Yang Yi-Mei, Fekete Adam, Arsenault Jason, Sengar Ameet S, Aitoubah Jamila, Grande Giovanbattista, Li Angela, Salter Eric W, Wang Alex, Mark Melanie D, Herlitze Stefan, Egan Sean E, Salter Michael W, Wang Lu-Yang
Neurosciences and Mental Health, SickKids Research Institute, Toronto, Ontario, Canada.
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
J Physiol. 2024 Oct 9. doi: 10.1113/JP286462.
Intersectin-1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild-type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre- and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca entry via voltage-gated Ca channels (VGCCs), transmitter release or short-term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca-dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH-PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1-mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high-frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca-dependent RRP replenishment and sustaining high-fidelity neurotransmission. KEY POINTS: Itsn1 is expressed in the pre- and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter-1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca-dependent recovery from short-term depression in mature synapses. Itsn1 mediates the recovery through its DH-PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca channels during development. Itsn1 supports high-fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity.
相交蛋白-1（Itsn1）是一种支架蛋白，在突触小泡（SVs）的胞吐作用和胞吞作用偶联过程中起关键作用。然而，目前尚不清楚Itsn1是否以及如何调节这些过程以支持发育过程中的高效神经传递。为了解决这个问题，我们在野生型和Itsn1突变型小鼠听觉脑干中，在听力开始前（未成熟）和开始后（成熟）检查了Held壶腹突触。在两个发育阶段，Itsn1都存在于突触前和突触后区室。在任何一个年龄组中，Itsn1功能丧失均未改变突触前动作电位、通过电压门控钙通道（VGCCs）的钙内流、递质释放或由易释放池（RRP）中SVs耗竭诱导的短期抑郁（STD）。然而，成熟突变突触中从STD的快速钙依赖性恢复减弱，而未成熟突变突触中则未改变。通过将Itsn1的DH-PH结构域引入突触前终末，可挽救成熟突触中的这一缺陷。抑制在胞吞作用期间与Itsn1相互作用的发动蛋白，对STD恢复没有影响。有趣的是，我们发现在VGCCs附近Itsn1在发育过程中富集，这可能是Itsn1介导的RRP快速补充的基础。因此，成熟突触中缺乏Itsn1导致高频突触传递期间突触后放电的失败率更高。综上所述，我们的研究结果表明，发育过程中Itsn1向VGCCs附近的易位对于加速钙依赖性RRP补充和维持高保真神经传递至关重要。要点：Itsn1在Held壶腹突触的突触前和突触后区室中表达。囊泡谷氨酸转运体-1的发育上调是Itsn1依赖性的。Itsn1在不同发育阶段不影响基础突触传递。成熟突触中从短期抑郁的钙依赖性恢复需要Itsn1。Itsn1通过其DH-PH结构域介导恢复，独立于其相互作用伙伴发动蛋白。发育过程中Itsn1易位到突触前钙通道附近。Itsn1通过在重复活动期间从囊泡耗竭中快速恢复来支持高保真神经传递。
