Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

生きた動物脳内で発現する神経伝達物質受容体に目印を付ける新手法を開発 --遺伝子操作を伴わず、生体内でたんぱく質の機能解析が可能に--. 京都大学プレスリリース. 2024-02-05.Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse–chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions

Effects of in vivo sodium azide administration on the immunohistochemical localization of kynurenine aminotransferase in the rat brain

Endogenous excitotoxins that act on receptors of cerebral excitatory amino acids play important roles in the pathogenesis of excitotoxic brain diseases. Activation of excitatory amino acid receptors results in neuronal death characteristic of these disorders. Kynurenic acid, a powerful endogenous excitatory amino acid receptor antagonist, which is therefore widely regarded as a potent neuroprotective agent, is produced from its biological precursor, L-kynurenine, by the action of the enzyme kynurenine aminotransferase-I. The chemical hypoxia induced by mitochondrial toxins produces a secondary excitotoxicity, leading to the activation of N-methyl-D-aspartate receptors. Accordingly, sodium azide, an inhibitor of cytochrome oxidase, induces the release of excitotoxins via an energy impairment and this, in turn, results in neurodegeneration. Since energy-dependent secondary excitotoxic mechanisms also account for the pathogenesis of neurodegenerative diseases, a study was made of the effects of sodium azide on the immunohistochemical localization of kynurenine aminotransferase-I. After in vivo administration of sodium azide for five days, a markedly decreased glial kynurenine aminotransferase-I immunoreactivity was found by immunohistochemical techniques in the glial cells of the striatum, hippocampus, dentate gyrus and temporal cortex; at the same time, kynurenine aminotransferase-I started to be expressed by nerve cells which had not been immunoreactive previously. The accumulation of kynurenine aminotransferase-I reaction product around the ribosomes of neuronal endoplasmic reticulum suggests de novo synthesis of kynurenine aminotransferase-l in the reactive nerve cells. (C) 1999 IBRO. Published by Elsevier Science Ltd

Expression of kynurenine aminotransferase in the subplate of the rat and its possible role in the regulation of programmed cell death

The neurons of the transient subplate zone, considered important for the prenatal development of the cerebral cortex, were shown here to express kynurenine aminotransferase (KAT)-I from embryonic day (E) 16 until postnatal day (P) 7 in the rat. No other cells of brain tissue exerted KAT-I immunoreactivity during this period. From P3 on, the neurons of the subplate gave rise to KAT-I immunoreactive, varicose axons, which entered the thalamus and terminated around thalamic nerve cells that are devoid of KAT-I immunoreactivity. Other subplate markers displayed a different expression pattern during development. Thus, subplate neurons displayed parvalbumin (PV) immunoreactivity from E16 to P10 and an intense NPY immunoreaction from P7 to P1. They also exhibited nitric oxide synthase immunoreactivity from E16 to P10, whereas on the surface of the subplate neurons, the 0 subunit of the nicotinic acetylcholine receptor (nAChR) was present from P1 to P10. The cells of Cajal-Retzius were nAChR-immunoreactive during this period. Between P1 and P7, the perikarya of subplate neurons also showed an intense immunoreaction with the N-methyl-D-aspartate (NMDA) receptor subtype R2A. After the first postnatal week, many of the KAT-I positive subplate neurons display a gradual decrease of immunoreactivity and undergo programmed cell death. Since KAT-I persists in the subplate through the period E16-P7, we conclude that KAT-I is a useful and reliable subplate marker in the rat. Since it is assumed that migration of nerve cells is regulated by NMDA receptors, and since kynurenic acid - the only naturally occurring NMDA receptor antagonist - is synthesized by KAT, we suggest that a temporary breakdown of the delicate equilibrium between NMDA and KAT might induce abnormal neuronal migration, giving rise to developmental abnormalities

Effect of 3-nitropropionic acid on kynurenine aminotransferase in the rat brain

Activation of excitatory amino acid receptors by endogenous excitotoxins results in degenerative changes characteristic of neurodegenerative brain diseases such as Huntington's disease. Excitatory amino acid receptors are present in the highest concentration in the striatum, the hippocampal region, and the temporal lobe. The most potent, naturally occurring excitatory amino acid receptor antagonist is kynurenic acid (KYNA) which acts preferentially on N-methyl-D-aspartate (NMDA)receptors. KYNA is produced from L-kynurenine, by the action of the enzymes kynurenine aminotransferases (KMAT I and KAT 11). Several inhibitors of mitochondrial energy metabolism result in an indirect excitotoxic neuronal degeneration. We examined whether systemic administration of the mitochondrial toxin 3-nitroproprionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, which also acts by an indirect excitotoxic mechanism, would produce alterations in the immunohistochemical pattern of KAT I. Our present investigations demonstrate that after 15 days of administration of 3-NP, an inhibitor of mitochondrial Complex 11, the most severe depletion of KAT I occurred in the striatum; less severe depletion occurred in other brain areas investigated, following a striatum > hippocampus > temporal cortex gradient. The alterations induced by 15 days of 3-NP treatment were less conspicuous in 6-week-old (young) animals than in 3-month-old adults. In these adult animals, 3-NP induced necrotic cores in the striatum, characterized by destruction of neuronal and glial elements, similar to that seen in the histologic and neurochemical features of Huntington's disease. It appears that immunohistochemical depletion of KAT after administration of 3-NP to adult animals may contribute to the pathological processes that characterize Huntington's disease. (C) 2002 Elsevier Science (USA)

Kynurenine aminotransferase in the supratentorial dura mater of the rat: effect of stimulation of the trigeminal ganglion

Electrical stimulation of the trigeminal ganglion has been widely used as a model of nociception, characterizing migraine. This treatment is known to evoke release of neuropeptides and neurotransmitters from nerve fibers of the dura mater. On the basis of immunocytochemical investigations, we found that under normal conditions, surface membranes of Schwann cells surrounding nerve fibers in the supratentorial dura mater display kynurenine aminotransferase-immunoreaction (KAT-IR); also KAT-IR are the granules of mast cells and the cytoplasms of macrophages (histiocytes). In consequence of stimulation of the trigeminal ganglion, Schwann cells in the dura mater became conspicuously swollen while their KAT-IR decreased considerably; also KAT-IR of mast cells and macrophages decreased significantly. At the same time, nitric oxide synthase (NOS)-IR of nerve fibers in the dura mater increased, suggesting release of nitric oxide (NO), this is-known to be involved in NMDA receptor activation leading to vasodilation followed by neurogenic inflammation. Because kynurenic acid (KYNA) is an antagonist of NMDA receptors, we hypothesize that KYNA and its synthesizing enzyme, KAT, may play a role in the prevention of migraine attacks. (C) 2004 Elsevier Inc. All rights reserved