Kyoko Ito

Glutamate receptors: RNA editing and death of motor neurons

The aetiology of sporadic amyotrophic lateral sclerosis (ALS), a fatal paralytic disease, is largely unknown. Here we show that there is a defect in the editing of the messenger RNA encoding the GluR2 subunit of glutamate AMPA receptors in the spinal motor neurons of individuals affected by ALS. This failure to swap an arginine for a glutamine residue at a crucial site in the subunit, which occurs normally in the affected brain areas of patients with other neurodegenerative diseases, will interfere with the correct functioning of the glutamate receptors and may be a contributory cause of neuronal death in ALS patients.

Underediting of GluR2 mRNA, a neuronal death inducing molecular change in sporadic ALS, does not occur in motor neurons in ALS1 or SBMA.

Deficient RNA editing of the AMPA receptor subunit GluR2 at the Q/R site is a primary cause of neuronal death and recently has been reported to be a tightly linked etiological cause of motor neuron death in sporadic amyotrophic lateral sclerosis (ALS). We quantified the RNA editing efficiency of the GluR2 Q/R site in single motor neurons of rats transgenic for mutant human Cu/Zn-superoxide dismutase (SOD1) as well as patients with spinal and bulbar muscular atrophy (SBMA), and found that GluR2 mRNA was completely edited in all the motor neurons examined. It seems likely that the death cascade is different among the dying motor neurons in sporadic ALS, familial ALS with mutant SOD1 and SBMA.

Low editing efficiency of GluR2 mRNA is associated with a low relative abundance of ADAR2 mRNA in white matter of normal human brain

The ionotropic glutamate receptor (GluR) subunits GluR2, GluR5 and GluR6 are subject to RNA editing at their Q/R sites, resulting in significant alterations in the channel properties of the receptors. RNA editing at the Q/R site of GluRs is both developmentally and regionally regulated. Here we provide the first quantitative measurements of both mRNAs of the GluR subunits and mRNAs of the RNA editing enzymes ADAR1–ADAR3 in a comparison of the efficiency of editing at the Q/R site with the expression levels of ADAR mRNA in human brain. We demonstrate that the Q/R site of GluRs in white matter is edited significantly less than in grey matter. In addition, by means of quantitative reverse transcription‐polymerase chain reaction methods, we demonstrate that the relative abundance of ADAR2 mRNA to GluR2 mRNA is significantly lower in white matter than in grey matter and that the GluR2 Q/R site editing decreased only when the ratio of ADAR2 mRNA (not that of ADAR1 mRNA) to GluR2 mRNA dropped below a threshold (20 × 10−3). These results suggest that Q/R site of GluRs editing is regulated in a regional, and hence presumably cell‐specific, manner and that the GluR2 Q/R site editing is critically regulated by ADAR2 in human brain.

Slow and selective death of spinal motor neurons in vivo by intrathecal infusion of kainic acid: implications for AMPA receptor-mediated excitotoxicity in ALS

Excitotoxicity mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptors has been proposed to play a major role in the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), and motor neurons are more vulnerable to AMPA receptor‐mediated excitotoxicity than are other neuronal subclasses. On the basis of the above evidence, we aimed to develop a rat model of ALS by the long‐term activation of AMPA receptors through continuous infusion of kainic acid (KA), an AMPA receptor agonist, into the spinal subarachnoid space. These rats displayed a progressive motor‐selective behavioral deficit with delayed loss of spinal motor neurons, mimicking the clinicopathological characteristics of ALS. These changes were significantly ameliorated by co‐infusion with 6‐nitro‐7‐sulfamobenso(f)quinoxaline‐2,3‐dione (NBQX), but not with d(–)‐2‐amino‐5‐phosphonovaleric acid (APV), and were exacerbated by co‐infusion with cyclothiazide, indicative of an AMPA receptor‐mediated mechanism. Among the four AMPA receptor subunits, expression of GluR3 mRNA was selectively up‐regulated in motor neurons but not in dorsal horn neurons of the KA‐infused rats. The up‐regulation of GluR3 mRNA in this model may cause a molecular change that induces the selective vulnerability of motor neurons to KA by increasing the proportion of GluR2‐lacking (i.e. calcium‐permeable) AMPA receptors. This rat model may be useful in investigating ALS etiology.

Dequenching of Cu(I)-bathocuproine disulfonate complexes for high-performance liquid chromatographic determination of phytochelatins, heavy-metal-binding peptides produced by the primitive red alga Cyanidioschyzon merolae.

A novel method has been devised for the determination of phytochelatins (PCs), heavy-metal-tolerant peptides produced by higher plants and algae. The method is based on the facts that fluorescence of bathocuproine disulfonate (BCS) is quenched by Cu(I) ions as a result of Cu(I)-BCS complex formation and that PCs compete with BCS for Cu(I). Detection of PCs via recovered fluorescence of BCS using the Cu(I)-BCS complex as a postcolumn reagent, following separation of peptides on an octyldecylsilane column, demonstrated a highly sensitive method for determination of PCs. PCs in the primitive red alga, Cyanidioschyzon merolae, grown in the presence or absence of added Cd(II) were successfully determined by this protocol. Unlike other methods for the determination of PCs, which rely on the SH groups in the peptides, the proposed method is unique in that detection is based on the chemical nature of PCs, which favors the formation of complexes with Cu(I). In this context, the new method yields chromatograms based on the strength of binding Cu(I) ions.

High-performance liquid chromatography method for ferric iron chelators using a post-column reaction with Calcein Blue

Iron (Fe) is an essential element for higher plants, which take it up from the soil at the root surface and transport it to shoots through the xylem. Fe(III) chelators, such as organic acids and phytosiderophores, play important roles in the acquisition and transportation of Fe(III). Therefore, a selective and sensitive method for analyzing Fe(III) chelators is required to study the many Fe-related physiological mechanisms in plants. A novel analytical approach employing a high-performance liquid chromatography post-column method with fluorescence detection was developed to separate and detect Fe(III) chelators. This method takes advantage of the quenching of the fluorescence of Calcein Blue (CB) that occurs with the formation of an Fe(III)-CB complex and the dequenching that occurs with the release of CB as a result of competition for Fe(III) between CB and an Fe(III) chelator. This simple experimental method does not require complicated pretreatments and can selectively detect Fe(III) chelators according to their Fe(III)-chelating ability. The detection limit for citric acid using this method was 72pmol. Furthermore, this method can also detect unknown Fe(III) chelators that exhibit a high affinity for Fe(III). The method was evaluated with xylem sap of barley, which was shown to contain several Fe(III) chelators.