Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression.

Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate (KA) stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis.

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.

Corrigendum: Protein Interactors and Trafficking Pathways That Regulate the Cannabinoid Type 1 Receptor (CB1R).

[This corrects the article DOI: 10.3389/fnmol.2020.00108.].

Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting.

Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans-Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex - a key regulator of retrograde CI-MPR sorting - revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.

Protein Interactors and Trafficking Pathways That Regulate the Cannabinoid Type 1 Receptor (CB1R).

The endocannabinoid system (ECS) acts as a negative feedback mechanism to suppress synaptic transmission and plays a major role in a diverse range of brain functions including, for example, the regulation of mood, energy balance, and learning and memory. The function and dysfunction of the ECS are strongly implicated in multiple psychiatric, neurological, and neurodegenerative diseases. Cannabinoid type 1 receptor (CB1R) is the most abundant G protein-coupled receptor (GPCR) expressed in the brain and, as for any synaptic receptor, CB1R needs to be in the right place at the right time to respond appropriately to changing synaptic circumstances. While CB1R is found intracellularly throughout neurons, its surface expression is highly polarized to the axonal membrane, consistent with its functional expression at presynaptic sites. Surprisingly, despite the importance of CB1R, the interacting proteins and molecular mechanisms that regulate the highly polarized distribution and function of CB1R remain relatively poorly understood. Here we set out what is currently known about the trafficking pathways and protein interactions that underpin the surface expression and axonal polarity of CB1R, and highlight key questions that still need to be addressed.

The C-terminal helix 9 motif in rat cannabinoid receptor type 1 regulates axonal trafficking and surface expression.

Cannabinoid type one receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

Exciting Times: New Advances Towards Understanding the Regulation and Roles of Kainate Receptors.

Kainate receptors (KARs) are glutamate-gated ion channels that play fundamental roles in regulating neuronal excitability and network function in the brain. After being cloned in the 1990s, important progress has been made in understanding the mechanisms controlling the molecular and cellular properties of KARs, and the nature and extent of their regulation of wider neuronal activity. However, there have been significant recent advances towards understanding KAR trafficking through the secretory pathway, their precise synaptic positioning, and their roles in synaptic plasticity and disease. Here we provide an overview highlighting these new findings about the mechanisms controlling KARs and how KARs, in turn, regulate other proteins and pathways to influence synaptic function.

ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity.

Kainate receptors (KARs) regulate neuronal excitability and network function. Most KARs contain the subunit GluK2 (also known as GRIK2), and the properties of these receptors are determined in part by ADAR2 (also known as ADARB1)-mediated mRNA editing of GluK2, which changes a genomically encoded glutamine residue into an arginine residue (Q/R editing). Suppression of synaptic activity reduces ADAR2-dependent Q/R editing of GluK2 with a consequential increase in GluK2-containing KAR surface expression. However, the mechanism underlying this reduction in GluK2 editing has not been addressed. Here, we show that induction of KAR upscaling, a phenomenon in which surface expression of receptors is increased in response to a chronic decrease in synaptic activity, results in proteasomal degradation of ADAR2, which reduces GluK2 Q/R editing. Because KARs incorporating unedited GluK2(Q) assemble and exit the ER more efficiently, this leads to an upscaling of KAR surface expression. Consistent with this, we demonstrate that partial ADAR2 knockdown phenocopies and occludes KAR upscaling. Moreover, we show that although the AMPA receptor (AMPAR) subunit GluA2 (also known as GRIA2) also undergoes ADAR2-dependent Q/R editing, this process does not mediate AMPAR upscaling. These data demonstrate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing are key determinants of KAR, but not AMPAR, trafficking and upscaling.This article has an associated First Person interview with the first author of the paper.

Assembly, Secretory Pathway Trafficking, and Surface Delivery of Kainate Receptors Is Regulated by Neuronal Activity.

Ionotropic glutamate receptor (iGluR) trafficking and function underpin excitatory synaptic transmission and plasticity and shape neuronal networks. It is well established that the transcription, translation, and endocytosis/recycling of iGluRs are all regulated by neuronal activity, but much less is known about the activity dependence of iGluR transport through the secretory pathway. Here, we use the kainate receptor subunit GluK2 as a model iGluR cargo to show that the assembly, early secretory pathway trafficking, and surface delivery of iGluRs are all controlled by neuronal activity. We show that the delivery of de novo kainate receptors is differentially regulated by modulation of GluK2 Q/R editing, PKC phosphorylation, and PDZ ligand interactions. These findings reveal that, in addition to short-term regulation of iGluRs by recycling/endocytosis and long-term modulation by altered transcription/translation, the trafficking of iGluRs through the secretory pathway is under tight activity-dependent control to determine the numbers and properties of surface-expressed iGluRs.

SENP3-mediated deSUMOylation of Drp1 facilitates interaction with Mff to promote cell death.

The GTPase dynamin-related protein 1 (Drp1) is essential for physiological and pathophysiological mitochondrial fission. DeSUMOylation of Drp1 by the enzyme SENP3 promotes cell death during reperfusion after ischaemia by enhancing Drp1 partitioning to the mitochondrial outer membrane (MOM), which causes cytochrome c release and apoptosis. However, how deSUMOylation recruits Drp1 to the MOM is unknown. Here we show that deSUMOylation selectively promotes Drp1 binding to the MOM resident adaptor protein mitochondrial fission factor (Mff). Consistent with this, preventing Drp1 SUMOylation by mutating the SUMO acceptor sites enhances binding to Mff. Conversely, increasing Drp1 SUMOylation by knocking down SENP3 reduces both Drp1 binding to Mff and stress-induced cytochrome c release. Directly tethering Drp1 to the MOM bypasses the need for Mff to evoke cytochrome c release, and occludes the effect of SENP3 overexpression. Thus, Drp1 deSUMOylation promotes cell death by enhancing Mff-mediated mitochondrial recruitment. These data provide a mechanistic explanation for how the SUMOylation status of Drp1 acts as a key switch in cell death/survival decisions following extreme cell stress.

SUMOylation of Syntaxin1A regulates presynaptic endocytosis.

Neurotransmitter release from the presynaptic terminal is under very precise spatial and temporal control. Following neurotransmitter release, synaptic vesicles are recycled by endocytosis and refilled with neurotransmitter. During the exocytosis event leading to release, SNARE proteins provide most of the mechanical force for membrane fusion. Here, we show one of these proteins, Syntaxin1A, is SUMOylated near its C-terminal transmembrane domain in an activity-dependent manner. Preventing SUMOylation of Syntaxin1A reduces its interaction with other SNARE proteins and disrupts the balance of synaptic vesicle endo/exocytosis, resulting in an increase in endocytosis. These results indicate that SUMOylation regulates the emerging role of Syntaxin1A in vesicle endocytosis, which in turn, modulates neurotransmitter release and synaptic function.