Ras-GRF1 和 Ras-GRF2 在 L-DOPA 诱导的运动障碍中的差异参与。
Differential involvement of Ras-GRF1 and Ras-GRF2 in L-DOPA-induced dyskinesia.
机构信息
Section of Pharmacology, Department of Medical Sciences, University of Ferrara Ferrara, Italy.
Division of Neuroscience, Institute of Experimental Neurology, IRCSS San Raffaele Scientific Institute Milan, Italy ; Istituto di Ricerche Farmacologiche "Mario Negri" Milan, Italy.
出版信息
Ann Clin Transl Neurol. 2015 Jun;2(6):662-78. doi: 10.1002/acn3.202. Epub 2015 Apr 24.
OBJECTIVE
Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates.
METHODS
We used both Ras-GRF1 knockout mice and lentiviral vectors (LVs) delivering short-hairpin RNA sequences (shRNAs) to obtain striatum-specific gene knockdown of Ras-GRF1 and Ras-GRF2. The consequences of these genetic manipulations were evaluated in the 6-hydroxydopamine mouse model of Parkinson's disease. Escalating doses of l-DOPA were administered and then behavioral analysis with immunohistochemical assays and in vivo microdialysis were performed.
RESULTS
Ras-GRF1 was found essential in controlling ERK signaling in dMSNs, but its ablation did not prevent ERK activation in ChIs. Moreover, striatal injection of LV-shRNA/Ras-GRF1 attenuated dyskinesia development and ERK-dependent signaling, whereas LV-shRNA/Ras-GRF2 was without effect, ruling out the involvement of Ras-GRF2 in LID expression. Accordingly, Ras-GRF1 but not Ras-GRF2 striatal gene-knockdown reduced l-DOPA-induced GABA and glutamate release in the substantia nigra pars reticulata, a neurochemical correlate of dyskinesia. Finally, inactivation of Ras-GRF1 provided a prolonged anti-dyskinetic effect for up to 7 weeks and significantly attenuated symptoms in animals with established LID.
INTERPRETATION
Our results suggest that Ras-GRF1 is a promising target for LID therapy based on Ras-ERK signaling inhibition in the striatum.
目的
最近的研究结果表明，通过对 Ras-ERK 通路的遗传操作来治疗 l-3,4-二羟基苯丙氨酸（l-DOPA）诱导的运动障碍（LID）是一种有前途的治疗方法。首先，我们研究了长期 l-DOPA 治疗是否会对直接通路（dMSNs）中的 ERK 信号和胆碱能无棘突中间神经元（ChIs）中的 ERK 信号产生不同的影响，并评估了 Ras-GRF1 在这两个亚群中的作用。其次，我们使用病毒辅助技术研究了 Ras-GRF1 和 Ras-GRF2 作为该通路的潜在靶点。我们研究了选择性阻断纹状体 Ras-GRF1 或 Ras-GRF2 表达如何影响 LID（诱导、维持和逆转）及其神经化学相关性。
方法
我们使用 Ras-GRF1 基因敲除小鼠和携带短发夹 RNA 序列（shRNAs）的慢病毒载体（LVs）来获得纹状体特异性 Ras-GRF1 和 Ras-GRF2 的基因敲低。在帕金森病的 6-羟多巴胺小鼠模型中评估了这些遗传操作的结果。给予递增剂量的 l-DOPA，然后进行行为分析、免疫组织化学检测和体内微透析。
结果
Ras-GRF1 被发现对控制 dMSNs 中的 ERK 信号至关重要，但它的缺失并不能阻止 ChIs 中的 ERK 激活。此外，纹状体注射 LV-shRNA/Ras-GRF1 可减弱运动障碍的发展和 ERK 依赖性信号，但 LV-shRNA/Ras-GRF2 没有效果，排除了 Ras-GRF2 参与 LID 表达的可能性。因此，Ras-GRF1 而非 Ras-GRF2 的纹状体基因敲低可减少 l-DOPA 诱导的黑质网状部 GABA 和谷氨酸释放，这是运动障碍的神经化学相关性。最后，Ras-GRF1 的失活可提供长达 7 周的抗运动障碍作用，并显著减轻已建立 LID 动物的症状。
结论
我们的研究结果表明，基于 Ras-ERK 信号在纹状体中的抑制，Ras-GRF1 是治疗 LID 的一个有前途的靶点。
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