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.

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.

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.

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.

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.

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.

Metabotropic glutamate receptor trafficking.

The metabotropic glutamate receptors (mGlu receptors) are G protein-coupled receptors that bind to the excitatory neurotransmitter glutamate and are important in the modulation of neuronal excitability, synaptic transmission, and plasticity in the central nervous system. Trafficking of mGlu receptors in and out of the synaptic plasma membrane is a fundamental mechanism modulating excitatory synaptic function through regulation of receptor abundance, desensitization, and signaling profiles. In this review, we cover the regulatory mechanisms determining surface expression and endocytosis of mGlu receptors, with particular focus on post-translational modifications and receptor-protein interactions. The literature we review broadens our insight into the precise events defining the expression of functional mGlu receptors at synapses, and will likely contribute to the successful development of novel therapeutic targets for a variety of developmental, neurological, and psychiatric disorders.

Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) contributes to impaired glucose tolerance, leading to type 2 diabetes (T2D); however, the precise mechanisms and target molecules that are involved remain unclear. Activating transcription factor 3 (ATF3) is associated with ß-cell dysfunction that is induced by severe stress signals in T2D. We aimed to explore the exact functional role of ATF3 as a mechanistic link between hepatic steatosis and T2D development. METHODS: Zucker diabetic fatty (ZDF) rats were utilized for animal experiments. An in vivo-jetPEI siRNA delivery system against ATF3 was used for loss-of-function experiments. We analyzed the baseline cross-sectional data derived from the biopsy-proven NAFLD registry (n=322). Human sera and liver tissues were obtained from 43 patients with biopsy-proven NAFLD and from seven healthy participants. RESULTS: ATF3 was highly expressed in the livers of ZDF rats and in human participants with NAFLD and/or T2D. Insulin resistance and hepatic steatosis were associated with increased ATF3 expression and decreased fatty acid oxidation via mitochondrial dysfunction and were attenuated by in vivo ATF3 silencing. Knockdown of ATF3 also ameliorated glucose intolerance, impaired insulin action, and inflammatory responses in ZDF rats. In patients with NAFLD and/or T2D, a significant positive correlation was observed between hepatic ATF3 expression and surrogate markers of T2D, mitochondrial dysfunction, and macrophage infiltration. CONCLUSIONS: Increased hepatic ATF3 expression is closely associated with hepatic steatosis and incident T2D; therefore, ATF3 may serve as a potential therapeutic target for NAFLD and hepatic steatosis-induced T2D. LAY SUMMARY: Hepatic activating transcription factor 3 (ATF3) may play an important role in oxidative stress-mediated hepatic steatosis and the development of type 2 diabetes (T2D) in a Zucker diabetic fatty (ZDF) rat model and in human patients with non-alcoholic fatty liver disease (NAFLD). Therefore, ATF3 may be a useful biomarker for predicting the progression of NAFLD and the development of T2D. Furthermore, given the significant association between hepatic ATF3 expression and both hepatic steatosis and impaired glucose homeostasis, in vivo ATF3 silencing may be a potential central strategy for preventing and managing NAFLD and T2D.

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum.

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.

Inhibitory RNA Aptamers of Tau Oligomerization and Their Neuroprotective Roles against Proteotoxic Stress.

Tau is a cytosolic protein that functions in the assembly and stabilization of axonal microtubule networks. Its oligomerization may be the rate-limiting step of insoluble aggregate formation, which is a neuropathological hallmark of Alzheimer's disease (AD) and a number of other tauopathies. Recent evidence indicates that soluble tau oligomers are the toxic species for tau-mediated pathology during AD progression. Herein, we describe novel RNA aptamers that target human tau and were identified through an in vitro selection process. These aptamers significantly inhibited the oligomerization propensity of tau both in vitro and in cultured cell models of tauopathy without affecting the half-life of tau. Tauopathy model cells treated with the aptamers were less sensitized to proteotoxic stress induced by tau overexpression. Moreover, the tau aptamers significantly alleviated synthetic tau oligomer-mediated neurotoxicity and dendritic spine loss in primary hippocampal neurons. Thus, our study demonstrates that delaying tau assembly with RNA aptamers is an effective strategy for protecting cells under various neurodegenerative stresses originating from pathogenic tau oligomerization.

TARP γ-8 glycosylation regulates the surface expression of AMPA receptors.

TARP [transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor regulatory protein] γ-8 is an auxiliary subunit of AMPA receptors that is widely distributed in the hippocampus. It has been shown that TARP γ-8 promotes surface expression of AMPA receptors; however, how TARP γ-8 regulates the expression of AMPA receptors remains unclear. In the present study, we examined the effect of TARP glycosylation on AMPA receptor trafficking. We first showed that TARP γ-8 is an N-glycosylated protein, which contains two glycosylation sites, Asn53 and Asn56, and compared this with the glycosylation of TARP γ-2 and the AMPA receptor auxiliary protein CNIH-2 (cornichon homologue 2). We next examine the effect of TARP glycosylation on TARP trafficking and also on AMPA receptor surface expression. We find that TARP γ-8 glycosylation is critical for surface expression of both TARP γ-8 and GluA1 in heterologous cells and neurons. Specifically, knockdown of TARP γ-8 causes a decrease in both total and surface AMPA receptors. We find that the expression of unglycosylated TARP γ-8 in cultured neurons is unable to restore GluA1 expression fully. Furthermore, when the maturation of TARP γ-8 is impaired, a large pool of immature GluA1 is retained intracellularly. Taken together, our data reveal an important role for the maturation of TARP γ-8 in the trafficking and function of the AMPA receptor complex.

DJ-1 associates with lipid rafts by palmitoylation and regulates lipid rafts-dependent endocytosis in astrocytes.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disease. Several genes have been associated with familial type PD, providing tremendous insights into the pathogenesis of PD. Gathering evidence supports the view that these gene products may operate through common molecular pathways. Recent reports suggest that many PD-associated gene products, such as α-synuclein, LRRK2, parkin and PINK1, associate with lipid rafts and lipid rafts may be associated with neurodegeneration. Here, we observed that DJ-1 protein also associated with lipid rafts. Palmitoylation of three cysteine residues (C46/53/106) and C-terminal region of DJ-1 were required for this association. Lipopolysaccharide (LPS) induced the localization of DJ-1 into lipid rafts in astrocytes. The LPS-TLR4 signaling was more augmented in DJ-1 knock-out astrocytes by the impairment of TLR4 endocytosis. Furthermore, lipid rafts-dependent endocytosis including the endocytosis of CD14, which play a major role in regulating TLR4 endocytosis was also impaired, but clathrin-dependent endocytosis was not. This study provides a novel function of DJ-1 in lipid rafts, which may contribute the pathogenesis of PD. Moreover, it also provides the possibility that many PD-related proteins may operate through common molecular pathways in lipid rafts.

Distance-dependent scaling of AMPARs is cell-autonomous and GluA2 dependent.

The extensive dendritic arbor of a pyramidal cell introduces considerable complexity to the integration of synaptic potentials. Propagation of dendritic potentials is largely passive, in contrast to regenerative axonal potentials that are maintained by voltage-gated sodium channels, leading to a declination in amplitude as dendritic potentials travel toward the soma in a manner that disproportionally affects distal synaptic inputs. To counteract this amplitude filtering, Schaffer collateral synapses onto CA1 pyramidal cells contain a varying number of AMPA receptors (AMPARs) per synapse that increases with distance from the soma, a phenomenon known as distance-dependent scaling. Here, we undertake an investigation into the molecular mechanisms of distance-dependent scaling. Using dendritic recordings from rat pyramidal neurons, we confirm the basic scaling phenomenon and find that it is expressed and can be manipulated cell autonomously. Finally, we show that it depends on the presence of both a reserve pool of AMPARs and the AMPAR subunit GluA2.

Regulation of metabotropic glutamate receptor 7 (mGluR7) internalization and surface expression by Ser/Thr protein phosphatase 1.

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Red emissive two-photon probe for real-time imaging of mitochondria trafficking.

Mitochondria trafficking plays an essential role for supplying energy in the neuronal system. We report here a red emissive two-photon probe for mitochondria (CMT-red) that showed high selectivity and robust staining ability for mitochondria, high photostability under a two-photon microscopy imaging condition, and low cytotoxicity. This probe can be easily loaded into live cells and tissue and used for real-time, high resolution imaging of the mitochondria trafficking in primary cortical neurons as well as in rat hippocampal tissue.

Astrocytes, but not microglia, rapidly sense H2O2via STAT6 phosphorylation, resulting in cyclooxygenase-2 expression and prostaglandin release.

Emerging evidence has established that astrocytes, once considered passive supporting cells that maintained extracellular ion levels and served as a component of the blood-brain barrier, play active regulatory roles during neurogenesis and in brain pathology. In the current study, we demonstrated that astrocytes sense H(2)O(2) by rapidly phosphorylating the transcription factor STAT6, a response not observed in microglia. STAT6 phosphorylation was induced by generators of other reactive oxygen species (ROS) and reactive nitrogen species, as well as in the reoxygenation phase of hypoxia/reoxygenation, during which ROS are generated. Src-JAK pathways mediated STAT6 phosphorylation upstream. Experiments using lipid raft disruptors and analyses of detergent-fractionated cells demonstrated that H(2)O(2)-induced STAT6 phosphorylation occurred in lipid rafts. Under experimental conditions in which H(2)O(2) did not affect astrocyte viability, H(2)O(2)-induced STAT6 phosphorylation resulted in STAT6-dependent cyclooxygenase-2 expression and subsequent release of PGE(2) and prostacyclin, an effect also observed in hypoxia/reoxygenation. Finally, PGs released from H(2)O(2)-stimulated astrocytes inhibited microglial TNF-α expression. Accordingly, our results indicate that ROS-induced STAT6 phosphorylation in astrocytes can modulate the functions of neighboring cells, including microglia, through cyclooxygenase-2 induction and subsequent release of PGs. Differences in the sensitivity of STAT6 in astrocytes (highly sensitive) and microglia (insensitive) to phosphorylation following brief exposure to H(2)O(2) suggest that astrocytes can act as sentinels for certain stimuli, including H(2)O(2) and ROS, refining the canonical notion that microglia are the first line of defense against external stimuli.

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.

DJ-1 facilitates the interaction between STAT1 and its phosphatase, SHP-1, in brain microglia and astrocytes: A novel anti-inflammatory function of DJ-1.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra. Importantly, altered astrocyte and microglial functions could contribute to neuronal death in PD. In this study, we demonstrate a novel mechanism by which DJ-1 (PARK7), an early onset autosomal-recessive PD gene, negatively regulates inflammatory responses of astrocytes and microglia by facilitating the interaction between STAT1 and its phosphatase, SHP-1 (Src-homology 2-domain containing protein tyrosine phosphatase-1). Astrocytes and microglia cultured from DJ-1-knockout (KO) mice exhibited increased expression of inflammatory mediators and phosphorylation levels of STAT1 (p-STAT1) in response to interferon-gamma (IFN-γ) compared to cells from wild-type (WT) mice. DJ-1 deficiency also attenuated IFN-γ-induced interactions of SHP-1 with p-STAT1 and STAT1, measured 1 and 12h after IFN-γ treatment, respectively. Subsequent experiments showed that DJ-1 directly interacts with SHP-1, p-STAT1, and STAT1. Notably, DJ-1 bound to SHP-1 independently of IFN-γ, whereas the interactions of DJ-1 with p-STAT1 and STAT1 were dependent on IFN-γ. Similar results were obtained in brain slice cultures, where IFN-γ induced much stronger STAT1 phosphorylation and inflammatory responses in KO slices than in WT slices. Moreover, IFN-γ treatment induced neuronal damage in KO slices. Collectively, these findings suggest that DJ-1 may function as a scaffold protein that facilitates SHP-1 interactions with p-STAT1 and STAT1, thereby preventing extensive and prolonged STAT1 activation. Thus, the loss of DJ-1 function may increase the risk of PD by enhancing brain inflammation.

Functional comparison of the effects of TARPs and cornichons on AMPA receptor trafficking and gating.

Glutamate receptors of the AMPA subtype (AMPARs) mediate fast synaptic transmission in the brain. These ionotropic receptors rely on auxiliary subunits known as transmembrane AMPAR regulatory proteins (TARPs) for both trafficking and gating. Recently, a second family of AMPAR binding proteins, referred to as cornichons, were identified and also proposed to function as auxiliary subunits. Cornichons are transmembrane proteins that modulate AMPAR function in expression systems much like TARPs. In the present study we compare the role of cornichons in controlling AMPA receptor function in neurons and HEK cells to that of TARPs. Cornichons mimic some, but not all, of the actions of TARPs in HEK cells; their role in neurons, however, is more limited. Although expressed cornichons can affect the trafficking of AMPARs, they were not detected on the surface of neurons and failed to alter the kinetics of endogenous AMPARs. This neuronal role is more consistent with that of an endoplasmic reticulum (ER) chaperone rather than a bona fide auxiliary subunit.