突触囊泡循环障碍与帕金森病

帕金森病是人类第二大神经退行性疾病,虽然通过对帕金森病遗传家族的研究已发现不少与之相关的致病基因,然而其病因尚不清楚。在已发现的与帕金森病相关基因中,α-synuclein、LRRK2、Parkin、PINK1以及DJ-1也在调节神经元突触前囊泡的神经递质释放以及突触前囊泡循环过程中发挥重要作用。最近的一些研究表明突触前囊泡循环障碍在帕金森病发病过程中扮演重要角色。本文对上述基因在突触囊泡循环以及其突变导致帕金森病病程中的作用作一概述,并推测囊泡循环障碍在帕金森病发病过程中的作用机理,最后指出目前在该研究领域需要解决的一些问题。     

神经元突触囊泡循环的分子机理

神经末梢突触囊泡循环包括锚靠、出胞、入胞及囊泡再生等步骤,由囊泡、轴浆及突触前膜的多种蛋白质的级联反应介导,其关键步骤的分子模型的确立,为进一步了解神经系统高级活动奠定了基础。     

植物蛋白磷酸化的检测方法

蛋白磷酸化是一种重要的蛋白质翻译后修饰方式,几乎参与植物所有生命过程的调节。蛋白磷酸化过程主要指在蛋白激酶的催化作用下,将三磷酸腺苷(ATP)上的γ位磷酸基团转移到底物蛋白特定氨基酸残基上的过程。底物蛋白上被磷酸化的常见氨基酸有丝氨酸、苏氨酸及酪氨酸,磷酸基团与氨基酸中的羟基通过酯键连接。该文详细描述了几种常用的蛋白质体外及体内磷酸化的检测方法及注意事项。     

神经末梢突触囊泡释放神经递质过程的调控蛋白

神经末梢突触囊泡释放神经递质是一个复杂且受到精细调控的过程,涉及多种蛋白质间的相互作用。位于突触囊泡膜上的突触囊泡蛋白／突触囊泡相关膜蛋白（synaptobrevin/VAMP),与位于突触前膜上的syntaxin和突触小体相关蛋白SNAP－25,三者聚合形成的可溶性N－甲基马来酰胺敏感因子（NSF）附着蛋白受体（SNARE）核心复合物是突触囊泡胞吐过程中的核心成分。本文主要围绕参与空触囊泡胞吐过程,以及调节SNARE核心复合物的形成,解离及其功能的蛋白质,并对突触囊泡胞吐过程的分子模型作一概述。     

组蛋白磷酸化修饰与精子发生

组蛋白磷酸化是组蛋白氨基酸残基的磷酸化修饰,是一类重要的翻译后修饰,与有丝分裂和减数分裂的染色质压缩、染色质功能调节、转录的激活与抑制、DNA损伤修复以及物质代谢等多种机制相关。文章对国内外近10年多种代表性生物精子发生(孢子形成)的相关文献进行总结,论述了组蛋白磷酸化在精子发生中调控蛋白质作用因子的结合位点、调控减数分裂过程中的DNA复制与重组、保障正确的染色质重塑、对减数分裂后的成熟精子核的完全包装等重要功能。这些发现加深了人们对于组蛋白及其翻译后修饰在精子发生及分化中作用的理解。     

循环光合磷酸化和光合作用

光合作用被称为"地球上最重要的化学反应",其二氧化碳同化是由还原辅酶II(NADPH)和腺三磷(ATP)来推动的。ATP主要来源于非循环光合磷酸化和循环光合磷酸化,但以往研究集中在前者。21世纪以来,随着测定技术的发展和多条与循环光合磷酸化有关的电子传递途径的发现,循环光合磷酸化的重要性和功能引起了极大地关注。该文作者结合自己实验室的相关的研究,围绕循环光合磷酸化的发现和重要性、同化力两个组分的比例与促进光合磷酸化提高光合作用的途径进行探讨,为进一步深入研究提供参考。     

循环光合磷酸化

文章在回顾循环光合磷酸化和循环电子传递链发现的基础上,分析了循环光合磷酸化在光合作用中的地位,并对影响循环光合磷酸化的内外因素及其调控作了述评,为进一步开展相关研究提供参考.     

败血症休克大鼠肝细胞质膜钙ATP酶磷酸化、去磷酸化的改变

本实验观察了早期、晚期败血症休克大鼠肝细胞质膜钙ＡＴＰ酶磷酸化、去磷酸化的改变。败血症休克模型由结扎大鼠盲肠并穿孔（ＣＬＰ）的方法复制,采用蔗糖密度梯度离心法分离肝细胞质膜,由聚丙烯酰胺凝胶电泳（ＳＯＳ－ＰＡＧＥ）鉴定肝细胞质膜钙ＡＴＰ酶磷酸化中间产物。结果发现,肝细胞膜钙ＡＴＰ酶磷酸化能力在败血症休克早期降低３２．３％,晚期降低４６．６％（Ｐ＜０．０１）。动力学分析发现,早期、晚期败血症休克时,钙离子使钙ＡＴＰ酶磷酸化的最大反应速度都明显降低,而Ｋｍ值无明显变化。ＳＤＳ－ＰＡＧＥ分析证实,肝细胞质膜钙ＡＴＰ酶磷酸化的中间产物是一个分子量为１０７ｋｕ的蛋白质。结论认为：败血症休克时,肝细胞质膜钙ＡＴＰ酶磷酸化能力明显减低,去磷酸化能力无明显变化,可能是败血症休克时肝纳胞钙稳态失衡,进而引起机体代谢改变的重要原因之一。     

The dystonia-associated protein torsinA modulates synaptic vesicle recycling

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.     

Visualizing recycling of synaptic vesicle proteins

The use of modern techniques (in particular, novel fluorescence markers of a few molecular participants of the exo-and endocytotic processes, including pH-sensitive agents, immuno-electron and laser-scanning microscopy) allows experimenters to visualize different stages of recycling of synaptic vesicle proteins. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 350–351, July–October, 2007.     

Mutations in synaptojanin disrupt synaptic vesicle recycling

Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in the budding of synaptic vesicles from the plasma membrane, in the uncoating of vesicles after fission, in the recovery of vesicles from endosomes, and in the tethering of vesicles to the cytoskeleton. Thus, these results confirm studies of the mouse synaptojanin 1 mutants, which exhibit defects in the uncoating of synaptic vesicles (Cremona, O., G. Di Paolo, M.R. Wenk, A. Luthi, W.T. Kim, K. Takei, L. Daniell, Y. Nemoto, S.B. Shears, R.A. Flavell, D.A. McCormick, and P. De Camilli. 1999. Cell. 99:179-188), and further demonstrate that synaptojanin facilitates multiple steps of synaptic vesicle recycling.     

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.     

The synaptic vesicle membrane: origin, axonal distribution, protein components, exocytosis and recycling

The paper discusses functional and molecular aspects of the synaptic vesicle membrane during its life cycle. The distribution of the synaptic vesicle membrane compartment in an entire cholinergic neuron is monitored using colloidal gold labelling and a monoclonal antibody against the synaptic vesicle membrane protein SV2. This provides new insights concerning vesicle origin and fate in the various compartments of the neuron. A new synaptic vesicle membrane protein (svp25) of Mr 25,000 with properties similar to synaptophysin as well as a synaptic vesicle binding phosphoprotein of the presynaptic membrane (Mr 92,000) likely to be involved in vesicle exocytosis are described. The membrane compartment recycled on induced transmitter release contains synaptic vesicle but not plasma membrane markers and encloses both newly synthesized transmitter and a sample of extracellular medium.     

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.     

Entering neurons: botulinum toxins and synaptic vesicle recycling

Botulinum toxins are metalloproteases that act inside nerve terminals and block neurotransmitter release through their cleavage of components of the exocytosis machinery. These toxins are used to treat human diseases that are characterized by hyperfunction of cholinergic terminals. Recently, evidence has accumulated that gangliosides and synaptic vesicle proteins cooperate to mediate toxin binding to the presynaptic terminal. The differential distribution of synaptic vesicle protein receptors, gangliosides and toxin substrates in distinct neuronal populations opens up the possibility of using different serotypes of botulinum toxins for the treatment of central nervous system diseases caused by altered activity of selected neuronal populations.     

The role of coated vesicles in recycling of synaptic vesicle membrane

The uptake of extracellular tracers into synaptic nerve terminals has been a phenomenon of persistent interest. Uptake is into synaptic vesicles, hence vesicles spend part of their life in continuity with the plasma membrane, as expected if exocytosis underlies the quantal discharge of neurotransmitters. However, exactly how or when synaptic vesicles acquire extracellular tracers has not been unambiguously determined. Two schools of thought have developed, one holding that vesicles acquire tracers directly via a reversible exo/endocytotic sequence in which they consistently maintain their biochemical identity during their transient continuity with the plasma membrane, the other holding that synaptic vesicles acquire tracers indirectly, via the formation of clathrin-coated vesicles which are spatially and temporally separate from exocytosis and reverse a temporary loss of the vesicles' individual identity upon merger with the plasma membrane. Efforts to distinguish between these two alternatives have generated an interesting diversity of electron microscopic experiments, many of which are reviewed here. However, definitive determination of which view is correct may ultimately require direct visualization of synaptic vesicle turnover in living nerve terminals. To this end, we here review the results of visualizing endocytosis in tissue cultured cells, where light microscopy can provide sufficient resolution to reveal membrane dynamics in living cells. This has allowed visual discrimination of two different types of endocytosis, one clathrin-mediated (coated vesicle formation) and the other actin-mediated (macropinocytosis). Current work is also reviewed which aims at determining experimental methods for inhibiting each type of endocytosis selectively. Hypertonicity and severe cytoplasmic acidification turn out to inhibit coated vesicle formation, while cytochalasin D and mild cytoplasmic acidification selectively inhibit macropinocytosis. Applied to nerves, these various treatments affect synaptic vesicle turnover in a manner that supports the notion that synaptic vesicle membrane recycles via the "indirect" route of coated vesicle formation.