Synaptophysin sustains presynaptic performance by preserving vesicular synaptobrevin-II levels

The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin-II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter-pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release

Altered synaptobrevin-II trafficking in neurons expressing a synaptophysin mutation associated with a severe neurodevelopmental disorder

textabstractFollowing exocytosis, synaptic vesicles (SVs) have to be reformed with the correct complement of proteins in the correct stoichiometry to ensure continued neurotransmission. Synaptophysin is a highly abundant, integral SV protein necessary for the efficient retrieval of the SV SNARE protein, synaptobrevin II (sybII). However the molecular mechanism underpinning synaptophysin-dependent sybII retrieval is still unclear. We recently identified a male patient with severe intellectual disability, hypotonia, epilepsy and callosal agenesis who has a point mutation in the juxtamembrane region of the fourth transmembrane domain of synaptophysin (T198I). This mutation had no effect on the activity-dependent retrieval of synaptophysin that was tagged with the genetically-encoded pH-sensitive reporter (pHluorin) in synaptophysin knockout hippocampal cultures. This suggested the mutant has no global effect on SV endocytosis, which was confirmed when retrieval of a different SV cargo (the glutamate transporter vGLUT1) was examined. However neurons expressing this T198I mutant did display impaired activity-dependent sybII retrieval, similar to that observed in synaptophysin knockout neurons. Interestingly this impairment did not result in an increased stranding of sybII at the plasma membrane. Screening of known human synaptophysin mutations revealed a similar presynaptic phenotype between T198I and a mutation found in X-linked intellectual disability. Thus this novel human synaptophysin mutation has revealed that aberrant retrieval and increased plasma membrane localisation of SV cargo can be decoupled in human disease

Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P-2), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P-2 results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death

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

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

BACKGROUND: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. RESULTS: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. CONCLUSIONS: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev’s activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-014-0121-9) contains supplementary material, which is available to authorized users

Exploiting endocytic pathways to prevent bacterial toxin infection

Many bacterial pathogens produce toxins that target intracellular molecules to alter or disrupt normal cellular function. To access the cell, these toxins often exploit endocytic pathways, recruit cellular machinery, or both to mediate their own internalization. Bacteria are becoming increasingly resistant to antibiotics, so alternatives to treat or prevent diseases caused by bacterial toxins are necessary. The advent of small molecule inhibitors, which target key proteins involved in endocytic trafficking pathways, provides an opportunity to develop these compounds as potential therapeutic inhibitors of bacterial toxicity. This chapter reviews the endocytic pathways commonly hijacked by these toxins and the drugs that are currently being developed to inhibit specific proteins involved in endocytosis. It further evaluates the feasibility of using these compounds to investigate the modalities of the endocytosis and trafficking of bacterial toxins, and the potential for the use of these drugs as therapeutics in the future

A role for SNAREs in neuronal survival?

Read the full article Botulinum protease-cleaved snare fragments induce cytotoxicity in neuroblastoma cells' on page