Regulation of the effects of antidepressants by sphingomyelin- and ceramide-controlled autophagy

我们以前的研究发现，酸性鞘磷脂酶(ASM)/神经酰胺系统是阿米替林和氟西汀等抗抑郁药物的靶点。我们证明了阿米替林和氟西汀降低了海马区ASM的活性，从而增加了神经元的增殖、成熟和存活率；并改善了应激性抑郁模型的行为。ASM的遗传缺陷剥夺了这些抗抑郁药物的作用。过度表达ASM的小鼠表现出与轻度重度抑郁障碍(MDD)相关的结构性变化。我们发现，阿米替林和氟西汀通过在溶酶体和高尔基体中缓慢积聚鞘磷脂以及在内质网中缓慢积聚神经酰胺来诱导海马神经元的自噬。内质网神经酰胺刺激磷酸酶2A(PP2A)、ULK、Beclin、PI3K/Vps34、P62和Lc3B，从而促进自噬和自噬溶酶体的形成。用D609直接抑制鞘磷脂合成酶可导致神经酰胺在内质网快速积聚，激活自噬，并迅速逆转应激诱导的MDD。抑制Beclin可阻断阿米替林和D609的抗抑郁作用，并诱导MDD典型的细胞和行为改变。我们现在将研究抗抑郁药通过调节内质网神经酰胺和溶酶体神经酰胺之间的平衡来发挥作用的假设，内质网神经酰胺诱导自噬并抵消MDD，溶酶体神经酰胺减少自噬从而诱导MDD：1.我们将通过一组基因敲除小鼠和转基因小鼠阐明自噬溶酶体形成在MDD模型治疗中的作用。我们将确定自噬溶酶体的形成是否在体内的海马体中发生，在MDD诱导后改变，并被抗抑郁药纠正；我们还将确定抗抑郁药的生化和行为效应是否需要自噬溶酶体的形成。自噬决定抗抑郁药物效果的分子和细胞机制将被分析。2.我们将确定溶酶体神经酰胺是否影响自噬小体-溶酶体融合或溶酶体重组，或两者兼而有之，从而导致MDD。最终，我们的目标是通过靶向鞘磷脂来诱导自噬溶酶体的形成来开发新型的、快速有效的抗抑郁药物。

Our previous studies identified the acid sphingomyelinase (Asm)/ceramide system as target for antidepressants such as amitriptyline and fluoxetine. We demonstrated that amitriptyline and fluoxetine reduce Asm activity in the hippocampus and thereby increase neuronal proliferation, maturation, and survival; and improve behaviour in models of stress-induced depression. Genetic deficiency of Asm abrogates the effects of these antidepressants. Mice overexpressing Asm show constitutive changes associated with mild major depressive disorder (MDD). We showed that amitriptyline and fluoxetine induce autophagy in hippocampal neurons via a slow accumulation of sphingomyelin in lysosomes and Golgi bodies and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A (PP2A), Ulk, Beclin, PI3K/Vps34, p62 and Lc3B and thereby autophagy and the formation of autophagolysosomes. Direct inhibition of sphingomyelin synthases with D609 results in rapid accumulation of ceramide in the ER, activation of autophagy and rapid reversal of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioural changes typical of MDD. We will now investigate the hypothesis that antidepressants act by regulating the balance between ceramide in the ER, which induces autophagy and counteracts MDD, and ceramide in lysosomes, which reduces autophagy and thereby induces MDD:1. We will elucidate the role of autophagolysosome formation in the treatment of MDD (models) by using a panel of knock-out mice and transgenic mice. We will determine whether autophagolysosome formation occurs in vivo in the hippocampus, is altered upon induction of MDD, and is corrected by antidepressants; we will also determine whether autophagolysosome formation is required for the biochemical and behavioural effects of antidepressants. Molecular and cellular mechanisms by which autophagy determines the effects of antidepressants will be analyzed. 2. We will determine whether lysosomal ceramide affects autophagosome-lysosome fusion, lysosomal reformation, or both and thereby induces MDD.Ultimately, we aim to develop novel, fast-acting antidepressants by targeting sphingolipids to induce the formation of autophagolysosomes.