The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-channels

The presynaptic proteome controls neurotransmitter release and the short and long term structural and functional dynamics of the nerve terminal. Using a monoclonal antibody against synaptic vesicle protein 2 we immunopurified a presynaptic compartment containing the active zone with synaptic vesicles docked to the presynaptic plasma membrane as well as elements of the presynaptic cytomatrix. Individual protein bands separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were subjected to nanoscale-liquid chromatography electrospray ionization-tandem mass spectrometry. Combining this method with 2-dimensional benzyldimethyl- n -hexadecylammonium chloride/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight and immunodetection we identified 240 proteins comprising synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the pre-synapse. Four maxi-channels, three isoforms of voltage-dependent anion channels and the tweety homolog 1 were co-isolated with the docked synaptic vesicles. As revealed by in situ hybridization, tweety homolog 1 reveals a distinct expression pattern in the rodent brain. Our results add novel information to the proteome of the presynaptic active zone and suggest that in particular proteins potentially involved in the short and long term structural modulation of the mature presynaptic compartment deserve further detailed analysis.     

Oligomerized liprin-α promotes phase separation of ELKS for compartmentalization of presynaptic active zone proteins

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Rebuilding essential active zone functions within a synapse

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Molecular organization of the presynaptic active zone

The exocytosis of neurotransmitter-filled synaptic vesicles is under tight temporal and spatial control in presynaptic nerve terminals. The fusion of synaptic vesicles is restricted to a specialized area of the presynaptic plasma membrane: the active zone. The protein network that constitutes the cytomatrix at the active zone (CAZ) is involved in the organization of docking and priming of synaptic vesicles and in mediating use-dependent changes in release during short-term and long-term synaptic plasticity. To date, five protein families whose members are highly enriched at active zones (Munc13s, RIMs, ELKS proteins, Piccolo and Bassoon, and the liprins-α), have been characterized. These multidomain proteins are instrumental for the diverse functions performed by the presynaptic active zone.In our laboratories, work on the molecular organization of the active zone is supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Fellowship, SFB645/A4 to S.S., SFB426/A1 to E.D.G.), the European Commission (SynScaff Consortium), the Land Sachsen-Anhalt (LSA-N2), the Fonds der Chemischen Industrie, and a Max Planck Research Award by the Max Planck Society, the Alexander von Humboldt Society, and local funding (BONFOR to S.S.).     

Transient active zone remodeling in the Drosophila mushroom body supports memory

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Amyloid precursor protein expression in the rat hippocampal dentate gyrus modulates during memory consolidation

Despite advances in our understanding of the basic biology of amyloid precursor protein (APP), the normal physiological function(s) of APP in learning and memory remains unclear. Here we show increased APP degradation in the hippocampus to be associated with the consolidation of a passive avoidance response. Neurone-specific APP695 expression became transiently reduced 2-4 h post-training through association with endosomal adaptin proteins and enhanced internalization. By contrast, internalization of glial-associated APP containing a Kunitz protease inhibitor-like domain (APP-KPI) was dependent on the low-density lipoprotein receptor-related protein (LRP). In addition, LRP expression and association with apolipoprotein E increased in the 2-4 h post-training period. The LRP antagonist receptor-associated protein prevented the APP-KPI internalization and LRP-apolipoprotein E association and this resulted in amnesia. Degradation of APP695 and APP-KPI did not appear to be related to alpha-secretase activity, as no learning-associated increase of secreted APP was observed in the CSF. Moreover, as internalization of APP isoforms was observed only in dentate gyrus, it probably relates to the learning-associated restructuring of the perforant path terminals. Memory-associated APP processing in both neuronal and glial compartments points to a role for glial unsheathing of synaptic connections, an event required for the synaptic restructuring that accompanies memory consolidation. These observations may have a direct relevance to understanding the pathophysiology of Alzheimer's disease as beta/gamma-secretase-derived beta-amyloid is formed following internalization of cell surface APP into the endosomal compartment.     

Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface

Alzheimer's disease is characterized by brain deposition of extracellular amyloid beta-peptide (Abeta)-containing plaques. The cellular site of gamma-secretase activity, which releases Abeta and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for gamma-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by gamma-secretase, and AICD-GFP is released into the cytosol. Inhibiting gamma-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that gamma-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another gamma-secretase substrate, NotchDeltaE. Our results suggest that intracellular domains are generated by gamma-secretase at the plasma membrane and/or early endosomes.     

microRNA 对淀粉样前体蛋白调控的研究进展

淀粉样前体蛋白(APP)参与了神经肌肉的信号传导、突触的可塑性及空间学习等生理过程,APP在阿兹海默病(AD)人脑组织中高表达,其切割产物β淀粉样蛋白(Aβ)则在AD的发生发展中起到重要作用。2011年4月,美国阿兹海默病协会将Aβ的聚集程度列入了新版AD诊疗指南中,通过减少APP的表达或降低其以β切割方式进行代谢来延缓AD的进展已成为很多学者的共识。microRNA(miRNA)是一类内生的、长度约19-24个核苷酸的小RNA,其在细胞内具有多种重要的调节作用,据推测,miRNA调控着人类约三分之一的基因。自2008年首次明确miRNA对APP表达存在调控作用之后,miRNA对APP的调控和相关机制的研究以及其对AD诊断和治疗潜在价值的探索已成为AD研究领域的热点之一,本文主要就miRNA对APP的表达、剪切和切割的调控及Aβ对miRNA的影响做一综述。     

Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor-Like Protein 2

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.     

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of γ-secretase are discussed.     

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a γ-secretase inhibitor, N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes α- and γ-secretase cleavage and subsequently induces CHOP-mediated cell death.     

Assembling the presynaptic active zone: a characterization of an active one precursor vesicle

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.     

Circadian expression of the presynaptic active zone protein bruchpilot in the lamina of Drosophila melanogaster

In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild‐type Canton‐S flies in constant darkness (DD), nor in norpA⁷ phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per⁰ arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton‐S flies in DD, as well as in the norpA⁷ mutant in LD, but is absent in per⁰¹, tim,⁰¹ and cry⁰¹ mutants in LD. In addition both peaks seem to depend on clock gene‐expressing photoreceptors and glial cells of the visual system. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Role of synaptic proteins in neurotransmitter release-related vesicular trafficking

As is known, regulated exocytosis of synaptic vesicles constitutes a primary means of communication between neurons, and it is subjected to substantial alterations in a number of brain pathologies. Recent investigations showed that vesicular transport events in neuroendocrine cells and presynaptic terminals are realized by a family of specialized membrane proteins of the vesicle (v-SNAREs) and another family located in the target cytoplasmic membrane (t-SNAREs). A variety of such proteins has already been described in different preparations; however, their precise localization and role in vesicular trafficking during functional changes in the cells remain ambiguous. In addition, new synaptic proteins appear to be involved in the vesicular cycle; the functions of these proteins remain unclear. The role of synaptic proteins in the course of cell excitation, in particular functions of core SNARE synaptic proteins (vesicular synaptobrevin/VAMPs and plasma membrane syntaxins/SNAP-25), as well as those of novel presynaptic proteins (Munc-13, Munc-18, CAPS proteins, and others), are discussed in this review. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 155–159, March–April, 2008.     

Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.     

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.     

Chemotactically active proteins of neutrophils

Polymorphonuclear leukocytes (neutrophils) are the first cells that arrive at sites of infection or injury. There, besides their microorganism-targeted effector functions, activated neutrophils secrete numerous chemoattractants that recruit other leukocyte subtypes into the inflamed tissue. First, neutrophil activation leads to the upregulation of the gene expression of several classical chemokines of the CXC and CC families. Second, neutrophil granules contain preformed intracellular storage pools of chemotactically active proteins that are rapidly released upon neutrophil degranulation. The third pathway of generation of chemotactically active proteins by activated neutrophils--shedding and concomitant proteolytic processing of a membrane protein--has recently been demonstrated in our laboratory. In this review, we summarize the essential features of chemoattractant production by neutrophils and their contribution to orchestrating the recruitment of leukocyte subtypes during inflammatory response.     

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13-1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13-1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.