Parkinson disease-linked Vps35 R524W mutation impairs the endosomal association of retromer and induces α-synuclein aggregation

Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease

Structure and membrane binding properties of the endosomal tetratricopeptide repeat (TPR) domain-containing sorting nexins SNX20 and SNX21

Sorting nexins (SNX) orchestrate membrane trafficking and signaling events required for the proper distribution of proteins within the endosomal network. Their phox homology (PX) domain acts as a phosphoinositide (PI) recognition module that targets them to specific endocytic membrane domains. The modularity of SNX proteins confers a wide variety of functions from signaling to membrane deformation and cargo binding, and many SNXs are crucial modulators of endosome dynamics and are involved in a myriad of physiological and pathological processes such as neurodegenerative diseases, cancer, and inflammation. Here, we have studied the poorly characterized SNX20 and its paralogue SNX21, which contain an N-terminal PX domain and a C-terminal PX-associated B (PXB) domain of unknown function. The two proteins share similar PI-binding properties and are recruited to early endosomal compartments by their PX domain. The crystal structure of the SNX21 PXB domain reveals a tetratricopeptide repeat (TPR)-fold, a module that typically binds short peptide motifs, with three TPR α-helical repeats. However, the C-terminal capping helix adopts a highly unusual and potentially self-inhibitory topology. SAXS solution structures of SNX20 and SNX21 show that these proteins adopt a compact globular architecture, and membrane interaction analyses indicate the presence of overlapping PI-binding sites that may regulate their intracellular localization. This study provides the first structural analysis of this poorly characterized subfamily of SNX proteins, highlighting a likely role as endosome-associated scaffolds

SNT-1 functions as the Ca2+ sensor for tonic and evoked neurotransmitter release in Caenorhabditis Elegans

Synaptotagmin-1 (Syt1) binds Ca2+ through its tandem C2 domains (C2A and C2B) and triggers Ca2+-dependent neurotransmitter release. Here, we show that snt-1, the homolog of mammalian Syt1, functions as the Ca2+ sensor for both tonic and evoked neurotransmitter release at the Caenorhabditis elegans neuromuscular junction. Mutations that disrupt Ca2+ binding in double C2 domains of SNT-1 significantly impaired tonic release, whereas disrupting Ca2+ binding in a single C2 domain had no effect, indicating that the Ca2+ binding of the two C2 domains is functionally redundant for tonic release. Stimulus-evoked release was significantly reduced in snt-1 mutants, with prolonged release latency as well as faster rise and decay kinetics. Unlike tonic release, evoked release was triggered by Ca2+ binding solely to the C2B domain. Moreover, we showed that SNT-1 plays an essential role in the priming process in different subpopulations of synaptic vesicles with tight or loose coupling to Ca2+ entry

Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

Munc 18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc 18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc 18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc 18-1 in vitro and in cells. Surprisingly, Munc 18-1 EIEE mutants also form Lewy body like structures that contain a-synuclein (alpha-Syn). We reveal that Munc 18-1 binds alpha-Syn, and its EIEE mutants coaggregate alpha-Syn. Likewise, removal of endogenous Munc 18-1 increases the aggregative propensity of alpha-Syn(wT) and that of the Parkinson's disease-causing a-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the alpha-Syn(A30P) mutant with Munc 18-1 reduced the number of alpha-Syn(A30P) aggregates. Munc 18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc 18-1 and a perturbed chaperone activity for a-Syn leading to aggregation-induced neurodegeneration

SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27-Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27-Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans

Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2

The endosome-associated cargo adaptor sorting nexin-27 (SNX27) is linked to various neuropathologies through sorting of integral proteins to the synaptic surface, most notably AMPA receptors. To provide a broader view of SNX27-associated pathologies, we performed proteomics in rat primary neurons to identify SNX27-dependent cargoes, and identified proteins linked to excitotoxicity, epilepsy, intellectual disabilities, and working memory deficits. Focusing on the synaptic adhesion molecule LRFN2, we established that SNX27 binds to LRFN2 and regulates its endosomal sorting. Furthermore, LRFN2 associates with AMPA receptors and knockdown of LRFN2 results in decreased surface AMPA receptor expression, reduced synaptic activity, and attenuated hippocampal long-term potentiation. Overall, our study provides an additional mechanism by which SNX27 can control AMPA receptor-mediated synaptic transmission and plasticity indirectly through the sorting of LRFN2 and offers molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions

Molecular characterization of caveolin-induced membrane curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events

Phosphoinositide binding by the SNX27 FERM domain regulates localisation at the immune synapse of activated T-cells

Sorting nexin 27 (SNX27) controls the endosomal to cell-surface recycling of diverse transmembrane protein cargos. Critical to this function is the recruitment of SNX27 to endosomes through the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by the phox-homology (PX) domain. In T cells, SNX27 is polarized to the immunological synapse (IS) in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide lipid-binding capabilities of full-length SNX27, and discovered a novel PtdInsP binding site within the C-terminal 4.1/ezrin/radixin/moesin (FERM) domain. This binding site showed a clear preference for di and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the IS of activated T-cells cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters its distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during IS assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking