Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

Altered spectrum of retrogradely transported axonal proteins in p-bromophenylacetylurea neuropathy

The composition of retrogradely transported axonal proteins was examined by acrylamide gel electrophoresis and gel autoradiography in the experimental neuropathy induced in rats by p-bromophenylacetylurea (BPAU). Protein composition was normal during the early phase of retrograde transport but showed significant abnormalities during a later phase. The early phase consisted of proteins collected distal to a mid-thigh ligature of sciatic nerve between 15 and 24 hours after injection of [³⁵S] methionine into lumbar ventral horn of the spinal cord. In terms of their relative labeling and electrophoretic mobility, these proteins were almost identical in experimental and control rats. Most of the labeled protein bands were also identical in the later phase, collected between 24 and 48 hours, but there were some consistent omissions and additions. Present in controls but missing in BPAU treated rats were three bands at 42, 41, and 25 KDa. In contrast, 4 bands (63, 56, 50, 26 KDa) were more prominent in the experimental rats than in controls. We suspect abnormal post-translational modification or proteolysis of rapidly transported proteins in the terminal or preterminal portion of the neurons exposed to BPAU. This abnormality, in addition to a previously reported premature processing of transported organelles, may underlie the development of peripheral neuropathy.     

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.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.     

Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.     

Studies of synaptic vesicle endocytosis in the nematode C. elegans

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.     

Dense core vesicle dynamics in Caenorhabditis elegans neurons and the role of kinesin UNC-104

We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes. The post-translational processing of IDA-1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL-3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time-lapse epifluorescence microscopy of IDA-1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC-104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 microm/s-2.7 microm/s.     

Analysis of Caenorhabditis elegans acetylcholine synthesis mutants reveals a temperature-sensitive requirement for cholinergic neuromuscular function

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits.     

Key role of clathrin-mediated endocytosis in synaptic vesicle recycling

Using novel fluorescent markers, virus-induced modulation of amphiphysin 1 expression, and electron microscopy, we demonstrated that clathrin-mediated endocytosis is the main mechanism of synaptic vesicle retrieval; a hypothesis on the role of a fast “kiss-and-run” mechanism has not been supported. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 388–389, July–October, 2007.     

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.     

Synaptotagmin-1 interacts with PI(4,5)P2 to initiate synaptic vesicle docking in hippocampal neurons

1. Download : Download high-res image (190KB)
2. Download : Download full-size image

     

Molecular structures of proteins involved in vesicle fusion

We present a summary of the structures of 13 proteins involved in the docking and fusion of intracellular transport vesicles to their target membranes.     

Changing synaptic specificities in the nervous system of Caenorhabditis elegans: Differentiation of the DD motoneurons

During postembryonic development, the DD motoneurons in the nematode Caenorhabditis elegans completely reorganize their pattern of synapses. Ablation of a pair of embryonic precursors results in the absence of this entire class of motoneurons. In their absence animals exhibit two developmentally distinct locomotory defects. The transition period from one defect to the other is correlated with the synaptic reorganization of the DD mns. Mutations in a gene (unc-123) have been isolated that exhibit locomotory defects similar to those of the ablated adult animals. Genetic and cellular analyses of one of these alleles suggest that the unc-123 gene product may be involved in the reestablishment of functional synapses in these neurons. © 1993 John Wiley & Sons, Inc.     

海马神经元突触囊泡释放特征及可释放囊泡含量定量分析(英文)

中枢神经系统突触前的神经末梢只有少量的突触囊泡存在,突触囊泡数目的多少和融合模式将影响突触传递的效率。对突触囊泡数目的多少和释放模式的研究依赖于有效的研究方法。在本研究中,与膜亲和力不同的荧光染料用于标记体外培养的海马神经元的功能性突触囊泡。通过场电位和高钾刺激,动态观察荧光强度的变化,结果显示在第一轮刺激中,与膜亲和力低的染料FM2-10脱色的比例(93.0%±5.9%)显著大于与膜亲和力高的染料FM1-43(57.9%±3.5%)。但是,第二和第三轮刺激中FM1-43脱色的比例分别为(24.0±2.3)%,(8.6±1.5)%,显著大于FM2-10的脱色比例[(1.4±3.8)%,(2.3±1.6)%]。这个结果提示快速内吞模式不仅存在于囊泡的第一次释放,同时还存在于囊泡回收后的再次释放。另一方面,高频刺激和高渗蔗糖溶液这两种方法同时用于检测体外混合培养13~14天的抑制性神经元的可释放囊泡池(readily releasable pool,RRP)的大小。结果显示,用高渗蔗糖溶液估计的RRP的大小[(200±23.0)pC]显著大于用高频刺激估计的RRP的大小[(51.1±10.5)pC]。分析其可能的...     

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.     

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.     

Aberrant expression of centractin and capping proteins, integral constituents of the dynactin complex, in fetal down syndrome brain

Down syndrome (DS, trisomy 21) is the most frequent genetic cause of mental retardation. Although known for more than a hundred years the underlying pathomechanisms for the phenotype and impaired brain functions remain elusive. Performing protein hunting in fetal DS brain, we detected a series of cytoskeleton proteins with aberrant expression in fetal DS cortex. Fetal brain cortex samples of controls and DS of the early second trimenon of gestation were used for the experiments. We applied two-dimensional electrophoresis with in-gel digestion of protein spots, subsequent mass spectroscopical (MALDI) identification, and quantification of spots using specific software. Centractin alpha, F-actin capping protein alpha-1, alpha-2 and beta subunits were significantly reduced in fetal DS cortex, whereas dynein intermediate clear 2, dynein intermediate chain 2, and kinesin light chain protein levels were unchanged. Centractins and F-actin capping proteins are major determinants of the cytoskeleton and are involved in pivotal functions including cellular, organelle, and nuclear motility. Deranged centractins and F-actin capping proteins may represent or induce deficient axonal transport and may well contribute to deterioration of the cytoskeleton's mitotic functions in trisomy 21.     

Short-term enhancement of motor neuron synaptic exocytosis during early aging extends lifespan in Caenorhabditis elegans

Age-related mobility decline is often associated with negative physical and psychological outcomes, such as frailty, in the elderly population. In C. elegans, during the early stage of the aging process, a progressive deficit of synaptic exocytosis in the motor neurons results in a functional decline at the neuromuscular junctions, which eventually leads to degeneration of both neurons and muscles. This age-dependent functional decline can be ameliorated by pharmacological interventions, such as arecoline, a muscarinic AChR agonist known to promote synaptic exocytosis at the neuromuscular junctions. In this study, we found that a short-term treatment of arecoline during the early stage of aging, when the NMJ functional decline begins, not only slows muscle tissue aging, but also extends lifespan in C. elegans. We have also demonstrated that arecoline acts on the GAR-2/PLCβ pathway in the motor neurons to increases longevity. Together, our findings suggest that synaptic transmission in aging motor neurons may serve as a potential target for pharmacological interventions to promote both health span and lifespan, when applied at the early stage aging.Impact statementThe functional decline of motor activity is a common feature in almost all aging animals that leads to frailty, loss of independence, injury, and even death in the elderly population. Thus, understanding the molecular mechanism that drives the initial stage of this functional decline and developing strategies to increase human healthspan and even lifespan by targeting this process would be of great interests to the field. In this study, we found that by precisely targeting the motor neurons to potentiate its synaptic releases either genetically or pharmacologically, we can not only delay the functional aging at NMJs but also slow the rate of aging at the organismal level. Most importantly, we have demonstrated that a critical window of time, that is the early stage of NMJs functional decline, is required for the beneficial effects. A short-term treatment within this time period is sufficient to extend the animals’ lifespan.