Ferdos Shima, Brockhaus Johannes, Missler Markus, Rohlmann Astrid
Institute of Anatomy and Molecular Neurobiology, Westfälische Wilhelms-University, Münster, Germany.
Front Neuroanat. 2022 Jan 31;15:757017. doi: 10.3389/fnana.2021.757017. eCollection 2021.
Communication between neurons through synapses includes the release of neurotransmitter-containing synaptic vesicles (SVs) and of neuromodulator-containing dense-core vesicles (DCVs). Neurexins (Nrxns), a polymorphic family of cell surface molecules encoded by three genes in vertebrates (Nrxn1-3), have been proposed as essential presynaptic organizers and as candidates for cell type-specific or even synapse-specific regulation of synaptic vesicle exocytosis. However, it remains unknown whether Nrxns also regulate DCVs. Here, we report that at least β-neurexins (β-Nrxns), an extracellularly smaller Nrxn variant, are involved in the distribution of presynaptic DCVs. We found that conditional deletion of all three β-Nrxn isoforms in mice by lentivirus-mediated Cre recombinase expression in primary hippocampal neurons reduces the number of ultrastructurally identified DCVs in presynaptic boutons. Consistently, colabeling against marker proteins revealed a diminished population of chromogranin A- (ChrgA-) positive DCVs in synapses and axons of β-Nrxn-deficient neurons. Moreover, we validated the impaired DCV distribution in cerebellar brain tissue from constitutive β-Nrxn knockout (β-TKO) mice, where DCVs are normally abundant and β-Nrxn isoforms are prominently expressed. Finally, we observed that the ultrastructure and marker proteins of the Golgi apparatus, responsible for packaging neuropeptides into DCVs, seem unchanged. In conclusion, based on the validation from the two deletion strategies in conditional and constitutive KO mice, two neuronal populations from the hippocampus and cerebellum, and two experimental protocols in cultured neurons and in the brain tissue, this study presented morphological evidence that the number of DCVs at synapses is altered in the absence of β-Nrxns. Our results therefore point to an unexpected contribution of β-Nrxns to the organization of neuropeptide and neuromodulator function, in addition to their more established role in synaptic vesicle release.
神经元之间通过突触进行的通讯包括释放含神经递质的突触小泡（SVs）和含神经调质的致密核心小泡（DCVs）。神经连接蛋白（Nrxns）是脊椎动物中由三个基因（Nrxn1 - 3）编码的多态性细胞表面分子家族，已被认为是突触前的重要组织者，也是突触小泡胞吐作用的细胞类型特异性甚至突触特异性调节的候选分子。然而，Nrxns是否也调节DCVs仍不清楚。在这里，我们报告至少β-神经连接蛋白（β-Nrxns），一种细胞外较小的Nrxn变体，参与突触前DCVs的分布。我们发现，通过慢病毒介导的Cre重组酶在原代海马神经元中表达，有条件地删除小鼠中的所有三种β-Nrxn异构体，可减少突触前终扣中超微结构鉴定的DCVs数量。一致地，针对标记蛋白的共标记显示，β-Nrxn缺陷神经元的突触和轴突中嗜铬粒蛋白A（ChrgA）阳性DCVs的数量减少。此外，我们在组成型β-Nrxn基因敲除（β-TKO）小鼠的小脑脑组织中验证了DCV分布受损，在那里DCVs通常丰富且β-Nrxn异构体显著表达。最后，我们观察到负责将神经肽包装到DCVs中的高尔基体的超微结构和标记蛋白似乎没有变化。总之，基于条件性和组成型基因敲除小鼠的两种缺失策略、海马体和小脑的两个神经元群体以及培养神经元和脑组织中的两个实验方案的验证，本研究提供了形态学证据，表明在没有β-Nrxns的情况下，突触处DCVs的数量会发生改变。因此，我们的结果表明，除了它们在突触小泡释放中更确定的作用外，β-Nrxns对神经肽和神经调质功能的组织有意外贡献。
