Functional integration of neurons derived from reprogrammed pericytes isolated from the adult human brain

体细胞重编程成诱导神经元可能为目前无法治愈的神经退行性疾病的新疗法铺平道路。我们之前可以证明，从成人大脑中分离的周细胞可以通过强制表达两种转录因子Sox2和Ascl1而被重新编程为诱导神经元。这项提议旨在研究这种重编程是否也可以在体内发生，以及由此产生的新神经元是否可以在功能上整合到神经元网络中。我们将通过巴西北格兰德联邦大学Marcos R Costa教授建立的移植模型来检验这一点。因此，将体外逆转录病毒转导的人周细胞移植到小鼠胚胎的侧脑室中。逆转录病毒编码Sox2和/或Ascl1以及报告基因（GFP或RFP）。在不同的存活时间后，移植细胞将根据其报告细胞的表达定位在宿主组织中，并检测其标记物的表达（神经元与周细胞标记物）。如果我们能够证明重编程也可以在体内发生，我们接下来将通过一种基于狂犬病病毒的追踪技术来研究突触前输入是否以及哪种类型的输入会影响来自周细胞的神经元。本研究将辅以经典的膜片钳电生理学研究突触活动。这里描述的实验将提供证据，证明来自人类起源的重编程周细胞的神经元是否可以被真正的神经元识别为靶细胞，从而证明支持或反对其突触整合的原则证据。这代表了我们对这种基于细胞的方法的潜力的理解的重大进步。该项目将与Marcos R. Costa教授密切合作，Marcos R. Costa教授已向CNPq申请资助，研究重编程小鼠星形胶质细胞神经元的整合。这两个项目将高度互补，需要两个实验室的专业知识。

Cellular reprogramming of somatic cells into induced neurons could pave the way for new therapies of currently uncurable neurodegenerative diseases. We could previously show that pericytes isolated from the adult human brain can be reprogrammed into induced neurons by forced expression of the two transcription factors Sox2 and Ascl1. This proposal aims at investigating whether such reprogramming can also take place in vivo and whether thereby newly generated neurons can functionally integrate into a neuronal network. We will examine this by using a transplantation model established by Prof. Marcos R Costa at the Federal University of Rio Grande do Norte (Brazil). Hereby in vitro retrovirus-transduced human pericytes are transplanted into the lateral ventricle of mouse embryos. The retroviruses encode Sox2 and/or Ascl1 as well as a reporter gene (GFP or RFP). After different survival times, the transplanted cell will be located in the host tissue based on their reporter expression and examined for their marker expression (neuronal versus pericyte markers). If we can show that reprogramming can also occur in the in vivo context, we will next examine by means of a rabies virus-based tracing technique whether and which type of presynaptic inputs impinge on neurons derived from pericytes. This study will be complemented by classical patch-clamp electrophysiology for synaptic activity. The experiments described here will provide evidence whether neurons derived from reprogrammed pericytes of human origin can be recognized by authentic neurons as target cells and thus proof of principle evidence for or against their synaptic integration. This represents a major advancement in our understanding of the potential of this cell-based approach. This project will be conducted in close collaboration with Prof. Marcos R. Costa, who has applied for a grant at the CNPq to study the integration of neurons of reprogrammed mouse astrocytes. Both project will be highly complementary and require the expertise of both laboratories.