Parker Michael J, Zhao Shengli, Bredt David S, Sanes Joshua R, Feng Guoping
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Neurosci. 2004 Jan 14;24(2):378-88. doi: 10.1523/JNEUROSCI.3865-03.2004.
Neuronal cholinergic synapses play important roles in both the PNS and CNS. However, the mechanisms that regulate the formation, maturation, and stability of neuronal cholinergic synapses are poorly understood. In this study, we use the readily accessible mouse superior cervical ganglion (SCG) and submandibular ganglion (SMG) to examine the assembly of the postsynaptic complex of neuronal cholinergic synapses. We find that novel splicing forms of PSD93 (postsynaptic density 93) are expressed in SCG. By immunostaining, we show that PSD93 proteins precisely colocalize with neuronal nicotinic acetylcholine receptors (nAChRs) at synapses of the SCG and SMG. Subcellular fractionation demonstrates that PSD93 is enriched in the PSD fraction of SCG, and coimmunoprecipitation shows that PSD93 and neuronal nAChRs form a complex in vivo. Furthermore, two additional components of the well characterized glutamatergic postsynaptic complex, GKAP/SAPAP (guanylate kinase domain-associated protein/synapse-associated protein-associated protein) and Shank/ProSAP family proteins, are also present at neuronal cholinergic synapses. To assess the function of this protein complex at neuronal cholinergic synapses in vivo, we examined ganglia in mice that lack PSD93. We find that neuronal cholinergic synapses form properly in PSD93 null mice. After denervation, however, synaptic clusters of nAChRs disassemble much faster in mice lacking PSD93 than those in wild-type mice. These results demonstrate that PSD93 is a key component of the postsynaptic scaffold at neuronal cholinergic synapses and plays an important role in synaptic stability. In addition, these results suggest that the mechanism of postsynaptic scaffolding is conserved between neuronal cholinergic and glutamatergic synapses.
神经元胆碱能突触在周围神经系统（PNS）和中枢神经系统（CNS）中均发挥着重要作用。然而，调节神经元胆碱能突触形成、成熟和稳定性的机制仍知之甚少。在本研究中，我们利用易于获取的小鼠颈上神经节（SCG）和下颌下神经节（SMG）来研究神经元胆碱能突触后复合体的组装。我们发现，PSD93（突触后致密蛋白93）的新型剪接形式在SCG中表达。通过免疫染色，我们表明PSD93蛋白在SCG和SMG的突触处与神经元烟碱型乙酰胆碱受体（nAChRs）精确共定位。亚细胞分级分离表明，PSD93在SCG的突触后致密部分中富集，免疫共沉淀显示PSD93和神经元nAChRs在体内形成复合物。此外，特征明确的谷氨酸能突触后复合体的另外两个组分，GKAP/SAPAP（鸟苷酸激酶结构域相关蛋白/突触相关蛋白相关蛋白）和Shank/ProSAP家族蛋白，也存在于神经元胆碱能突触中。为了评估这种蛋白复合体在体内神经元胆碱能突触中的功能，我们检查了缺乏PSD93的小鼠的神经节。我们发现，在PSD93基因敲除小鼠中，神经元胆碱能突触能够正常形成。然而，去神经支配后，缺乏PSD93的小鼠中nAChRs的突触簇解体速度比野生型小鼠快得多。这些结果表明，PSD93是神经元胆碱能突触后支架的关键组分，在突触稳定性中发挥重要作用。此外，这些结果表明，突触后支架机制在神经元胆碱能突触和谷氨酸能突触之间是保守的。
