Aß42 oligomers impair the bioenergetic activity in hippocampal synaptosomes derived from APP-KO mice.

Employing hippocampal synaptosomes from amyloid precursor protein (APP)-deleted mice we analyzed the immediate effects of amyloid beta peptide 42 (Aß42) peptide in its oligomeric or fibrillar assembly or of soluble amyloid precursor protein alpha (sAPPα) protein on their bioenergetic activity. Upon administration of oligomeric Aß42 peptide for 30 min we observed a robust decrease both in mitochondrial activity and in mitochondrial membrane potential (MMP). In contrast the respective fibrillary or scrambled peptides showed no effect, indicating that inhibition strictly depends on the oligomerization status of the peptide. Hippocampal synaptosomes from old APP-KO mice revealed a further reduction of their already impaired bioenergetic activity upon incubation with 10 µm Aß42 peptide. In addition we evaluated the influence of the sAPPα protein on mitochondrial activity of hippocampal synaptosomes derived from young or old APP-KO animals. In neither case 20 nm nor 200 nm sAPPα protein had an effect on mitochondrial metabolic activity. Our findings demonstrate that hippocampal synaptosomes derived from APP-KO mice are a most suitable model system to evaluate the impact of Aß42 peptide on its bioenergetic activity and to further elucidate the molecular mechanisms underlying the impairments by oligomeric Aß42 on mitochondrial function. Our data demonstrate that extracellular Aß42 peptide is taken up into synaptosomes where it immediately attenuates mitochondrial activity.

Egg-laying behavior and morphological and chemical characterization of egg surface and egg attachment glue of the digger wasp Ampulex compressa (Hymenoptera, Ampulicidae).

For providing their offspring females of the digger wasp species Ampulex compressa hunt cockroaches, paralyze them and attach as a rule one egg to the coxa of one of the mid legs of their prey. We observed the egg-laying behavior and examined with light- and scanning microscopy (i) nearly mature eggs from ovaries of freshly dissected females and (ii) eggs immediately after their deposition on the coxae of their prey. The length of the white bean-shaped eggs varied between 2.2 and 3.0 mm, their diameter between 0.66 and 0.72 mm, and their weight between 345 and 832 µg. The surface of fresh, untreated eggs shows even at higher magnifications (>20.000×) a smooth appearance. However, after conventional fixation, dehydration with ethyl-alcohol and critical-point drying the egg-surface exhibited a little bit texture. The eggs are at two-third of their underside glued to the coxa of the prey. With the naked eye the glue appears as a compact mass. The eggs may be mechanically removed from the substrate (their attachment site); however, in doing so the viscous attachment glue appears in a more fibrous consistence. The polypeptide composition washed off the egg surface and the glue revealed no similarities, whereas the molecular mass of two polypeptides were similar between glue and the Dufour's gland contents.

APP-A Novel Player within the Presynaptic Active Zone Proteome.

The amyloid precursor protein (APP) was discovered in the 1980s as the precursor protein of the amyloid A4 peptide. The amyloid A4 peptide, also known as A-beta (Aß), is the main constituent of senile plaques implicated in Alzheimer's disease (AD). In association with the amyloid deposits, increasing impairments in learning and memory as well as the degeneration of neurons especially in the hippocampus formation are hallmarks of the pathogenesis of AD. Within the last decades much effort has been expended into understanding the pathogenesis of AD. However, little is known about the physiological role of APP within the central nervous system (CNS). Allocating APP to the proteome of the highly dynamic presynaptic active zone (PAZ) identified APP as a novel player within this neuronal communication and signaling network. The analysis of the hippocampal PAZ proteome derived from APP-mutant mice demonstrates that APP is tightly embedded in the underlying protein network. Strikingly, APP deletion accounts for major dysregulation within the PAZ proteome network. Ca2+-homeostasis, neurotransmitter release and mitochondrial function are affected and resemble the outcome during the pathogenesis of AD. The observed changes in protein abundance that occur in the absence of APP as well as in AD suggest that APP is a structural and functional regulator within the hippocampal PAZ proteome. Within this review article, we intend to introduce APP as an important player within the hippocampal PAZ proteome and to outline the impact of APP deletion on individual PAZ proteome subcommunities.

APP Deletion Accounts for Age-Dependent Changes in the Bioenergetic Metabolism and in Hyperphosphorylated CaMKII at Stimulated Hippocampal Presynaptic Active Zones.

Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.

The synaptic vesicle protein SV31 assembles into a dimer and transports Zn2.

The integral synaptic vesicle protein SV31 has been shown to bind divalent cations. Here, we demonstrate that SV31 protein synthesized within a cell-free system binds Zn2+ and to a lower extent Ni2+ and Cu2+ ions. Expression with Zn2+ stabilized the protein and increased solubility. SV31 was preferentially monomeric in detergent and revealed specific binding of Zn2+ . When co-translationally inserted into defined nanodisc bilayers, SV31 assembled into dimeric complexes, resulting in increased binding of Zn2+ . Putative Zn2+ -binding motifs within SV31 comprise aspartic acid and histidine residues. Site-directed mutagenesis of two conserved aspartic acid residues leads to a potent decrease in Zn2+ binding but did not affect dimerization. Chemical modification of histidine residues abolished some of the Zn2+ -binding capacity. We demonstrate proton-dependent transport of Zn2+ as by accumulation of fluorescent FluoZin-1 inside of SV31-containing proteoliposomes. Transport activity has a Km value of 44.3 µM and required external Zn2+ and internal acidic pH. Our results demonstrate that the synaptic vesicle-integral protein SV31 functions as a proton-dependent Zn2+ transporter. SV31 may attribute specific and yet undiscovered functions to subsets of synapses.

APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone.

The hallmarks of Alzheimer's disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aß-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.

Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta, Hymenoptera, Sphecidae): structural and biochemical aspects.

The digger wasp species Ampulex compressa produces its venom in two branched gland tubules. They terminate in a short common duct, which is bifurcated at its proximal end. One leg is linked with the venom reservoir, the other one extends to the ductus venatus. Each venom gland tubule possesses, over its entire length, a cuticle-lined central duct. Around this duct densely packed class 3 gland units each composed of a secretory cell and a canal cell are arranged. The position of their nuclei was demonstrated by DAPI staining. The brush border of the secretory cells surrounds the coiled end-apparatus. Venom is stored in a bladder like reservoir, which is surrounded by a thin reticulated layer of muscle fibres. The reservoir as a whole is lined with class 3 gland units. The tubiform Dufour's gland has a length of about 350 µm (∅ 125 µm) only and is surrounded by a network of pronounced striated muscle fibres. The glandular epithelium is mono-layered belonging to the class 1 type of insect epidermal glands. The gland cells are characterized by conspicuous lipid vesicles. Secretion of material via the gland cuticle into the gland lumen is apparent. Analysis of the polypeptide composition demonstrated that the free gland tubules and the venom reservoir contain numerous proteins ranging from 3.4 to 200 kDa. The polypeptide composition of the Dufour's gland is completely different and contains no lectin-binding glycoproteins, whereas a dominant component of the venom droplets is a glycoprotein of about 80 kDa. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets revealed that all of the major proteinaceous constituents are secreted. The secreted venom contains exclusively proteins present in the soluble contents of the venom gland. The most abundant compound class in the Dufour's gland consisted of n-alkanes followed by monomethyl-branched alkanes and alkadienes. Heptacosane was the most abundant n-alkane. Furthermore, a single volatile compound, 2-methylpentan-3-one, was identified in various concentrations in the lipid extract of the Dufour's gland.

The Amyloid Precursor Protein-A Novel Player within the Molecular Array of Presynaptic Nanomachines.

More than 20 years ago the amyloid precursor protein (APP) was identified as the precursor protein of the Aß peptide, the main component of senile plaques in brains affected by Alzheimer's disease (AD). The pathophysiology of AD, characterized by a massive loss of synapses, cognitive decline, and behavioral changes was in principle attributed to the accumulation of Aß. Within the last decades, much effort has gone into understanding the molecular basis of the progression of AD. However, little is known about the actual physiological function of APPs. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone (PAZ) highlights APP as a hitherto unknown player within the setting of the presynapse. The molecular array of presynaptic nanomachines comprising the life cycle of synaptic vesicles, exo- and endocytosis, cytoskeletal rearrangements, and mitochondrial activity provides a balance between structural and functional maintenance and diversity. The generation of genetically designed mouse models further deciphered APP as an essential player in synapse formation and plasticity. Deletion of APP causes an age-dependent phenotype: while younger mice revealed almost no physiological impairments, this condition was changed in the elderly mice. Interestingly, the proteomic composition of neurotransmitter release sites already revealed substantial changes at young age. These changes point to a network that incorporates APP into a cluster of nanomachines. Currently, the underlying mechanism of how APP acts within these machines is still elusive. Within the scope of this review, we shall construct a network of APP interaction partners within the PAZ. Furthermore, we intend to outline how deletion of APP affects this network during space and time leading to impairments in learning and memory. These alterations may provide a molecular link to the pathogenesis of AD and the physiological function of APP in the central nervous system.

The synaptic proteome.

Synapses are focal hot spots for signal transduction and plasticity in the brain. A synapse comprises an axon terminus, the presynapse, the synaptic cleft containing extracellular matrix proteins as well as adhesion molecules, and the postsynaptic density as target structure for chemical signaling. The proteomes of the presynaptic and postsynaptic active zones control neurotransmitter release and perception. These tasks demand short- and long-term structural and functional dynamics of the synapse mediated by its proteinaceous inventory. This review addresses subcellular fractionation protocols and the related proteomic approaches to the various synaptic subcompartments with an emphasis on the presynaptic active zone (PAZ). Furthermore, it discusses major constituents of the PAZ including the amyloid precursor protein family members. Numerous proteins regulating the rearrangement of the cytoskeleton are indicative of the functional and structural dynamics of the pre- and postsynapse. The identification of protein candidates of the synapse provides the basis for further analyzing the interaction of synaptic proteins with their targets, and the effect of their deletion opens novel insights into the functional role of these proteins in neuronal communication. The knowledge of the molecular interactome is also a prerequisite for understanding numerous neurodegenerative diseases.

Regional Specializations of the PAZ Proteomes Derived from Mouse Hippocampus, Olfactory Bulb and Cerebellum.

Neurotransmitter release as well as structural and functional dynamics at the presynaptic active zone (PAZ) comprising synaptic vesicles attached to the presynaptic plasma membrane are mediated and controlled by its proteinaceous components. Here we describe a novel experimental design to immunopurify the native PAZ-complex from individual mouse brain regions such as olfactory bulb, hippocampus, and cerebellum with high purity that is essential for comparing their proteome composition. Interestingly, quantitative immunodetection demonstrates significant differences in the abundance of prominent calcium-dependent PAZ constituents. Furthermore, we characterized the proteomes of the immunoisolated PAZ derived from the three brain regions by mass spectrometry. The proteomes of the release sites from the respective regions exhibited remarkable differences in the abundance of a large variety of PAZ constituents involved in various functional aspects of the release sites such as calcium homeostasis, synaptic plasticity and neurogenesis. On the one hand, our data support an identical core architecture of the PAZ for all brain regions and, on the other hand, demonstrate that the proteinaceous composition of their presynaptic active zones vary, suggesting that changes in abundance of individual proteins strengthen the ability of the release sites to adapt to specific functional requirements.

Amyloid precursor protein knockout diminishes synaptic vesicle proteins at the presynaptic active zone in mouse brain.

The amyloid precursor protein (APP) has previously been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a presynaptic active zone-localized pool. By analyzing homozygous APP knockout mice we evaluated the impact of APP on synaptic vesicle protein abundance at synaptic release sites. Following immunopurification of synaptic vesicles and the attached presynaptic plasma membrane, individual proteins were subjected to quantitative Western blot analysis. We demonstrate that APP deletion in knockout animals reduces the abundance of the synaptic vesicle proteins synaptophysin, synaptotagmin-1, and SV2A at the presynaptic active zone. Conversely, deletion of the additional APP family members, APLP1 and APLP2 resulted in an increase in synaptophysin, synaptogamin-1, and SV2A abundance. When transmembrane APP is lacking in APPsα-KI/APLP2-KO mice synaptic vesicle protein abundance corresponds to that in APP -KO mice. Deletion of the synaptic vesicle protein 2 (SV2) A and B had no effect on APP and synaptophysin abundance but decreased synaptotagmin-1. Our data suggest that APP controls the abundance of synaptic vesicle proteins at the presynaptic release sites and thus impacts synaptic transmission.

The proteome of the presynaptic active zone from mouse brain.

Neurotransmitter release as well as the structural and functional dynamics of the presynaptic active zone is controlled by proteinaceous components. Here we describe for the first time an experimental approach for the isolation of the presynaptic active zone from individual mouse brains, a prerequisite for understanding the functional inventory of the presynaptic protein network and for the later analysis of changes occurring in mutant mice. Using a monoclonal antibody against the ubiquitous synaptic vesicle protein SV2 we immunopurified synaptic vesicles docked to the presynaptic plasma membrane. Enrichment studies by means of Western blot analysis and mass spectrometry identified 485 proteins belonging to an impressive variety of functional categories. Our data suggest that presynaptic active zones represent focal hot spots that are not only involved in the regulation of neurotransmitter release but also in multiple structural and functional alterations the adult nerve terminal undergoes during neural activity in adult CNS. They furthermore open new avenues for characterizing alterations in the active zone proteome of mutant mice and their corresponding controls, including the various mouse models of neurological diseases.

The Proteome of the Murine Presynaptic Active Zone.

The proteome of the presynaptic active zone controls neurotransmitter release and the short- and long-term structural and functional dynamics of the nerve terminal. The proteinaceous inventory of the presynaptic active zone has recently been reported. This review will evaluate the subcellular fractionation protocols and the proteomic approaches employed. A breakthrough for the identification of the proteome of the presynaptic active zone was the successful employment of antibodies directed against a cytosolic epitope of membrane integral synaptic vesicle proteins for the immunopurification of synaptic vesicles docked to the presynaptic plasma membrane. Combining immunopurification and subsequent analytical mass spectrometry, hundreds of proteins, including synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular and extracellular signaling and a large variety of adhesion molecules, were identified. Numerous proteins regulating the rearrangement of the cytoskeleton are indicative of the functional and structural dynamics of the presynapse. This review will critically discuss both the experimental approaches and prominent protein candidates identified. Many proteins have not previously been assigned to the presynaptic release sites and may be directly involved in the short- and long-term structural modulation of the presynaptic compartment. The identification of proteinaceous constituents of the presynaptic active zone provides the basis for further analyzing the interaction of presynaptic proteins with their targets and opens novel insights into the functional role of these proteins in neuronal communication.

Amyloid precursor proteins are constituents of the presynaptic active zone.

The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface-localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock-out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.

SV31 is a Zn2+-binding synaptic vesicle protein.

We recently identified in a proteomic screen a novel synaptic vesicle membrane protein of 31 kDa (SV31) of unknown function. According to its membrane topology and its phylogenetic relation SV31 may function as a vesicular transporter. Based on its amino acid sequence similarity to a prokaryotic heavy metal ion transporter we analyzed its metal ion-binding properties and show that recombinant SV31 binds the divalent cations Zn(2+) and Ni(2+) and to a minor extent Cu(2+), but not Fe(2+), Co(2+), Mn(2+), or Ca(2+). Zn(2+)-binding of SV31 in viable cells was verified following heterologous transfection of pheochromocytoma cells 12 (PC12) with recombinant red fluorescent SV31 (SV31-RFP) and the fluorescent zinc indicator FluoZin-3. Sucrose density gradient fractionation of SV31-RFP-transfected PC12 cells revealed a partial overlap of SV31-RFP with synaptic-like vesicle markers and the early endosome marker rab5. Immunocytochemical analysis demonstrated a punctuate distribution in the cell soma and in neuritic processes and in addition in a compartment in vicinity to the plasma membrane that was immunopositive also for synaptosomal-associated protein 25 (SNAP-25) and syntaxin1A. Our data suggest that SV31 represents a novel Zn(2+) -binding protein that in PC12 cells is targeted to endosomes and subpopulations of synaptic-like microvesicles.

Proteomic investigations of the synaptic vesicle interactome.

Synaptic vesicles are key organelles in chemical signal transmission allowing neurons to communicate with each other and neighboring cells. The numerous tasks of synaptic vesicles are governed by a unique set of proteins. Recently, proteomic studies have been performed by several laboratories employing mass spectrometry and immunoblotting in order to identify the complete proteinaceous inventory of the purified synaptic vesicle compartment. Surprisingly, several fold more proteins were assigned to the organelle than previously anticipated. Despite several novel candidates, a large variety of proteins assumed to be only transiently associated with the vesicular compartment turned out to be constitutive components of the synaptic vesicle proteome. In recent years, the focus on protein-protein interactions has led to a deeper understanding of functional aspects in cellular trafficking. Several proteins acting in concert in defined cellular processes build an interactome. This article will survey the interacting partners during the entire synaptic vesicle life cycle identified by proteomic approaches. This includes anterograde and retrograde axonal transport of the synaptic vesicle membrane compartment, transport within the presynapse to the active zone, priming, docking, exocytosis, endocytosis, recycling and neurotransmitter reuptake to replenish the pool of exocytosis-competent synaptic vesicles.

Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes.

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.

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.

Evaluation of small hairpin RNA silencing efficiency in live cells by cotransfection of two fluorescent probes.

During recent years, the selected knockdown of protein expression by RNA interference has received rapidly growing interest. Although short interfering RNA (siRNA) target designers apply strict selection parameters, the deduced small RNAs need to be tested for their silencing potency. Here we describe a fast and efficient method for evaluating the silencing efficiency of target gene by small hairpin RNAs (shRNAs) in mammalian cells. Cells were cotransfected with two vectors: one containing shRNAs as well as the coding region for green fluorescent protein and one containing a chimeric fusion construct encoding red fluorescent protein coupled to the synaptic vesicle protein SV31. The efficiency of various shRNAs was directly monitored in vivo by fluorescence microscopy.

Identification and characterization of SV31, a novel synaptic vesicle membrane protein and potential transporter.

Synaptic vesicle proteins govern all relevant functions of the synaptic vesicle life cycle, including vesicle biogenesis, vesicle transport, uptake and storage of neurotransmitters, and regulated endocytosis and exocytosis. In spite of impressive progress made in the past years, not all known vesicular functions can be assigned to defined protein components, suggesting that the repertoire of synaptic vesicle proteins is still incomplete. We have identified and characterized a novel synaptic vesicle membrane protein of 31 kDa with six putative transmembrane helices that, according to its membrane topology and phylogenetic relation, may function as a vesicular transporter. The vesicular allocation is demonstrated by subcellular fractionation, heterologous expression, immunocytochemical analysis of brain sections and immunoelectron microscopy. The protein is expressed in select brain regions and contained in subpopulations of nerve terminals that immunostain for the vesicular glutamate transporter 1 and the vesicular GABA transporter VGaT (vesicular amino acid transporter) and may attribute specific and as yet undiscovered functions to subsets of glutamatergic and GABAergic synapses.