从小鼠记忆缺陷表型出发鉴定参与学习记忆的新基因

Molecular basis of learning and memory (L&M) is one of the central questions in neuroscience field. To understand it will not only gain our knowledge on the mechanisms underlying L&M per se, but also provide insights into the pathogenesis of neurological diseases, including Alzheimer’s disease and autism spectrum disorders. Here, we describe using mouse forward genetic approach to identify novel genes involved in L&M by two memory tests. A mutagen, N-ethyl N-nitrosourea (ENU) was injected into C57BL/6J mouse to induce random point mutations, and two well-established experimental paradigms, contextual fear conditioning and novel object recognition, were used for evaluating hippocampus-dependent long-term memory ability in the mutant mice. In our G1 dominant screening, we identified 3 mutant strains carrying inheritable L&M deficits out of 38 ENU-induced mutant families. By the combination of exon enrichment and high though-put sequencing, we narrowed down the candidates to 2 genes in one family, and 3 genes in another. One of the candidate genes, Mark2 (Microtubule affinity-regulating kinase 2), is highly expressed in mouse hippocampus, and functions in neuron polarity establishment and dendritic spine morphogenesis. Therefore, our approach is feasible to identify novel genes in L&M, and from the entry point of these genes, we hope to gain new insights into the molecular basis of the processes and etiology of neurological diseases with L&M deficits.

面对衰老过程伴有的学习记忆功能减退，和神经精神疾病过程中的学习记忆功能紊乱我们束手无策的现状，加深理解学习记忆分子机制迫在眉睫。本课题利用致突变剂诱导小鼠可遗传随机基因突变，并采用经典的场景恐惧记忆和新物体识别，两种海马依赖的行为范式，筛选伴有长时程记忆能力缺陷表型的G1突变小鼠。在38个突变家系中，我们鉴定到3个家系伴有稳定的可遗传记忆缺陷。结合外显子捕获和高通量测序， 我们将2个突变小鼠家系的候选基因分别缩小至2和3个。在候选基因中，我们发现既往研究报道在海马高表达，参与神经元极性建立和树突棘形成的Mark2 (Microtubule affinity-regulating kinase 2)基因，提示本研究策略的可靠性和可行性。我们期望本课题的预期结果可以加深我们对哺乳动物的学习记忆分子机制的认识，并为改善衰老和神经精神疾病过程中的学习记忆缺陷，提供新的理论依据和干预靶点。

通过ENU筛选和外显子捕获技术结合高通量测序从三个稳定遗传家系中筛选出三个参与学习记忆的基因，其中两个已被报道参与学习记忆另外一个为从未报道的新基因M。基于Cas9基因敲除系统，我们构建了M基因敲除小鼠，通过CFC测试进一步确定此基因参与学习记忆；通过一系列的其它行为学测试全面评估了M基因缺失对小鼠行为学的影响；通过RNA原为杂交和免疫染色等分子生物学手段，确定了M基因的表达图谱以及细胞类型；在分子水平上通过质谱鉴定等找到M基因相互作用蛋白，暗示了其可能参与的分子信号通路；在电生理水平上检测了M基因对细胞兴奋性和神经可塑性方面的影响；并通过化学遗传揭示了M基因表达的这群细胞的生理功能。综上所述，本研究为阐明M基因参与学习记忆的机制提供了坚实的实验证据，并为加深对于学习记忆的理解提供新的研究思路和理论依据。