The higher-order brain dysfunction in TRH knockout mice

据报道，TRH在大脑中具有几种神经生理作用。为了深入了解这些影响的分子机制，特别是在小脑中，我们试图用TRH敲除小鼠和减法cDNA分析克隆受TRH调节的cDNA。对野生型和TRH^<-/->小脑进行减法杂交分析得到的100多个无性系进行了分析。其中4个克隆是相同的cdc2相关激酶（PFTAIRE蛋白激酶1 (PFTK1)） cDNA，此前报道仅在脑和睾丸中表达。与野生型相比，添加甲状腺激素的正常甲状腺TRH^<-/->小脑中PFTK1 mRNA水平显著降低。在表达TRH受体亚型1 mRNA的人髓母细胞瘤来源的HTB-185细胞中，TRH也以时间和剂量依赖的方式诱导PFTK1 mRNA。此外，8-Br-cGMP处理显著增加了PFTK1 mRNA水平，一种特定的cGMP产生抑制剂ODQ完全阻断了trh诱导的PFTK1 mRNA表达。此外，TRH对PFTK1 mRNA的诱导可被NOS特异性抑制剂L-NAME显著抑制，而MEK抑制剂PD98059或钙通道抑制剂尼莫地平则没有作用。这些发现首次证明了大脑中神经肽和细胞周期相关肽之间的新途径，PFTK1可能通过NO-cGMP途径在小脑中发挥TRH作用的关键调节作用。

TRH has been reported to possess several neurophysiological actions in the brain. To gain insights into the molecular mechanisms underlying these effects, particularly in the cerebellum, we attempted to clone a cDNA that was regulated by TRH using TRH knockout mice and subtractive cDNA analysis. Over 100 clones obtained by subtractive hybridization analysis between the wild-type and TRH^<-/-> cerebellum were analyzed. Four clones among them were identical and cdc2-related kinase (PFTAIRE protein kinase 1 (PFTK1)) cDNA, which was previously reported to be expressed only in the brain and testis. PFTK1 mRNA levels in the euthyroid TRH^<-/-> cerebellum supplemented with thyroid hormone were significantly decreased compared with those in the wild-type. Induction of PFTK1 mRNA by TRH was also observed in a time- and dose-dependent manner in human medulloblastoma-derived HTB-185 cells that expressed TRH receptor subtype 1 mRNA. In addition, treatment of 8-Br-cGMP significantly increased PFTK1 mRNA levels, and a specific inhibitor of cGMP production, ODQ, completely blocked TRH-induced expression of PFTK1 mRNA. Furthermore, induction of PFTK1 mRNA by TRH was significantly inhibited by a NOS specific inhibitor, L-NAME, but not by a MEK inhibitor, PD98059 or a calcium channel inhibitor, nimodipine. These findings demonstrated, for the first time, a novel pathway between a neuropeptide and a cell cycle related peptide in the brain, and PFTK1 may be a key regulator for TRH action in the cerebellum through the NO-cGMP pathway.