Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain
Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain
复制标题
DOI：
10.1002/cne.24792
复制
发表时间：
2019-11-14
影响因子：
2.5
通讯作者：
Watanabe, Masahiko
中科院分区：
文献类型：
--
作者：
Nakamoto, Chihiro;Konno, Kohtarou;Watanabe, Masahiko
在小脑中，GluD 2仅在浦肯野细胞中表达，在那里它调节突触形成和再生、突触可塑性和运动学习。GluD 2基因突变的人类认知发育延迟表明GluD 2的小脑外功能。然而，小脑外表达的GluD 2及其与GluD 1的关系知之甚少。GluD 2 mRNA和蛋白在嗅球层、内侧前额叶皮质、扣带皮质、压后颗粒皮质、嗅结节、下托、纹状体、外侧隔、丘脑前背核和下丘脑弓状核中表达量较高。这些区域也富含GluD 1，许多单个神经元共表达这两种GluDs。在压后颗粒皮质中，GluD 1和GluD 2选择性地表达在PSD-95表达的突触上，SDS消化的冷冻断裂复制标记显示它们在相同突触上的共表达。在生物化学上，GluD 1和GluD 2在HEK 293 T细胞和大脑皮层和海马中形成免疫共沉淀复合物。我们使用GluA 2/GluD 2和GluA 2/GluD 1嵌合蛋白作为GluD 1和GluD 2抗体滴定的标准，通过定量免疫印迹进一步估计相对蛋白量。有趣的是，从大脑皮层和海马制备的突触后密度部分中GluD 2的相对量几乎与GluD 1相当。相比之下，GluD 2在小脑中占压倒性优势。因此，我们已经确定了区域，神经元和突触水平上的谷氨酸D1和谷氨酸D2的相对小脑外表达。这些数据提供了一个可能的竞争和合作的相互作用的GluD家族成员在突触在不同的大脑区域的分子解剖学基础。
In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.