Abstract：
The heterotrimeric G-protein α subunit, Gαolf, acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gαolf, have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gαolf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS. Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gαolf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL, with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gαolf signaling in vivo in neurons that highly express Gnal.
摘要：
异源三聚体G蛋白α亚基，Gαolf,通过G蛋白偶联受体（GPCRs）转导细胞外信号，并刺激腺苷酸环化酶介导的第二信使环磷酸腺苷的产生。大量的GNAL基因突变，编码Gαolf，已被确定为成人发作性肌张力障碍的病因。这些突变通过几种机制破坏体外测定中的GPCR信号级联，据推测，这种信号中断会导致患者出现肌张力障碍运动症状。然而,对GNAL中突变的细胞和电路的了解还不清楚。纹状体之外的Gαolf表达的已发布模式是稀疏的，冲突,通常缺乏细胞类型特异性，并且可能被GNAS的紧密GNAL同源物的表达所混淆。这里,我们使用RNAScope原位杂交定量表征野生型C57BL/6J成年小鼠脑组织中GnalmRNA的表达。我们观察到Gnalpuncta在整个大脑中广泛表达，这表明Gαolf在更多的大脑结构和神经元类型中表达，而不是以前的解释。我们在单细胞水平上量化转录本，并使用神经元类型特异性标记来进一步分类和理解GNAL表达的模式。我们的数据表明，传统上与运动控制相关的大脑区域，initiation,和调节显示GNAL的最高表达，小脑的Purkinje细胞显示所检查的任何神经元类型的最高表达。随后在浦肯野细胞中条件性Gnal敲除导致细胞内cAMP水平显着降低和下游cAMP依赖性酶激活。我们的工作提供了整个大脑中Gnal表达的详细表征，以及高度表达Gnal的神经元体内Gαolf信号传导丧失的生化后果。
