敲除Efr3b对小鼠PV神经元发育的影响和机制研究

Parvalbumin (PV) neurons regulate the activity of local neural circuits and cognitive functions. Dysfunction of PV neurons is associated with neurological and psychiatry disorders. Therefore, it is extremely important to elucidate the pathways and mechanisms underlying the development of PV neurons. Efr3 (pro Eighty-Five Requiring 3) was identified as an anchor protein to locate the PI4KIIIα to the plasma membrane and therefore plays important roles in maintaining the plasmalemmal level of phosphatidylinositol. Two isoforms of Efr3 (Efr3a and Efr3b) were found in mammalian cells. Although Efr3b was highly expressed in the brain, its roles in the brain remain unknown. Our preliminary data indicated that regulating the expression of Efr3b in the brain affected the number and morphology of PV neurons. Meanwhile, the BDNF-TrkB signaling pathway was affected as well, suggesting that Efr3b might regulate the development of PV neurons via BDNF-TrkB pathway. In the present project, we will cross Nestin-Cre, PV-Cre and CaMK2-Cre mice with Efr3bf/f mice to regulate the expression of Efr3b in the brain, PV neuron itself, and pyramidal neurons, respectively. After that, multiple approaches including immunostaining, western blot and electrophysiological recordings will be used to investigate the effects of ablating Efr3b on the development of PV neurons in the mouse cortex and hippocampus. We will also investigate the development of PV neurons in Efr3b-deleting mice after treatment with TrkB agonist and antagonist to regulate the BDNF-TrkB signaling pathway. We want to know whether the BDNF-TrkB pathway medicates the effects of deleting Efr3b on PV neuron development. Finally, behavioral tests will be performed to test the behaviors of Efr3b deficiency mice. Our results will provide new insights into the pathways for the regulation of PV neuron development.

PV神经元调控局部神经环路活动及认知功能，其发育和功能异常与多种神经和精神疾病相关。因此，阐明其发育的调控机制至关重要。Efr3参与细胞膜上脂类的形成和维持，在哺乳动物中有a和b两个亚型。Efr3b在脑中高度表达，但其在脑中的作用鲜有报道。我们前期发现，调控脑中Efr3b的表达影响PV神经元的数量和形态，同时影响BDNF-TrkB信号通路，提示Efr3b可能通过BDNF-TrkB影响PV神经元的发育。在此基础上，本项目我们拟将Nestin-Cre、PV-Cre和CaMK2-Cre小鼠和Efr3bf/f小鼠杂交，结合免疫染色、Western blot、电生理等，进一步研究敲除Efr3b对PV神经元发育的影响，并通过调控BDNF-TrkB信号通路探究Efr3b影响PV神经元发育的机制。然后，利用行为学检测调控Efr3b对小鼠认知行为的影响。研究结果将为全面了解PV神经元的发育调控提供新的认识。

Efr3（Eighty-five requiring）作为锚定蛋白帮助磷脂酰肌醇-4-磷酸激酶（PI4K）定位在细胞质膜上，从而为维持质膜中磷脂酰肌醇-4-磷酸（PI4P）和磷脂酰肌醇-4，5-二磷酸（PIP2）等的水平并介导相关信号通路起重要作用。哺乳动物中，Efr3包括Efr3a和Efr3b两个亚型。文献报道定量PCR结果显示Efr3b在小鼠脑中的表达量最高，但其在脑中的具体分布及作用尚不清楚。..我们利用RNAscope检测结果发现，Efr3b mRNA在小鼠脑中的多个脑区中均有表达，但在海马的CA2/CA3区域富集最明显。我们将nestin-cre小鼠与Efr3bf/f小鼠杂交以获得在脑中特异性敲除Efr3b的条件性敲除小鼠（cKO）。系列行为学检测发现，cKO小鼠中，海马依赖的空间学习和记忆、运动及焦虑样行为、恐惧性学习和记忆等均无明显异常。然而，在三箱社交等实验中，cKO小鼠表现出明显的社交障碍，提示Efr3b在小鼠社交行为中具有重要作用。通过在正常小鼠中敲降Efr3b的表达或在cKO小鼠中恢复Efr3b的表达，我们进一步证实了Efr3b在社交行为中的作用。..生化检测发现，cKO小鼠海马中BDNF-TrkB-Akt信号通路异常，但MAPK的表达没有显著变化。免疫染色及RNAscope结果发现，cKO小鼠海马中GABA能抑制性神经元（PV+、SST+和5-HT3aR+神经元）数量增多。进一步的脑片电生理和在体光纤钙成像检测表明，Efr3b缺失导致CA2锥体神经元兴奋性下降，而Golgi染色实验结果提示，Efr3b缺失可影响CA2锥体神经元的形态（树突棘、树突分支等）。化学遗传学特异性激活CA2锥体神经元可改善Efr3b条件性敲除小鼠的社交行为。..结论：我们的实验发现Efr3b在小鼠脑中广泛表达，尤其在海马的CA2/CA3区高度富集。Efr3b缺失可导致CA2锥体神经元兴奋性下降，这一结果可能与BDNF-TrkB-Akt信号通路增强并进而导致GABA能神经元数量增多相关。CA2区锥体神经元兴奋性下降是导致Efr3b条件性敲除小鼠社交损伤的重要原因。我们的数据表明，Efr3b对于海马CA2神经元的活动和功能是必需的，且Efr3b的缺失可能与自闭症和精神分裂症等神经精神类疾病有关系。