Zolboot, N. | bioRxiv Science

bioRxiv2023

MicroRNAs are necessary for the emergence of Purkinje cell identity

Brain computations are dictated by the unique morphology and connectivity of neuronal subtypes, features established by closely timed developmental events. MicroRNAs (miRNAs) are critical for brain development, but current technologies lack the spatiotemporal resolution to determine how miRNAs instruct the steps leading to subtype identity. Here, we developed new tools to tackle this major gap. Fast and reversible miRNA loss-of-function revealed that miRNAs are necessary for cerebellar Purkinje cell (PC) differentiation, which previously appeared miRNA-independent, and resolved distinct miRNA critical windows in PC dendritogenesis and climbing fiber synaptogenesis, key determinants of PC identity. To identify underlying mechanisms, we generated a mouse model, which enables precise mapping of miRNAs and their targets in rare cell types. With PC-specific maps, we found that the PC-enriched miR-206 drives exuberant dendritogenesis and modulates synaptogenesis. Our results showcase vastly improved approaches for dissecting miRNA function and reveal that many critical miRNA mechanisms remain largely unexplored. HighlightsO_LIFast miRNA loss-of-function with T6B impairs postnatal Purkinje cell development C_LIO_LIReversible T6B reveals critical miRNA windows for dendritogenesis and synaptogenesis C_LIO_LIConditional Spy3-Ago2 mouse line enables miRNA-target network mapping in rare cells C_LIO_LIPurkinje cell-enriched miR-206 regulates its unique dendritic and synaptic morphology C_LI

neuroscience↗

bioRxiv2022

MicroRNA-218 instructs proper assembly of hippocampal networks

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. A key aspect of this developmental program is mediated at the post-transcriptional level by microRNAs (miRNAs). Deletion of neuronal enriched miRNAs induces strong developmental phenotypes, and multiple reports have found altered levels of miRNAs in patients with neurodevelopmental disorders. However, cellular and molecular mechanisms used by miRNAs to instruct proper brain development remain largely unexplored. Here, through multiple screens, we identified miR-218 as a critical regulator of hippocampal assembly in mice. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons and GABAergic inhibitory interneurons. Transient inhibition of miR-218 in early life results in an adult brain with heightened network activity and a predisposition to seizures. We used RNA-seq and FACS-seq (fluorescence-activated cell sorting followed by RNA-seq) to identify global and cell type-specific changes in gene expression in the absence of miR-218 and narrow down which altered developmental processes would lead to long-term network instability. 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. Finally, conditional knockout of miR-218 in interneurons, but not pyramidal neurons is sufficient to recapitulate the effects on long-term stability. Taken together, the data suggest that miR-218 orchestrates hippocampal network assembly to produce a stable network in the adult, primarily by regulating interneuron function in early postnatal life.

neuroscience↗