MicroRNA-218 instructs proper assembly of hippocampal networks. MicroRNA-218 instructs proper assembly of hippocampal networks

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. Post-transcriptional regulation of gene expression by microRNAs (miRNAs) is a key aspect of this program. Indeed, deletion of neuron-enriched miRNAs induces strong developmental phenotypes, and miRNA levels are altered in patients with neurodevelopmental disorders. However, the mechanisms used by miRNAs to instruct brain development remain largely unexplored. Here, we identified miR-218 as a critical regulator of hippocampal assembly. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons (PNs) and GABAergic inhibitory interneurons (INs). Early life inhibition of miR-218 results in an adult brain with a predisposition to seizures. Changes in gene expression in the absence of miR-218 suggest that network assembly is impaired. Indeed, we find that miR-218 inhibition results in the disruption of early depolarizing GABAergic signaling, structural defects in dendritic spines, and altered intrinsic membrane excitability. Conditional knockout of miR-218 in INs, but not PNs, is sufficient to recapitulate long-term instability. Finally, de-repressing Kif21b and Syt13, two miR-218 targets, phenocopies the effects on early synchronous network activity induced by miR-218 inhibition. Taken together, the data suggest that miR-218 orchestrates formative events in PNs and INs to produce stable networks. Overall design: To identify targets of miR-218, we conducted three different RNA-sequencing experiments 1) Locked nuclei acid miR antagomiRs targeting either miR-218 or a scrambled sequence were injected into the dorsal hippocampus of P2 mice (3/condition). The dorsal hippocampus was dissected at P8 for RNA extraction. 2) The dorsal hippocampi of P15 miR-218-1-/-; miR-218-2+/- mice (n=5) and miR-218-1+/+; miR-218-2+/+ (n=3) mice were dissected and RNA extracted for RNA-sequencing 3) We used FACS to isolate VGluT2+ neurons from the dorsal hippocampus of 4 P8 Ai14; VGluT2-Cre mice and to isolate VGAT+ neurons from the dorsal hippocampus of 3 Ai14; VGAT-IRES-Cre mice.

哺乳动物大脑的组装由时序协调的基因表达波所精准调控。基因表达的转录后调控中，微小RNA（microRNAs, miRNAs）是该发育程序的关键环节。事实上，神经元富集型miRNAs的敲除会引发严重的发育表型，且神经发育障碍患者体内的miRNA水平存在显著异常。然而，miRNAs指导大脑发育的具体分子机制仍未得到充分探索。

本研究鉴定出miR-218是海马体组装的关键调控因子。miR-218在海马体中高表达，同时富集于兴奋性主神经元（excitatory principal neurons, PNs）与GABA能抑制性中间神经元（GABAergic inhibitory interneurons, INs）中。早期抑制miR-218会导致成年大脑出现癫痫易感性。缺失miR-218后出现的基因表达变化提示神经网络组装过程受损。具体而言，我们发现抑制miR-218会破坏早期去极化GABA能信号通路、造成树突棘结构缺陷，并改变神经元内在膜兴奋性。在INs中条件性敲除miR-218（而非PNs）即可重现长期网络不稳定的表型。最后，解除miR-218的两个靶基因Kif21b与Syt13的表达抑制，可重现miR-218抑制所引发的早期同步网络活动异常。综上，实验数据表明miR-218通过调控PNs与INs的发育形成过程，从而构建稳定的神经网络。

整体实验设计：为鉴定miR-218的靶基因，我们开展了三组不同的RNA测序实验：1）将靶向miR-218或乱序序列的锁核酸（Locked Nucleic Acid, LNA）miRNA拮抗物注射至P2（出生后第2天）小鼠的背侧海马体中（每组3只），于P8（出生后第8天）时分离背侧海马体以提取RNA进行测序；2）分离P15（出生后第15天）月龄miR-218-1-/-; miR-218-2+/-小鼠（n=5）与野生型对照小鼠（miR-218-1+/+; miR-218-2+/+，n=3）的背侧海马体，提取RNA进行RNA测序；3）我们通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）从4只P8月龄Ai14; VGluT2-Cre小鼠的背侧海马体中分离VGluT2+神经元，并从3只Ai14; VGAT-IRES-Cre小鼠的背侧海马体中分离VGAT+神经元。