3.1.1 Editing patterns in whole brain minus cerebellum
The first set of experiments was designed to assay the molecular and behavioral phenotypes of transgenic mice carrying a genomic copy of the human ADAR2 gene. For the molecular phenotype, two lines, Tg14Dn and Tg15Dn, were verified to express the human ADAR2 mRNA in addition to normal levels of mouse Adar2 (Supplementary Figure 1
). However, while levels and proportions of alternatively spliced variants of the mouse transcripts were normal, those for the human transcripts showed some abnormalities. In particular, inclusion of the Alu containing exon5a was increased to >95% in the transgenic brains, in contrast to approximately 50% seen in normal brain (Supplementary Figure 1
). This is noteworthy because exon 5a transcripts result in a protein with a 50% reduction in deaminase activity. Western blots of whole brain lysates using two different antibodies, however, detected no increases (<15%) in Adar2 protein levels. Lastly, no alterations in editing patterns of Htr2c mRNA or of the glutamate receptors (GluR2, 5 and 6) were detected in naive mice of either line (data not shown). Together, these data suggest that mechanisms for regulating Adar2 activity compensate for gene dosage effects.
Because exposure to the MWM has been shown to alter Htr2c mRNA levels in rats (Cavallaro et al 2002
), and because stimulation/stress might induce transgene-related phenotypes not apparent in naive mice, we next tested for possible transgene effects in mice exposed to swimming only and exposed to swimming plus learning the location of a hidden platform. As shown in , there were no significant differences in performance between wild type and either line of transgenic mice. All were equally successful in learning the location of the platform, as measured by the decreased time to find the platform with increasing number of trials (p<0.01, Two-Way Repeated Measure ANOVA, block) and by the amount of time spent searching the training quadrant when the platform was removed (p=0.84, One Way ANOVA). The presence of the transgene, therefore, has no consequences for performance in the MWM.
Figure 2 Results of Morris Water Maze with wild type (WT) C57BL/6J and two lines of ADAR2 transgenic mice, Tg14 and Tg15. a) Latency: learning in the MWM is indicated by the decreased amount of time (in seconds) required to find the hidden platform with increasing (more ...)
Mice were euthanized at one, six and 24 hours after the final swimming session (probe test) and Ht2cr editing patterns were assayed by primer extension as detailed in and represented in (adapted from Chen et al 2000
; Niswender et al 2001
). No differences in editing levels at any site or combination of sites were detected in transgenic mice of either line when compared to wild type controls, regardless of treatment or time (Supplementary Table 1
). We next compared editing patterns between different treatment groups (N, SC and WM) and between time points (1, 6 and 24 hours). Based on the observation of no Adar2 protein increases, no abnormalities in editing patterns, and no deficit in the MWM, we concluded that the presence of the transgene does not affect the parameters of interest here. Accordingly, data for the three genotypes (wild type, tg14, tg15) were pooled to improve statistical significance for comparative analysis. summarizes the significant similarities and differences. Note that there is little variation in measured values between replicates and between mice of the same treatment, as reflected in the typical SEM of 1%-2% and as shown in Supplementary Table 1
Editing levels of individual sites in brains minus cerebellum.
As shown in , editing levels at the A site did not change with either treatment or time (F2, 45 = 2.03, p=0.14). However, levels of A+B+, that averaged 75% in N mice, were significantly increased in both SC and WM mice at one hour, where they averaged 81%, remaining high in WM mice at six hours (82%; F2, 45 = 20.29, p<0.0001). In contrast, no differences in the levels of A−B+ were detected in any comparison (F2, 45 = 1.18, p=0.32).
Analysis of editing at the E and C sites is complicated in that the method used here couples editing at the E and C sites to the editing status at the D site. To distinguish measurements with and without editing at the D site, levels of the E and C site editing are expressed in two columns (; ddT, Rxn 4; ddC, Rxn 5). Editing of the E site is measured when the C and D sites are both edited (E+C+D+), or when the C and D sites are both unedited (E+C−D−). Similarly, editing of the C site is measured when the D site is edited (C+D+ or unedited (C+D−).
Levels of E+C+D+ were significantly affected by treatment (F2, 44 = 9.42, p<0.001). In naïve mice, the level of E+C+D+ averaged 5%. While this was not significantly different in SC mice (averaging 7%), it was significantly increased in WM mice at one hour, where it averaged 9% (p<0.01). Levels of E+C−D− were significantly affected by time (F2, 45 = 4.70, p<0.05), however, significant interactions between treatment and time were also observed (F4, 45 = 4.09, p<0.01), indicating that treatment had different effects across the three time points. In naïve mice, levels of E+C−D− averaged 10%. This was significantly increased, to 19%, in SC mice at six hours (p<0.01) which was also significantly higher than in WM mice, at 12%, at six hours (p<0.05).
Levels of C+D+ also were significantly affected by time (F2, 45 = 3.26, p<0.05), while levels of C+D− did not change with either time or treatment (F2, 45 = 2.00, p=0.15, treatment; F2, 45 = 2.14, p= 0.13, time).
Levels of editing at the D site changed with treatment (in ddT reactions, F2, 45 = 8.99, p<0.001; in ddC reactions, F2, 45 = 5.07, p<0.05). In naïve mice, they averaged 86%, but, in WM mice, they decreased to 82% and 80% at one and six hours (p<0.05 and p<0.01, respectively)
To determine if Htr2c editing patterns were altered at least in part because of changes in levels of Htr2c mRNA , Real Time PCR was used to quantitate amounts of the full length and truncated splice variants of Htr2c. There were no significant differences between naive mice and SC or WM mice, although there was a trend towards a decrease with swimming ().
Expression of 5HT2CR splice variants in whole brain minus cerebellum.
3.1.2 Alterations in Htr2c mRNA isoforms in whole brain minus cerebellum
In order to deduce the consequences for protein isoforms, mRNA sequences were analyzed in two segments, the segment containing the A and B sites and the segment containing the E, C and D sites. Editing at the A site, with or without editing at the B site, results in valine, and therefore the frequency of mRNA variants encoding valine at position 157 will be reflected in the level of editing at the A site, regardless of editing at the B site (Rxn 1, ). Consistent with editing at the A site, the frequency of mRNA isoforms encoding valine at position 157 did not change with treatment or time. Similarly, levels of transcripts encoding isoleucine (A−B−) and methionine (A−B+) were unchanged (p=0.71 and 0.37, treatment and time; p=0.24 and 0.77, treatment and time, respectively).
Editing at the E, C and D sites creates eight amino acid combinations at positions 159 and 161. As shown in , the proportions of transcripts encoding GV, SV and NV+DV and NI, DI and SI+GI can be calculated. Significant changes are shown in . Treatment had an overall significant effect on the proportion of transcripts encoding NI (F2, 45 = 3.82, p<0.05). In contrast, the proportion of transcripts encoding SV did not change with either treatment or time (F2, 45 = 1.57, p=0.22, treatment; F2, 45 = 2.29, p=0.11, time). The proportions of transcripts encoding NV+DV and SI+GI both changed with treatment (F2, 45 = 3.85, p<0.05; F2, 45 = 11.62, p<0.0001). In WM mice at one hour, levels of NV+DV (65% in N mice) decreased significantly to 58% (p<0.05), and levels of SI+GI (4% in N mice) increased significantly to 8% (p<0.01). The latter remained high at 6 hours, where they averaged 7% (p<0.05).
Levels of partial mRNA isoforms in whole brains minus cerebellum.
3.2.1 Editing in hippocampus and cortex
The significant changes in editing patterns seen in whole brain minus cerebellum are assumed to be a summation of changes that may vary among brain regions. Because the Morris Water Maze tests spatial learning and because swimming induces stress in mice, we next assayed editing in hippocampus and cortex from a second set of mice composed entirely of wild type C57BL/6J mice. This set included 13 naive mice (3, 5 and 5 sacrificed at one, six and 24 hours) and three SC and WM mice sacrificed at each time point. Results for individual mice are presented in Supplemental Tables 2
. Examination of the data for hippocampus unexpectedly showed that there were significant differences among individual mice, unrelated to treatment or time. Specifically, mice can be divided into two groups: (1) the majority, 25 of the total of 31 exhibited high levels of editing at the A site, averaging 78%, similar to those seen in whole brain minus cerebellum of naive mice (), and (2) the remaining 6 mice showed significantly lower levels of editing at the A site, five averaging ~25%, and one at 55% (). As shown in , editing levels at additional sites, most often the B site, were also affected. This variation cannot be attributed to differences in the time of day at which the mice were sacrificed (note that while all three N mice at one hour showed low levels, five N mice at 24 hours, the same time of day, did not), the order in which the mice were euthanized at any time point (which might affect stress levels), or to quality of the isolated RNA or RT-PCR product. Because the mice are genetically identical, differences due to polymorphisms in either the Htr2c gene or the adenosine deaminases that carry out editing can be ruled out. These points suggest that stochastic processes play a role, a conclusion that is further supported by the observation that editing levels in the cortex of the same mice do not show these patterns of individual variation (), with the exception of the N1-3 mouse at the A site (55%). In addition, of the 42 mice analyzed in whole brain in the first experiments, only one showed a moderately low level of editing at the A site (54%).
Figure 3 A subset of mice show low levels of editing at the A and B sites in hippocampus. a) Proportion of transcripts edited at the A site in 13 naive (N), 9 swimming control (SC) and 9 mice trained to find the platform (WM). Five mice are edited at <30% (more ...)
Site-specific low editing levels in a subset of mice.
The appearance of mice with low and high editing levels complicates the comparisons between naive mice, and SC and WM mice and could obscure time/treatment dependent differences. Therefore, we restricted analysis in hippocampus to only those mice with the (predominant) high levels of editing at the A site, deleting the five mice with A site editing <30%.
3.2.2. Editing in hippocampus
Significant changes in hippocampus of SC and WM mice are shown in (additional data are provided in Supplementary Tables 2
). In hippocampus, levels of editing at the A site did not change with either treatment or time (treatment, F2, 39
= 0.33, p=0.72; time, F2, 39
= 0.79, p=0.46) and nor did those of A+B+ (treatment, F2,39
= 1.24, p=0.30; time, F2, 39
= 0.62, p=0.54). However, levels of A−B+ significantly changed with treatment (F2, 39
= 5.45, p<0.01) (). In naive mice, levels of A−B+ averaged 31%, which decreased in SC and WM mice, at one, six and 24 hours, to 23%, 21% and 23% and 17%, 18% and 21%, respectively.
Figure 4 Significant changes in editing patterns in hippocampus. a) Levels of A−B+ were affected by treatment (p<0.01). b) Levels of E+C+D+ were affected by treatment (p<0.05) and time (p<0.01). c) Levels of C+D+ were affected by (more ...)
Levels of E+C+D+ were significantly affected by both treatment and time (treatment, F2, 39 = 4.51, p<0.05; time, F2, 39 = 5.52, p<0.01) (). In naïve mice, levels averaged 9%, and were decreased to 5% in both SC and WM mice at 6 hours (p<0.05). In contrast, levels of E+C−D− did not change with either treatment or time (treatment, F2, 39 = 0.03, p=0.97; time, F2, 39 = 0.41, p=0.67).
Levels of C+D+ were significantly affected by time (F2, 39 = 5.00, p<0.05) (), while levels of C+D− and levels of the D+ did not change with either time or treatment.
Because of the significant effect of treatment on levels of A−B+, levels of transcripts encoding M at the position 157, compared to those encoding I or V, changed with treatment (F2,39 = 3.70, p<0.05) (). Although it did not reach significance, levels of transcripts encoding methionine were decreased in SC and WM mice (4%-5%) compared to naïve mice (7%).
The proportion of transcripts encoding SV (E−C+D+) changed with time (time, F2,39 = 3.57, p<0.05) (), however, the proportion of transcripts encoding NI (E−C−D−), NV+DV (C−D+) or SI+GI (C+D−) did not change with either treatment or time.
3.2.3. Editing in cortex
Site-specific editing and amino acid combinations with significant changes in cortex are shown in (see also Supplementary Tables 3
). Levels of A−B+ changed significantly with treatment (F2, 48
= 7.24, p<0.01), and a showed significant interaction between treatment and time (treatment has different effects at each time point) (F4, 48
= 2.88, p<0.05) (). Relative to 35% in naive mice, levels of A−B+ were increased, to 54%, in SC mice at 1hour (p<0.05) and to 46%, in WM mice at 1 hour.
Figure 5 Significant changes in editing patterns in cortex. a) Levels of A−B+ were affected by time (p<0.01); time and treatment show an interaction (p<0.05). b) Levels of E+C+D+ are affected by time (p<0.05). c) Levels of D+ were (more ...)
Levels of E+C+D+ were significantly affected by time (F2,48
= 3.66, p<0.05) (), increasing in SC and WM mice, particularly at 6 hours. Treatment had a significant effect on levels of DT
= 3.70, p<0.05) (). (Note that DC
values were not analyzed; see Supplementary Table 6
Time had significant effects on the proportion of transcripts encoding methionine (A−B+) compared to those encoding valine and isoleucine (F2,48 = 4.38, p<0.05) (), increasing in SC and WM mice at 1 hour (14% and 9%, respectively), compared to naïve mice at 7%. The proportions of transcripts encoding SV, NI, NV+DV or SI+GI did not change with either treatment or time.