Title

Deficient RNA editing at the GluR2 Q/R site is a direct cause of neuronal death in ALS motoneurons

Authors

Yukio Kawahara1, Kyoko Ito1, Hui Sun1, Hitoshi Aizawa2, Ichiro Kanazawa1,3, Shin Kwak1

Affiliations

1 Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Tel.: +81-3-5800-8672, Fax.: +81-3-5800-6548

2 The First Department of Internal Medicine, Asahikawa Medical College, 1-1-1 Midorigaoka Higashinijyo, Asahikawa-shi, Hokkaido 078-8510, Japan

Tel.: +81-166-68-2449

3 National Center of Neurology and Psychiatry, SORST, Japan Science and Technology Corporation, 4-1-1 Ogawa-Higashi, Kodaira-shi, Tokyo 187-8502, Japan

Tel.: +81-42-346-1711, Fax.: +81-42-346-1741

E-mails

Yukio Kawahara:

Kyoko Ito:

Hui Sun:

Hitoshi Aizawa:

Ichiro Kanazawa:

Shin Kwak:

Corresponding author: Shin Kwak

Department of Neurology, Graduate School of Medicine, University of Tokyo

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Tel.: +81-3-5800-8672, Fax: +81-3-5800-6548

E-mail:

Competing financial interests: declared none.
RNA editing is a genetic mechanism through which gene-specified codons are altered by a posttranscriptional modification of the base sequence of mRNA. The change in amino acid from glutamine (Q) to arginine (R) caused at the Q/R site in the putative second membrane domain of GluR2, a subunit of -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, results in alternative properties of AMPA receptors, including a decrease in Ca2+ permeability{Sommer, 1991 #202}. Here we show for the first time that RNA editing at the Q/R site of GluR2 is significantly reduced to an extent sufficient to induce neuronal death in a considerable proportion of the spinal motoneurons examined from individuals with sporadic amyotrophic lateral sclerosis (ALS). This molecular change, a direct cause of neuronal death, is highly relevant to the as yet unresolved aetiology of the most devastating human neurological disease ALS.

GluR2 Q/R site editing in neurons occurs with virtually 100% efficiency throughout life from an embryonic stage, and the early death of mice deficient in GluR2 mRNA editing caused by neuronal death{Brusa, 1995 #204} can be rescued by restoring RNA editing{Higuchi, 2000 #279}, indicating that GluR2 modification by RNA editing is a biologically crucial event for neuronal survival and its deficiency is a direct cause of neuronal death.

We extracted RNA from single motoneurons isolated with a laser microdissector{Kawahara, 2003 #437} from five individuals with ALS and five normal control subjects (Supplementary Table 1). Written informed consent was obtained from all subjects. The Ethics Committee of the University of Tokyo approved the experimental procedures. After nested RT-PCR, mRNA products including the Q/R site of GluR2 were digested with a restriction enzyme BbvI, and the editing efficiency was calculated by measuring the difference in digestion patterns, which depends on the occurrence of editing{Takuma, 1999 #269;Kawahara, 2003 #432} (Fig. 1a). As a non-ALS disease control, the editing efficiency of cerebellar Purkinje cells (PKCs) was also quantified in two individuals with Dentatorubral-pallidoluysian atrophy (DRPLA) and compared with normal subjects.

The frequency of GluR2 mRNA positivity was not significantly different between the ALS and the control groups (two-sample test for equality of proportions, p > 0.05). The editing efficiency varied greatly, from 0% to 100%, among the motoneurons of each individual with ALS, and was not complete in 44 of them (56%); this was in marked contrast to the control motoneurons, of which all 76 examined showed 100% editing efficiency (Fig. 1b). Other factors that might influence the properties of AMPA receptors, including absolute expression level and the relative proportion of GluR2 mRNA to total GluRs mRNA, did not differ between ALS motoneurons and control motoneurons{Kawahara, 2003 #437}.

In accordance with our results, mice transgenic for an artificial Ca2+-permeable GluR2, whose expression level was a quarter of that of intrinsic GluR2, develop motor neuron disease late in life{Feldmeyer, 1999 #259}, indicating that motoneurons are specifically vulnerable to deficient RNA editing and that a moderate decrease in GluR2 RNA editing can induce an ALS-like syndrome in mice. The editing efficiency in PKCs was nearly complete in both the DRPLA and the normal groups (Fig. 1b). Albeit at a tissue level, GluR2 Q/R site editing has been reported to be preserved in the severely pathological brain areas of other neurodegenerative diseases{Akbarian, 1995 #216}. Taken together, the significant reduction in GluR2 RNA editing at the Q/R site is highly relevant to ALS aetiology, thereby providing a therapeutic target specific to ALS.
References

Figure legends

Figure 1 Editing efficiency at the Q/R site of GluR2 mRNA in single neurons. a, Three representatives of the editing pattern in individual A3 are shown. Nested PCR for GluR2 was conducted with the following primers: for the first PCR, hG2F1 (5'-TCTGGTTTTCCTTGGGTGCC-3') and hG2R1 (5'-AGATCCTCAGCACTTTCG-3'); for the nested PCR, hG2F2 (5'-GGTTTTCCTTGGGTGCCTTTAT) and hG2R2 (5'-ATCCTCAGCACTTTCGATGG-3'). We confirmed that these primer pairs did not amplify products originating from other GluR subunits (data not shown). The PCR products (182 bp) originating from completely edited GluR2 mRNA were digested into two bands (116 bp, 66 bp) (left panel), whereas the products originating from unedited GluR2 mRNA were digested into three bands (81 bp, 66 bp, 35 bp) (right panel). Thus, the editing efficiency of the partially edited GluR2 mRNA was calculated by the quantitative analysis of each fragment using a 2100 Bioanalyser (Agilent Technology) (middle panel). LM, lower marker (15 bp); UM, upper marker (600 bp). b, Each small circle represents the editing efficiency at the Q/R site of GluR2 mRNA of one cell, whereas each large circle represents five cells in which editing was complete. For each individual, the mean ± SEM and the number (n) of the examined cells are also shown. GluR2 mRNA was significantly decreased in motoneurons from all patients with ALS (A1-A5) and showed high variability even within individuals (from 75.3 ± 9.9% of A5 to 38.1 ± 13.1% of A1), whereas editing was complete in all of the control motoneurons (C1; n = 28, C2; n = 12, C3; n = 13, C4; n = 12, C5; n = 11) (Mann-Whitney U test, p < 0.001). By contrast, the editing efficiency in Purkinje cells from individuals with DRPLA (D1; 98.8 ± 0.5%, D2; 99.8 ± 0.2%) was the same as that of control cells (C1; 99.8 ± 0.1%, C2; 99.9 ± 0.05%) (p > 0.05).
Supplementary information

Supplemental Table 1. Profiles of the individuals analysed in this study

Individual / Sex/Age (years) / Disease / Duration of illness (years) / Postmortem delay (hours)
A1 / M/79 / ALS / 2.0 / 15
A2 / M/71 / ALS / 2.0 / 12
A3 / F/24 / ALS / 0.7 / 4.5
A4 / M/52 / ALS / 13.0 / 3
A5 / F/79 / ALS / 1.5 / 10
D1 / M/65 / DRPLA / 16 / 15
D2 / F/47 / DRPLA / 26 / 812
C1 / F/28 / Hypovolemia / ─ / 17
C2 / F/31 / Hypovolemia / ─ / 6
C3 / M/78 / Heart failure / ─ / 12
C4 / M/57 / Hypovolemia / ─ / 17
C5 / F/27 / Hypovolemia / ─ / 28

Age, age of patient at death; A1-A5, individuals with amyotrophic lateral sclerosis (ALS); D1-D2, individuals with Dentatorubral-pallidoluysian atrophy (DRPLA); C1-C5, normal controls.

Supplementary Information contents

Supplementary Text: This contains supplementary methods and one supplementary table. (DOC; 64KB)

Supplementary Information Text

Methods

To quantify the editing efficiency at the Q/R site of GluR2 mRNA, nested PCR for GluR2 was conducted after cDNA synthesis with the following primers: for the first PCR, hG2F1 (5'-TCTGGTTTTCCTTGGGTGCC-3') and hG2R1 (5'-AGATCCTCAGCACTTTCG-3'); for the nested PCR, hG2F2 (5'-GGTTTTCCTTGGGTGCCTTTAT-3’) and hG2R2 (5'-ATCCTCAGCACTTTCGATGG-3'). We confirmed that these primer pairs were situated in two distinct exons with an intron between them and did not amplify products originating from other GluR subunits (data not shown). The PCR products (182 bp) originating from completely edited GluR2 mRNA were digested into two bands (116 bp, 66 bp) with the restriction enzyme BbvI, whereas the products originating from unedited GluR2 mRNA were digested into three bands (81 bp, 66 bp, 35 bp). Thus, the editing efficiency of the partially edited GluR2 mRNA was calculated by the quantitative analysis of each fragment (116 bp, 81 bp, 66 bp, 35 bp) using a 2100 Bioanalyser (Agilent Technology). In order to show that the GluR2 mRNA, both edited and unedited, was derived from single-neuron tissue and not from glial tissue, we confirmed the presence of microtubule-associated protein-2 (MAP-2) mRNA, coupled with the absence of glial fibrillary acidic protein (GFAP) mRNA in each RNA sample by a PCR method. The PCR primer sets were shown in Supplementary Table 1b.

Supplementary Table 1a. Profiles of the individuals analysed in this study

Individual / Sex/Age (years) / Disease / Duration of illness (years) / Postmortem delay (hours)
A1 / M/79 / ALS / 2.0 / 15
A2 / M/71 / ALS / 2.0 / 12
A3 / F/24 / ALS / 0.7 / 4.5
A4 / M/52 / ALS / 13.0 / 3
A5 / F/79 / ALS / 1.5 / 10
D1 / M/65 / DRPLA / 16 / 15
D2 / F/47 / DRPLA / 26 / 812
C1 / F/28 / Hypovolemia from trauma / ─ / 17
C2 / F/31 / Hypovolemia from trauma / ─ / 6
C3 / M/78 / Heart failure, not otherwise specified / ─ / 12
C4 / M/57 / Hypovolemia from rupture of splenic artery, liver cirrhosis / ─ / 17
C5 / F/27 / Hypovolemia from trauma / ─ / 28

Age, age of patient at death; A1-A5, individuals with amyotrophic lateral sclerosis (ALS); D1-D2, individuals with dentatorubral-pallidoluysian atrophy (DRPLA); C1-C5, normal controls with no neurological disorders.

Supplementary Table 1b. Sequences of primers used for PCR

Oligonucleotide sequence / Amplified product length (bp)
MAP-2 / 198
Forward primer / 5’-CCAAGGAGTCTGATTGCAGGA-3’
Reverse primer / 5’-CCTCAACCACAGCTCAAATGC-3’
GFAP / 182
Forward primer / 5’-CTTGCGGTCCCTTCTTACTCAC-3’
Reverse primer / 5’-CTCAGTCAAAGCAGAGTGGGTG-3’

MAP-2, microtubule-associated protein-2 (GenBank accession no. NM031846); GFAP, glial fibrillary acidic protein (GenBank accession no. NM002055).

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