SGIP1 binding to the α-helical H9 domain of cannabinoid receptor 1 promotes axonal surface expression.

Endocannabinoid signalling mediated by cannabinoid receptor 1 (CB1R, also known as CNR1) is critical for homeostatic neuromodulation of both excitatory and inhibitory synapses. This requires highly polarised axonal surface expression of CB1R, but how this is achieved remains unclear. We previously reported that the α-helical H9 domain in the intracellular C terminus of CB1R contributes to axonal surface expression by an unknown mechanism. Here, we show in rat primary neuronal cultures that the H9 domain binds to the endocytic adaptor protein SGIP1 to promote CB1R expression in the axonal membrane. Overexpression of SGIP1 increases CB1R axonal surface localisation but has no effect on CB1R lacking the H9 domain (CB1RΔH9). Conversely, SGIP1 knockdown reduces axonal surface expression of CB1R but does not affect CB1RΔH9. Furthermore, SGIP1 knockdown diminishes CB1R-mediated inhibition of presynaptic Ca2+ influx in response to neuronal activity. Taken together, these data advance mechanistic understanding of endocannabinoid signalling by demonstrating that SGIP1 interaction with the H9 domain underpins axonal CB1R surface expression to regulate presynaptic responsiveness.

Aggregation-prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity.

Mitochondrial protein import is essential for organellar biogenesis, and thereby for the sufficient supply of cytosolic ATP - which is particularly important for cells with high energy demands like neurons. This study explores the prospect of import machinery perturbation as a cause of neurodegeneration instigated by the accumulation of aggregating proteins linked to disease. We found that the aggregation-prone Tau variant (TauP301L) reduces the levels of components of the import machinery of the outer (TOM20, encoded by TOMM20) and inner membrane (TIM23, encoded by TIMM23) while associating with TOM40 (TOMM40). Intriguingly, this interaction affects mitochondrial morphology, but not protein import or respiratory function; raising the prospect of an intrinsic rescue mechanism. Indeed, TauP301L induced the formation of tunnelling nanotubes (TNTs), potentially for the recruitment of healthy mitochondria from neighbouring cells and/or the disposal of mitochondria incapacitated by aggregated Tau. Consistent with this, inhibition of TNT formation (and rescue) reveals Tau-induced import impairment. In primary neuronal cultures, TauP301L induced morphological changes characteristic of neurodegeneration. Interestingly, these effects were mirrored in cells where the import sites were blocked artificially. Our results reveal a link between aggregation-prone Tau and defective mitochondrial import relevant to disease.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

The SUMO protease SENP3 regulates mitochondrial autophagy mediated by Fis1.

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.

Phosphorylation on Ser-359 of the α2 subunit in GABA type A receptors down-regulates their density at inhibitory synapses.

GABA type A receptors (GABAARs) mediate fast synaptic inhibition and are trafficked to functionally diverse synapses. However, the precise molecular mechanisms that regulate the synaptic targeting of these receptors are unclear. Whereas it has been previously shown that phosphorylation events in α4, ß, and γ subunits of GABAARs govern their function and trafficking, phosphorylation of other subunits has not yet been demonstrated. Here, we show that the α2 subunit of GABAARs is phosphorylated at Ser-359 and enables dynamic regulation of GABAAR binding to the scaffolding proteins gephyrin and collybistin. We initially identified Ser-359 phosphorylation by MS analysis, and additional experiments revealed that it is regulated by the activities of cAMP-dependent protein kinase (PKA) and the protein phosphatase 1 (PP1) and/or PP2A. GST-based pulldowns and coimmunoprecipitation experiments demonstrate preferential binding of both gephyrin and collybistin to WT and an S359A phosphonull variant, but not to an S359D phosphomimetic variant. Furthermore, the decreased capacity of the α2 S359D variant to bind collybistin and gephyrin decreased the density of synaptic α2-containing GABAAR clusters and caused an absence of α2 enrichment in the axon initial segment. These results suggest that PKA-mediated phosphorylation and PP1/PP2A-dependent dephosphorylation of the α2 subunit play a role in the dynamic regulation of GABAAR accumulation at inhibitory synapses, thereby regulating the strength of synaptic inhibition. The MS data have been deposited to ProteomeXchange, with the data set identifier PXD019597.

Neuronal SUMOylation: mechanisms, physiology, and roles in neuronal dysfunction.

Protein SUMOylation is a critically important posttranslational protein modification that participates in nearly all aspects of cellular physiology. In the nearly 20 years since its discovery, SUMOylation has emerged as a major regulator of nuclear function, and more recently, it has become clear that SUMOylation has key roles in the regulation of protein trafficking and function outside of the nucleus. In neurons, SUMOylation participates in cellular processes ranging from neuronal differentiation and control of synapse formation to regulation of synaptic transmission and cell survival. It is a highly dynamic and usually transient modification that enhances or hinders interactions between proteins, and its consequences are extremely diverse. Hundreds of different proteins are SUMO substrates, and dysfunction of protein SUMOylation is implicated in a many different diseases. Here we briefly outline core aspects of the SUMO system and provide a detailed overview of the current understanding of the roles of SUMOylation in healthy and diseased neurons.

SUMOylation of synaptic and synapse-associated proteins: An update.

SUMOylation is a post-translational modification that regulates protein signalling and complex formation by adjusting the conformation or protein-protein interactions of the substrate protein. There is a compelling and rapidly expanding body of evidence that, in addition to SUMOylation of nuclear proteins, SUMOylation of extranuclear proteins contributes to the control of neuronal development, neuronal stress responses and synaptic transmission and plasticity. In this brief review we provide an update of recent developments in the identification of synaptic and synapse-associated SUMO target proteins and discuss the cell biological and functional implications of these discoveries.

Phosphorylation of Syntaxin-1a by casein kinase 2α regulates pre-synaptic vesicle exocytosis from the reserve pool.

The t-soluble NSF-attachment protein receptor protein Syntaxin-1a (Stx-1a) is abundantly expressed at pre-synaptic terminals where it plays a critical role in the exocytosis of neurotransmitter-containing synaptic vesicles. Stx-1a is phosphorylated by Casein kinase 2α (CK2α) at Ser14, which has been proposed to regulate the interaction of Stx-1a and Munc-18 to control of synaptic vesicle priming. However, the role of CK2α in synaptic vesicle dynamics remains unclear. Here, we show that CK2α over-expression reduces evoked synaptic vesicle release. Furthermore, shRNA-mediated knockdown of CK2α in primary hippocampal neurons strongly enhanced vesicle exocytosis from the reserve pool, with no effect on the readily releasable pool of primed vesicles. In neurons in which endogenous Stx-1a was knocked down and replaced with a CK2α phosphorylation-deficient mutant, Stx-1a(D17A), vesicle exocytosis was also increased. These results reveal a previously unsuspected role of CK2α phosphorylation in specifically regulating the reserve synaptic vesicle pool, without changing the kinetics of release from the readily releasable pool.

Synaptic AMPA receptor composition in development, plasticity and disease.

AMPA receptors (AMPARs) are assemblies of four core subunits, GluA1-4, that mediate most fast excitatory neurotransmission. The component subunits determine the functional properties of AMPARs, and the prevailing view is that the subunit composition also determines AMPAR trafficking, which is dynamically regulated during development, synaptic plasticity and in response to neuronal stress in disease. Recently, the subunit dependence of AMPAR trafficking has been questioned, leading to a reappraisal of this field. In this Review, we discuss what is known, uncertain, conjectured and unknown about the roles of the individual subunits, and how they affect AMPAR assembly, trafficking and function under both normal and pathological conditions.

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.

Guanosine modulates SUMO2/3-ylation in neurons and astrocytes via adenosine receptors.

SUMOylation is a post-translational modification (PTM) whereby members of the Small Ubiquitin-like MOdifier (SUMO) family of proteins are conjugated to lysine residues in target proteins. SUMOylation has been implicated in a wide range of physiological and pathological processes, and much attention has been given to its role in neurodegenerative conditions. Due to its reported role in neuroprotection, pharmacological modulation of SUMOylation represents an attractive potential therapeutic strategy in a number of different brain disorders. However, very few compounds that target the SUMOylation pathway have been identified. Guanosine is an endogenous nucleoside with important neuromodulatory and neuroprotective effects. Experimental evidence has shown that guanosine can modulate different intracellular pathways, including PTMs. In the present study we examined whether guanosine alters global protein SUMOylation. Primary cortical neurons and astrocytes were treated with guanosine at 1, 10, 100, 300, or 500 µM at four time points, 1, 6, 24, or 48 h. We show that guanosine increases global SUMO2/3-ylation in neurons and astrocytes at 1 h at concentrations above 10 µM. The molecular mechanisms involved in this effect were evaluated in neurons. The guanosine-induced increase in global SUMO2/3-ylation was still observed in the presence of dipyridamole, which prevents guanosine internalization, demonstrating an extracellular guanosine-induced effect. Furthermore, the A1 adenosine receptor antagonist DPCPX abolished the guanosine-induced increase in SUMO2/3-ylation. The A2A adenosine receptor antagonist ZM241385 increased SUMOylation per se, but did not alter guanosine-induced SUMOylation, suggesting that guanosine may modulate SUMO2/3-ylation through an A1-A2A receptor interaction. Taken together, this is the first report to show guanosine as a SUMO2/3-ylation enhancer in astrocytes and neurons.

Sorting nexin 27 rescues neuroligin 2 from lysosomal degradation to control inhibitory synapse number.

Retromer is an evolutionarily conserved endosomal trafficking complex that mediates the retrieval of cargo proteins from a degradative pathway for sorting back to the cell surface. To promote cargo recycling, the core retromer trimer of VPS (vacuolar protein sorting)26, VPS29 and VPS35 recognises cargo either directly, or through an adaptor protein, the most well characterised of which is the PDZ [postsynaptic density 95 (PSD95), disk large, zona occludens] domain-containing sorting nexin SNX27. Neuroligins (NLGs) are postsynaptic trans-synaptic scaffold proteins that function in the clustering of postsynaptic proteins to maintain synaptic stability. Here, we show that each of the NLGs (NLG1-3) bind to SNX27 in a direct PDZ ligand-dependent manner. Depletion of SNX27 from neurons leads to a decrease in levels of each NLG protein and, for NLG2, this occurs as a result of enhanced lysosomal degradation. Notably, while depletion of the core retromer component VPS35 leads to a decrease in NLG1 and NLG3 levels, NLG2 is unaffected, suggesting that, for this cargo, SNX27 acts independently of retromer. Consistent with loss of SNX27 leading to enhanced lysosomal degradation of NLG2, knockdown of SNX27 results in fewer NLG2 clusters in cultured neurons, and loss of SNX27 or VPS35 reduces the size and number of gephyrin clusters. Together, these data indicate that NLGs are SNX27-retromer cargoes and suggest that SNX27-retromer controls inhibitory synapse number, at least in part through trafficking of NLG2.

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.

Prospecting Peptides Isolated From Black Soldier Fly (Diptera: Stratiomyidae) With Antimicrobial Activity Against Helicobacter pylori (Campylobacterales: Helicobacteraceae).

Helicobacter pylori (Marshall & Goodwin) is a widespread human pathogen that is acquiring resistance to the antibiotics used to treat it. This increasing resistance necessitates a continued search for new antibiotics. An antibiotic source that shows promise is animals whose immune systems must adapt to living in bacteria-laden conditions by producing antibacterial peptides or small molecules. Among these animals is the black soldier fly (BSF; Hermetia illucens Linnaeus), a Diptera that colonizes decomposing organic matter. In order to find anti-H. pylori peptides in BSF, larvae were challenged with Escherichia coli (Enterobacteriales: Enterobacteriaceae). Small peptides were extracted from hemolymph and purified using solid-phase extraction, molecular weight cutoff filtration and two rounds of preparative high performance liquid chromatography (HPLC). The anti-H. pylori fraction was followed through the purification process using the inhibition zone assay in brain-heart infusion agar, while peptides from uninoculated larvae had no activity. The inhibition halo of the active sample was comparable to the action of metronidazole in the inhibition zone assay. The purified sample contained four peptides with average masses of approximately 4.2 kDa that eluted together when analyzed by HPLC-mass spectrometry. The peptides likely have similar sequences, activity, and properties. Therefore, BSF produces inducible antibacterial peptides that have in vitro activity against H. pylori, which highlights BSF's position as an important target for further bioprospecting.

The transcription factor MEF2A plays a key role in the differentiation/maturation of rat neural stem cells into neurons.

Neural stem cells (NSCs) are self-renewing multipotent stem cells that can be proliferated in vitro and differentiated into neuronal and/or glial lineages, making them an ideal model to study the processes involved in neuronal differentiation. Here we have used NSCs to investigate the role of the transcription factor MEF2A in neuronal differentiation and development in vitro. We show that although MEF2A is present in undifferentiated NSCs, following differentiation it is expressed at significantly higher levels in a subset of neuronal compared to non-neuronal cells. Furthermore, shRNA-mediated knockdown of MEF2A reduces the number of NSC-derived neurons compared to non-neuronal cells after differentiation. Together, these data indicate that MEF2A participates in neuronal differentiation/maturation from NSCs.

SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia.

Global increases in small ubiquitin-like modifier (SUMO)-2/3 conjugation are a neuroprotective response to severe stress but the mechanisms and specific target proteins that determine cell survival have not been identified. Here, we demonstrate that the SUMO-2/3-specific protease SENP3 is degraded during oxygen/glucose deprivation (OGD), an in vitro model of ischaemia, via a pathway involving the unfolded protein response (UPR) kinase PERK and the lysosomal enzyme cathepsin B. A key target for SENP3-mediated deSUMOylation is the GTPase Drp1, which plays a major role in regulating mitochondrial fission. We show that depletion of SENP3 prolongs Drp1 SUMOylation, which suppresses Drp1-mediated cytochrome c release and caspase-mediated cell death. SENP3 levels recover following reoxygenation after OGD allowing deSUMOylation of Drp1, which facilitates Drp1 localization at mitochondria and promotes fragmentation and cytochrome c release. RNAi knockdown of SENP3 protects cells from reoxygenation-induced cell death via a mechanism that requires Drp1 SUMOylation. Thus, we identify a novel adaptive pathway to extreme cell stress in which dynamic changes in SENP3 stability and regulation of Drp1 SUMOylation are crucial determinants of cell fate.

SUMOylation of Argonaute-2 regulates RNA interference activity.

Post-translational modification of substrate proteins by small ubiquitin-like modifier (SUMO) regulates a vast array of cellular processes. SUMOylation occurs through three sequential enzymatic steps termed E1, E2 and E3. Substrate selection can be determined through interactions between the target protein and the SUMO E2 conjugating enzyme Ubc9 and specificity can be enhanced by substrate interactions with E3 ligase enzymes. We used the putative substrate recognition (PINIT) domain from the SUMO E3 PIAS3 as bait to identify potential SUMO substrates. One protein identified was Argonaute-2 (Ago2), which mediates RNA-induced gene silencing through binding small RNAs and promoting degradation of complimentary target mRNAs. We show that Ago2 can be SUMOylated in mammalian cells by both SUMO1 and SUMO2. SUMOylation occurs primarily at K402, and mutation of the SUMO consensus site surrounding this lysine reduces Ago2-mediated siRNA-induced silencing in a luciferase-based reporter assay. These results identify SUMOylation as a potential regulator of Ago2 activity and open new avenues for research into the mechanisms underlying the regulation of RNA-induced gene silencing.

In vivo characterization of the properties of SUMO1-specific monobodies.

Monobodies are small recombinant proteins designed to bind with high affinity to target proteins. Monobodies have been generated to mimic the SIM [SUMO (small ubiquitin-like modifier)-interacting motif] present in many SUMO target proteins, but their properties have not been determined in cells. In the present study we characterize the properties of two SUMO1-specific monobodies (hS1MB4 and hS1MB5) in HEK (human embyronic kidney)-293 and HeLa cells and examine their ability to purify SUMO substrates from cell lines and rat brain. Both hS1MB4 and hS1MB5 compared favourably with commercially available antibodies and were highly selective for binding to SUMO1 over SUMO2/3 in pull-down assays against endogenous and overexpressed SUMO and SUMOylated proteins. Monobodies expressed in HeLa cells displayed a nuclear and cytosolic distribution that overlaps with SUMO1. Expression of the monobodies effectively inhibited protein SUMOylation by SUMO1 and, surprisingly, by SUMO2/3, but were not cytotoxic for at least 36 h. We attribute the effects on SUMO2/3 to the role of SUMO1 in chain termination and/or monobody inhibition of the SUMO-conjugating E1 enzyme complex. Taken together, these data provide the first demonstration that monobodies represent useful new tools both to isolate SUMO conjugates and to probe cell SUMOylation pathways in vivo.

Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis.

The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.

An essential role for the RNA helicase DDX6 in NMDA receptor-dependent gene silencing and dendritic spine shrinkage.

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins in the RNA-induced silencing complex (RISC) to modulate protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by repressing the translation of proteins involved in dendritic spine morphogenesis. Rapid NMDAR-dependent silencing of Limk1 is essential for spine shrinkage and requires Ago2 phosphorylation at S387. Not all gene silencing events are modulated by S387 phosphorylation, and the mechanisms that govern the selection of specific mRNAs for silencing downstream of S387 phosphorylation are unknown. Here, we show that NMDAR-dependent S387 phosphorylation causes a rapid and transient increase in the association of Ago2 with Limk1, but not Apt1 mRNA. The specific increase in Limk1 mRNA binding to Ago2 requires recruitment of the helicase DDX6 to RISC. Furthermore, we show that DDX6 is required for NMDAR-dependent silencing of Limk1 via miR-134, but not Apt1 via miR-138, and is essential for NMDAR-dependent spine shrinkage. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression of specific genes to control dendritic spine morphology.