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.     

卒中后痉挛与上运动神经元综合征

卒中后肢体痉挛是影响功能恢复的主要因素之一，一直是康复治疗的重点。目前对痉挛的认识不够统一，在痉挛的发生机制、鉴别和治疗观念上也存在很多争议。文章对卒中后痉挛的危害、发生机制、鉴别诊断和治疗进展，以及与上运动神经元综合征之间的关系进行了综述。     

钾离子通道相互作用蛋白1与γ-氨基丁酸能神经元在戊四唑致癫痫大鼠海马部位的改变

目的研究钾离子通道相互作用蛋白1（KChIP1）在癫痫发生过程中的变化及其与γ-氨基丁酸（GABA）能神经元的关系。方法成年大鼠制作急性戊四唑癫痫模型,免疫组织化学法结合激光扫描共聚焦显微镜技术观察大鼠脑中KChIP1免疫阳性神经元与GABA能阳性神经元在海马部位的表达及改变。结果急性戊四唑癫痫大鼠海马部KChIP1阳性神经元数较对照组明显增加（P〈0.05）,GABA能阳性神经元、KChIP1/GABA双标阳性神经元数与对照组无显著性差异（P〉0.05）;KChIP1和GABA能阳性神经元在海马部位的共存率约为63.9%。结论KChIP1在癫痫发生中可能起重要作用;KChIP1与GABA能神经元虽有密切的共存关系,但在功能上并不完全一致。     

小鼠脊髓损伤组织高表达的microRNA-21对neuro-2a细胞PDCD4的影响

目的验证小鼠损伤脊髓中高表达的microRNA-21(miR-21)在neuro-2a细胞中对程序性细胞死亡因子4(PDCD4)的作用,初步探讨miR-21在脊髓损伤过程中的作用。方法将neuro-2a细胞在体外培养16h,分别按相应体系转染miR-21 mimics,继续培养48h,提取6孔板内细胞总蛋白和总RNA行蛋白印记和实时定量PCR检测,利用HEK293细胞行荧光素酶报告基因检测。结果 neuro-2a细胞转染miR-21 mimics后,其内源性程序性细胞死亡因子4(PDCD4)的表达在蛋白和mRNA水平均明显降低。双荧光素酶报告基因检测结果显示,miR-21对PDCD4 3'非翻译区(3'-UTR)的预测位点有直接抑制作用,而将此位点突变后,miR-21对PDCD4 3'-UTR预测位点的抑制作用消失。结论 miR-21通过直接作用于PDCD4 3'-UTR靶位点从而在mRNA和蛋白水平抑制PDCD4的表达。miR-21可能在脊髓损伤过程中具有重要的保护作用。     

动作观察疗法对亚急性期脑卒中患者上肢运动功能的影响

目的:探讨动作观察疗法对亚急性期脑卒中患者上肢运动功能的影响。方法:将31例脑卒中患者按随机数字表法分为观察组(16例)和对照组(15例)。对照组采用常规康复治疗,观察组在对照组基础上辅以动作观察疗法,每周6次,每次20min,共治疗4周。分别于治疗前、治疗4周后对两组患者采用上肢Fugl-Meyer运动功能评分法(FMA)并使用量角器对肩关节前屈、肘关节伸展和腕背伸主动活动度进行测量,以评定患者的上肢运动功能;并分别检测治疗前后FMA与肩关节前屈、肘关节伸展和腕背伸主动活动度之间的相关性。结果:治疗前,两组患者的FMA评分及肩关节前屈、肘关节伸展和腕背伸主动活动度评定差异无显著性意义(P0.05);治疗4周后,两组患者上述指标较治疗前均有所改善(P0.05),且与对照组相比,观察组的FMA评分(44.81±8.86)、肩关节前屈(150.88°±21.32°)、肘关节伸展(135.56°±17.22°)主动活动度的改善程度显著(P0.05);两组患者,除治疗后的对照组FMA评分与肩关节主动前屈活动度、FMA评分与肘关节主动伸展活动度不相关(P0.05)外,其余FMA评分均与肩关节主动前屈活动度、肘关节主动伸展活动度及腕主动背伸活动度之间存在正相关性(P0.05)。结论:基于镜像神经元理论的动作观察疗法可改善亚急性期脑卒中患者的上肢运动功能,且FMA与肩关节前屈、肘关节伸展和腕背伸主动活动度存在相关性。     

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP‐ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β‐DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve. J. Comp. Neurol. 523:479–494, 2015. © 2014 Wiley Periodicals, Inc.     

Memories as bifurcations: Realization by collective dynamics of spiking neurons under stochastic inputs

How the neural system proceeds from sensory stimuli to generate appropriate behaviors is a basic question that has not yet been fully answered. In contrast to the conventional viewpoint, in which the external stimulus dominantly drives the response behavior, recent studies have revealed that not only external stimuli, but also intrinsic neural dynamics, contribute to the generation of response behavior. In particular, spontaneous activity, which is neural activity without extensive external stimuli, has been found to exhibit similar patterns to those evoked by external inputs, from time to time. In order to further understand the role of this spontaneous activity on the response, we propose a viewpoint, memories-as-bifurcations, that differs from the traditional memories-as-attractors viewpoint. According to this viewpoint, memory is recalled when spontaneous neural activity is changed to an appropriate output activity upon the application of an input. After reviewing the previous rate-coding model embodying this viewpoint, we employ a model of a spiking neuron network that can embed input/output associations, and study the dynamics of collective neural activity. The organized neural activity, which matched the target pattern, is shown to be generated even under application of stochastic input, while the spontaneous activity, which apparently shows noisy dynamics, is found to exhibit selectively higher similarity with evoked activities corresponding to embedded target patterns. These results suggest that such an intrinsic structure in the spontaneous activity might play a role in generating the higher response. The relevance of these results to biological neural processing is also discussed.