The results of our experiments with the AR2 mice indicate that deficient-ADAR2-mediated death of motor neurons in the spinal cord and the cranial motor nerve nuclei is specifically mediated by the failure to edit the GluA2 Q/R site. In the ALS spinal cord, some motor neurons express unedited GluA2, but others express only edited GluA2. The majority of motor neurons expressing unedited GluA2 also express edited GluA2 (Kawahara et al., 2004
). Furthermore, a recent immunohistochemical study demonstrated that both ADAR2-positive and ADAR2-negative motor neurons coexist in patients with sporadic ALS, whereas all motor neurons are ADAR2-positive in control subjects (Aizawa et al., 2010
). However, we do not know the expression level of ADAR2 that is required to edit all GluA2 mRNA or the proportion of unedited GluA2 that is not harmful to motor neurons.
To answer these questions, we investigated the extent of GluA2 Q/R site editing in motor neurons lacking one ADAR2 allele in the heterozygous ADAR2flox/+/VAChT-Cre (HeteroAR2) mice. Additionally, we investigated whether motor neurons lacking one ADAR2 allele can survive in HeteroAR2 mice compared to AR2 and control mice. In HeteroAR2 mice, the proportion of motor neurons that express Cre is the same as in AR2 mice; the Cre-expressing motor neurons express only one ADAR2 allele. Therefore, if the expression level of ADAR2 in normal motor neurons is sufficiently above the requirement to edit the Q/R site of all GluA2 mRNAs expressed (i.e., threefold or more), all motor neurons would express only edited GluA2. However, if normal motor neurons express ADAR2 at a level that is only sufficient to edit GluA2 (i.e., less than twofold), motor neurons with one ADAR2 allele would express abundant unedited GluA2 and die. Furthermore, if motor neurons expressing one ADAR2 allele express both edited and unedited GluA2 and undergo degeneration in HeteroAR2 mice, we would expect to find the proportion of unedited GluA2 that is toxic to motor neurons.
When the extent of GluA2 Q/R site editing was examined in the lysates of three laser-captured motor neurons of 2-month-old HeteroAR2 mice, GluA2 Q/R site editing was incomplete in approximately 20% of the lysates (Figure ). The proportion of edited GluA2 was above 70% in all the lysates; however, in the AR2 mice of the same age, unedited GluA2 was detected in more than 60% of the lysates of three motor neurons, and the editing efficiency was 0 in 7% of the lysates examined (Hideyama et al., 2010
). Therefore, it is likely that motor neurons exhibit considerable editing activity in the expression of only one ADAR2 allele; however, this is not sufficient to edit the Q/R site of all GluA2 mRNA in HeteroAR2 mice.
Figure 10 Some of the motor neurons from HeteroAR2 mice express Q/R site-unedited GluA2 mRNA. A frequency histogram of editing efficiency at the GluA2 Q/R site in the lysates of three motor neurons obtained from heterozygous ADAR2flox/+/VAChT-Cre.Fast (HeteroAR2) (more ...)
We also examined whether there was a loss of AHCs in HeteroAR2 mice. The extent of AHC loss in HeteroAR2 mice was approximately half (26%) of that observed in AR2 mice (46%) at 12
months of age (Figure A). A moderate increase in GFAP- and MAC2-immunoreactivity was detected in the anterior horns of HeteroAR2 mice at 12
months of age (Figure B). HeteroAR2 mice did not exhibit significant behavioral changes until 12
months of age, indicating that mild loss of motor neurons would not affect motor function at least until 1
year of age.
These results show that one ADAR2 gene allele is sufficient to edit all GluA2 mRNA in half of the motor neurons but is insufficient in the other half of motor neurons. Because the editing efficiency was above 70% in the motor neuron lysates of HeteroAR2 mice, it is likely that the minimal expression level of ADAR2 required for complete GluA2 editing is slightly higher than half of the normal level in mouse motor neurons.
More than a quarter of the motor neurons in HeteroAR2 mice underwent degeneration by the age of 1
year. Furthermore, from the results of our experiments with AR2 and AR2res (Hideyama et al., 2010
), it is likely that only the motor neurons expressing unedited GluA2 have undergone degeneration. The proportions of unedited GluA2 are 30% at the maximum and less than 10% in the majority of motor neurons in HeteroAR2 mice, indicating that expression of unedited GluA2, even in a small proportion, is not favorable for the survival of motor neurons in mice.