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 polyglutamylation—TTLL glutamylases vs. CCP deglutamylases—accelerates 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).

发育性重塑通过神经元活动依赖性的突触与轴突修剪，塑造神经环路。细胞骨架是该过程的关键调控因子，经酶解切断引发的微管丢失，是众多物种与神经环路中修剪过程的早期标志性事件。然而，微管切断酶（如斯帕斯汀（spastin））如何在特定神经元区室中被激活，目前仍无定论。本研究发现，多谷氨酰化修饰——一种富集于神经元内的微管翻译后修饰——可通过标记待切断的微管，在发育性重塑过程中发挥指导性调控作用。对运动神经元特异性的微管多谷氨酰化修饰相关酶——即负责添加修饰的TTLL谷氨酰转移酶与负责去除修饰的CCP去谷氨酰化酶——进行基因敲除，会分别以神经递质传递依赖性的方式，加速或延缓神经肌肉接头突触的重塑过程。该机制并非仅局限于外周突触，同样可在中枢神经环路（如海马体）中发挥功能。综上，微管多谷氨酰化修饰可作为神经元活动依赖性的重塑调控变阻器，精准塑造神经元形态与连接模式。本数据集来源于对不同出生后发育阶段（第5、7、9、11、14天）的小鼠脊髓开展核糖体标签（ribotagging）技术后的RNA测序。核糖体上的HA标签（HA-tag）通过杂交繁育RiboTag（又名Rpl22tm1.1Psam，杰克逊实验室品系编号JAX #011029）、ChAT-Cre（Chattm2(cre)Lowl，JAX #006410）以及Spast条件性敲除小鼠（Brill等，《神经元》，2016年）得以构建。