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

我们之前的研究发现，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的靶点。我们证明阿米替林和氟西汀降低海马中Asm的活性，从而增加神经元的增殖、成熟和存活；改善应激性抑郁模型的行为。Asm的遗传缺陷取消了这些抗抑郁药的作用。过表达Asm的小鼠表现出与轻度重度抑郁障碍（MDD）相关的结构变化。我们发现阿米替林和氟西汀通过溶酶体和高尔基体中鞘磷脂和内质网（ER）中神经酰胺的缓慢积累诱导海马神经元自噬。内质网神经酰胺刺激磷酸酶2A （PP2A）、Ulk、Beclin、PI3K/Vps34、p62和Lc3B，从而产生自噬和自噬溶酶体的形成。D609直接抑制鞘磷脂合成酶可导致内质网神经酰胺的快速积累，激活自噬，快速逆转应激诱导的MDD。Beclin的抑制阻断了阿米替林和D609的抗抑郁作用，诱导了典型的重度抑郁症的细胞和行为改变。我们现在将研究一种假设，即抗抑郁药通过调节内质网中的神经酰胺和溶酶体中的神经酰胺之间的平衡来起作用，内质网中的神经酰胺诱导自噬并抵消MDD，溶酶体中的神经酰胺减少自噬从而诱导MDD。我们将通过敲除小鼠和转基因小鼠来阐明自噬溶酶体形成在MDD治疗中的作用（模型）。我们将确定体内海马体中的自噬溶酶体形成是否在MDD诱导下发生改变，并通过抗抑郁药物纠正；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体-溶酶体融合，溶酶体重组，或两者兼而有之，从而诱导MDD。最终，我们的目标是通过针对鞘脂诱导自噬溶酶体的形成来开发新的，快速的抗抑郁药。

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.