A new frontier in pharmacology: the endoplasmic reticulum as a regulated export pathway in health and disease

The endoplasmic reticulum (ER), the first secretory compartment of eukaryotic cells, co-ordinates the biogenesis and export of all membrane-bound and soluble cargo molecules to the cell surface. ER function is now recognised to have unprecedented links with signalling pathways regulating cell growth and differentiation and host physiology. Misfolding and aggregation of newly synthesised proteins in the ER or alterations in ER processing of cargo mediated by pathogens is responsible for a broad range of diseases including cystic fibrosis, emphysema and neuropathies such as Alzheimer’s disease. The central, integrative role of the ER in determining cell physiology in health and disease represents an untapped area for pharmacological intervention. This review focuses on the potential use of pharmacological agents to modulate cargo selection, folding and degradation in the ER with the goal of alleviating ER export disease. In addition, implementation of novel technologies that utilise normal ER function to store and release biologically active substances of therapeutic relevance are presented as a new frontier in drug delivery.     

Pre‐ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress‐dependent autophagy via PERK and IRE1 signalings

Melatonin has demonstrated a potential protective effect in central nervous system. Thus, it is interesting to determine whether pre‐ischemia melatonin administration could protect against cerebral ischemia/reperfusion (IR)‐related injury and the underlying molecular mechanisms. In this study, we revealed that IR injury significantly activated endoplasmic reticulum (ER) stress and autophagy in a middle cerebral artery occlusion mouse model. Pre‐ischemia melatonin treatment was able to attenuate IR‐induced ER stress and autophagy. In addition, with tandem RFP‐GFP‐LC3 adeno‐associated virus, we demonstrated pre‐ischemic melatonin significantly alleviated IR‐induced autophagic flux. Furthermore, we showed that IR induced neuronal apoptosis through ER stress related signalings. Moreover, IR‐induced autophagy was significantly blocked by ER stress inhibitor (4‐PBA), as well as ER‐related signaling inhibitors (PERK inhibitor, GSK; IRE1 inhibitor, 3,5‐dibromosalicylaldehyde). Finally, we revealed that melatonin significantly alleviated cerebral infarction, brain edema, neuronal apoptosis, and neurological deficiency, which were remarkably abolished by tunicamycin (ER stress activator) and rapamycin (autophagy activator), respectively. In summary, our study provides strong evidence that pre‐ischemia melatonin administration significantly protects against cerebral IR injury through inhibiting ER stress‐dependent autophagy. Our findings shed light on the novel preventive and therapeutic strategy of daily administration of melatonin, especially among the population with high risk of cerebral ischemic stroke.     

Advance of autophagy in chronic kidney diseases

Autophagy, a highly conserved mechanism for cell survival, emerges as an important pathway in many biological processes and diseases conditions. Studies of cultured renal cells, human kidney tissues and experimental animal models implicate that autophagy regulation is the critical aspects in chronic kidney diseases (CKD). Here, we summarize the current studies on the role of autophagy in CKD. Unveiling the precise regulation mechanism of autophagy in CKD is essential for developing potential prevention, diagnostic and therapeutic targets of these sticky clinical challenges.     

Caspase-12在D-氨基半乳糖联合脂多糖诱导小鼠急性肝功能衰竭中的表达及作用

目的探讨Caspase-12在D-氨基半乳糖(D-Gal)/脂多糖(LPS)诱导小鼠急性肝功能衰竭发生发展过程中表达水平的变化及Caspase-12介导的内质网应激肝细胞凋亡途径在急性肝功能衰竭中的作用。方法以D-Gal/LPS联合腹腔注射诱导小鼠急性肝功能衰竭建立实验模型,在不同时间点动态检测血清氨基转移酶水平和观察肝组织病理变化,评估肝细胞凋亡和肝坏死演变过程;应用琼脂糖凝胶电泳检测肝细胞DNA凋亡条带;用半定量逆转录聚合酶链反应检测肝组织中Caspase-12 mRNA表达水平;west- ern blot检测Caspase-12、Bip/GRP78蛋白表达。结果药物诱导5h时肝组织中典型凋亡细胞增多,血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)开始升高,Caspase-12 mRNA表达明显增加, Caspase-12蛋白量表达反而减少;7h镜下出现大量肝细胞凋亡和坏死,ALT、AST水平达高峰,分别为(2564±1384)U/L和(1198±497)U/L,正常对照组为(59±17)U/L和(135±12)U/L,Caspase- 12 mRNA表达仍增加,Caspase-12蛋白表达量继续降低;9h 肝细胞坏死明显,伴散在凋亡细胞,ALT、AST水平明显下降,Caspase-12 mRNA表达较7 h下降。Bip/GRP78蛋自表达从5 h开始,至7 h逐渐增加。结论D-Gal/LPS诱导小鼠急性肝功能衰竭早期Caspase-12 mRNA表达水平逐渐升高,后期(7-9 h)降低,与肝细胞凋亡发生的时相一致;Caspase-12蛋白酶因内质网应激而被大量活化,提示Caspase-12介导的内质网应激肝细胞凋亡参与炎症性急性肝功能衰竭的发生发展,是急性肝功能衰竭中肝细胞损伤的重要机制之一,提示早期干预Caspase-12表达及活化对急性肝功能衰竭可能具有保护作用。     

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Autophagy is a conserved catabolic process characterized by the de novo generation of a double-membrane structure called an autophagosome with a fundamental function in the bulk turnover of cytoplasmic components, including proteins, RNAs, and organelles. Genetic studies in yeast have elucidated the molecular machinery of autophagy, whereby 42 autophagy-related (ATG) genes have been identified (1–3). These ATG genes are highly conserved among eukaryotes but often have multiple isoforms in other higher organisms, in particular in sessile plants. Albeit increasing understanding on the molecular function of Atg proteins in acting hierarchically on the phagophore assembly site (PAS) to produce autophagosomes, the origin of the autophagosomal membrane remains unclear in higher eukaryotes. Furthermore, the dedication of other membranes and machineries in the autophagy pathway remains under investigation.Plant autophagy is known to play important roles in the sessile lifestyle of plants, participating in seed germination, seedling establishment, plant development, hormone responses, lipid metabolism, and reproductive development (4). Plant autophagy research is advancing with findings not only on the counterparts of the yeast/mammalian Atg proteins but also dealing with some plant-unique factors functioning in different steps of autophagosome biogenesis, thereby uncovering novel mechanisms that might or might not be conserved in nonplant species (5). More interestingly, higher plants possess multiple protein isoforms of ATG machinery, whose functional heterogeneity in the autophagy pathway has only recently been unveiled (6).The coat protein complex II (COPII) machinery consists of five cytosolic components: the small GTPase Sar1, the inner coat protein dimer Sec23-Sec24, and the outer coat proteins Sec13-Sec31. These proteins are essential for COPII-coated vesicle formation, which buds from specialized regions of the ER, namely ER exit sites (ERESs) (7). Under nutrient-rich conditions, COPII vesicles mediate anterograde ER to Golgi transport. However, increasing evidence from yeast and mammals suggests that the COPII machinery or even COPII vesicles themselves may contribute to autophagosome formation when cells are starved for nutrients (8–16). Gene duplication events have occurred substantially in sessile plants during evolution, and the importance of distinct paralogs in environmental stress adaptation during plant development has been implied (17). Arabidopsis encodes multiple COPII paralogs in its genome, including five Sar1s, seven Sec23s, three Sec24s, two Sec13s, and two Sec31s (17). Increasing numbers of studies have pinpointed the functional diversity and importance of distinct COPII paralogs in ER protein export (18–23). Nonetheless, the mechanism by which COPII vesicles are redirected to the autophagy pathway upon nutrient starvation, and their roles in autophagosome biogenesis, remains unclear. Furthermore, the participation of specific COPII paralogs in autophagy regulation remains unknown in plants.Here, we report on a role of a specific Sar1 homolog, AtSar1d, that modulates plant autophagosome biogenesis in concert with AtRabD2a. Large-scale proteomic analysis of the ATG interactome has revealed possible mechanistic connections between the ATG machinery and specific COPII components in plants. Cellular and biochemical analyses have shown that the dominant negative (DN) mutant of AtSar1d (AtSar1dDN) specifically perturbs YFP-ATG8 vacuolar degradation upon autophagic induction. Consistently, a transfer DNA (T-DNA) insertion mutant of AtSar1d exhibited starvation-related phenotypes. Notably, AtSar1d regulates autophagosome progression through specific recognition of ATG8e by a previously uncharacterized noncanonical motif. We further identify a plant-unique Rab1/Ypt1 homolog AtRabD2a that colocalizes with AtSar1d and ATG8 upon starvation by transient expression in Arabidopsis protoplasts. A DN mutant of AtRabD2a (AtRabD2aNI) perturbs autophagy flux, while AtRabD2a is indispensable for bridging the AtSar1d-positive COPII vesicles with the ATG1 complex, thus contributing to autophagosome biogenesis in plants. Our study therefore unequivocally demonstrates that the plant-specific COPII machinery regulates autophagosome biogenesis and sheds light on the evolutionary importance of gene duplication events in the plant autophagy pathway.     

异丙肾上腺素对心肌细胞内钙释放和肌浆网内钙容量的影响

目的 :心肌细胞膜上的 β-肾上腺素受体的激活对兴奋 -收缩耦联 （ECC）过程有重要的调节作用。本课题利用异丙肾上腺素 （ISO,1μmol/ L）激活 β-肾上腺素受体从而研究其对源自心肌细胞肌浆网的胞内钙释放 （ECC的重要环节 ）和肌浆网内钙容量的影响 ,进而分析钙释放与钙容量之间的关系。方法 :局部场刺激作用于成年大鼠心肌细胞 ,促使后者产生动作电位 ,进而诱发胞内钙瞬变 （ACT） ,由 ACT可估测胞内钙释放。肌浆网内钙容量则由咖啡因 （2 0 m mol/ L）诱发的钙瞬变 （CCT）估测。实验结果均由 Zeiss L SM- 5 10激光共聚焦显微镜系统记录。结果 :ISO作用下的 ACT峰值为 10 .2 9± 0 .35 （n=13）比正常情况下的 5 .74± 0 .2 7（n=18）高 （P<0 .0 1）。 ISO作用下的CCT峰值为 11.2 3± 0 .2 9（n=13）比正常情况下的 7.6 2± 0 .2 4 （n=18）高 （P<0 .0 1）。结论 :ISO可明显地提高心肌细胞内钙释放量和肌浆网内的钙容量。不管有无 ISO存在 ,胞内钙释放量总是只占肌浆网内钙容量的一部分。在正常情况下 ,心肌的钙释放量有较大的储备能力 ,且此储备可因β-肾上腺素受体的激活而动员。     

裙带菜多糖保护异丙肾上腺素诱导的心脏纤维化研究

目的：探讨裙带菜多糖在异丙肾上腺素（ISO）诱导的心脏纤维化中的作用。方法将40只C57BL／6J小鼠随机分为对照组、ISO组、ISO＋裙带菜多糖组、裙带菜多糖组。ISO组连续皮下注射ISO 14 d（前3 d 10 mg·kg－1·d－1,后11 d 5 mg· kg－1·d－1）,ISO＋裙带菜多糖组除做上述ISO处理外,ISO处理前7 d开始给予裙带菜多糖200 mg·kg－1·d－1灌胃,持续到ISO皮下注射第14天;裙带菜多糖组连续21 d裙带菜多糖200 mg·kg－1·d－1灌胃;对照组以生理盐水代替ISO皮下注射。心脏超声检测各组小鼠心功能的改变,病理染色检测心脏纤维化程度,实时定量PCR检测转化生长因子-β（TGF-β）、Ⅰ型胶原α（CollagenⅠα）和Ⅲ型胶原（CollagenⅢ）的mRNA表达量的变化,Western Blot检测各组小鼠心脏自噬的改变。结果裙带菜多糖明显改善心功能,减少ISO诱导的心脏纤维化程度,心脏胶原蛋白CollagenⅠα和CollagenⅢ的mRNA表达量比ISO组显著降低（P＜0．05）,且裙带菜多糖能减少ISO诱导的心脏自噬。结论裙带菜能减轻ISO诱导的心脏纤维化,其主要是通过降低ISO诱导的心脏自噬而发挥作用的。     

Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation

Aim:

Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro.

Methods:

HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells.

Results:

Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 μg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells.

Conclusion:

The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.