Cholesterol accumulation is associated with lysosomal dysfunction and autophagic stress in Npc1 -/- mouse brain

Niemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1(-/-) mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1(-/-) mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1(-/-) mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC.     

The effect and mechanism of miR-210 in down-regulating the autophagy of lung cancer cells

This project aims to investigate the roles of miR-210 in autophagy of lung cancer cells and the related mechanism. The expressions of miR-210 and ATG7 in 30 cancer tissues and the adjacent tissues in patients with lung cancer were compared using RT-qPCR methods, Western Blot assay was carried out to test the expression of ATG7 in protein. Moreover, the dual luciferase reporter gene assay system was used to confirm ATG7 is a target gene of miR-210. Furthermore, lung cancer cell line A549 was transfected with either miR-210 mimics or inhibitors and RT-qPCR methods was used to detect the expression of miR-210 and ATG7. Next, MTT assay was used to examine the effect of miR-210 on the growth of the lung cancer cells, and finally, the expression of autophagy related genes, ATG7, LC3-II/LC3-I and Beclin-1 were detected by Western Blot and ICC assay. We observed that miR-210 was significantly increased and ATG7 was markedly decreased in cancer tissue of patients with lung cancer compared with normal tissue. Moreover, results of dual luciferase reporter assay indicated that ATG7 is a direct target of miR-210. Next, transfection of miR-210 mimics in lung cancer cells induced significant increase in cell proliferation, and transfection of miR-210 inhibitors lead to inhibited cell proliferation. Furthermore, over-expression of miR-210 induced marked decrease in the expression of ATG7, LC3-II/LC3-I and Beclin-1, while transfection of miR-210 inhibitors induced significant increase in the expression of ATG7, LC3-II/LC3-I and beclin-1. Our results suggested that miR-210 plays a great role in autophagy of lung cancer cell by targeting ATG7.     

Drp1 knockdown represses apoptosis of rat retinal endothelial cells by inhibiting mitophagy

Diabetic retinopathy (DR) is the leading clinical cause of blindness in diabetic patients. Mitophagy participates in the pathogenesis of DR. Dynamin related protein 1 (Drp1) is associated with mitophagy. Here, we investigated whether Drp1 can regulate mitophagy to affect the progression of DR. We constructed DR rat model by administration of streptozocin. Primary rat retinal endothelial cells (RECs) were treated with high glucose (HG) as a DR cell model. Drp1 was highly expressed in the retinal tissues of DR rats and HG-treated RECs. Drp1 knockdown inhibited HG-mediated increase of reactive oxygen species (ROS) levels and apoptosis in RECs. Moreover, Drp1 silencing inhibited the expression of autophagy-related proteins LC3-II/LC3–1 and Beclin-1 and reduced LC3 puncta in HG-treated RECs. The expression of mitochondrial marker Tom20 was reduced and the levels of mitophagy were increased in the HG-treated RECs, which was rescued by Drp1 silencing. Drp1 knockdown repressed LC3-II expression in HG-treated RECs, indicating that autophagy flux was inhibited. Rapamycin (autophagy activator) enhanced ROS levels and apoptosis in HG-treated RECs by activating autophagy, which was rescued by Drp1 knockdown. In conclusion, these data demonstrated that Drp1 knockdown repressed apoptosis of rat retinal endothelial cells by inhibiting mitophagy. Thus, this work suggests that targeted regulation of Drp1 may become a treatment for DR.     

Disruption and therapeutic rescue of autophagy in a human neuronal model of Niemann Pick type C1

An unresolved issue about many neurodegenerative diseases is why neurons are particularly sensitive to defects in ubiquitous cellular processes. One example is Niemann Pick type C1, caused by defects in cholesterol trafficking in all cells, but where neurons are preferentially damaged. Understanding this selective failure is limited by the difficulty in obtaining live human neurons from affected patients. To solve this problem, we generated neurons with decreased function of NPC1 from human embryonic stem cells and used them to test the hypothesis that defective cholesterol handling leads to enhanced pathological phenotypes in neurons. We found that human NPC1 neurons have strong spontaneous activation of autophagy, and, contrary to previous reports in patient fibroblasts, a block of autophagic progression leading to defective mitochondrial clearance. Mitochondrial fragmentation is an exceptionally severe phenotype in NPC1 neurons compared with fibroblasts, causing abnormal accumulation of mitochondrial proteins. Contrary to expectation, these abnormal phenotypes were rescued by treatment with the autophagy inhibitor 3-methyladenine and by treatment with the potential therapeutic cyclodextrin, which mobilizes cholesterol from the lysosomal compartment. Our findings suggest that neurons are especially sensitive to lysosomal cholesterol accumulation because of autophagy disruption and accumulation of fragmented mitochondria, thus defining a new route to effective drug development for NPC1 disease.     

Endosomal/lysosomal processing of gangliosides affects neuronal cholesterol sequestration in Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (β1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently co-localized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal β-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol.     

Runx2通过抑制细胞巨自噬以诱导C2C12细胞向成骨细胞分化

 目的：探讨细胞巨自噬与Runx2诱导C2C12细胞成骨分化的关系。方法： 在强力霉素(doxycycline, Dox)诱导Runx2表达的细胞系C2C12/Runx2Dox中进行研究。Dox (10 mg/L) 处理0 d、1 d、3 d及6 d后,real-time qPCR检测LC3b、Beclin-1、p62和LAMP-2表达情况,Western blotting分析LC3-I/LC3-II比值。设置不同的3-甲基腺嘌呤(3-methyladenine, 3-MA)或雷帕霉素(rapamycin, Rap)浓度,Dox处理14 d后分析碱性磷酸酶(alkaline phosphatase,ALP)活性。用3-MA (5 mmol/L)或Rap (10 μmol/L)与Dox共同处理1 d、3 d及6 d后检测ALP及骨钙素 (osteocalcin,OC)表达情况。结果： (1) C2C12细胞向成骨分化时,LC3b 与Beclin-1显著下调,p62与LAMP-2无明显变化;(2) LC3-I向LC3-II转换的过程被抑制;(3) 3-MA (5 mmol/L)可增强ALP 活性,而Rap(10 μmol/L)则抑制其活性;(4) 3-MA可上调ALP及OC表达,Rap则下调二者表达。结论： Runx2通过下调LC3和Beclin-1、抑制LC3-I向LC3-II转换的方式阻碍自噬体形成,以诱导C2C12细胞分化为成骨细胞。     

The Role of Autophagy in Kidney Inflammatory Injury via the NF-κB Route Induced by LPS

Acute kidney injury (AKI) is a systemic inflammatory response syndrome associated with poor clinical outcomes. No treatments effective for AKI are currently available. Thus, there is an urgent need of development of treatments effective for AKI. Autophagy, an intracellular proteolytic system, is induced in renal cells during AKI. However, whether autophagy is protective or injurious for AKI needs to be clearly clarified. We addressed this question by pharmacological inhibition of autophagy using a mouse model of lipopolysaccharide (LPS) induced-AKI. We found that autophagy was induced in renal cortex of mice during LPS-induced AKI as reflected by a dose-and time-dependent increased accumulation of light chain 3-II (LC3-II), the common marker of autophagy, compared to that of control group; 2) the occurrence of intensive, punctate and increased immunohistochemical staining image of LC3-II in renal cortex; 3) the significant increase in the expression levels of Beclin-1, another key marker of autophagy; 4) the significantly increased levels of plasma urea and serum creatinine and 5) the significant increase in autophagagosome area ratio. We observed that 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy, blocked autophagy flux, alleviated AKI and protected against LPS-induced AKI. LPS triggered kidney inflammation by activation of the canonical NF-κB pathway. This route can be modulated by autophagy. Activation of the canonical NF-κB pathway was reduced in 3-MA+LPS as compared to that in LPS-treated group of mice. Mice pretreated with 3-MA before exposure to LPS showed a reduction in p65 phosphorylation, resulting in the accumulation of ubiquitinated IκB. In conclusion, impairment of autophagy ameliorates LPS-induced inflammation and decreases kidney injury. The accumulation of ubiquitinated IκB may be responsible for this effect.     

Intracellular accumulation of amyloidogenic fragments of amyloid-beta precursor protein in neurons with Niemann-Pick type C defects is associated with endosomal abnormalities

Niemann-Pick type C disease (NPC) is characterized by neurodegeneration secondary to impaired cholesterol trafficking and excessive glycosphingolipid storage. Abnormal cholesterol and ganglioside metabolism may influence the generation and aggregation of amyloidogenic fragments (ie, C99 and Abeta) from amyloid-beta precursor protein (APP), crucial factors causing neurodegeneration in Alzheimer's disease. To reveal whether abnormal accumulation and aggregation of APP fragments also occurs in NPC, we studied their expression in cultured cortical neurons treated with U18666A, a compound widely used to induce NPC defects, and also in brain tissues from NPC patients. U18666A treatment resulted in increased intraneuronal levels of C99 and insoluble Abeta42, which were distributed among early and late endosomes, in compartments distinct from where endogenous cholesterol accumulates. Analyses of NPC brains revealed that C99 or other APP C-terminal fragments (APP-CTF), but not Abeta42, accumulated in Purkinje cells, mainly in early endosomes. In contrast, in hippocampal pyramidal neurons, the major accumulated species was Abeta42, in late endosomes. Similar to what has been shown in Alzheimer's disease, cathepsin D, a lysosomal hydrolase, was redistributed to early endosomes in NPC Purkinje cells, where it co-localized with C99/APP-CTF. Our results suggest that endosomal abnormalities related to abnormal lipid trafficking in NPC may contribute to abnormal APP processing and Abeta42/C99/APP-CTF deposition.     

Astragaloside IV attenuates orbital inflammation in Graves’ orbitopathy through suppression of autophagy

Introduction

Graves’ orbitopathy (GO) is an autoimmune inflammatory disorder affecting the orbit around the eye. Astragaloside IV (AS-VI) is the main active ingredient of the Chinese herbal medicine Huangqi (Radix Astragali Mongolici). AS-IV exhibits antioxidant and anti-inflammatory properties, and shows therapeutic potential in a number of ischemic and inflammatory diseases; however, its pharmaceutical activities in GO remain undefined.

Materials and methods

In this study, we investigated the effects of AS-IV on interleukin (IL)-1β-induced orbital fibroblast inflammation in vitro and GO orbital inflammation and ocular histopathological changes in vivo, as well as the underlying mechanisms responsible for these effects.

Results and conclusion

The results show that IL-1β increased mRNA expression of the inflammatory cytokines IL-6, IL-8, TNF-α, and MCP-1 in cultured orbital fibroblasts. This IL-1β-induced inflammation was accompanied by increased autophagic activity as reflected in increased Beclin-1 and Agt-5 expression, as well as LC3-I to LC3-II conversion. Pretreatment with the autophagy inhibitors 3-MA and bafilomycin A1, or silencing of autophagy-related proteins Beclin-1 and Atg-5, prevented IL-1β-induced orbital fibroblast inflammation, while pretreatment with the autophagy activator rapamycin had the opposite effects. These data suggested that autophagy was involved in GO orbital inflammation. AS-IV treatment significantly decreased IL-1β-induced inflammatory cytokine production in orbital fibroblasts in vitro and attenuated GO orbital inflammation, fat accumulation, collagen deposition, and macrophage infiltration in vivo. These in vitro and in vivo protective effects of AS-IV against GO were accompanied by decreased autophagic activities in orbital fibroblasts and GO orbital tissues, respectively. Collectively, our findings suggested that AS-IV protects against GO through suppression of autophagy. Thus, AS-IV may have preventive benefits for GO.

     

自噬水平变化对心脏骤停心肺复苏后大鼠海马神经元凋亡的影响

目的探讨自噬水平变化对心脏骤停心肺复苏(CA/CPR)后大鼠海马神经元凋亡的影响。方法将40只大鼠随机分为假手术组(sham)、CA/CPR模型组(model)、雷帕霉素(Rapa)组(CA/CPR+Rapa)及3-甲基腺嘌呤(3-MA)组(CA/CPR+3-MA)。采用呼气末夹闭气管窒息法复制大鼠CA/CPR动物模型,分别给予自噬激动剂Rapa 0.2 mg/kg及自噬抑制剂3-MA 10 mg/kg进行干预。采用神经功能缺陷评分(NDS)评价CA/CPR大鼠神经功能;用TUNEL染色法检测大鼠海马神经元的凋亡变化;用RT-PCR和Western blotting法检测大鼠海马内微管蛋白轻链3(LC3)、Beclin-1、Bax、Bcl-2及Caspase-3 mRNA和蛋白的表达水平。结果与假手术组比较,CA/CPR模型组大鼠NDS评分明显降低;海马神经元TUNEL染色阳性细胞数明显增多,凋亡率显著升高;海马内LC3、Beclin-1、Caspase-3、Bax表达上调,Bcl-2表达下调(P<0.05,P<0.01)。与模型组比较,CA/CPR+Rapa大鼠NDS评分明显降低,而海马神经元凋亡率明显有所增加,海马内LC3、Beclin-1、Caspase-3、Bax表达明显上调,而Bcl-2表达则明显有所下降;CA/CPR+3-MA大鼠NDS评分明显升高,而海马神经元凋亡率下降,海马内LC3、Beclin-1、Caspase-3、Bax表达明显下调,而Bcl-2表达则有所升高(P<0.05,P<0.01)。结论 CA/CPR后自噬水平升高促进海马神经元凋亡,自噬水平降低抑制海马神经凋亡,两者相互作用共同参与CA/CPR的病理过程。     

Stimulation of autophagy in the liver by lipopolysaccharide-induced systemic inflammation in a rat model of diabetes mellitus

The dysregulated metabolism associated with diabetes mellitus (DM) impairs membrane trafficking events in the liver, including the process of autophagy, which is an essential ongoing cellular process that is highly regulated by nutrients, endocrine factors, and signaling pathways. High-mobility group box 1 (HMGB1) is a nuclear protein with a known role in systemic inflammation and the related various organ injuries. However, its relationship to autophagy is not well understood. The aim of this study was to investigate the effects of inflammation injury on autophagy in the liver in a rat model of DM. DM was induced in animals with streptozotocin, followed four weeks later by induction of inflammation by LPS injection. At 12 h after LPS administration, autophagy was assessed by immunohistochemistry and Western blot analysis of microtubule-associated protein light chain 3 (LC3)-II, as well as transmission electron microscopy. Expression of HMGB1 was also examined by immunohistochemistry and Western blot analysis. Western blot analysis of liver tissue revealed that levels of LC3-II and HMGB1 protein increased in DM rats subjected to LPS-induced inflammation compared with non-DM rats. Autophagy was particularly enhanced in DM rats. Thus, autophagy might be related to progression to organ injury in patients with DM, and inflammation in these patients might be associated with over-induction of autophagy and increased HMGB1 expression.     

电针联合跑轮训练对 MCAO 模型大鼠自噬、 神经细胞凋亡和神经发生的影响

目的 探究电针联合跑轮训练对大脑中动脉栓塞 (middle cerebral artery embolism, MCAO) 模型 大鼠自噬、 神经细胞凋亡和发生的影响。 方法 30 只 SD 大鼠随机分为 MCAO 模型组、 联合干预组和假手术 组, 每组10 只。 测量神经系统评分和脑梗塞体积。 检测自噬效应蛋白 (Beclin1) 和膜型微管相关蛋白 1A/ 1B-轻 链3 (LC3) (LC3B-Ⅱ) / 胞浆型微管相关蛋白1A/ 1B-轻链3 (LC3) (LC3B-Ⅰ) 表达以及凋亡相关蛋白 B 淋巴细胞 瘤-2 基因 (bcl-2)、 BCL2 关联 X 蛋白 (BAX) 和半胱氨酸蛋白酶-3 (Caspase-3) 表达。 检测神经细胞凋亡和超 氧化物歧化酶 (SOD) 和过氧化氢酶 (CAT) 的活性以及丙二醛 (MDA)、 p-AMPK 和 p-mTOR 的表达。 结果 MCAO 模型组 Tunel 阳性细胞、 神经系统评分、 脑梗塞体积、 BAX、 Caspase-3、 Beclin-1、 LC3B-Ⅱ/ LC3B-I、 p-AMPK 表达、 MDA 含量增加, SOD 和 CAT 活性、 p-mTOR、 Bcl-2 表达降低 (P<0. 05)。 联合干预组 Tunel 阳性细 胞、 神经系统评分、 脑梗塞体积、 BAX、 Caspase-3、 Beclin-1 和 LC3B-Ⅱ/ LC3B-I、 p-AMPK 表达、 MDA 含量降低, SOD 和 CAT 活性、 Bcl-2、 p-mTOR 表达增加 (P<0. 05)。 结论 联合干预抑制氧化应激、 自噬相关蛋白 Beclin-1 和 LC3B-Ⅱ/ LC3B-I 以及 BAX 和 Caspase-3 表达并促进 Bcl-2 表达。     

饥饿诱导增生性瘢痕成纤维细胞自噬发生

 ［摘要］目的:探讨饥饿诱导的增生性瘢痕成纤维细胞自噬的发生。方法:原代培养增生性瘢痕成纤维细胞至对数生长期,用EBSS（Earle’s balanced salt solution）代替培养液,分别饥饿0 h、1 h、2 h、3 h,应用Western blotting和定量PCR(qRT-PCR)方法分别检测成纤维细胞微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3, LC-3）和自噬相关蛋白Beclin-1的蛋白和mRNA表达,单丹磺酰戊二胺(monodansylcadaverine,MDC)染色和透射电镜观察自噬体。结果:Western blotting和qRT-PCR 检测LC3和Beclin-1 蛋白和mRNA表达在饥饿1 h开始增加,2 h达到高峰,3 h逐渐下降,均明显大于对照组（P<0.05）。荧光显微镜和电镜下观察到饥饿2 h诱导的成纤维细胞出现自噬囊泡。结论: 饥饿可诱导增生性瘢痕成纤维细胞发生自噬,可能与增生性瘢痕的形成有关。     

Bone marrow mesenchymal stem cell-derived exosomes inhibit chondrocyte apoptosis and the expression of MMPs by regulating Drp1-mediated mitophagy

Osteoarthritis (OA) is a joint degenerative disease commonly seen in the elderly. Bone marrow mesenchymal stem cell-exosomes (BMSC-exosomes) are closely associated with the progression of OA. Here, we investigated whether BMSC-exosomes can affect OA development by regulating mitophagy. Primary rat chondrocytes were treated with advanced glycation end products (AGEs) to induce cell damage. The results of flow cytometry showed that AGEs treatment significantly promoted apoptosis of chondrocytes. AGEs treatment also enhanced the expression of matrix metalloproteinases (MMPs), MMP-3 and MMP-13, and dynamin-related protein 1 (Drp1) in chondrocytes. To investigate the impact of BMSC-exosomes on chondrocytes, chondrocytes were treated with BMSC-exosomes. AGEs-mediated increase of apoptosis and up-regulation of MMP-3, MMP-13, and Drp1 in chondrocytes were abrogated by BMSC-exosomes. Western blot analysis of autophagy-related proteins and Mito-Keima assay revealed that BMSC-exosome treatment elevated the expression of autophagy-related proteins, LC3-II/LC3-I and Beclin-1, and promoted mitophagy in the AGEs-treated chondrocytes. Moreover, Drp1 overexpression repressed the expression of LC3-II/LC3-I and Beclin-1, and enhanced apoptosis and the expression of MMP-3 and MMP-13 in AGEs-treated chondrocytes. BMSC-exosomes reversed the impact of Drp1 overexpression on AGEs-treated chondrocytes. In conclusion, this work demonstrates that BMSC-exosomes inhibit chondrocyte apoptosis and the expression of MMPs, which attributes to regulate Drp1-mediated mitophagy. Thus, BMSC-exosomes may be a potential treatment for OA.     

Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of B. abortus and also contributes to the establishment of bacterial infection in mice. The internalization of B. abortus was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL). Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of B. abortus. Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of B. abortus in macrophages. The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted B. abortus infection. NPC1-deficient mice were resistant to the bacterial infection. Molecules associated with cholesterol-rich microdomains, "lipid rafts," accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of B. abortus. Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of B. abortus infection.     

The development of an immunohistochemical method to detect the autophagy-associated protein LC3-II in human tumor xenografts

Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.     

ERK1/2和PI3-K通路在蛛网膜下腔出血大鼠海马区对神经细胞自噬的调控作用

目的:探讨细胞外信号调节激酶(extracellular regulated protein kinases,ERK1/2)和磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3-K)通路对大鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后神经细胞自噬的调控作用。方法:雄性SD大鼠160只,分为假手术(Sham)组、模型(SAH)组、ERK1/2抑制剂U0126组和PI3-K抑制剂LY294002组。采用二次注血法制作SAH大鼠模型,HE染色观察海马区神经细胞的形态变化;免疫组织化学染色法检测海马区磷酸化ERK1/2、PI3-K、自噬相关蛋白Beclin-1和微管相关蛋白1轻链(LC3)-II的表达实时荧光定量PCR检测海马区ERK1/2、PI3-K、自噬相关蛋白Beclin-1和微管相关蛋白LC3的表达。结果:SAH组海马区神经细胞存活率低于Sham组,ERK1/2、PI3-K、Beclin-1和LC3的表达水平高于Sham组(P0.05);U0126组和LY294002组海马区神经细胞存活率均高于SAH组,ERK1/2、PI3-K、Beclin-1和LC3表达均低于SAH组(P0.05);U0126组和LY294002组两组间海马区神经细胞存活率差异无统计学意义(P0.05),U0126组ERK1/2、Beclin-1和LC3表达水平低于LY294002组(P0.05),PI3-K表达水平高于LY294002组(P0.05)。结论:ERK1/2和PI3-K通路激活共同参与SAH后神经细胞自噬的调节,且以PI3-K通路更为主要。     

Development of a Rab9 Transgenic Mouse and Its Ability to Increase the Lifespan of a Murine Model of Niemann-Pick Type C Disease

Niemann-Pick, type C (NP-C) disease is an autosomal recessive neurovisceral storage disorder in which cholesterol and sphingolipids accumulate. There is no specific treatment for this disease, which is characterized by progressive neurological deterioration, sometimes accompanied by hepatosplenomegaly. We and others have shown that overexpression of certain Rab GTPases corrects defective membrane trafficking and reduces lipid storage in cultured NP-C fibroblasts. Here, we tested the possibility that Rab protein overexpression might also have beneficial effects in vivo using a murine model of NP-C. We first generated several lines of transgenic mice that ubiquitously overexpress Rab9 up to ~30-fold more than endogenous levels and found that the transgene expression had no obvious effects on fertility, behavior, or lifespan in normal mice. These transgenic strains were then crossed with NP-C mutant mice to produce NP-C homozygous recessive mice with and without the Rab9 transgene. Life expectancy of the NPC1 homozygous recessive animals was extended up to 22% depending on gender and the transgenic strain that was used. Histological studies and lipid analysis of brain sections indicated that the NP-C mice carrying the Rab9 transgene had dramatically reduced storage of GM₂ and GM₃ gangliosides relative to NP-C animals lacking the transgene. These results demonstrate that Rab9 overexpression has the potential to reduce stored lipids and prolong lifespan in vivo.     

脂多糖通过PI3K/Akt/mTOR通路调控巨噬细胞自噬*

 目的： 观察脂多糖对巨噬细胞自噬活化的影响及相关信号通路的探讨。方法: 体外培养巨噬细胞株RAW264.7,分为对照组、饥饿状态激活自噬组、单纯脂多糖（LPS）刺激组、LPS+PI3K抑制剂（hVps34）组和LPS+mTOR抑制剂（雷帕霉素）组。构建荧光真核表达载体pcDNA3.1-GFP-LC3,转染巨噬细胞,通过荧光显微镜观察各组细胞中自噬体形成情况。qRT-PCR方法检测各组中与细胞自噬相关的Atg5、Atg7、LC3-II和Bnip3 mRNA表达水平的改变。利用Western blotting检测LC3-II、p-Akt和p-mTOR蛋白在各组中的表达情况,以评价LPS激活巨噬细胞自噬的分子通路。结果: 成功构建稳定表达GFP-LC3的巨噬细胞,在荧光显微镜下可以观察到自噬在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组均有明显增强;qRT-PCR检测到Atg5、LC3-II和Bnip3 mRNA的表达在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组均有明显增强,而在LPS组中略微下降;Western blotting 检测发现p-Akt在饥饿状态组、LPS组和LPS+雷帕霉素组中表达明显升高;p-mTOR在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组表达明显下降;LC3-II的表达在饥饿状态组、LPS+hVps34组和LPS+雷帕霉素组中表达要高于对照组和LPS组。结论: LPS参与巨噬细胞自噬的调控,其可能的信号通路为PI3K/Akt/mTOR通路,但仍存在其它有效的调控通路。