Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi syndrome

Abstract

RNA transcripts encoding the 2C-subtype of serotonin (5HT(2C)) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT(2C) editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT(2C) receptor expression and 5HT(2C)-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT(2C) gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT(2C) expression, but also demonstrate the importance of normal patterns of 5HT(2C) RNA editing in vivo.

Fig. 1

Targeting strategy and genotype analysis for 5HT_2C-VGV mice. (A) The predicted secondary structure for 5HT_2C pre-mRNA near the distal end of exon 5 is presented. The positions of the five editing sites (A-E) are shown with site-specific adenosine to guanosine mutations indicated in red. (B) Schematic diagram and abbreviated restriction map of the mouse 5HT_2C gene before and after targeted gene modification; the location of exon 5, the loxP sites (▶) flanking the PGK/neomycin resistance cassette, the negative selectable marker (PGK/TK) outside the region of homology, the approximate position of the introduced mutations (*) and sequences outside the region of homology (dotted line) are indicated; Av, Avr II; N, Not I; A, Acc I; K, Kpn I; S, Sfi I. (C) Genotype analysis of offspring from a hemizygous 5HT_2C-VGV male (VGV/Y) x heterozygous mutant female (VGV/+) mating; migration positions of PCR amplicons corresponding to the wild-type (371 bp) or mutant (468 bp) 5HT_2C alleles and animal genotypes are indicated. (D) Sequence electropherogram traces of 5HT_2C receptor-derived RT-PCR products generated from wild-type and 5HT_2C-VGV hemizygous male mice. The positions of the five editing sites and the corresponding nucleotide sequences are indicated; R, purine.

Fig. 2

Analysis of 5HT_2C mRNA expression in wild-type and 5HT_2C-VGV mice. (A) A schematic diagram of a portion of the 5HT_2C gene is shown in which 5′-splice site selection can generate three alternatively spliced 5HT_2C transcripts (RNA 1, RNA 2 and RNA 3). The relative positions of contiguous antisense riboprobes used for ribonuclease protection analysis (bold lines) to quantify either total 5HT_2C mRNA (1+2+3) or the three individual 5HT_2C splice variants is presented with the size of the expected protected fragment (nucleotides, nt) for each mRNA isoform; the location of the 5 editing sites is indicated in red. (B) Ribonuclease protection analysis of total 5HT_2C mRNA expression (1+2+3) in whole brain and dissected brain regions for 5HT_2C-VGV (■) male mice is presented as a percentage of mean wild-type (□) expression; mean ± SEM, n ≥ 4 animals/genotype for whole brain samples and n ≥ 6 animals/genotype for dissected brain regions. Inset, Representative ribonuclease protection analysis of total 5HT_2C RNA expression in whole brain and dissected brain region samples from individual mice; WB, whole brain; Hy, hypothalamus; OB, olfactory bulb; Cx, frontal cortex; Hi, hippocampus. (C) Quantitative analysis of the relative expression levels for 5HT_2C alternative splicing variants (RNA 1 and RNA 2) in whole brain samples from wild-type (□) and 5HT_2C-VGV (■) male mice (mean ± SEM; n=7; **p ≤ 0.01). Inset, Representative ribonuclease protection analysis of alternatively spliced 5HT_2C RNAs in whole brain samples from individual mice. (D) Semi-quantitative analysis of 5HT_2C mRNA splicing patterns in wild-type and 5HT_2C-VGV male mice. Quantification of each 5HT_2C splice variant is presented as a percentage of total 5HT_2C mRNA expression (n=7; mean ± SEM; ***p ≤ 0.001); inset, Representative RT-PCR amplification using common primers in exons 5 and 6 to detect three 5HT_2C mRNA isoforms; the expected migration positions of amplicons corresponding to each 5HT_2C splice variant are indicated.

Fig. 3

Analysis of 5HT_2C protein expression in wild-type and 5HT_2C-VGV mice. (A) Saturation binding isotherms demonstrating 5HT_2C receptor-specific binding for total membranes prepared from dissected cortex, striatum and hippocampus in wild-type (□) and 5HT_2C-VGV (●) male mice (wild-type, n=4 groups of five pooled mice per brain region; 5HT_2C-VGV, n=7 brain regions from individual animals; mean ± SEM). (B) Western blotti ng analysis of 5HT_2C receptor and β-actin expression in whole brain samples isolated from individual wild-type and 5HT_2C-VGV male mice at postnatal day 2 (P2) or in adulthood (14-16 weeks); n ≥ 8 for mice of each genotype, two representative samples are shown for each age and genotype. Representative Western blotting controls from NIH-3T3 cells stably expressing either 5HT_2A or 5HT_2C receptors are presented.

Fig. 4

Body weight analysis of wild-type and 5HT_2C-VGV mice. (A) Pre- and (B) post-weaning growth curves are shown for wild-type and 5HT_2C-VGV mutant mice (mean ± SEM; n=6 for each genotype and gender; p ≤ 0.0001 using two-way ANOVA). Inset, A representative photograph of wild-type and mutant (VGV/Y) littermates at postnatal day 21 is presented.

Fig. 5

Feeding behavior and metabolic analysis of wild-type and 5HT_2C-VGV mice. (A) Food intake for 15-week old male wild-type (□) and 5HT_2C-VGV (■) mice per 24-hours (hr), 12-hr light, or 12-hr dark period, corrected for total body mass (mean ± SEM, n=6, *p ≤ 0.05). (B) Average daily activity detected by infrared sensors for wild-type (□) and 5HT_2C-VGV adult (■) mice maintained in a 12hr-12hr light/dark environment (mean ± SEM; n=6). (C) Indirect calorimetric analysis of energy expenditure for wild-type (□) and 5HT_2C-VGV (■) mice per 24-hr, while resting, or while active, corrected for total body mass (mean ± SEM, n=6, *p ≤ 0.05). (D) Quantitative analysis of hypothalamic POMC and NPY mRNA expression at 0.5 weeks (n=4), 3 weeks (n=8), 14 weeks (n=12) and 36 weeks (n=11) in wild-type and 5HT_2C-VGV male mice (mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Fig. 6

Analysis of muscle strength in wild-type and 5HT_2C-VGV mice. (A) Motor coordination and endurance of post-weaning (P22-P28, n=10) and adult (25 weeks, n=12) wild-type (□) and 5HT_2C-VGV mutant (■) male mice (mean ± SEM; *p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001). (B) Average grip strength of recently weaned (P21-P28; n=5) or adult (n=7) wild-type (□) and mutant (■) mice (mean ± SEM; ***p ≤ 0.001).

Fig. 7

Strain-specific lethality in 5HT_2C-VGV mice. Cumulative survival rate for offspring resulting from the mating of heterozygous mutant 129S6 females (VGV/+) to wild-type C57BL/6 males (n=28, +/Y; n=32, VGV/Y; n=33, +/+; n=17, VGV/+).