中国科学院昆明动物研究所机构知识库

自噬是溶酶体介导的细胞降解自身细胞器和蛋白的过程，主要包含巨型自噬、微型自噬和分子伴侣介导的自噬。自噬和细胞存活、细胞分化、发育和体内稳态密切相关。大量研究表明，自噬异常与很多疾病相关，包括肿瘤、免疫性疾病和神经退行性疾病等。因此，研究自噬在疾病中的生物学机制，对于未来疾病的治疗以及新药的研发都具有十分重要的作用。我们主要致力于研究自噬在毒品成瘾，特别是吗啡成瘾和镇痛中的作用机制。毒品成瘾是一种慢性易复发脑病，表现为强制性用药、敏感化和依赖，其机制至今未明。吗啡是阿片类毒品的典型代表，研究吗啡成瘾的作用机制能为阐明阿片类毒品成瘾的分子机制以及临床治疗提供新的思路和理论依据。研究发现，自噬可能与成瘾相关。我们前期研究显示，成瘾过程中吗啡诱导线粒体功能异常，进而诱发自噬。褪黑素是一种靶向线粒体的抗氧化剂。预先用褪黑素处理细胞和小鼠能够拯救吗啡诱导的线粒体功能异常，进而拯救吗啡诱导的自噬，最终阻断吗啡诱导的小鼠行为敏感化和光热疼痛耐受。但自噬是否参与成瘾，亦或是成瘾的副产物没有阐释。我们希望在本研究中解决这一问题，并阐述吗啡诱导自噬的分子机制，明确自噬在吗啡成瘾和镇痛过程中的作用。为了验证自噬在吗啡成瘾中的地位，我们通过侧脑室注射自噬抑制剂和自噬诱导剂，检测吗啡诱导的小鼠运动行为敏感化。发现预先通过侧脑室注射自噬抑制剂3-甲基腺嘌呤，能够抑制吗啡诱导的小鼠运动行为敏感化。相反，预先注射自噬诱导剂雷帕霉素，会促进吗啡诱导小鼠运动行为敏感化。这些结果说明自噬在成瘾中发挥重要作用，并非成瘾副产物。随后，我们开始深入研究吗啡诱导自噬的分子机制。发现慢性吗啡诱导的自噬依赖于自噬相关基因5（autophagy-related 5，Atg5）和自噬相关基因7（autophagy-related 7，Atg7）。在小鼠中脑组织中，吗啡诱导的自噬小体大部分与多巴胺能神经元共定位，与GABA能神经元共定位的自噬小体很少。多巴胺能神经元特异性敲除Atg5或者Atg7能拯救吗啡诱导的自噬，说明吗啡诱导Atg5和Atg7依赖的自噬具有多巴胺能神经元特异性。树突的伸展和树突棘的形成对神经元功能十分重要，前期研究表明，树突形态改变与毒品成瘾相关。为了确认吗啡和自噬对神经元的形态变化，我们用吗啡处理原代神经元，用酪氨酸羟化酶（tyrosine hydroxylase，TH）和微管相关蛋白2（microtubule-associated protein 2，MAP2）分别标记多巴胺能神经元和神经元树突。吗啡处理原代中脑神经元，导致多巴胺能神经元树突总长度和树突复杂度显著降低，siRNA干扰Atg5或者Atg7能够拯救吗啡诱导的多巴胺能神经元树突形态变化。在体内实验中，我们也发现了相似的结果，通过高尔基染色显示，慢性吗啡注射诱导小鼠中脑和海马神经元的树突棘密度显著减少，条件性敲除Atg5或者Atg7能够拯救吗啡神经毒性。这些结果表明，Atg5和Atg7依赖的自噬调控吗啡诱导的神经元的树突形态变化。Atg5和Atg7依赖的自噬是否能通过调控神经元树突可塑性参与成瘾行为的调控呢？我们的检测结果发现，多巴胺能神经元中特异性敲除Atg5或者Atg7基因的小鼠，吗啡诱导的奖赏效应、行为敏感化、疼痛耐受和药物依赖的行为会受到抑制。与此同时，我们发现多巴胺能特异性敲除Atg5或者Atg7基因的小鼠的疼痛耐受阈值比野生型小鼠升高。而且中枢神经系统特异性敲除Atg5或者Atg7基因能够减轻福尔马林和醋酸诱导的小鼠急性痛行为。进一步的研究表明，吗啡诱导的自噬可能通过调控阿片受体的内吞和降解参与吗啡镇痛。综上所述，本研究从小鼠成瘾模型出发，首先确认吗啡诱导的自噬参与成瘾而非成瘾副产物，随后在原代神经元上研究吗啡诱导自噬的分子机制，吗啡会导致ATG5和ATG7蛋白表达显著上调，从而促进多巴胺能神经元中ATG12-ATG5 complex形成和Atg5和Atg7依赖的自噬活性增强。Atg5和Atg7依赖的自噬通过调控多巴胺能神经元的树突棘密度、树突复杂度和树突总长度进而调控成瘾行为学。多巴胺能神经元中特异性敲除Atg5或Atg7基因，能拯救吗啡诱导的自噬介导的树突可塑性改变，最终阻断吗啡诱导的成瘾行为学，包括吗啡奖赏效应、行为敏感化、疼痛耐受和戒断症状。自噬可能通过调控阿片受体内吞和降解参与吗啡镇痛。我们的研究结果深入阐述了吗啡诱导自噬产生的分子机制，有望为成瘾的治疗和临床镇痛提供新的思路与科学依据。 

Autophagy is a lysosome-mediated degradation pathway that controls the turnover of cytoplasmic contents and organelles through the engulfment of cargo into double-membrane autophagosomes. It is divided into macroautophagy, microautophagy and chaperone-mediated autophagy. Basal levels of autophagy are important for maintaining cell survival, differentiation, development, and normal cellular homeostasis. Growing evidence reveals that deregulation of autophagy is involved in numerous human diseases, such as tumor, infection and immunity, and neurodegenerative diseases. Therefore, an improved understanding of the biological changes of autophagy underlying the development of diseases will be essential for providing important clues for exploring potential targets for novel therapeutics of these diseases. In this study, we focus on the potential role of autophagy in drug addiction, especially for morphine addiction and analgesia.Drug addiction is a chronic, relapsing brain disease that characterized by compulsive drug use, sensitization and dependence. Albeit there are many studies, the exact mechanism of addiction remains insufficiently resolved. As morphine is a typical of opiate, exploring the molecular mechanism of morphine may provide some helpful information for opiate withdrawal therapy. Previous studies have suggested that autophagy may be involved in drug addiction. Recently, we found that morphine could induce mitochondrial dysfunction and led to autophagy. Pretreatment with melatonin, which is a mitochondria-targeted antioxidant, restored the abnormal mitochondrial function and prevented the increase of autophagy induced by morphine. In mice, pre-injection of melatonin with morphine ameliorated morphine-induced behavioral sensitization and analgesic tolerance. However, we have no idea about whether autophagy is actively involved in addiction, or just a by-product of the addiction process. In this study, we aim to answer this question, and to elucidate the molecular mechanism of autophagy induced by morphine. Our results clarify the crucial role for autophagy in the process of morphine addiction and analgesia.To assess whether autophagy played active roles in morphine addiction, we analyzed the behavioral sensitization changes in mice with the intracerebroventricular injection of 3-methyladenine (3-MA) or rapamycin (Rap) before morphine administration. We found that pretreatment with 3-MA significantly reduced behavioral sensitization compared to the morphine group, whilst pretreatment with Rap significantly promoted the behavioral sensitization compared to the morphine group. This result suggested that autophagy plays a critical role in drug addiction but not just a by-product. Then we investigated the molecular mechanism of autophagy induced by morphine. Our results showed that chronic morphine exposure caused autophagy-related 5 (Atg5)- and autophagy-related 7 (Atg7)-dependent autophagy in the mouse midbrain neurons. A large number of microtubule-associated protein 1 light chain 3 beta (LC3) puncta were found in the midbrain slices of mice which had been given morphine by injection. Many of the puncta were co-localized with dopaminergic neurons, whereas a few were co-localized with GABAergic interneurons. These effects induced by morphine could be reversed by conditional knockout of Atg5 or Atg7 specifically in the dopaminergic neurons of mice. These results indicated that morphine-induced Atg5- and Atg7-dependent autophagy might be specific to dopaminergic neurons but not GABAergic interneurons.Dendritic arborization and spine formation are critical for neuron function. There are some studies implicated that dendritic changes have been implicated in drug addiction. To confirm the impact of morphine treatment and autophagy on the branching of neurons, mouse primary midbrain neurons were treated with morphine, followed by staining with microtubule-associated protein 2 (MAP2) to evaluate the changes of total neuron dendritic length and complexity and with tyrosine hydroxylase (TH) to recognize the dopaminergic neurons, respectively. As expected, both total dendritic length and dendritic complexity were significantly reduced in morphine-treated dopaminergic neurons relative to untreated neurons. The decreased total dendritic length and dendritic complexity induced by morphine could be reversed by knockdown of Atg5 or Atg7 using siRNAs. We got a similar result in vivo, Golgi-Cox impregnated striatal neurons showed that morphine given by injection dramatically decreased the number of neuronal dendritic spines in the midbrain and hippocampus compared to the untreated WT mice. Deficiency of Atg5 or Atg7 in dopaminergic neurons rescued the detrimental effects of morphine. These findings indicated a critical role of the Atg5- and Atg7-dependent autophagy in regulating neuronal morphological changes triggered by morphine.We further explored whether the Atg5- and Atg7-dependent autophagy participated in the development of addictive behaviors by regulating morphological changes of neurons. We measured the behavioral changes in mice with deficiency of the Atg5- and Atg7 gene specifically in the dopaminergic neurons. We found that mice deficient for Atg5- and Atg7 specifically in the dopaminergic neurons were less sensitive to develop morphine reward, behavioral sensitization, analgesic tolerance and physical dependence compared to wild-type mice. At the same time, we found that Th-Cre/Atg5flox/flox and Th-Cre/Atg7flox/flox mice showed a higher nociceptive threshold than the WT littermates in the tail-flick and the hot-plate tests. Conditional knockout Atg5 or Atg7 specific for central nervous system attenuated the acute pain behavior in mice induced by formaldehyde and acetic acid. Our preliminary results further showed that autophagy might play a key role in morphine analgesia by regulating the endocytosis and degradation of opioid receptors.Taken together, based on the mouse animal models, we clarified the role of autophagy in addiction. We then explored the molecular mechanism of autophagy induced by morphine. We found that repeated morphine administration led to upregulation of ATG5 and ATG7, which facilitated the formation of the ATG12-ATG5 conjugate and Atg5- and Atg7-dependent autophagy in the dopaminergic neurons. The detrimental effects of morphine-induced autophagy could be counteracted by a specific deficiency of ATG5 or ATG7 in dopaminergic neurons, and this protected neuron function and prevented the development of addictive behaviors. Autophagy also participated in morphine analgesia by regulating the endocytosis and degradation of opioid receptors. These convergent lines of evidence uncovered the key role of autophagy in morphine addiction. Manipulation autophagy might be explored as a potential therapeutic strategy in the future treatment of drug addiction and morphine analgesia.