Department of Psychology, Section of Neuroscience, Istituto Pasteur-Fondazione Cenci Bolognetti and D. Bovet Research Center, Sapienza University of Rome, Rome, Italy.
Cerebellum. 2012 Mar;11(1):28-40. doi: 10.1007/s12311-010-0211-8.
We previously demonstrated that TSC22D4, a protein encoded by the TGF-β1-activated gene Tsc22d4 (Thg-1pit) and highly expressed in postnatal and adult mouse cerebellum with multiple post-translationally modified protein forms, moves to nucleus when in vitro differentiated cerebellum granule neurons (CGNs) are committed to apoptosis by hyperpolarizing KCl concentrations in the culture medium. We have now studied TSC22D4 cytoplasmic/nuclear localization in CGNs and Purkinje cells: (1) during CGN differentiation/maturation in vivo, (2) during CGN differentiation in vitro, and (3) by in vitro culturing ex vivo cerebellum slices under conditions favoring/inhibiting CGN/Purkinje cell differentiation. We show that TSC22D4 displays both nuclear and cytoplasmic localizations in undifferentiated, early postnatal cerebellum CGNs, irrespectively of CGN proliferation/migration from external to internal granule cell layer, and that it specifically accumulates in the somatodendritic and synaptic compartments when CGNs mature, as indicated by TSC22D4 abundance at the level of adult cerebellum glomeruli and apparent lack in CGN nuclei. These features were also observed in cerebellum slices cultured in vitro under conditions favoring/inhibiting CGN/Purkinje cell differentiation. In vitro TSC22D4 silencing with siRNAs blocked CGN differentiation and inhibited neurite elongation in N1E-115 neuroblastoma cells, pinpointing the relevance of this protein to CGN differentiation.
我们之前证明，TSC22D4 是 TGF-β1 激活基因 Tsc22d4（Thg-1pit）编码的一种蛋白质，在出生后和成年小鼠小脑中有多种翻译后修饰的蛋白质形式高度表达，当体外分化的小脑颗粒神经元（CGNs）通过培养基中高浓度的 KCl 而被诱导凋亡时，它会转移到细胞核。我们现在研究了 TSC22D4 在 CGNs 和浦肯野细胞中的细胞质/核定位：（1）在体内 CGN 分化/成熟期间，（2）在体外 CGN 分化期间，以及（3）通过在体外培养有利于/抑制 CGN/浦肯野细胞分化的小脑切片。我们表明，在未分化的、早期出生后的小脑 CGNs 中，TSC22D4 显示出核质和细胞质定位，无论 CGN 是从外部向内部颗粒细胞层增殖/迁移，而且当 CGNs 成熟时，它特异性地积累在体细胞树突和突触区，如成年小脑肾小球水平的 TSC22D4 丰度和 CGN 核中明显缺乏所示。这些特征也在体外培养的小脑切片中观察到，在有利于/抑制 CGN/浦肯野细胞分化的条件下。用 siRNAs 进行 TSC22D4 沉默阻断了 CGN 的分化，并抑制了 N1E-115 神经母细胞瘤细胞中神经突的伸长，这表明该蛋白与 CGN 的分化有关。
