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.
Highlights
The specification of cell identity during central nervous system development is regulated by positive and negative transcriptional regulators that act simultaneously to shape the cell-specific transcriptome
To study the impact of RE1-silencing transcription factor (REST) modulation on epileptogenesis, we developed a tool for its specific inhibition in vivo
We previously described that a chimera of the minimal inhibitory peptide paired-amphipathic helix 1 (PAH1) with the switchable AsLOV domain was able to inhibit REST activity and de-repress the transcription of REST target genes in primary neurons when AsLOV was in the open state and the PAH1 peptide was exposed (Paonessa et al, 2016)
Summary
The specification of cell identity during central nervous system development is regulated by positive and negative transcriptional regulators that act simultaneously to shape the cell-specific transcriptome. REST represses the expression of various channels, such as sodium (Chong et al, 1995; Pozzi et al, 2013), calcium (Ariano et al, 2010; van Loo et al, 2012) and potassium channels (Cheong et al, 2005). It mediates the transcriptional downregulation of the KCC2 chloride transporter, which is involved in the GABAergic switch from excitatory to inhibitory transmission during neuronal maturation (Yeo et al, 2009). REST is involved in the control of neurotransmitter release, whereby it represses several genes involved in neurosecretion, like SNAREs (D’Alessandro et al, 2009), and in synaptic vesicle trafficking, like synapsin 1 (Paonessa et al, 2013)
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