Polyglutamylation of microtubules drives neuronal remodeling

Developmental remodeling shapes neural circuits via activity-dependent pruning of synapses and axons. The cytoskeleton is critical for this process, as microtubule loss via enzymatic severing is an early step of pruning across many circuits and species. However, how microtubule-severing enzymes, such as spastin, are activated in specific neuronal compartments remains unknown. Here, we reveal that polyglutamylation, a posttranslational tubulin modification that is enriched in neurons, plays an instructive role in developmental remodeling by tagging microtubules for severing. Motor neuron-specific gene deletion of enzymes that add or remove tubulin polyglutamylationTTLL glutamylases vs. CCP deglutamylasesaccelerates or delays neuromuscular synapse remodeling in a neurotransmission-dependent manner. This mechanism is not specific to peripheral synapses but also operates in central circuits, e.g., the hippocampus. Thus, tubulin polyglutamylation acts as an activity-dependent rheostat of remodeling and shapes neuronal morphology and connectivity. RNA sequencing following ribotagging from murine spinal cord at different postnatal ages (day 5, 7, 9, 11, 14). HA-tag on ribosomes was induced by crossbreeding RiboTag (also known as Rpl22tm1.1Psam, JAX #011029), ChAT-Cre (Chattm2(cre)Lowl, JAX #006410), and Spast flox mice (Brill et al., Neuron, 2016).发育重塑通过依赖活动的突触与轴突修剪塑造神经环路。细胞骨架对于该过程至关重要，因为依赖酶解的微管剪切（microtubule severing）导致的微管丢失，在多种神经环路与物种中均为修剪过程的早期事件。然而，微管剪切酶（如spastin）如何在特定神经元区室中被激活，目前仍不明确。本研究发现，多谷氨酰化（polyglutamylation）——一种在神经元中富集的微管翻译后修饰——通过标记微管以供剪切，在发育重塑中发挥指导性作用。对运动神经元特异性敲除添加或去除微管多谷氨酰化的酶：TTLL谷氨酰转移酶与CCP去谷氨酰酶，会以依赖神经递质传递的方式加速或延迟神经肌肉突触重塑。该机制并非仅局限于外周突触，同样可在中枢环路（如海马体）中发挥作用。综上，微管多谷氨酰化可作为依赖活动的重塑动态调控器，塑造神经元形态与连接网络。本数据集源自对不同产后日龄（第5、7、9、11、14天）的小鼠脊髓进行核糖体标签（ribotagging）后的RNA测序。核糖体上的HA标签通过杂交RiboTag（亦称Rpl22tm1.1Psam，JAX编号#011029）、ChAT-Cre（Chattm2(cre)Lowl，JAX编号#006410）以及Spast条件性敲除小鼠（Brill等，《神经元》，2016）诱导获得。