Carminati Emanuele, Buffolo Federica, Rocchi Anna, Michetti Caterina, Cesca Fabrizia, Benfenati Fabio
Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Genova, Italy.
IRCCS Ospedale Policlinico San Martino, Genova, Italy.
Front Cell Neurosci. 2020 Jan 10;13:580. doi: 10.3389/fncel.2019.00580. eCollection 2019.
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 . 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 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 . 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 and the potential impact of REST modulation on epileptogenesis.
RE-1沉默转录因子（REST）通过调节多种神经基因的表达来控制神经发育的多个步骤。REST表达的改变与癫痫的发作有关；然而，这种改变是有害的还是代表一种保护性的稳态反应仍不清楚。为了研究REST调节对癫痫易感性的影响，我们开发了一种对其进行负调节的工具。该工具由双嗜性螺旋1（PAH1）结构域组成，它是mSin3激活REST的竞争性抑制剂，与光敏色素1的光氧电压传感2（LOV2）结构域融合，光敏色素1是一种分子开关，可交替隐藏或暴露PAH1抑制剂。我们分别使用C450A和I539E光独立的AsLOV2变体来模拟LOV2-PAH1的关闭（无活性）和开放（有活性）状态。重组腺相关病毒1/2病毒颗粒（rAAVs）使LOV2-PAH1在HEK293T细胞和原代神经元中表达，并有效地转导海马神经元。mRNA表达分析显示，在表达开放探针的小鼠海马中，几种神经元基因的表达增加。腺相关病毒转导的小鼠接受单剂量的海藻酸（KA），已知这种处理会导致海马中REST水平短暂升高。值得注意的是，表达活性变体的小鼠KA诱导的癫痫发作次数减少，与携带无活性探针的小鼠相比，发作程度较轻。这些数据支持了我们调节REST活性工具的有效性以及REST调节对癫痫发生的潜在影响。
