Masayuki Sekiguchi

Stargazin modulates AMPA receptor gating and trafficking by distinct domains

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors mediate fast excitatory synaptic transmission in the brain. These ion channels rapidly deactivate and desensitize, which determine the time course of synaptic transmission. Here, we find that the AMPA receptor interacting protein, stargazin, not only mediates AMPA receptor trafficking but also shapes synaptic responses by slowing channel deactivation and desensitization. The cytoplasmic tail of stargazin determines receptor trafficking, whereas the ectodomain controls channel properties. Stargazin alters AMPA receptor kinetics by increasing the rate of channel opening. Disrupting the interaction of stargazin ectodomain with hippocampal AMPA receptors alters the amplitude and shape of synaptic responses, establishing a crucial function for stargazin in controlling the efficacy of synaptic transmission in the brain.

Identification of muscle-specific microRNAs in serum of muscular dystrophy animal models: promising novel blood-based markers for muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMDJ), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMDJ. Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy.

Heterogeneity and potentiation of AMPA type of glutamate receptors in rat cultured microglia.

α‐amino‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop‐ and flip‐preferring allosteric modulators of AMPA receptors, 4‐[2‐(phenylsulfonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)‐ or kainite (KA)‐induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu‐receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu‐ and KA‐induced currents were potentiated by PEPA and CTZ in a concentration‐dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell‐to‐cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT‐PCR revealed that GluR1‐3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu‐induced release of tumor necrosis factor‐α (TNF‐α) unpredictably. The increase in TNF‐α release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen‐activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF‐α release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.

N‐Acetylaspartylglutamate acts as an agonist upon homomeric NMDA receptor (NMDAR1) expressed in Xenopus oocytes

The electrophysiological effects of N‐acetylaspartylglutamate (NAAG), an endogenous peptide restrictively distributed in the central nervous system, were studied using, Xenopus oocytes injected with RNAs transcribed from cloned glutamate receptor cDNAs. NAAG induced an inward current, dose dependently, in oocytes injected with RNA for an N‐methyl‐D‐aspartate receptor subunit (NMDAR1), In contrast, the oocytes injected with RNAs for AMPA‐selective glutamate receptors (GluR1, GluR3, GluR1+GluR2 and GluR2+GluR3) scarcely responded to NAAG, and the oocytes injected with RNA for kainate receptor (GluR6) did not respond to NAAG. The half‐maximal response (ED50) value of NAAG on expressed NMDAR1 was 185 μM, which shows that NAAG is about 115‐times less potent than L‐glutamate (Glu), the ED50 of which value was 1.6, μM. The maximal current amplitude induced by NAAG was about 70% of that by Glu. NAAG‐induced current in NMDAR1‐injected oocytes was potentiated by glycine, dose‐dependently antagonized by DL‐2‐amino‐5‐phosphonovaleric acid, and blocked by magnesium ions in a voltage‐dependent fashion. These results suggest that NAAG is one of the endogenous agonists selective for NMDAR1.

Ubiquitin C-terminal hydrolase L1 regulates the morphology of neural progenitor cells and modulates their differentiation

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a component of the ubiquitin system, which has a fundamental role in regulating various biological activities. However, the functional role of the ubiquitin system in neurogenesis is not known. Here we show that UCH-L1 regulates the morphology of neural progenitor cells (NPCs) and mediates neurogenesis. UCH-L1 was expressed in cultured NPCs as well as in embryonic brain. Its expression pattern in the ventricular zone (VZ) changed between embryonic day (E) 14 and E16, which corresponds to the transition from neurogenesis to gliogenesis. At E14, UCH-L1 was highly expressed in the ventricular zone, where neurogenesis actively occurs; whereas its expression was prominent in the cortical plate at E16. UCH-L1 was very weakly detected in the VZ at E16, which corresponds to the start of gliogenesis. In cultured proliferating NPCs, UCH-L1 was co-expressed with nestin, a marker of undifferentiated cells. In differentiating cells, UCH-L1 was highly co-expressed with the early neuronal marker TuJ1. Furthermore, when UCH-L1 was induced in nestin-positive progenitor cells, the number and length of cellular processes of the progenitors decreased, suggesting that the progenitor cells were differentiating. In addition, NPCs derived from gad (UCH-L1-deficient) mice had longer processes compared with controls. The ability of UCH-L1 to regulate the morphology of nestin-positive progenitors was dependent on its binding affinity for ubiquitin but not on hydrolase activity; this result was also confirmed using gad-mouse-derived NPCs. These results suggest that UCH-L1 spatially mediates and enhances neurogenesis in the embryonic brain by regulating progenitor cell morphology.

Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures

In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4‐[2‐(phenylsulphonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. PEPA dose‐dependently potentiated AMPA‐induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell‐to‐cell variation in rat hippocampal cultures. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. Reverse transcriptase‐polymerase chain reaction analysis suggested that GluR2‐flip, GluR1‐flip, GluR2‐flop, and GluR1‐flop were abundantly expressed (in this rank order) in the cultures used. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice‐variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. These results suggest that the potentiation by PEPA alone reveals cell‐to‐cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

β-Lactotensin derived from bovine β-lactoglobulin exhibits anxiolytic-like activity as an agonist for neurotensin NTS2 receptor via activation of dopamine D1 receptor in mice

J. Neurochem. (2011) 119, 785–790.

Heightened Amygdala Long-Term Potentiation in Neurotensin Receptor Type-1 Knockout Mice

Neurotensin receptor type-1 (Ntsr1) is the main receptor subtype that underlies neurotensin (NT)-mediated modulation of the dopamine (DA) system. Although NT and DA coexist in the basolateral nucleus of the amygdala (BLA), the function of Ntsr1 in the amygdala is not well characterized. In the present study, we utilized Ntsr1 knockout (Ntsr1-KO) mice to examine the role of Ntsr1 in the amygdala. In acute brain slices of Ntsr1-KO mice, synaptic currents elicited in BLA pyramidal neurons by electrical stimulation of the lateral nucleus of the amygdala (LA) were greatly potentiated by tetanic stimulation (BLA-long-term potentiation (LTP)). Such potentiation was not evident in pyramidal neurons of wild-type mice. In the presence of an antagonist of Ntsr1, SR48692, BLA-LTP was consistently observed in the neurons of wild-type mice, suggesting that both inherited deletion and acute pharmacological blockade of Ntsr1 induce BLA-LTP. BLA-LTP in Ntsr1-KO mice was impaired by sulpiride, a DA D2-like receptor antagonist. Conversely, quinpirole, a D2-like receptor agonist, induced pronounced BLA-LTP in wild-type mice, suggesting the upregulation of D2-like receptor activity in Ntsr1-KO mice. The ratio of NMDA receptor-mediated to non-NMDA receptor-mediated synaptic currents in Ntsr1-KO mouse BLA neurons was approximately double that measured in wild-type mouse neurons. Furthermore, quinpirole potentiated NMDA receptor-mediated synaptic currents in the BLA of wild-type mice. These results suggest that, without Ntsr1, synaptic responses from the LA to BLA pyramidal neurons undergo LTP in response to tetanus stimulation through facilitation of D2-like receptor-induced activation of NMDA receptors.

Age-dependent sensitivity to glucocorticoids in the developing mouse basolateral nucleus of the amygdala

Experiences of severe trauma during childhood are thought to be risk factors for developing mental disorders, such as anxiety and mood disorders, later in life. Correspondingly, exposure of rodents to early-life stress has been shown to affect neuronal circuitry and emotional behavior in adulthood, indicating a significant impact of stress on brain development. One current hypothesis proposes that the developing central nervous system is more sensitive to environmental influences, such as stress, than the adult. To test this hypothesis, we compared long-lasting effects of systemic corticosterone (CORT) administrations in two distinct early developmental periods. Mice exposed to early-neonatal CORT treatment on postnatal days (PD) 2-4 exhibited strongly enhanced excitability of neurons of the basolateral nucleus of the amygdala (BLA) in early adolescence and displayed impaired extinction of contextually conditioned fear memory, a type of behavior in which the BLA plays an important role. Furthermore, gene-expression of NMDA receptor subunits as well as calcium-activated K(+)-channels was reduced in the amygdala. In contrast, exposure to the same CORT concentrations in a late-neonatal period (PD17-19) did not significantly affect BLA electrophysiology or extinction learning in adolescence. These results suggest age-dependent consequences of neonatal CORT exposure in amygdala neurons and provide evidence for a detrimental influence of early-neonatal stress on adolescent fear-memory processing.

Reduction of abnormal behavioral response to brief restraint by information from other mice in dystrophin-deficient mdx mice

We recently reported that dystrophin-deficient mdx mice exhibited a hypersensitive freezing response to fearful events such as brief restraint. In the present study, we ethologically characterized the restraint-induced freezing response in mdx mice. This response was evident when restrained mdx mice were released into a new cage or their home cage, but it was remarkably reduced in cages in which other individuals (wild-type mice that had never been reared with the tested mice) had been reared (the resident mice were removed prior to testing). Reciprocally, exploratory behaviors of restrained mdx mice were outstandingly enhanced in the cages in which other individuals had been reared, suggesting the possibility that scent deposited by residents induced exploration in mdx mice. These results suggest that restraint-induced freezing response in mdx mice is influenced by the attention state of the mouse.