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.

Establishment of a reference interval for glycated albumin based on medical check-up data from multiple medical institutions.

Objectives: Data were collected to establish a reference interval for glycated albumin (GA), as well as to calculate a cutoff value for diagnosing diabetes mellitus and the GA level corresponding to a 75-g oral glucose tolerance test (OGTT) 2 h plasma glucose (2h-PG) level of 200 mg/dL.Methods: This study involved 1,843 subjects who were undergoing medical check-ups at several medical institutions and whose HbA1c and GA levels had been measured by OGTT.Results: The GA reference interval that was calculated based on the data obtained from study subjects with normal glucose tolerance was 12.1-17.1%. Using standardized major axis regression, the levels that corresponded to an OGTT 2h-PG level of 11.1 mmol/L were a GA level of 17.5% and an HbA1c level of 47.5 mmol/mol. A receiver-operating characteristic curve analysis was used to calculate the points at which sensitivity and specificity matched as the cutoff values, and the results yielded a GA level of 15.0% (sensitivity 69.3%).Conclusions: The GA reference interval was calculated to be 12.1-17.1%. We propose a GA level of 17.4% as a cutoff value to diagnose diabetes mellitus and a GA level of 15.0% as a screening cutoff value for diabetes mellitus, taking previous reports into account.

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.

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.

Partnership Between Japan and the United States for Early Development of Pediatric Medical Devices　- Harmonization By Doing for Children.

BACKGROUND: The Harmonization By Doing (HBD) program was established in 2003 as a partnership among stakeholders of academia, industry and regulatory agencies in Japan and the United States, with a primary focus on streamlining processes of global medical device development for cardiovascular medical devices. While HBD has traditionally focused on development of devices intended to treat conditions prevalent in adults, in 2016, HBD established the "HBD-for-Children" program, which focuses on the development of pediatric devices as the development of medical devices for pediatric use lags behind that of medical devices for adults in both countries.Methods and Results:Activities of the program have included: (1) conducting a survey with industry to better understand the challenges that constrain the development of pediatric medical devices; (2) categorizing pediatric medical devices into five categories based on global availability and exploring concrete solutions for the early application and regulatory approval in both geographies; and (3) facilitating global clinical trials of pediatric medical devices in both countries. CONCLUSIONS: The establishment of the HBD-for-Children program is significant because it represents a global initiative for the introduction of pediatric medical devices for patients in a timely manner. Through the program, academia, industry and regulatory agencies can work together to facilitate innovative pediatric device development from a multi-stakeholder perspective. This activity could also encourage industry partners to pursue the development of pediatric medical devices.

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.

Ginkgolic acid promotes autophagy-dependent clearance of intracellular alpha-synuclein aggregates.

The accumulation of intracytoplasmic inclusion bodies (Lewy bodies) composed of aggregates of the alpha-synuclein (α-syn) protein is the principal pathological characteristic of Parkinson's disease (PD) and may lead to degeneration of dopaminergic neurons. To date there is no medication that can promote the efficient clearance of these pathological aggregates. In this study, the effect on α-syn aggregate clearance of ginkgolic acid (GA), a natural compound extracted from Ginkgo biloba leaves that inhibits SUMOylation amongst other pathways, was assessed in SH-SY5Y neuroblastoma cells and rat primary cortical neurons. Depolarization of SH-SY5Y neuroblastoma cells and rat primary cortical neurons with KCl was used to induce α-syn aggregate formation. Cells pre-treated with either GA or the related compound, anacardic acid, revealed a significant decrease in intracytoplasmic aggregates immunopositive for α-syn and SUMO-1. An increased frequency of autophagosomes was also detected with both compounds. GA post-treatment 24 h after depolarization also significantly diminished α-syn aggregate bearing cells, indicating the clearance of pre-formed aggregates. Autophagy inhibitors blocked GA-dependent clearance of α-syn aggregates, but not increased autophagosome frequency. Western analysis revealed that the reduction in α-syn aggregate frequency obtained with GA pre-treatment was accompanied by little change in the abundance of SUMO conjugates. The current findings show that GA can promote autophagy-dependent clearance of α-syn aggregates and may have potential in disease modifying therapy.

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.

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.

The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, ß, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors.

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.

A case of Dravet syndrome complicated by human herpesvirus-6 infection-associated acute encephalopathy and choreoathetosis.

We report the case of a 14-month-old girl with Dravet syndrome carrying a splice-site mutation of c. 1170+1G>A on neuronal sodium channel alpha 1 subunit (SCN1A). She had a history of recurrent febrile or afebrile seizures since 4 months of age and developed acute encephalopathy due to infection with human herpesvirus-6, which presented with high grade fever, severe consciousness disturbances, and prolonged and clustered seizures. Electroencephalography showed a generalized slow activity. Intensive treatments, including mechanical ventilation, continuous thiopental infusion, and high-dose steroid therapy were initiated, and she gradually recovered. During the recovery phase, choreoathetosis-like involuntary movements appeared on the face and limbs, which were treated successfully with haloperidol. MRI findings during the acute phase were normal; however, diffuse cerebral atrophy became evident during the recovery phase. Single photon emission computed tomography (SPECT) of the brain revealed decreased cerebral perfusion over bilateral frontal and temporal lobes; however, perfusion of the occipital lobes, basal ganglia, and cerebellum remained normal. The patient showed serious developmental regression at discharge, with the loss of head control and meaningful words. Patient's clinical course and the findings of SPECT resembled those of acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), with the exception of lack of reduced diffusion of the subcortical white matter on the acute phase MRI and the prolonged and severe clinical symptoms. It has been reported that patients with Dravet syndrome are prone to complications, including various types of acute encephalopathies. Therefore, clinicians should carefully manage prolonged febrile seizures in patients with Dravet syndrome.

Glutamine synthetase stability and subcellular distribution in astrocytes are regulated by γ-aminobutyric type B receptors.

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity.

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.

Identification of two novel Shank3 transcripts in the developing mouse neocortex.

SHANK3 is a synaptic scaffolding protein enriched in the post-synaptic density of excitatory synapses. Since several SHANK3 mutations have been identified in a particular phenotypic group of patients with autism spectrum disorder (ASD), SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Several SHANK3 isoforms are known to be produced in the developing brain, but they have not been fully investigated. Here, we identified two different amino-terminus truncated Shank3 transcripts. One transcript, designated as Shank3c-3, produces an isoform that contains the entire carboxyl-terminus, but the other transcript, designated as Shank3c-4, produces a carboxyl-terminus truncated isoform. During development, expression of the novel Shank3 transcripts increased after birth, transiently decreased at P14 and then gradually increased again thereafter. We also determined that methyl CpG-binding protein 2 (MeCP2) is involved in regulating expression of the novel Shank3 transcripts. MeCP2 is a transcriptional regulator that has been identified as the causative molecule of Rett syndrome, a neurodevelopmental disorder that includes autistic behavior. We demonstrated a difference between the expression of the novel Shank3 transcripts in wild-type mice and Mecp2-deficient mice. These findings suggest that the SHANK3 isoforms may be implicated in the synaptic abnormality in Rett syndrome. SHANK3 is a synaptic scaffolding protein and is suspected of being implicated in the pathogenesis and neuropathology of ASD. We here identified two different amino-terminus truncated Shank3 transcripts, Shank3c-3 and Shank3c-4, expressed from the intron 10 of the Shank3 gene, and also suggested the epigenetic regulation of their expression via methyl CpG-binding protein 2 (MeCP2) that has been identified as the causative molecule of Rett syndrome.

Disrupted Cl(-) homeostasis contributes to reductions in the inhibitory efficacy of diazepam during hyperexcited states.

The K(+) -Cl(-) cotransporter type 2 is the major Cl(-) extrusion mechanism in most adult neurons. This process in turn leads to Cl(-) influx upon activation of γ-aminobutyric acid type A (GABAA ) receptors and the canonical hyperpolarising inhibitory postsynaptic potential. Several neurological disorders are treated with drugs that target and enhance GABAA receptor signaling, including the commonly used benzodiazepine diazepam and the anesthetic propofol. Some of these disorders are also associated with deficits in GABAA signaling and become less sensitive to therapeutic drugs that target GABAA receptors. To date, it is unknown if alterations in the neuronal Cl(-) gradient affect the efficacies of diazepam and propofol. We therefore used the in vitro model of glutamate-induced hyperexcitability to test if alterations in the Cl(-) gradient affect the efficacy of GABAA modulators. We exclusively utilised the gramicidin perforated-patch-clamp configuration to preserve the endogenous Cl(-) gradient in rat neurons. Brief exposure to glutamate reduced the inhibitory efficacy of diazepam within 5 min, which was caused by the collapse of the Cl(-) gradient, and not due to reductions in GABAA receptor number. Unlike diazepam, propofol retained its efficacy by shunting the membrane conductance despite the glutamate-induced appearance of depolarising GABAA -mediated currents. Similarly, pharmacological inhibition of K(+) -Cl(-) cotransporter type 2 by furosemide disrupted Cl(-) homeostasis and reduced the efficacy of diazepam but not propofol. Collectively our results suggest that pathological hyperexcitable conditions could cause the rapid accumulation of intracellular Cl(-) and the appearance of depolarising GABAA -mediated currents that would decrease the efficacy of diazepam.

Influences of cycloplegia with topical atropine on ocular higher-order aberrations.

PURPOSE: To investigate the influence of cycloplegia with topical atropine on higher-order aberrations (HOAs) of the eye in children. DESIGN: Prospective, noncomparative study. PARTICIPANTS: Twenty-three eyes of 23 subjects (11 boys and 12 girls) with hyperopia, ranging in age from 3 to 12 years (average, 5.74 years), were enrolled in this study. METHODS: Refraction and wavefront aberration were evaluated before and after cycloplegia with topical instillation of 1% atropine solution administered twice daily for 1 week. Ocular and corneal HOAs were measured simultaneously, and individual Zernike components were analyzed for a 6-mm pupil up to the 6th order. All these parameters were compared before and after cycloplegia. MAIN OUTCOME MEASURES: Ocular and corneal HOAs before and after cycloplegia. RESULTS: Cycloplegia with topical atropine significantly increased spherical equivalent refraction from +1.92±1.53 diopters (D) to +3.10±1.61 D (P<0.01, paired t test). Ocular HOAs significantly increased from 0.282±0.086 µm to 0.316±0.087 µm for coma-like aberrations (P=0.02), from 0.169±0.058 µm to 0.192±0.076 µm for spherical-like aberrations (P=0.02), and from 0.333±0.093 µm to 0.377±0.095 µm for total HOAs (P=0.01). Corneal HOAs did not change after cycloplegia. As for individual Zernike components, significant changes were found in C3(1) and C4(0) after cycloplegia (P<0.05). The induced changes in C4(0) were correlated significantly with those in spherical equivalent refraction (Pearson's correlation coefficient, R=0.45; P=0.03). CONCLUSIONS: Cycloplegia with atropine induced significant hyperopic shift and increases in ocular HOAs in children, leading to reduction in optical quality of the eye. It may be that physiologic tonic accommodation plays a role in improving retinal image quality by decreasing HOAs and refractive errors. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

[Comparison between subtraction skin electrodes and corneal-contact electrodes in flash electroretinograms].

PURPOSE: To compare electroretinogram (ERG) responses measured with a skin electrode with those from a corneal-contact electrode in healthy adults. METHOD: Sixty two eyes of 31 healthy adults (8 men and 23 women) were enrolled (mean age 34.0 +/- 8.1 years). ERG responses for full-field and single flash stimuli was recorded with skin electrodes and corneal-contact electrodes. Morphology of the ERG waveform, a-wave and b-wave amplitudes, b/a ratios, and a-wave and b-wave implicit times were compared between the two electrode measurements. Correlations between the two measurements were also examined. RESULTS: All features of the ERG waveform were identifiable using the skin electrodes. In the skin electrode ERGs, a-wave and b-wave amplitudes, and the b/a ratios were smaller (p<0.001) and the a-wave and b-wave implicit times were shorter (p<0.001) than those in the corneal-contact electrode ERGs. The B-wave amplitudes, b/a ratios and the a-wave and b-wave implicit times showed significant correlations between the two electrodes (p<0.01). In 16 eyes the OP4 was detected as the b-wave. CONCLUSION: The skin electrode ERG had shorter amplitudes and shorter implicit times than the corneal electrode ERGs, but corresponded well in its morphology to the corneal-contact electrode ERGs. Skin electrodes ERG would be useful for the examination of child patients and patients with corneal diseases. Further study is required for clinical application.

Involvement of activated microglia in increased vulnerability of motoneurons after facial nerve avulsion in presymptomatic amyotrophic lateral sclerosis model rats.

Activated microglia are observed in various neurodegenerative diseases and are thought to be involved in the processes of neuronal cell death. Motoneuron damage in the facial nuclei after facial nerve avulsion is accelerated in presymptomatic transgenic rats expressing human mutant Cu(2+) /Zn(2+) superoxide dismutase 1 (SOD1), compared with that in wild-type rats. To reveal the functional role of microglia in motoneuronal death, we investigated the microglial response after facial nerve avulsion in presymptomatic mutant SOD1(H46R) (mSOD1(H46R) ) rats. At 3 days after avulsion, microglial clusters were observed in the facial nuclei of both wild-type and mSOD1(H46R) rats. The numbers of microglial clusters, proliferating microglia, and microglial attachments to motoneurons were significantly higher in mSOD1(H46R) rats, compared with those in wild-type rats. Immunopositive signals for the phagocytic marker ED1 were significantly stronger in mSOD1(H46R) rats, compared with that in wild-type rats, at 2 weeks after avulsion. Furthermore, primary microglia prepared from mSOD1(H46R) rats showed enhanced phagocytic activity, compared with that in wild-type rats. The expression of P2Y(12) mRNA was higher in the facial nuclei of mSOD1(H46R) rats, compared with that in wild-type rats. A laser microdissection system revealed that the expression of ATF3 mRNA was higher in the motoneurons of mSOD1(H46R) rats, compared with that in wild-type rats, at 2 days after avulsion. These results indicate that microglial activation in response to early neuronal damage increased in mSOD1(H46R) rats and suggest that the enhanced activation of microglia may lead to an increase in the vulnerability of motoneurons after avulsion in mSOD1(H46R) rats.

Adenosine A3 receptor is involved in ADP-induced microglial process extension and migration.

The extension of microglial processes toward injured sites in the brain is triggered by the stimulation of the purinergic receptor P2Y(12) by extracellular ATP. We recently showed that P2Y(12) stimulation by ATP induces microglial process extension in collagen gels. In the present study, we found that a P2Y(12) agonist, 2-methylthio-ADP (2MeSADP), failed to induce the process extension of microglia in collagen gels and that co-stimulation with adenosine, a phosphohydrolytic derivative of ATP, and 2MeSADP restored the chemotactic process extension. An adenosine A3 receptor (A3R)-selective agonist restored the chemotactic process extension, but other receptor subtype agonists did not. The removal of adenosine by adenosine deaminase and the blocking of A3R by an A3R-selective antagonist inhibited ADP-induced process extension. The A3R antagonist inhibited ADP-induced microglial migration, and an A3R agonist promoted 2MeSADP-stimulated migration. ADP and the A3R agonist activated Jun N-terminal kinase in microglia, and a Jun N-terminal kinase inhibitor inhibited the ADP-induced process extension. An RT-PCR analysis showed that A1R and A3R were expressed by microglia sorted from adult rat brains and that the A2AR expression level was very low. These results suggested that A3R signaling may be involved in the ADP-induced process extension and migration of microglia.