RHEB1 insufficiency in aged male mice is associated with stress-induced seizures

Abstract

The mechanistic target of rapamycin (mTOR), a protein kinase, is a central regulator of mammalian metabolism and physiology. Protein mTOR complex 1 (mTORC1) functions as a major sensor for the nutrient, energy, and redox state of a cell and is activated by ras homolog enriched in brain (RHEB1), a GTP-binding protein. Increased activation of mTORC1 pathway has been associated with developmental abnormalities, certain form of epilepsy (tuberous sclerosis), and cancer. Clinically, those mTOR-related disorders are treated with the mTOR inhibitor rapamycin and its rapalogs. Because the effects of chronic interference with mTOR signaling in the aged brain are yet unknown, we used a genetic strategy to interfere with mTORC1 signaling selectively by introducing mutations of Rheb1 into the mouse. We created conventional knockout (Rheb1 ^+/- ) and gene trap (Rheb1 ^Δ/+ ) mutant mouse lines. Rheb1-insufficient mice with different combinations of mutant alleles were monitored over a time span of 2 years. The mice did not show any behavioral/neurological changes during the first 18 months of age. However, after aging (> 18 months of age), both the Rheb1 ^+/- and Rheb1 ^{Δ /-} hybrid males developed rare stress-induced seizures, whereas Rheb1 ^+/- and Rheb1 ^{Δ /-} females and Rheb1 ^Δ/+ and Rheb1 ^Δ/Δ mice of both genders did not show any abnormality. Our findings suggest that chronic intervention with mTORC1 signaling in the aged brain might be associated with major adverse events.

Keywords: Gene targeting; Mice; Rapamycin; Rheb1; mTOR inhibitor; mTORC1.

Fig. 1

Targeting of the Rheb1 gene. a The eight exons of Rheb1 coding the carboxyl-terminal motif CSVM were deleted after homologous recombination. b Southern blot analysis after digestion of ES cell DNA using BamHI results in 11.4 and 7.3 kb WT and KO fragments, respectively. Position of the external probe is indicated. c PCR genotyping of ES cells and Rheb1 KO mice; 400 and 200 b.p. wild-type and KO fragments, correspondingly. d PCR genotyping of the embryos at E9.5 after mating of Rheb1 ^+/− mice. e Whole-mount lateral views of wild-type (left) and abnormal Rheb1 ^−/− (right) embryos at E11.5

Fig. 2

Comparison of Rheb1 mRNA expression by qRT-PCR in Rheb1-insufficient ES cells and embryos. a Rheb1 mRNA expression in ES cells using a pair of primers covering the Ex 2-Ex 5 region of Rheb1 mRNA. b Rheb1 mRNA expression in ES cells using a pair of primers covering the Ex7-Ex 8 region of Rheb1 mRNA. c Rheb1 mRNA expression in ES cells and 9.5-days-old embryos using a pair of primers covering Exon 1. No significant change of mRNA levels in Rheb1 ^+/− embryos; however, a decrease is visible in Rheb1 ^−/− embryos. d Rheb1 mRNA expression in ES cells and 9.5-day-old embryos using a pair of primers covering exon 8. The number of dishes with ES cells or embryos of each genotype is indicated. *Statistical difference versus wild type control (p < 0.05)

Fig. 3

Analysis of RHEB1 protein expression in tissues of Rheb1-insufficient mice. a RHEB1 expression in MEFs produced from 13.5-day-old Rheb1 KO embryos. c RHEB1 expression in MEFs produced from Rheb1-insufficient embryos with combinations of different mutant Rheb1 alleles. e RHEB1 expression in the brain of Rheb1-insufficient mice of different age. b, d, f The quantification of RHEB1expression by ImageJ. The number of the dishes with MEFs or mice of each genotype is indicated. *Statistical difference versus wild-type control (p < 0.05)

Fig. 4

Stress-induced seizures in Rheb1 ^Δ/− mutant males at age 20 months evaluated according to the Racine scale. a–d neurological phenotype of Rheb1 ^Δ/− mutant males. a Mouse reaction (stage 1) on the opening of cage. b Mouse reaction (stage 2) on transfer into new cage. c Reaction on transfer in a novel environment (open space). Both reactions (c, b) characterized by aberrant tail position (resembling Straub tail). d Severe reaction of Rheb1 mutant male in a novel environment characterized by generalized clonic convulsions; the mouse fell onto its side (stage 4). e, f Normal exploratory behavior of Rheb1 ^{Δ /+} mutant male and a wild-type female in the novel environment (open space) correspondingly