New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism

Abstract

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes.

Keywords: AgRP neurons; GABA; eating disorders; feeding behavior; inducible gene knockout.

Fig. 1.

Designing and validation of a novel inducible nsCre transgene. (A) A schematic diagram showing the aminoglycoside (AG)-mediated nonsense suppression of the PTC. Without treatment of AG, the majority of PTC-bearing mRNAs are rapidly degraded by the process of NMD, whereas only a small fraction of the transcripts can be translated into truncated, nonfunctional peptides. In contrast, AG, such as geneticin (G418), promotes read-through of PTCs during the translation, thereby restoring full-length functional proteins. Of note, a random amino acid (blue spheres) is incorporated into the PTC locus through AG-mediated nonsense suppression. (B) A PTC, TGA, was inserted into position 9 or 837 of the wild-type Cre gene. (C) A diagram illustrating the functional validation of nsCre transgene in a cell culture-based assay. Neo-resistant HEK293 cells were transfected with CMV-flox-Neo-dsRed2 and Pgk-myc-nls-nsCre plasmids. (D–F) Representative images showing DsRed fluorescence in nsCre2-expressing HEK293 cells 4 d after treatment of vehicle (D) or G418 at a concentration of either 1 mg/mL (E) or 2 mg/mL (F). These results indicated that nsCre-mediated homologous recombination is restored by G418 in a dose-dependent manner. (Scale bar, 200 µm.)

Fig. 2.

Engineering an inducible Agrp^nsCre transgenic mouse line. (A) A schematic diagram showing the targeting construct for generation of Agrp^nsCre mice. Specifically, the nsCre2-containing cassette was cloned into Agrp gene locus immediately 5′ of the translation start codon. (B) Fluorescence image shows no leaky expression of tdTomato (red) in Agrp^nsCre/+::Rosa26^tdTomato mice. Dotted circles indicate the ARC region of the hypothalamus where AgRP-expressing neurons are located. (C) Fluorescence image shows tdTomato expression profile (red) in the Agrp^nsCre/+::Rosa26^tdTomato mice 4 d after injection of NB124 into the third ventricle. (D) Immunostaining image shows expression profile of AgRP (green) in the same mice as described in C. (E) Merged image of C and D illustrates the colocalization of tdTomato marker and AgRP peptides. (F) Quantification of AgRP neurons expressing tdTomato under vehicle or NB124 treatment. Asterisks in B and C indicate the third ventricle. (Scale bar: B–E, 300 µm.)

Fig. S1.

Screening of the lead compounds that mediate nonsense suppression of nsCre transgene in vivo. Fluorescence image shows tdTomato expression profile (red) in the ARC region of Agrp^nsCre/+::Rosa26^tdTomato mice 4 d after injection of either G418 (A and E), NB124 (B and F), NB127 (C and G), or NB128 (D and H) into the third ventricle. Each compound was delivered either once (A–E) or twice 48 h apart (F–H). Asterisks indicate the third ventricle. (Scale bar: 150 µm.)

Fig. S2.

Chemical structures of the aminoglycoside G418 and its synthetic derivatives NB124, NB127, and NB128 that were investigated in this study. The common part of all of the structures is highlighted in blue color. The ring numbers are in capital roman numerals (circled). The identity of a particular pharmacophore and its attachment site in the synthetic derivatives are highlighted: (S)-4-amino-2-hydroxybutanoyl (AHB; red), (S)-5″-Me (green), and (R)-5″-Me (green).

Fig. 3.

Deletion of GABA neurotransmitter from the AgRP neurons upon NB124 treatment. (A and B) Immunostaining images show anti-GABA (A; green) and tdTomato (B; red) in the ARC of control group mice 4 d after NB124 treatment. (C) Merged image of A and B shows colocalization of GABA and tdTomato in the ARC. (D) High-magnification image of the dotted area in C shows that almost all AgRP neurons coexpress GABA neurotransmitter (yellow) in control mice. (E and F) Immunostaining images show anti-GABA (E) and tdTomato (F) in the ARC of mutant-group mice 4 d after NB124 treatment. (G) Merged image of E and F shows GABA and tdTomato in the ARC. (H) Higher-magnification image of the dotted area in G shows that almost all AgRP neurons are depleted of GABA in mutant mice. Asterisks in A and E indicate the third ventricle. (I and J) Real-time qPCR analysis of transcript levels of Gad1 (I) and Gad2 (J) expressed in AgRP neurons that were isolated by a flow cytometry approach from Agrp^Cre::Rosa26^tdTomato mice (WT group) and control and mutant group mice treated with NB124. (K) Abundance of GABA in the ARC was measured by HPLC in wild-type mice, DT-treated Agrp^DTR/+ mice, and NB124-treated control and mutant groups. Values represent group means and SEM; n = 5 or 6 mice per group. *P < 0.05 [analysis of variance (ANOVA) with SNK post hoc]. Control group, Agrp^nsCre::Rosa26^tdTomato mice; mutant group, Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice. (Scale bars: A–C and E–G, 100 µm; D and H, 60 µm.)

Fig. S3.

Measurement of glutamic acid and Agrp mRNA. (A) Abundance of glutamic acid in the ARC was measured by HPLC in wild-type mice, DT-treated Agrp^DTR/+ mice, and NB124-treated control and mutant groups. (B) qPCR results showed relative abundance of Agrp transcripts from the ARC in the NB124-treated control and mutant groups as well as DT-treated Agrp^DTR/+ mice. Control group, Agrp^nsCre::Rosa26^tdTomato mice; mutant group, Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice. Values represent group means and SEM; n = 5 or 6 mice per group. *P < 0.05 (ANOVA with SNK post hoc).

Fig. S4.

Fos activation in postsynaptic neurons after rapid deletion of GABA signaling from AgRP neurons at different age. (A–C) Representative immunostaining pictures of Fos in postsynaptic regions of AgRP neurons, including the BNST (A), the PVN (B), and the PBN (C), 5 d after injection of NB124 into the third ventricle of 3-mo-old control group Agrp^nsCre/+::Gad1^lox/+::Gad2^lox/+::Rosa26^tdTomato mice. (D–F), Fos in postsynaptic regions of AgRP neurons, including the BNST (D), the PVN (E), and the PBN (F), 5 d after injection of NB124 (80 µg × 2, i.c.v.) into 3-mo-old mutant group Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice. (G–I) Fos in postsynaptic regions of AgRP neurons, including the BNST (G), the PVN (H), and the PBN (I), 5 d after injection of NB124 (80 µg × 2, i.c.v.) into 8-mo-old mutant group Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice. BNST, the bed nucleus of the stria terminalis; PVN, the paraventricular nucleus of hypothalamus. (Scale bar: 250 µm.)

Fig. S5.

GABA level in other brain regions after deletion of GABA from AgRP neurons. (A–C) Immunostaining images show anti-GABA in the LH (A), DMH (B), and CeA (C) of control group mice 4 d after NB124 treatment. (D–F) Immunostaining images show anti-GABA in the LH (D), DMH (E), and CeA (F) of mutant group mice 4 d after NB124 treatment. Control group, Agrp^nsCre::Rosa26^tdTomato mice; mutant group, Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice. (Scale bar: 250 µm.)

Fig. 4.

Acute inactivation of GABA signaling from AgRP neurons in 3-mo-old adult mice leads to reduction of feeding, severe weight loss, enhanced energy expenditure, and glucose intolerance. (A and B) Body weight (A) and daily calorie intake (B) of the following four groups of mice with drug or vehicle delivered into the third ventricle: control mice treated with vehicle, control mice treated with NB124 (80 µg × 2, i.c.v.), mutant mice treated with vehicle, and mutant mice treated with NB124 (80 µg × 2, i.c.v.). (C–E) Immediately after the second treatment of NB124, O₂ consumption (C), CO₂ production (D), and locomotor activity (E) of the mice as described in A and B. *P < 0.05 (between mutant+NB124 group and the control groups; ANOVA with SNK post hoc). (F) Glucose tolerance test (GTT) was performed in control mice treated with NB124, mutant mice pair-fed to the mutant group treated with NB124, and mutant group treated with NB124. *P < 0.05 (between mutant+NB124 group and control+NB124 group; Student t test); ^#P < 0.05 (between mutant+NB124 group and mutant+pair-fed group; Student t test). Values represent group means and SEM; n = 8–10 mice per group. Control group, Agrp^nsCre/+::Gad1^lox/+::Gad2^lox/+::Rosa26^tdTomato mice; mutant group, Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice.

Fig. 5.

Metabolic characterization of 8-mo-old mice upon acute disruption of GABA signaling from AgRP neurons. (A and B) Body weight (A) and daily calorie intake (B) of the following two groups of mice with drug or vehicle delivered into the third ventricle: control mice treated with NB124 and mutant mice treated with NB124 (80 µg × 2, i.c.v.). (C–E) Immediately after the second treatment of NB124, O₂ consumption (C), CO₂ production (D), and locomotor activity (E) of the mice as described in A and B. (F) GTT was performed in control mice treated with NB124, mutant mice treated with vehicle, and mutant group treated with NB124. *P < 0.05 (between mutant+NB124 group and control+NB124 group; Student t test). Values represent group means and SEM; n = 8–10 mice per group. Control group, Agrp^nsCre/+::Gad1^lox/+::Gad2^lox/+::Rosa26^tdTomato mice; mutant group, Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice.

Fig. S6.

Treatment of NB124 leads to acute activation of Cre recombinase and subsequent removal of GABA signaling from AgRP neurons in 8-mo-old transgenic mice. (A) Immunostaining image showed tdTomato expression profile in the ARC of 8-mo-old mutant Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice 4 d after i.c.v. injection of NB124. (B and C) qPCR analysis of transcripts level of Gad1 (B) and Gad2 (C) expressed in tdTomato-positive AgRP neurons that were isolated through a flow cytometry approach 6 d after i.c.v. injection of NB124 or vehicle into 8-mo-old mutant group Agrp^nsCre/+::Gad1^lox/lox::Gad2^lox/lox::Rosa26^tdTomato mice. Values represent means and SEM; n = 5 or 6 mice per group. *P < 0.05 (Student t test). (Scale bar, 300 µm.)