Neuroscience Institute, School of Medicine, Universitat Autònoma de Barcelona, Spain; Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Spain; Neurodegenerative Diseases Research Group, Vall d'Hebron Research Institute (VHIR)-Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), Spain.
Neuroscience Institute, School of Medicine, Universitat Autònoma de Barcelona, Spain; Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Spain.
Neuropharmacology. 2022 Jul 1;212:109058. doi: 10.1016/j.neuropharm.2022.109058. Epub 2022 Apr 13.
Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.
突触事件对定义大脑疾病的治疗策略很重要。在本论文中，我们将新鲜获得的大鼠脑纹状体组织在不同时间和条件下孵育，以确定多巴胺的生物合成、储存和酪氨酸羟化酶磷酸化。值得注意的是，我们发现内源性多巴胺在 37°C 下的组织孵育过程中会自发积累，而多巴胺的合成同时减少。我们分析了这些脑多巴胺生物合成和储存的变化是否与多巴胺对其合成限速酶酪氨酸羟化酶的反馈抑制有关。芳香族 L-氨基酸脱羧酶抑制剂 NSD-1015 阻止了这两种作用。正如预期的那样，添加 l-DOPA 或 VMAT2 过表达会增加多巴胺的积累，而添加多巴胺、VMAT2 抑制剂四苯嗪或 D 自动受体激动剂喹吡罗则会进一步降低多巴胺的合成，这与这些处理的已知突触效应一致。用 okadaic acid、Sp-cAMP 和 PD98059 分别对酪氨酸羟化酶 Ser31 和 Ser40 进行磷酸化激活和抑制，也产生了预期的效果。然而，通过 Western blot 和质谱分析，并没有发现多巴胺对自身生物合成的反馈抑制与酪氨酸羟化酶磷酸化变化之间有明显的关联。后一种技术还揭示了大鼠酪氨酸羟化酶上新的 Thr30 磷酸化。我们对脑多巴胺合成和储存动力学的体外评估方法可用于预测多巴胺相关疾病动物模型中药物干预的体内效应。
