Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.

CRISPR/Cas9 system-mediated impairment of synaptobrevin/VAMP function in postmitotic hippocampal neurons.

BACKGROUND: The use of the CRISPR/Cas9 system is becoming widespread, however current studies have predominantly focused on dividing cells. It is currently unknown if CRISPR/Cas9 can be used in a postmitotic setting to examine non-cell autonomous/presynaptic phenotypes in the resulting genetically heterogeneous cell population. NEW METHOD: A single CRISPR/Cas9 lentivirus was used to transfect a high percentage of primary cultured neurons and target synaptobrevin 2 (Syb2, also called VAMP2). RESULTS: Primary hippocampal cultures infected with the Syb2 targeting virus displayed dramatic reductions in Syb2 protein and immunocytochemical staining. In many boutons Syb2 was completely undetected. These cultures recapitulated the known functional phenotypes of Syb2 knockout neurons, which are non-cell autonomous and presynaptic in origin, indicating that Syb2 was knocked out in a large fraction of neurons. COMPARISON WITH EXISTING METHOD(S): Previous methods used multiple viruses or sparse transfection methods and only examined cell autonomous or postsynaptic phenotypes. The current method demonstrates that the CRISPR/Cas9 system can be used to alter network dynamics by removing or lowering the target gene from a majority of cells in the culture. CONCLUSIONS: A combination of CRISPR/Cas9 system and single high efficiency lentivirus infection can be used to examine non-cell autonomous and presynaptic phenotypes in postmitotic neurons.

SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium.

The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91(phox), and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function.

A cross-over inhibitor of the botulinum neurotoxin light chain B: a natural product implicating an exosite mechanism of action.

Clostridium botulinum produces the most lethal toxins known to man, as such they are high risk terrorist threats, and alarmingly there is no approved therapeutic. We report the first cross-over small molecule inhibitor of these neurotoxins and propose a mechanism by which it may impart its inhibitory activity.

Myosin Va controls oligodendrocyte morphogenesis and myelination.

A product of myosin Va mutations, Griscelli's syndrome type 1 (GS1) is characterized by several neurologic deficits including quadraparesis, mental retardation, and seizures. Although multiple studies have not clearly established a cause for the neurologic deficits linked with GS1, a few reports suggest that GS1 is associated with abnormal myelination, which could cause the neurologic deficits seen with GS1. In this report, we investigate whether myosin Va is critical to oligodendrocyte morphology and to myelination in vivo. We found that myosin Va-null mice exhibit significantly impaired myelination of the brain, optic nerve, and spinal cord. Oligodendrocytes express myosin Va and loss of myosin Va function resulted in significantly smaller lamellas and decreased process number, length, and branching of oligodendrocytes. Loss of myosin Va function also blocked distal localization of vesicle-associated membrane protein 2 (VAMP2), which is known to associate with myosin Va. When VAMP2 function was disrupted, oligodendrocytes exhibited similar morphologic deficits to what is seen with functional ablation of myosin Va. Our findings establish a role for both myosin Va and VAMP2 in oligodendrocyte function as it relates to myelination.