet syndrome, where loss of excitability in fast-spiking interneurons is thought to be the major disease mechanism, ¹⁰ activation of Kv3.1 may be a potential therapeutic avenue.