C57BL/6N mutation in cytoplasmic FMRP interacting protein 2 regulates cocaine response

Abstract

The inbred mouse C57BL/6J is the reference strain for genome sequence and for most behavioral and physiological phenotypes. However, the International Knockout Mouse Consortium uses an embryonic stem cell line derived from a related C57BL/6N substrain. We found that C57BL/6N has a lower acute and sensitized response to cocaine and methamphetamine. We mapped a single causative locus and identified a nonsynonymous mutation of serine to phenylalanine (S968F) in Cytoplasmic FMRP interacting protein 2 (Cyfip2) as the causative variant. The S968F mutation destabilizes CYFIP2, and deletion of the C57BL/6N mutant allele leads to acute and sensitized cocaine-response phenotypes. We propose that CYFIP2 is a key regulator of cocaine response in mammals and present a framework to use mouse substrains to identify previously unknown genes and alleles regulating behavior.

Fig. 1

Acute and sensitized cocaine response, measured by locomotor hyperactivity, is lower in C57BL/6N (B6N) substrain than in C57BL/6J (B6J). (A) Baseline locomotor velocity data of B6J (blue) and B6N (green) mice were measured for 30 minutes and then injected with cocaine (20 mg/kg, red arrow) and recorded for further 60 minutes. (B) Cocaine dose response for B6J and B6N. (C) Decreased methamphetamine response in B6N. Animals were injected with methamphetamine (2mg/kg, red arrow) and recorded for 3 hours. (D) Methamphetamine dose response for B6J and B6N. Mice were treated with 1mg/kg, 2mg/kg and 4mg/kg of the drug. Average response 60 minutes post injection is shown. (E, F) B6N have a lower sensitized response to cocaine at 10mg/kg (E) but respond similarly at 15mg/kg dose (F). Mice were measured for baseline and post injection response for one hour and injected with saline(S) or cocaine(C). 30-minute post injection response is shown. All data are shown as mean ± SEM, the number of animals in each group is indicated; *p<0.05,**p<0.01,***p<0.001,****p<0.0001 from Tukey post hoc test following two way ANOVA (B,D) or pair wise t-test (E,F).

Fig. 2

QTL on chromosome 11 regulates cocaine response. (A) Hyperactivity following intraperitoneal injection of cocaine (20 mg/kg) was quantitated. B6J (n = 73), B6N (n = 44), F1 (n = 124), F2 (n = 244) were tested. The blue box represents mean ± 1SD range and histogram shows normal distribution of the measures. (B) Locations of polymorphic markers between B6J and B6N. (C) Genome wide QTL scan revealed a single highly significant QTL on chromosome 11. The significance thresholds are established through 100,000 permutation tests. (D) The chromosome 11 QTL is significant for multiple cocaine response measures. (E) Genotype effect plot at maker rs13481014 shows the B6N allele has lower response than B6J allele, and the F1 is intermediate. Data are mean ± SEM.

Fig. 3

Next generation sequencing identifies a single nonsynonymous polymorphism in Cyfip2. Classification of SNPs (A), indels (B), and structural variants (C) sequencing reveals only a single SNP (top row of A) in Cyfip2 (D) that changes Serine 968 to phenylalanine in B6N. Only the QTL support interval is shown. (E, F) Comparison of C57BL/6 substrains shows that S968F variant was fixed in B6N lineage between 1961 and 1974.

Fig. 4

CYFIP2 S968F causes destabilization and knockout analyses of Cyfip2 shows heterozygous mice have cocaine response phenotypes. (A) Protein stability assay shows CYFIP2 S968F is less stable. 293T cells transfected with FLAG-CYFIP2 (green bands) were treated cycloheximide for the indicated times and western blot was performed (bottom). ß-tubulin was used as control (red bands). Replicate experiments were quantitated and half-life was calculated using nonlinear one phase decay. K, the half-file parameter is highly significant p < 0.0001 (top panel). (B) Quantitative IP experiments show CYFIP2 S968F interacts with WAVE complex members. IP (left) was conducted in replicate and binding was quantitated (right). (C) Mice generated from ES cells harboring the knockout first allele of Cyfip2 from the IKMC are on B6N background (designated Cyfip2^B6N/B6N for WT and Cyfip2^B6N/− for heterozygous knockout). The homozygous deletion is lethal at birth and therefore not shown. Cyfip2^B6N/B6N animals have lower cocaine response than the Cyfip2^B6N/− mice. (D) The Cyfip2 heterozygous mice show higher sensitized response to cocaine. Cyfip2^B6N/B6N (orange), Cyfip2^B6N/− (purple) were injected with saline (S) or cocaine (C, 10 mg/kg). Pairwise comparisons for genotype for each day were significant as indicated. (E) Diolistic labeling of nucleus accumbens neurons shows decrease in dendritic spine density in B6N. Sample images are shown with representative location of neurons (yellow on coronal section of brain). Quantitation and classification of spines are shown. At least 6 animals were used in each group with over 10 images collected from each animal. (F) B6N has a decrease in frequency of AMPAR mEPSCs. Sample traces of mEPSCs in NAc shell MSNs from B6J and B6N mice. Dot plots for mEPSC amplitude and frequency. Data are mean ± SEM and Student's t-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Two-way ANOVA with Tukey post hoc test (A).