An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2-arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer’s disease,European Journal of Neuroscience

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An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2-arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer’s disease
European Journal of Neuroscience ( IF 2.7 ) Pub Date : 2020-08-19 , DOI: 10.1111/ejn.14944
Matilde S Rodrigues ₁ , Cláudia Ferreira ₁ , Cândida Dias ₁ , Anna Pliássova ₁ , Lisiane Souza ₁ , Ana Ledo _{1,

2} , João Laranjinha _{1,

2} , Rodrigo A Cunha _{1,

3} , Attila Köfalvi _{1,

4}

Affiliation

The endocannabinoid system is implicated in a plethora of neuropsychiatric disorders. However, it is technically challenging to assess the turnover of 2-arachidonoyl glycerol (2-AG), the principal endocannabinoid molecule in the brain. Two recent studies showed that diacylglycerol lipase α (DAGLα), an enzyme chiefly responsible for the cerebral production of 2-AG, also accepts the surrogate chromogenic substrate 4-nitrophenyl butyrate (4-NPB). Here, we aimed to optimize this spectrophotometric assay for ex vivo brain tissue, in particular, rat cerebrocortical homogenates, to measure the activity of the major enzymes responsible for the production and degradation of 2-AG. The initial velocity of 4-NPB hydrolysis was dependent on protein, substrate, and Ca²⁺ concentrations, and was sensitive to the non-selective serine hydrolase inhibitor, methoxy arachidonyl fluorophosphonate, the DAGLα inhibitors, OMDM188, tetrahydrolipstatin, and RHC80267, as well as the monoacylglycerol lipase (MAGL) inhibitor, JZL184, respectively. Next, we tested the usefulness of this assay in ex vivo brain tissue of rat models of human health conditions known to affect cerebrocortical 2-AG production, i.e. pathological stress and sporadic Alzheimer's disease (AD). In rats submitted to chronic restraint stress, cortical CB₁R density was significantly decreased, as assessed with radioligand binding. Nevertheless, 4-NPB hydrolysis remained at control levels. However, in rats 4 weeks after intracerebroventricular injection with streptozotocin – an established model of sporadic AD –, both CB₁R levels and 4-NPB hydrolysis and its DAGL- and MAGL-dependent fractions were significantly increased. Altogether, we optimized a simple complementary ex vivo technique for the quantification of DAGL and MAGL activity in brain samples.

中文翻译：

优化的分光光度测定法显示阿尔茨海默病大鼠模型大脑皮层中参与 2-花生四烯酸甘油转换的酶的活性增加

内源性大麻素系统与过多的神经精神疾病有关。然而，评估大脑中主要的内源性大麻素分子 2-花生四烯酸甘油 (2-AG) 的营业额在技术上具有挑战性。最近的两项研究表明，二酰基甘油脂肪酶 α (DAGLα) 是一种主要负责大脑产生 2-AG 的酶，它也接受替代显色底物 4-硝基苯基丁酸 (4-NPB)。在这里，我们旨在优化这种分光光度法，用于体外脑组织，特别是大鼠脑皮质匀浆，以测量负责 2-AG 产生和降解的主要酶的活性。4-NPB 水解的初始速度取决于蛋白质、底物和 Ca ²⁺浓度，并且分别对非选择性丝氨酸水解酶抑制剂甲氧基花生四烯酰氟膦酸盐、DAGLα抑制剂OMDM188、四氢利普他汀和RHC80267以及单酰基甘油脂肪酶（MAGL）抑制剂JZL184敏感。接下来，我们在已知会影响脑皮质 2-AG 产生的人类健康状况大鼠模型的离体脑组织中测试了该测定法的有效性，即。病理性应激和散发性阿尔茨海默病（AD）。在遭受慢性约束压力的大鼠中，皮质 CB ₁用放射性配体结合评估，R 密度显着降低。尽管如此，4-NPB 水解仍保持在对照水平。然而，在大鼠脑室内注射链脲佐菌素（一种已建立的散发性 AD 模型）后 4 周，CB ₁ R 水平和 4-NPB 水解及其 DAGL 和 MAGL 依赖性部分均显着增加。总之，我们优化了一种简单的补充体外技术，用于量化脑样本中的 DAGL 和 MAGL 活性。

更新日期：2020-08-19

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