Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement [snRNA-Seq]. Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement [snRNA-Seq]

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelop- mental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brain’s capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. Overall design: Single nucleus RNA-sequencing was performed from WT and MeCP2 QKI littermates at P20. Six mice per genotype were sacrificed, followed by isolation of the dLGN. 3 dLGN per genotype were pooled, for total of two replicates.

MECP2基因突变会引发雷特综合征（Rett syndrome, RTT），这是一种X连锁神经发育障碍，可导致女性出现广泛的认知功能损伤。尽管雷特综合征症状的确切病因仍未明确，但针对其临床表型的一种潜在解释是：MeCP2缺失会导致神经回路连接紊乱，这源于大脑对神经元活动与感官经验变化的响应能力存在缺陷。本研究发现，大脑中的MeCP2会响应神经元活动，在四个氨基酸残基位点（S86、S274、T308及S421）发生磷酸化；我们构建了一种四位点敲入（quadruple knock-in, QKI）小鼠品系，该品系中四个活性依赖的磷酸化位点均突变为丙氨酸，从而阻止其磷酸化。QKI小鼠在两个脑区中未表现出明显的雷特综合征表型，也未检测到可观测的基因表达变化。然而，对QKI小鼠视网膜膝状体突触的电生理记录显示：尽管在出生后第14天（P14）突触消除过程初始正常，但在出生后第20天（P20）时该过程显著受损。值得注意的是，该表型与此前报道的Mecp2基因敲除小鼠的突触重塑缺陷不同——Mecp2敲除小鼠的突触初始可正常重塑，但在出生后第三周后会发生退行性变化。因此，我们提出一种模型：由神经元活动诱导的MeCP2磷酸化，对于视网膜膝状体突触的成熟时序至关重要，且这一作用仅发生在出生后早期阶段。实验整体设计：于出生后第20天（P20）采集野生型（wild type, WT）与MeCP2 QKI同窝小鼠的单细胞核RNA测序样本。每个基因型组处死6只小鼠，随后分离背外侧膝状体核（dorsolateral geniculate nucleus, dLGN）；每个基因型组取3个背外侧膝状体核样本进行混合，最终获得2个生物学重复。