PDF(Pubmed)
Abstract：
Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.
摘要：
神经元依赖于突触成分的远程运输来形成和维持编码人类经验的复杂神经网络。一个神经元能够沿着其轴突形成数千个不同的突触，必要的突触成分的空间精确传递是至关重要的。这些突触是如何被图案化的，以及如何调节突触成分的有效传递，仍然很大程度上未知。这里,我们揭示了微管（MT）-切断酶spastin在局部增强MT聚合以影响突触前货物沿轴突的暂停和保留中的新作用。在诱导多能干细胞（iPSCs）衍生的人类神经元中，在通过突触后接触并列的成熟突触前的稳健组装之前，我们确定了沿着轴突稳定富集突触前成分的位点。这些位点能够循环突触小泡，富含痉挛，并且是新MT生长和突触小泡前体（SVP）暂停/保留的热点。神经元痉挛水平或活动的破坏，通过CRISPRi介导的耗竭，瞬时过表达,或酶活性的药理学抑制，中断动态MT+末端的局部富集，减少SVP积累。使用创新的人类异源突触模型，其中微流体分离的人类轴突识别并与表达神经凝集素的非神经元细胞形成突触前连接，我们发现，缺乏痉挛的神经元没有达到与对照神经元相同的突触前成分积累水平。我们提出了一个模型，其中spastin在局部充当MT聚合的放大器，以图案化轴突的特定区域以进行突触发生并指导突触货物的传递。
