Maya Yamazaki

The Endocannabinoid 2-Arachidonoylglycerol Produced by Diacylglycerol Lipase α Mediates Retrograde Suppression of Synaptic Transmission

Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.

Motor Neuron-specific Disruption of Proteasomes, but not Autophagy, Replicates Amyotrophic Lateral Sclerosis.*

Background: It is not clear how protein degradation systems are involved in ALS pathogenesis. Results: Transgenic mice with motor neuron-specific knock-out of proteasomes, but not of autophagy showed ALS phonotypes. Conclusion: Dysfunction of proteasome may primarily contribute to the pathogenesis of ALS than that of autophagy. Significance: Modulation of proteasome function is a promising approach toward treatment of ALS. Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.

Anti-aquaporin 4 antibody in selected Japanese multiple sclerosis patients with long spinal cord lesions:

Multiple sclerosis (MS) in Asian populations is often characterized by the selective involvement of the optic nerve (ON) and spinal cord (SP) (OSMS) in contrast to classic MS (CMS), where frequent lesions are observed in the cerebrum, cerebellum or brainstem. In Western countries, inflammatory demyelinating disease preferentially involving the ON and SP is called neuromyelitis optica (NMO). Recently, Lennon et al. discovered that NMO-IgG, shown to bind to aquaporin 4 (AQP4), could be a specific marker of NMO and also of Japanese OSMS whose clinical features were identical to NMO having long spinal cord lesions extending over three vertebral segments (LCL). To examine this antibody in larger populations of Japanese OSMS patients in order to know its epidemiological and clinical spectra, we established an immunohistochemical detection system for the anti-AQP4 antibody (AQP4-Ab) using the AQP4-transfected human embryonic kidney cell line (HEK-293) and confirmed AQP4-Ab positivity together with the immunohistochemical staining pattern of NMO-IgG in approximately 60% of Japanese OSMS patients with LCL. Patients with OSMS without LCL and those with CMS were negative for this antibody. Our results accorded with those of Lennon et al. suggest that Japanese OSMS with LCL may have an underlying pathogenesis in common with NMO. Multiple Sclerosis 2007; 13: 850—855. http://msj.sagepub.com

Abundant distribution of TARP γ-8 in synaptic and extrasynaptic surface of hippocampal neurons and its major role in AMPA receptor expression on spines and dendrites

Transmembrane α‐amino‐3‐hydroxyl‐5‐isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP γ‐8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of γ‐8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of γ‐8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, γ‐8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of γ‐8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the γ‐8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35–37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber–CA3 synapses. Therefore, γ‐8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.

A novel action of stargazin as an enhancer of AMPA receptor activity

Stargazin (gamma-2) is disrupted in the ataxic and epileptic mutant mouse, stargazer (stg). The striking defect in the stg cerebellum is the lack of functional AMPA receptors on granule cells. Recently, it has been reported that gamma-2 and its related molecules are crucial for the surface expression, synaptic targeting and recycling of AMPA receptors, being termed collectively as the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is still unclear whether TARPs directly modulate AMPA receptor activity. Here we report that coexpression of GluRalpha1 (GluR1) with gamma-2 using HEK293 cells and Xenopus oocytes markedly enhanced glutamate-induced currents. This effect was far beyond the increase of AMPA receptor surface expression and accompanied by increased glutamate affinity and subunit cooperativity. Other member of TARPs (gamma-3, gamma-4, and gamma-8) also enhanced the current response through the AMPA receptors. The enhancing effect by gamma-2 coexpression was further observed for homomeric GluRalpha2 (GluR2) channels, which, when expressed alone, are known to produce only a small or negligible current response. These results suggest that gamma-2 not only promotes AMPA receptor surface expression but also directly modulates AMPA receptor activity.

Unique inhibitory synapse with particularly rich endocannabinoid signaling machinery on pyramidal neurons in basal amygdaloid nucleus

2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB1 receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB1 is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short- and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here, we clarified a unique molecular convergence of DGLα, CB1, and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGLα was preferentially recruited to concave somatic membrane of postsynaptic pyramidal neurons, whereas invaginating presynaptic terminals highly expressed CB1, MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGLα and CB1 were expressed weakly at axospinous excitatory synapses. Consistent with these morphological data, thresholds for DGLα-mediated depolarization-induced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of 2-AG-mediated control of the excitability of BA pyramidal neurons.

Postsynaptic P/Q-type Ca2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum

Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Cav2.1 P/Q-type Ca2+ channel, the major voltage-dependent Ca2+ channel in PCs. In the PC-selective Cav2.1 knockout mice, Ca2+ transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca2+ channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.

Roles of nitric oxide as a vasodilator in neurovascular coupling of mouse somatosensory cortex

Neural activities trigger regional vasodilation in the brain. Diffusible messengers such as nitric oxide (NO) and prostanoids are considered to work as vasodilators in neurovascular coupling. However, their roles are still controversial. In the present study, cortical images of neural activities and vasodilation were recorded through the intact skull of C57BL/6 mice anesthetized with urethane. Flavoprotein fluorescence responses elicited by vibratory hindpaw stimulation were followed by darkening of arteriole images reflecting vasodilation in the somatosensory cortex. Vasodilation was also observed in light reflection images at the wavelength of 570 nm in the same mice. We perfused the surface of the cortex under the skull with 100 microM N(G)-nitro-l-arginine (l-NA), an inhibitor of NO synthase (NOS), and 10 microM indomethacin, an inhibitor of cyclooxygenase (COX). These drugs suppressed vasodilation without changing flavoprotein fluorescence responses. A mixture of l-NA and indomethacin almost completely eliminated vasodilation. In mice lacking neuronal NOS (nNOS), activity-dependent vasodilation was significantly suppressed compared with that in littermate control mice, while that in mice lacking cytosolic phospholipase A2 alpha (cPLA2alpha) was unchanged. These results indicate that NO works as a vasodilator in neurovascular coupling of the mouse somatosensory cortex.

Molecular and Morphological Configuration for 2-Arachidonoylglycerol-Mediated Retrograde Signaling at Mossy Cell–Granule Cell Synapses in the Dentate Gyrus

2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)–granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase α (DGLα) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC–GC synapses, as they were abolished in DGLα-knock-out mice. Immunohistochemistry revealed that DGLα was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB1 receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC–GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC–GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC–GC synapses by escaping from enzymatic degradation. This molecular–anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.

Differential palmitoylation of two mouse glutamate receptor interacting protein 1 forms with different N-terminal sequences.

Glutamate receptor interacting protein (GRIP) is a member of the PDZ domain-containing protein family that is localized in the postsynaptic density area. This protein has been reported to interact specifically with the C-termini of AMPA-selective glutamate receptor channel subunits, GluRalpha2 and GluRalpha3 through its PDZ domains. To clarify the physiological functions of GRIP, we cloned mouse GRIP1, and found that there are three sites for alternative splicing and two putative translational start codons by characterizing GRIP1 cDNA clones and reverse transcription-polymerase chain reaction products. Metabolic labeling of COS-7 cells expressing two N-terminal GRIP1 proteins demonstrated that these proteins differed in their pattern of palmitoylation. These findings suggested that the molecular diversity of GRIP1 underlies the localization and functional heterogeneity of this protein.