Circadian analysis of the mouse cerebellum

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated here in mice using large-scale two-dimensional differences in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24-h based on cosinor regression. Notably, rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only 6 proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including 2 proteins involved in synapse activity (Synapsin 2 (Syn2) and vesicle-fusing ATPase (Nsf)), 2 others participating in carbohydrate metabolism (triosephosphate isomerase (Tpi1) and alpha-enolase (Eno1)), and Glutamine synthetase (Glul), as well as Tubulin alpha (Tuba4a). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. This study highlights extensive rhythmic aspects of the cerebellar proteome.

小脑拥有昼夜节律时钟，可产生内源性时间信号。本研究以小鼠为模型，采用大规模二维差异凝胶电泳（2D-DIGE）技术，对小脑蛋白质的每日振荡特征展开探究。通过对2D-DIGE凝胶的分析，基于余弦回归分析法（cosinor regression），研究人员发现588个蛋白点中有27个（占比5%）的信号强度在24小时周期内呈现节律性变化。值得注意的是，丰度较高的小脑蛋白的节律性表达主要集中在两个核心时相（正午与午夜），形成双峰分布特征。本研究鉴定出的具有小脑节律性表达的蛋白中，仅有6种同样在视交叉上核（suprachiasmatic nuclei）中存在振荡现象，其中包括2种参与突触活动的蛋白：突触蛋白2（Synapsin 2, Syn2）与囊泡融合ATP酶（vesicle-fusing ATPase, Nsf）；2种参与碳水化合物代谢的蛋白：磷酸丙糖异构酶（triosephosphate isomerase, Tpi1）与α-烯醇化酶（alpha-enolase, Eno1）；以及谷氨酰胺合成酶（Glutamine synthetase, Glul）和α微管蛋白（Tubulin alpha, Tuba4a）。绝大多数具有节律振荡特征的小脑蛋白，在此前任何组织的昼夜节律蛋白质组学分析中均未被鉴定发现。尤为引人注目的是，线粒体蛋白的每日积累量集中于静息中期，这与此前在肝脏中针对特定线粒体蛋白观察到的积累规律一致。本研究揭示了小脑蛋白质组广泛存在的节律性特征。