SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Abstract

Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicleassociated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.clathrin-mediated endocytosis | structure N eurotransmission in the brain depends on the calcium-triggered fusion and recycling of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic membrane at active zones (1). Following their exocytic insertion into the presynaptic membrane, SV proteins need to be retrieved at a precisely defined stoichiometry by endocytosis, a process involving clathrin, adaptors, and other endocytic proteins (2). Fast calcium-triggered SV fusion critically depends on the SV arginine (R)-soluble NSF attachment protein receptor (SNARE) synaptobrevin [or vesicle-associated membrane protein (VAMP)], which by forming a complex with the plasma membrane glutamine (Q)-SNAREs syntaxin and synaptosomal-associated protein (SNAP)-25 (3) drives neuroexocytosis (4, 5). Synapses lacking synaptobrevin 2 display <1% of wild-type release when stimulated by action potential (AP)-mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exoendocytically sorted with very high precision (8). Similar observations have been made for other SV proteins, including synaptotagmin and vesicular glutamate transporters (VGLUTs). How such precise sorting of synaptobrevin 2 is achieved has remained enigmatic. Synaptobrevin lacks recognizable linear sorting motifs (9), and unlike other SNARE proteins does not contain a folded N-terminal domain that serves as a targeting determinant in other VAMP family members (10-12).Genetic data have linked synaptobrevin sorting to the function of the AP180 N-terminal homology (...

P2‐030: Investigating the Role of Clu, Picalm, and Cr1 in Alzheimer's Disease

“…They did not find co-localisation of the two molecules, leading to speculation that any interaction must be weak or indirect , although later studies have indicated that PICALM and VAMP2 (as well as VAMP3 and VAMP8) do physically interact via the N-terminal region of the SNARE motif in VAMP2 and the ANTH domain common to both PICALM and AP180 (Koo et al 2011;Miller et al 2011). Koo et al similarly reported a link between PICALM expression and surface VAMP2 -again, suppression of PICALM (and AP180) expression was shown to lead to an increase in VAMP2 present at the neuronal surface, indicative that it is failing to be retrieved and recycled effectively; an effect which seemed specific to VAMP2, since other SV proteins were unaffected (Koo et al 2011). The effect was more pronounced when both PICALM and AP180 expression were suppressed, perhaps suggesting overlapping functionality in this context (Koo et al 2011).…”

“…This had previously been implicated in AD since Aβ can increase AMPA's rate of endocytosis, decreasing surface AMPA receptor presence, and leading to signalling abnormalities and structural changes in neurons (Hsieh et al 2006). RNAi knockdown of PICALM also affects the endocytosis of EGFR (Huang et al 2004) and R-SNARE proteins Koo et al 2011;Miller et al 2011). Changes in PICALM expression levels can also alter the intracellular distribution of many other molecules, such as AP1, mannose-6-phosphate receptor, and transferrin (Meyerholz et al 2005).…”

“…Harel et al first reported a link between R-SNARE protein VAMP2 (vesicle associated membrane protein 2, also known as synaptobrevin 2), the most abundant synaptic vesicle protein (Koo et al 2011), and PICALM when they demonstrated that over expression of PICALM lead to a reduction in surface VAMP2 by around 20%, while PICALM knockdown using siRNA increased the presence of surface VAMP2 by around 30% ). They did not find co-localisation of the two molecules, leading to speculation that any interaction must be weak or indirect , although later studies have indicated that PICALM and VAMP2 (as well as VAMP3 and VAMP8) do physically interact via the N-terminal region of the SNARE motif in VAMP2 and the ANTH domain common to both PICALM and AP180 (Koo et al 2011;Miller et al 2011).…”

“…Efficient recycling and sorting of SNAREs with high accuracy is crucial to ensure prolonged neurotransmitter release is possible. It was unclear how these SNARE proteins are endocytosed and sorted with such specificity, but it has recently reported that PICALM may be a key player in these processes (Koo et al 2011). …”

Unraveling the genes implicated in Alzheimer's disease

“…PICALM is located on chromosome 11q14.2 and is largely restricted to endothelial cells, with a low expression level in neurons and glial cells. PICALM promotes clathrin-mediated endocytosis and facilitates with presynaptic endocytosis (34). PICALM has been associated with Aβ production and APP processing (33,34).…”