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Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability

Gene Therapy volume 31, pages 144–153 (2024)Cite this article

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Abstract

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

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Fig. 1: Viral expression of KCNN4 (KCa3.1 channel) in thy1-positive L5 neocortical neurons that natively lacked its expression before transduction.
Fig. 2: TRAM-34, a selective blocker of KCa3.1 (KCNN4) channels blocks Ca2 + -induced outward current in KCNN4+ HEK293 cells but does not affect sAHP in KCNN4+ neurons.
Fig. 3: Comparison of frequency transfer functions of KCNN4+ neurons vs control neurons using injection of fluctuating noise.
Fig. 4: KCNN4 overexpression reduces ictal activity in the entorhinal cortex of C57BL/6 J mice in the 4-aminopyridine in vitro model.
Fig. 5: The viral transduction of a neuron by KCa3.1 channels significantly reduced the contribution of this neuron to the generation of epileptiform activity in 4-aminopyridine model in brain slices.

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All relevant data supporting the key findings of this study are available within the article or from corresponding authors on request.

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Acknowledgements

This research was funded by Russian Science Foundation; grant: RSF 20-15-00408. We would like to thank Dr. Alexander S. Chernyshev for his help in analyzing the field recordings.

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Authors and Affiliations

  1. Institute of Higher Nervous Activity and Neurophysiology, RAS, 117485, Moscow, Russia

    Evgeny S. Nikitin, Anastasia A. Borodinova, Violetta Ivanova, Matvey V. Roshchin, Maria P. Smirnova & Pavel M. Balaban

  2. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, 194223, Saint Petersburg, Russia

    Tatiana Y. Postnikova, Elena Y. Proskurina & Aleksey V. Zaitsev

  3. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, Moscow, Russia

    Ilya Kelmanson & Vsevolod V. Belousov

  4. Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, 117997, Moscow, Russia

    Ilya Kelmanson & Vsevolod V. Belousov

  5. Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, 117997, Moscow, Russia

    Vsevolod V. Belousov

  6. Life Improvement by Future Technologies (LIFT) Center, 143025, Moscow, Russia

    Vsevolod V. Belousov

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  1. Evgeny S. Nikitin
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Contributions

Conceptualization, ESN, VVB, PMB, and AVZ; Funding acquisition, PMB; ESN, MVR, TYP, VI, IK, AAB, EYP, MPS, and AVZ performed the experiments; Methodology, AAB, ESN, TYP, and AVZ; Project administration, ESN, PMB, and AVZ; Writing—original draft, ESN, TYP, EYP, and AVZ; Reviewing & editing, ESN, PMB, VVB, and AVZ. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Evgeny S. Nikitin or Aleksey V. Zaitsev.

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Nikitin, E.S., Postnikova, T.Y., Proskurina, E.Y. et al. Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability. Gene Ther 31, 144–153 (2024). https://doi.org/10.1038/s41434-023-00427-9

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