Mild Inactivation of RE-1 Silencing Transcription Factor (REST) Reduces Susceptibility to Kainic Acid-Induced Seizures
Abstract
RE-1 Silencing Transcription factor (REST) controls several steps in neural development by modulating the expression of a wide range of neural genes. Alterations in REST expression have been associated with the onset of epilepsy; however, whether such alterations are deleterious or represent a protective homeostatic response remains elusive. To study the impact of REST modulation on seizure propensity, we developed a tool for its negative modulation in vivo. The tool is composed of the paired-amphipathic helix 1 (PAH1) domain, a competitive inhibitor of REST activation by mSin3, fused to the light-oxygen-voltage sensing 2 (LOV2) domain of Avena sativa phototropin 1, a molecular switch to alternatively hide or expose the PAH1 inhibitor. We employed the C450A and I539E light-independent AsLOV2 variants to mimic the closed (inactive) and open (active) states of LOV2-PAH1, respectively. Recombinant AAV1/2 viral particles (rAAVs) allowed LOV2-PAH1 expression in HEK293T cells and primary neurons, and efficiently transduced hippocampal neurons in vivo. mRNA expression analysis revealed an increased expression of several neuronal genes in the hippocampi of mice expressing the open probe. AAV-transduced mice received a single dose of kainic acid (KA), a treatment known to induce a transient increase of REST levels in the hippocampus. Remarkably, mice expressing the active variant displayed a reduced number of KA-induced seizures, which were less severe compared to mice carrying the inactive probe. These data support the validity of our tool to modulate REST activity in vivo and the potential impact of REST modulation on epileptogenesis.
Acknowledgments
Dr. M. Cilli is gratefully acknowledged for his help with in vivo experiments. A. Mehilli is acknowledged for primary cell culture preparations, D. Moruzzo, R. Ciancio and I. Dallorto for technical and administrative support.
Footnotes
Funding. This study was supported by research grants from the European Commission and CURE Epilepsy (FP7 Integrating Project “DESIRE,” grant number 602531 and CURE Innovation Award to FBe, and by the Italian Ministry of Health Ricerca Finalizzata (GR-2013-02355540 to AR).