蛋白激脢CK2在多巴胺調控大鼠紋狀體細胞訊息機制中所扮演的角色：對DARPP-32、GAD蛋白和運動行為的影響

Abstract

蛋白激酶CK2（protein kinase CK2）在成鼠腦部的活性遠高於其他器官， 而且在紋狀體腦區主要是表現CK2，但目前僅知CK2 參與在保護多巴胺神經 細胞和與藥物成癮的相關機制中，並不知是否參與多巴胺所調控的運動行為神 經生理功能。CK2 磷酸化受質蛋白之一的DARPP-32 被證實在紋狀體medium spiny neuron（MSN）中有大量的表現，目前僅知DARPP-32 調控的訊息傳遞路 徑與神經生理的研究多與藥物成癮有相關，在藥物成癮機制中CK2 針對大鼠品 系DARPP-32 Ser102 的磷酸化作用可以增加PKA 對Thr34 的磷酸化作用，Thr34 磷酸化的DARPP-32 則會抑制PP-1 的活性。然而巴金森氏症動物長期L-DOPA 投藥處理引起的運動困難（dyskinesia）和DARPP-32 Thr34 磷酸化調控訊息傳 遞的活化有關，此外，施予L-DOPA 處理而產生dyskinesia 現象時，紋狀體負責 GABA 生合成酵素之一的Glutamic acid decarboxylase 67（GAD67）mRNA 和蛋 白質的表現量會伴隨有增加的情形，因此CK2/DARPP32/GAD67 的細胞訊息傳 遞機制與多巴胺控制運動行為神經生理的相關性有待釐清。此外，在GAD67 的 細胞調控中，已知PKA 和PP2A 參與在GAD67 磷酸化作用的細胞調控機制， 然而是否存在其他kinase 和phosphatase 的調控機制則還尚未定論，由於PKA、 PP2A 和CK2 皆與DARPP-32 磷酸化變化有相關性，DARPP-32 可承接來自上游 的細胞訊息經由抑制PP-1 活性影響改變細胞的生理狀態，因此CK2 調控 DARPP-32 Ser102 磷酸化的細胞機制可能也參與調控GAD67 酵素活性。此外， Histone H3 蛋白的磷酸化現象也被證實和許多基因表現改變的epigenetic effect 有相關性，CK2 和DARPP-32 亦被發現參與和藥物成癮相關之Histone H3 蛋白 磷酸化的改變，因此CK2/DARPP-32 的細胞機制與GAD67 基因表現的epigenetic effect 之間可能存在有相關性。除此之外，經由direct striatonigra pathway 或 indirect striatopallidal pathway，MSN 會以feedback regulation 方式參與黑質多巴 胺神經元的功能調控，而黑質腦區多巴胺的生成含量和運動行為影響的相關性 間接顯示負責多生合成的tyrosine hydroxylase（TH）功能受到影響，其中TH 胺 基酸ser19、ser31 和ser40 的磷酸化現象和增進TH 的酵素活性促使多巴胺的生 合成有關，在導致巴金森氏症有關之alpha-synuclein 蛋白的aggregation 現象的 細胞中，可以發現TH 蛋白磷酸化的減少；也有研究發現經由transcription factor CREB 磷酸化的訊息傳遞機制可以促進TH mRNA 的表現，但鮮少有研究探討紋 狀體細胞機制對黑質TH 蛋白功能的影響。本計畫的研究動機即是針對上述尚未 釐清之相關問題進行研究探討，三年的研究目的包括有：（1）建立紋狀體medium spiny neuron 中CK2/DARPP-32/GAD 訊息傳遞路徑影響和多巴胺對運動行為的 相關性；（2）建立CK2/DARPP-32 對GAD 基因之epigenetic effect 調控機制；（3） 建立紋狀體CK2/DARPP-32/GAD 細胞訊息機制與調控黑質多巴胺神經元功能 和運動行為神經生理的相關性。

Protein kinase CK2 is a heterotetrameric and ubiquitous serine/threonine protein kinase. Its protein levels and activity were found to be elevated in the brain when compared to other organs. The two catalytic subunits,  and ` are highly homologous but  isoform protein levels in the striatum are relatively high when compared with ` isoform. In the striatum, CK2 is known to involve in the neuroprotective effects of dopaminergic neurons and the process of drug abuse. Whether CK2 also regulates the neuronal function about motor behaviors is still unclear. DARPP-32 protein, which is highly expressed in the striatal medium spiny neurons responsible for dopamine stimulation, is known as one of the substrates for CK2. During the process of drug abuse, the phosphorylation of DARPP-32 at Ser102 by CK2 can stimulate Thr34 phosphorylation for PKA and thereby converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP-1). Although DARPP-32 plays a key role in integrating in antidepressants and drugs of abuse, the dyskinesia symptom induced by long-term L-DOPA treatments was found to correlate with Thr34 phosphorylation and then activation of DARPP-32 mediated signaling. The long-term L-DOPA treatment was also found to increase the Glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is one of the enzymes responsible for the synthesis of neurotransmitter GABA. But it is not clear whether the CK2/DARPP-32/ GAD signaling exists in the striatum responsive to dopamine-mediated motor functions. So far, at the post-translational regulation for GAD67 phosphorylation is known by PKA and PP2A, other kinase and/or phosphatase also might involve in this regulation mechanism. Because the signaling from CK2, PKA or PP2A can be integrated by DARPP-32 and then to its downstream molecule PP-1 to influence many neuronal functions, it is worthy to clarify the role of DARPP-32 at ser102 phosphorylation in the regulation mechanism for GAD67. Furthermore, the epigenetic regulation at transcription level for GAD67 cannot be excluded. CK2 and DARPP-32 were found to regulate the histone H3 phosphorylation at ser10 relative to drugs of abuse, whether the epigenetic regulation of CK2/DARPP-32 involves in regulating of GAD67 gene expression is also worthy to investigate. At the other side, the MSN can feedback-regulate the nigra dopaminergic functions through direct striatonigral pathway or indirect striatopallidal pathway. The activity of tyrosine hydroxylase (TH), which is the rate-limiting enzyme of dopamine biosynthesis, is correlative to dopamine contents and motor functions. At now, TH phosphorylation at ser19, ser31or ser40 is known to relative to its enzyme activity. The decrease in TH phosphorylation was found parallel with the aggregation of alpha-synuclein in the Parkinson’s model. It is also found that the TH mRNA can be increased through pCREB-mediated signaling. However, little is known about how strital function regulates nigra TH activity through feedback regulation. The purpose of this proposal is focused to investigate these unclear phenomena mentioned as above. The specific aims for the three-year proposal are as following: (1) to establish the CK2/DARPP-32/GAD signaling responsive for dopamine stimulation and the relationship with motor functions in the striatum; (2) to establish the epigenetic mechanism of CK2/DARPP-32 on GAD gene expression; and (3) to clarify the mechanisms of striatal feedback-regulation on substantia nigra dopaminergic functions.