Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

Amyloid-ß Peptides Disrupt Interactions Between VAMP-2 and SNAP-25 in Neuronal Cells as Determined by FRET/FLIM.

BACKGROUND: Synaptic dysfunction prevalent in Alzheimer's disease (AD) brain is closely associated with increased accumulation of amyloid-ß (Aß) peptides in the brain parenchyma. It is widely believed that Aß peptides trigger synaptic dysfunction by interfering with the synaptic vesicular fusion and the release of neurotransmitters, primarily facilitated by the SNARE protein complexes formed by VAMP-2, SNAP-25, and syntaxin-1. However, Aß interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood. OBJECTIVE: Our objective is to elucidate mechanisms by which Aß peptides perturb SNARE complexes. METHODS: Intensity (qualitative) and lifetime (quantitative) based measurements involving Forster (fluorescence) resonance energy transfer (FRET) followed by fluorescence lifetime imaging microscopy (FLIM) were employed to investigate the effect of Aß peptides on dynamic interactions between VAMP-2, labeled with cerulean (Cer) at the N-terminus (FRET donor), and SNAP-25 labeled with citrine (Cit) on the N-terminus (FRET acceptor). The FRET and FLIM interactions at the exocytosis locations on the pre-synaptic membrane were recorded under spontaneous and high potassium evoked conditions. Moreover, cellular accumulation of fluorescein labeled Aß (F-Aß) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy. RESULTS: The F-Aß40 and F-Aß42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aß40 and Aß42 decrease interactions between the N-termini of Cer-VAMP2 and Cit-SNAP25 in N2A cells, as determined by FRET/FLIM. CONCLUSION: By perturbing the N-terminal interactions between VAMP-2 and SNAP-25, Aß40 and Aß42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.

Age-Related Cognitive Impairment: Role of Reduced Synaptobrevin-2 Levels in Deficits of Memory and Synaptic Plasticity.

Cognitive impairment in the aging population is quickly becoming a health care priority, for which currently no disease-modifying treatment is available. Multiple domains of cognition decline with age even in the absence of neurodegenerative diseases. The cellular and molecular changes leading to cognitive decline with age remain elusive. Synaptobrevin-2 (Syb2), the major vesicular SNAP receptor protein, highly expressed in the cerebral cortex and hippocampus, is essential for synaptic transmission. We have analyzed Syb2 protein levels in mice and found a decrease with age. To investigate the functional consequences of lower Syb2 expression, we have used adult Syb2 heterozygous mice (Syb2+/-) with reduced Syb2 levels. This allowed us to mimic the age-related decrease of Syb2 in the brain in order to selectively test its effects on learning and memory. Our results show that Syb2+/- animals have impaired learning and memory skills and they perform worse with age in the radial arm water maze assay. Syb2+/- hippocampal neurons have reduced synaptic plasticity with reduced release probability and impaired long-term potentiation in the CA1 region. Syb2+/- neurons also have lower vesicular release rates when compared to WT controls. These results indicate that reduced Syb2 expression with age is sufficient to cause cognitive impairment.

Novel SNARE Complex Polymorphisms Associated with Multiple Sclerosis: Signs of Synaptopathy in Multiple Sclerosis

Background: It is well known that axonal degeneration plays a role in disability in patients with multiple sclerosis, and synaptopathy has recently become an important issue. Aims: To investigate the possible roles of selected synaptic and presynaptic membrane protein genetic polymorphisms (VAMP2, SNAP-25, synaptotagmin, and syntaxin 1A) in patients with multiple sclerosis. Study Design: Case-control study. Methods: A total of 123 patients with multiple sclerosis and 192 healthy controls were included. The functional polymorphisms of specific SNARE complex proteins (VAMP2, synaptotagmin XI, syntaxin 1A, and SNAP-25) were analyzed by polymerase chain reaction. Results: Significant differences were detected in the genotype and allele distribution of 26-bp Ins/Del polymorphisms of VAMP2 between patients with multiple sclerosis and control subjects; Del/Del genotype and Del allele of VAMP2 were more frequent in patients with multiple sclerosis (p=0.011 and p=0.004, respectively). Similarly, Ddel polymorphism of SNAP-25 gene C/C genotype (p=0.059), syntaxin 1A T/C and C/C genotypes (p=0.005), and synaptotagmin XI gene C allele (p=0.001) were observed more frequently in patients with multiple sclerosis. CC, syntaxin rs1569061 1A gene for 33-bp promoter region TC haplotypes, and synaptotagmin XI gene were found to be associated with an increased risk for multiple sclerosis (p=0.012). Similarly, GC haplotype for rs3746544 of SNAP-25 gene and rs1051312 of SNAP-25 gene were associated with an increased risk for multiple sclerosis (p=0.022). Conclusion: Genetic polymorphisms of SNARE complex proteins, which have critical roles in synaptic structure and communication, may play a role in the development of multiple sclerosis.

A Fine Balance of Synaptophysin Levels Underlies Efficient Retrieval of Synaptobrevin II to Synaptic Vesicles.

Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15-2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells.

Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.

Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells after stimulation with isoproterenol.

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after beta-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B-GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.

Synaptophysin as a probable component of neurotransmission occurring in taste receptor cells.

Taste signal is received in taste buds and transmitted via sensory afferent nerves to the brainstem. Although a signaling pathway involving phospholipase C-beta2 has been shown to transduce taste signals of bitterness, sweetness and umami in taste receptor cells (Type II cells), these taste receptor cells appear to be different from the presynaptic cells (Type III cells) containing afferent synapses associated with nerve processes. To elucidate the neurotransmission system in the taste receptor cells expressing phospholipase C-beta2, we searched for candidate molecules involved in the neurotransmission, and identified synaptophysin. Synaptophysin was expressed in the taste receptor cells expressing phospholipase C-beta2, as well as in the presynaptic cells harboring synaptic structures with taste nerves and containing serotonin. Synaptophysin-immunoreactive signals were not limited to gustducin-positive bitter taste receptor cells, and sweet/umami taste receptor cells were indicated to also express synaptophysin. Expression of synaptophysin was already initiated 6 days after cell division, almost in synchrony with the initiation of phospholipase C-beta2 expression. Synaptophysin-containing cells co-expressed vesicular-associated membrane protein 2, a v-SNARE molecule which is important for exocytosis. In addition, majority of the synaptophysin-expressing cells also expressed cholecystokinin, a neuropeptide expressed in taste buds. These results suggest that the taste receptor cells have a neurotransmission system involving synaptophysin, which occurs alternatively or additionally to a recently shown hemichannel system.

Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-expansion in the gene encoding the protein huntingtin. The disease is characterized by progressive motor disturbances, cognitive defects, dementia, and weight loss. Using western blotting and immunohistochemistry we have assessed the expression levels and patterns of a number of proteins involved in neurotransmitter release in post-mortem frontal cortex samples from 10 HD cases with different disease grades. We report a loss of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, synaptosome-associated protein 25 (SNAP 25) in HD brains of grades I-IV. Moreover, in brains of grade III and IV we found a reduction in rabphilin 3a, a protein involved in vesicle docking and recycling. These losses appear to be specific and not due to a general loss of synapses in the HD cortex. Thus, levels of synaptobrevin II, syntaxin 1, rab3a or synaptophysin are unaltered in the same patient samples. SNAP 25 and rabphilin 3a are crucial for neurotransmitter release. Therefore, we suggest that a deficient pre-synaptic transmitter release may underlie some of the symptoms of HD.

Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

Synapse-specific regulation of AMPA receptor subunit composition by activity.

We examined the changes that arise when neurotransmitter release is inhibited in a subpopulation of hippocampal neurons in coculture with normally active neighbors. Subsets of neurons were presynaptically silenced by chronic expression of tetanus toxin light chain tagged with cyan fluorescent protein (TNTCFP). Surprisingly, silenced neurons formed as many presynaptic terminals as their active neighbors when grown together on glial microislands. However, silenced neurons could not recruit the AMPA-type glutamate receptor subunit GluR1 as efficiently when competing with active neighbors. The immunofluorescence intensity ratio of GluR1 at synaptic puncta versus shaft was reduced by 22% opposite TNTCFP-expressing terminals compared with active neighbors. In contrast, this effect is abolished when vesicular release is blocked in all neurons. Local presynaptic inhibition by TNTCFP did not change the synaptic level of the AMPA receptor subunits GluR2 or GluR2/3 or of the PSD95 (postsynaptic density 95) family scaffolding proteins. Thus, neurotransmitter release selectively regulates the AMPA receptor population on a synapse-by-synapse basis but is not essential for an axon to efficiently compete for synaptic territory in a simple model system. These results demonstrate precise input specificity of postsynaptic receptor composition via differential activity among neighbor synapses.