SHP2 regulates GluA2 tyrosine phosphorylation required for AMPA receptor endocytosis and mGluR-LTD.

Posttranslational modifications regulate the properties and abundance of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that mediate fast excitatory synaptic transmission and synaptic plasticity in the central nervous system. During long-term depression (LTD), protein tyrosine phosphatases (PTPs) dephosphorylate tyrosine residues in the C-terminal tail of AMPA receptor GluA2 subunit, which is essential for GluA2 endocytosis and group I metabotropic glutamate receptor (mGluR)-dependent LTD. However, as a selective downstream effector of mGluRs, the mGluR-dependent PTP responsible for GluA2 tyrosine dephosphorylation remains elusive at Schaffer collateral (SC)-CA1 synapses. In the present study, we find that mGluR5 stimulation activates Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) by increasing phospho-Y542 levels in SHP2. Under steady-state conditions, SHP2 plays a protective role in stabilizing phospho-Y869 of GluA2 by directly interacting with GluA2 phosphorylated at Y869, without affecting GluA2 phospho-Y876 levels. Upon mGluR5 stimulation, SHP2 dephosphorylates GluA2 at Y869 and Y876, resulting in GluA2 endocytosis and mGluR-LTD. Our results establish SHP2 as a downstream effector of mGluR5 and indicate a dual action of SHP2 in regulating GluA2 tyrosine phosphorylation and function. Given the implications of mGluR5 and SHP2 in synaptic pathophysiology, we propose SHP2 as a promising therapeutic target for neurodevelopmental and autism spectrum disorders.

Targeted degradation of SNCA/α-synuclein aggregates in neurodegeneration using the AUTOTAC chemical platform.

Parkinson disease (PD) characterized by dopaminergic neuronal loss is caused by aggregation of misfolded SNCA/α-synuclein. We recently developed autophagy-targeting chimera (AUTOTAC), a targeted protein degradation (TPD) technology based on the macroautophagy/autophagy-lysosome pathway (ALP). In this study, we employed AUTOTAC to synthesize ATC161, a chimeric compound that adopts Anle138b as target-binding ligand (TBL) for SNCA aggregates. The autophagy-targeting ligand (ATL) of ATC161 was designed to allosterically activate the autophagy receptor SQSTSM1/p62 (sequestosome 1), a key step for targeting SNCA aggregates to the phagophore. The lysosomal degradation of SNCA aggregates by ATC161 acutely occurs at DC50 of 100-500 nM with no significant off-target degradation of monomeric SNCA. ATC161 protects cells from DNA and mitochondrial damage by SNCA aggregates. In PD model mice, oral administration of ATC161 decreases the level of SNCA aggregates and their propagation across brain regions, which mitigates glial inflammatory responses and improves muscle strength and locomotive activity. An Investigational New Drug (IND) was approved by the Korean Food and Drug Administration for a phase 1 clinical trial to treat PD, Alzheimer disease (AD), progressive supranuclear palsy (PSP), and amyotrophic lateral sclerosis (ALS). We suggest that AUTOTAC provides a platform for drug discovery in proteinopathies and other diseases.

Self-Aggregating Tau Fragments Recapitulate Pathologic Phenotypes and Neurotoxicity of Alzheimer's Disease in Mice.

In tauopathy conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic details of this process remain unclear. In the brains of AD patients, only a minor segment of tau forms ß-helix-stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, it is demonstrated that the tau AD nucleation core (tau-AC) sufficiently induced self-aggregation and recruited full-length tau to filaments. Unexpectedly, phospho-mimetic forms of tau-AC (at Ser324 or Ser356) show markedly reduced oligomerization and seeding propensities. Biophysical analysis reveal that the N-terminus of tau-AC facilitates the fibrillization kinetics as a nucleation motif, which becomes sterically shielded through phosphorylation-induced conformational changes in tau-AC. Tau-AC oligomers are efficiently internalized into cells via endocytosis and induced endogenous tau aggregation. In primary hippocampal neurons, tau-AC impaired axon initial segment plasticity upon chronic depolarization and is mislocalized to the somatodendritic compartments. Furthermore, it is observed significantly impaired memory retrieval in mice intrahippocampally injected with tau-AC fibrils, which corresponds to the neuropathological staining and neuronal loss in the brain. These findings identify tau-AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.

The N-degron pathway mediates lipophagy: The chemical modulation of lipophagy in obesity and NAFLD.

BACKGROUND AND AIMS: Central to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) is the accumulation of lipids in the liver and various fat tissues. We aimed to elucidate the mechanisms by which lipid droplets (LDs) in the liver and adipocytes are degraded by the autophagy-lysosome system and develop therapeutic means to modulate lipophagy, i.e., autophagic degradation of LDs. METHODS: We monitored the process in which LDs are pinched off by autophagic membranes and degraded by lysosomal hydrolases in cultured cells and mice. The autophagic receptor p62/SQSTM-1/Sequestosome-1 was identified as a key regulator and used as a target to develop drugs to induce lipophagy. The efficacy of p62 agonists was validated in mice to treat hepatosteatosis and obesity. RESULTS: We found that the N-degron pathway modulates lipophagy. This autophagic degradation initiates when the molecular chaperones including BiP/GRP78, retro-translocated from the endoplasmic reticulum, is N-terminally (Nt-) arginylated by ATE1 R-transferase. The resulting Nt-arginine (Nt-Arg) binds the ZZ domain of p62 associated with LDs. Upon binding to Nt-Arg, p62 undergoes self-polymerization and recruits LC3+ phagophores to the site of lipophagy, leading to lysosomal degradation. Liver-specific Ate1 conditional knockout mice under high fat diet developed severe NAFLD. The Nt-Arg was modified into small molecule agonists to p62 that facilitate lipophagy in mice and exerted therapeutic efficacy in obesity and hepatosteatosis of wild-type but not p62 knockout mice. CONCLUSIONS: Our results show that the N-degron pathway modulates lipophagy and provide p62 as a drug target to treat NAFLD and other diseases related with metabolic syndrome.

Targeted degradation of ⍺-synuclein aggregates in Parkinson's disease using the AUTOTAC technology.

BACKGROUND: There are currently no disease-modifying therapeutics for Parkinson's disease (PD). Although extensive efforts were undertaken to develop therapeutic approaches to delay the symptoms of PD, untreated α-synuclein (α-syn) aggregates cause cellular toxicity and stimulate further disease progression. PROTAC (Proteolysis-Targeting Chimera) has drawn attention as a therapeutic modality to target α-syn. However, no PROTACs have yet shown to selectively degrade α-syn aggregates mainly owing to the limited capacity of the proteasome to degrade aggregates, necessitating the development of novel approaches to fundamentally eliminate α-syn aggregates. METHODS: We employed AUTOTAC (Autophagy-Targeting Chimera), a macroautophagy-based targeted protein degradation (TPD) platform developed in our earlier studies. A series of AUTOTAC chemicals was synthesized as chimeras that bind both α-syn aggregates and p62/SQSTM1/Sequestosome-1, an autophagic receptor. The efficacy of Autotacs was evaluated to target α-syn aggregates to phagophores and subsequently lysosomes for hydrolysis via p62-dependent macroautophagy. The target engagement was monitored by oligomerization and localization of p62 and autophagic markers. The therapeutic efficacy to rescue PD symptoms was characterized in cultured cells and mice. The PK/PD (pharmacokinetics/pharmacodynamics) profiles were investigated to develop an oral drug for PD. RESULTS: ATC161 induced selective degradation of α-syn aggregates at DC50 of ~ 100 nM. No apparent degradation was observed with monomeric α-syn. ATC161 mediated the targeting of α-syn aggregates to p62 by binding the ZZ domain and accelerating p62 self-polymerization. These p62-cargo complexes were delivered to autophagic membranes for lysosomal degradation. In PD cellular models, ATC161 exhibited therapeutic efficacy to reduce cell-to-cell transmission of α-syn and to rescue cells from the damages in DNA and mitochondria. In PD mice established by injecting α-syn preformed fibrils (PFFs) into brain striata via stereotaxic surgery, oral administration of ATC161 at 10 mg/kg induced the degradation of α-syn aggregates and reduced their propagation. ATC161 also mitigated the associated glial inflammatory response and improved muscle strength and locomotive activity. CONCLUSION: AUTOTAC provides a platform to develop drugs for PD. ATC161, an oral drug with excellent PK/PD profiles, induces selective degradation of α-syn aggregates in vitro and in vivo. We suggest that ATC161 is a disease-modifying drug that degrades the pathogenic cause of PD.

Deneddylating enzyme SENP8 regulates neuronal development.

Neddylation is a cellular process in which the neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) is conjugated to the lysine residue of target proteins via serial enzymatic cascades. Recently, it has been demonstrated that neddylation is required for synaptic clustering of metabotropic glutamate receptor 7 (mGlu7) and postsynaptic density protein 95 (PSD-95), and the inhibition of neddylation impairs neurite outgrowth and excitatory synaptic maturation. Similar to the balanced role of deubiquitylating enzymes (DUBs) in the ubiquitination process, we hypothesized that deneddylating enzymes can regulate neuronal development by counteracting the process of neddylation. We find that the SUMO peptidase family member, NEDD8 specific (SENP8) acts as a key neuronal deneddylase targeting the global neuronal substrates in primary rat cultured neurons. We demonstrate that SENP8 expression levels are developmentally regulated, peaking around the first postnatal week and gradually diminishing in mature brain and neurons. We find that SENP8 negatively regulates neurite outgrowth through multiple pathways, including actin dynamics, Wnt/ß-catenin signaling, and autophagic processes. Alterations in neurite outgrowth by SENP8 subsequently result in the impairment of excitatory synapse maturation. Our data indicate that SENP8 plays an essential role in neuronal development and is a promising therapeutic target for neurodevelopmental disorders.

Development of diagnostic tests for pathogen identification and detection of antimicrobial resistance on WHO global priority pathogens using modular real-time nucleic acid amplification test.

BACKGROUND: Concerns regarding antimicrobial resistance (AMR) have resulted in the World Health Organization (WHO) designating so-called global priority pathogens (GPPs). However, little discussion has focused on the diagnosis of GPPs. To enable the simultaneous identification of pathogens and AMR, we developed a modular real-time nucleic acid amplification test (MRT-NAAT). METHODS: Sequence-specific primers for each modular unit for MRT-NAAT pathogen identification and AMR sets were designed. The composition of the reaction mixture and the real-time PCR program were unified irrespective of primer type so to give MRT-NAAT modularity. Standard strains and clinical isolates were used to evaluate the performance of MRT-NAAT by real-time PCR and melting curve analysis. Probit analysis for the MRT-NAAT pathogen identification set was used to assess the limit of detection (LoD). RESULTS: The MRT-NAAT pathogen identification set was made up of 15 modular units 109-199 bp in product size and with a Tms of 75.5-87.5 °C. The LoD was < 15.548 fg/µL, and nine modular units successfully detected the target pathogens. The MRT-NAAT AMR set included 24 modular units 65-785 bp in product size with a Tms of 75.5-87.5 °C; it showed high performance for detecting GPP target genes and variants. CONCLUSIONS: MRT-NAAT enables pathogen identification and AMR gene detection and is time-effective. By unifying the reaction settings of each modular unit, the modularity where combinations of primers can be used according to need could be achieved. This would greatly help in reflecting the researcher's need and the AMR status of a certain region while successfully detecting pathogens and AMR genes.

LRRK2 decreases microglial actin dynamics by filamentous actin depolymerization and Rac1 inhibition.

An active actin dynamic is a crucial feature of brain microglia. Here we report that LRRK2, a primary familial Parkinson's disease-associated gene, negatively regulates microglia's actin dynamics. LRRK2 depolymerized filamentous actin (F-actin) by directly binding to it or inhibiting microglia's Rac-PAK signaling. LRRK2 knockdown resulted in a reduced ruffle and enhanced lamellipodia formation of ADP-activated microglia, altering the microglia's physiological activity to vigorous migration toward damaged cells. These results suggest that LRRK2 is a negative regulator for the controlled actin dynamics in microglia.

CHIP-mediated hyperubiquitylation of tau promotes its self-assembly into the insoluble tau filaments.

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3ß and CHIP, respectively. The resulting phospho-ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one-three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho-ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho-ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a "multiple-hit model," where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.

Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals.

Neddylation is a posttranslational modification in which NEDD8 is conjugated to a target substrate by cellular processes similar to those involved in ubiquitination. Recent studies have identified PSD-95 and cofilin as substrates for neddylation in the brain and have shown that neddylation modulates the maturation and stability of dendritic spines in developing neurons. However, the precise substrates and functional consequences of neddylation at presynaptic terminals remain elusive. Here, we provide evidence that the mGlu7 receptor is a target of neddylation in heterologous cells and rat primary cultured neurons. We found that mGlu7 neddylation is reduced by agonist treatment and is required for the clustering of mGlu7 in the presynaptic active zone. In addition, we observed that neddylation is not required for the endocytosis of mGlu7, but it facilitates the ubiquitination of mGlu7 and stabilizes mGlu7 protein expression. Finally, we demonstrate that neddylation is necessary for the maturation of excitatory presynaptic terminals, providing a key role for neddylation in synaptic function.

Pathogenic GRM7 Mutations Associated with Neurodevelopmental Disorders Impair Axon Outgrowth and Presynaptic Terminal Development.

Metabotropic glutamate receptor 7 (mGlu7) is an inhibitory heterotrimeric G-protein-coupled receptor that modulates neurotransmitter release and synaptic plasticity at presynaptic terminals in the mammalian central nervous system. Recent studies have shown that rare mutations in glutamate receptors and synaptic scaffold proteins are associated with neurodevelopmental disorders (NDDs). However, the role of presynaptic mGlu7 in the pathogenesis of NDDs remains largely unknown. Recent whole-exome sequencing (WES) studies in families with NDDs have revealed that several missense mutations (c.1865G>A:p.R622Q; c.461T>C:p.I154T; c.1972C>T:p.R658W and c.2024C>A:p.T675K) or a nonsense mutation (c.1757G>A:p.W586X) in the GRM7 gene may be linked to NDDs. In the present study, we investigated the mechanistic links between GRM7 point mutations and NDD pathology. We find that the pathogenic GRM7 I154T and R658W/T675K mutations lead to the degradation of the mGlu7 protein. In particular, the GRM7 R658W/T675K mutation results in a lack of surface mGlu7 expression in heterologous cells and cultured neurons isolated from male and female rat embryos. We demonstrate that the expression of mGlu7 variants or exposure to mGlu7 antagonists impairs axon outgrowth through the mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling pathway during early neuronal development, which subsequently leads to a decrease in the number of presynaptic terminals in mature neurons. Treatment with an mGlu7 agonist restores the pathologic phenotypes caused by mGlu7 I154T but not by mGlu7 R658W/T675K because of its lack of neuronal surface expression. These findings provide evidence that stable neuronal surface expression of mGlu7 is essential for neural development and that mGlu7 is a promising therapeutic target for NDDs.SIGNIFICANCE STATEMENT Neurodevelopmental disorders (NDDs) affect brain development and function by multiple etiologies. Metabotropic glutamate receptor 7 (mGlu7) is a receptor that controls excitatory neurotransmission and synaptic plasticity. Since accumulating evidence indicates that the GRM7 gene locus is associated with NDD risk, we analyzed the functional effects of human GRM7 variants identified in patients with NDDs. We demonstrate that stable neuronal surface expression of mGlu7 is essential for axon outgrowth and presynaptic terminal development in neurons. We found that mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling and subsequent cytoskeletal dynamics are defective because of the degradation of mGlu7 variants. Finally, we show that the defects caused by mGlu7 I154T can be reversed by agonists, providing the rationale for proposing mGlu7 as a potential therapeutic target for NDDs.

The pathogenic S688Y mutation in the ligand-binding domain of the GluN1 subunit regulates the properties of NMDA receptors.

Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and D-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.

N-linked glycosylation of the mGlu7 receptor regulates the forward trafficking and transsynaptic interaction with Elfn1.

Metabotropic glutamate receptor 7 (mGlu7) regulates neurotransmitter release at the presynaptic active zone in the mammalian brain. The regulation of mGlu7 trafficking into and out of the plasma membrane by binding proteins within the C-terminal region of mGlu7 governs the bidirectional synaptic plasticity. However, the functional importance of the extracellular domain of mGlu7 has not yet been characterized. N-glycosylation is an abundant posttranslational modification that plays crucial roles in protein folding and forward trafficking, but the role of N-glycosylation in mGlu7 function remains unknown. In this study, we find that mGlu7 is N-glycosylated at four asparagine residues in heterologous cells and rat cultured neurons. We demonstrate that N-glycosylation is essential for forward transport and surface expression of mGlu7. Deglycosylated mGlu7 is retained in the ER, obstructing expression on the cell surface, and is degraded through the autophagolysosomal degradation pathway. In addition, we identify the binding domain of mGlu7 to Elfn1, a transsynaptic adhesion protein. We find that N-glycosylation of mGlu7 promotes its interaction with Elfn1, thereby enabling proper localization and stable surface expression of mGlu7 at the presynaptic active zone. These findings provide evidence that N-glycans act to modulate the surface expression, stability, and function of mGlu7.

Genome-Wide Analysis Identifies NURR1-Controlled Network of New Synapse Formation and Cell Cycle Arrest in Human Neural Stem Cells.

Nuclear receptor-related 1 (Nurr1) protein has been identified as an obligatory transcription factor in midbrain dopaminergic neurogenesis, but the global set of human NURR1 target genes remains unexplored. Here, we identified direct gene targets of NURR1 by analyzing genome-wide differential expression of NURR1 together with NURR1 consensus sites in three human neural stem cell (hNSC) lines. Microarray data were validated by quantitative PCR in hNSCs and mouse embryonic brains and through comparison to published human data, including genome-wide association study hits and the BioGPS gene expression atlas. Our analysis identified ~40 NURR1 direct target genes, many of them involved in essential protein modules such as synapse formation, neuronal cell migration during brain development, and cell cycle progression and DNA replication. Specifically, expression of genes related to synapse formation and neuronal cell migration correlated tightly with NURR1 expression, whereas cell cycle progression correlated negatively with it, precisely recapitulating midbrain dopaminergic development. Overall, this systematic examination of NURR1-controlled regulatory networks provides important insights into this protein's biological functions in dopamine-based neurogenesis.

Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation.

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in hippocampal GCs using male and female mice, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level determined by Western blot analysis was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low-frequency (LF) and high-frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 MΩ, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.SIGNIFICANCE STATEMENT The sparse activity of dentate granule cells (GCs), which is essential for pattern separation, is supported by high inhibitory inputs and low intrinsic excitability of GCs. Low excitability of GCs is thought to be attributable to a high K+ conductance at resting membrane potentials, but this study identifies Kv4.1, a depolarization-activated K+ channel, as a key ion channel that regulates firing of GCs without affecting resting membrane potentials. Kv4.1 expression is developmentally regulated and Kv4.1 currents are detected only in mature GCs that show low-frequency firing, but not in less mature high-frequency firing GCs. Furthermore, mice depleted of Kv4.1 transcripts in the dentate gyrus show impaired pattern separation, suggesting that Kv4.1 is crucial for sparse coding and pattern separation.

Nedd4 E3 ligase and beta-arrestins regulate ubiquitination, trafficking, and stability of the mGlu7 receptor.

The metabotropic glutamate receptor 7 (mGlu7) is a class C G protein-coupled receptor that modulates excitatory neurotransmitter release at the presynaptic active zone. Although post-translational modification of cellular proteins with ubiquitin is a key molecular mechanism governing protein degradation and function, mGlu7 ubiquitination and its functional consequences have not been elucidated yet. Here, we report that Nedd4 ubiquitin E3 ligase and ß-arrestins regulate ubiquitination of mGlu7 in heterologous cells and rat neurons. Upon agonist stimulation, ß-arrestins recruit Nedd4 to mGlu7 and facilitate Nedd4-mediated ubiquitination of mGlu7. Nedd4 and ß-arrestins regulate constitutive and agonist-induced endocytosis of mGlu7 and are required for mGlu7-dependent MAPK signaling in neurons. In addition, Nedd4-mediated ubiquitination results in the degradation of mGlu7 by both the ubiquitin-proteasome system and the lysosomal degradation pathway. These findings provide a model in which Nedd4 and ß-arrestin act together as a complex to regulate mGlu7 surface expression and function at presynaptic terminals.

The AMPA Receptor Subunit GluA1 is Required for CA1 Hippocampal Long-Term Potentiation but is not Essential for Synaptic Transmission.

AMPA receptors mediate the majority of excitatory glutamatergic transmission in the mammalian brain and are heterotetramers composed of GluA1-4 subunits. Despite genetic studies, the roles of the subunits in synaptic transmission and plasticity remain controversial. To address this issue, we investigated the effects of cell-specific removal of GluA1 in hippocampal CA1 pyramidal neurons using virally-expressed GluA1 shRNA in organotypic slice culture. We show that this shRNA approach produces a rapid, efficient and selective loss of GluA1, and removed > 80% of surface GluA1 from synapses. This loss of GluA1 caused a modest reduction (up to 57%) in synaptic transmission and when applied in neurons from GluA3 knock-out mice, a similar small reduction in transmission occurred. Further, we found that loss of GluA1 caused a redistribution of GluA2 to synapses that may compensate functionally for the absence of GluA1. We found that LTP was absent in neurons lacking GluA1, induced either by pairing or by a theta-burst pairing protocol previously shown to induce LTP in GluA1 knock-out mice. Our findings demonstrate a critical role of GluA1 in CA1 LTP, but no absolute requirement for GluA1 in maintaining synaptic transmission. Further, our results indicate that GluA2 homomers can mediate synaptic transmission and can compensate for loss of GluA1.

Attenuation of Experimental Autoimmune Encephalomyelitis in a Common Marmoset Model by Dendritic Cell-Modulating Anti-ICAM-1 Antibody, MD-3.

MD-3 is a novel anti-human ICAM-1 monoclonal antibody that induces T cell tolerance in humanized mice via modulation of dendritic cell differentiation and efficiently suppresses the development of collagen-induced arthritis. This effect has also been observed in xenograft rejection in nonhuman primates, where grafts survived for more than 2.5 years following MD-3 administration. Here, we show that MD-3 can attenuate experimental autoimmune encephalomyelitis (EAE) that was induced in common marmoset monkeys by immunization with human myelin oligodendrocyte glycoproteins. MD-3 administration was initiated 1 week after immunization and efficiently delayed the development of EAE phenotypes, although the disease was not completely prevented. Based on the results of histopathological examination, MD-3 treatment greatly suppressed total inflammation with respect to demyelination, as well as T cell and microglial infiltration in the brain. However, the antibody response against myelin oligodendrocyte glycoprotein was not suppressed with this treatment protocol. These observations suggest that the MD-3 antibody has beneficial effects on the treatment of EAE via the suppression of T cell-mediated cellular responses.

N-Glycosylation Regulates the Trafficking and Surface Mobility of GluN3A-Containing NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by N-glycosylation; however, limited information is available regarding the role of N-glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified N-glycan structures, including hybrid/complex forms of N-glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that N-glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of N-glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function.