Functional role of the N-terminal domain of ΔFosB in response to stress and drugs of abuse

Abstract

Previous work has implicated the transcription factor, ΔFosB, acting in the nucleus accumbens, in mediating the pro-rewarding effects of drugs of abuse such as cocaine as well as in mediating resilience to chronic social stress. However, the transgenic and viral gene transfer models used to establish these ΔFosB phenotypes express, in addition to ΔFosB, an alternative translation product of ΔFosB mRNA, termed Δ2ΔFosB, which lacks the N-terminal 78 aa present in ΔFosB. To study the possible contribution of Δ2ΔFosB to these drug and stress phenotypes, we prepared a viral vector that overexpresses a point mutant form of ΔFosB mRNA which cannot undergo alternative translation as well as a vector that overexpresses Δ2ΔFosB alone. Our results show that the mutant form of ΔFosB, when overexpressed in the nucleus accumbens, reproduces the enhancement of reward and of resilience seen with our earlier models, with no effects seen for Δ2ΔFosB. Overexpression of full length FosB, the other major product of the FosB gene, also has no effect. These findings confirm the unique role of ΔFosB in the nucleus accumbens in controlling responses to drugs of abuse and stress.

Keywords: FosB; cocaine; social defeat stress; viral-mediated gene transfer; Δ2ΔFosB; ΔFosB.

Figure 1. Expression levels of FosB gene products

A) Scheme showing that the FosB gene produces two mRNAs, FosB and ΔFosB. ΔFosB mRNA arises from alternative splicing within Exon 4 of the FosB primary transcript; this splicing generates a premature stop codon and the truncated ΔFosB protein. Δ2ΔFosB is generated from an alternative start codon corresponding to aa 79 in ΔFosB. The Δ1 and Δ3 ATGs, also present in the ΔFosB mRNA sequence, are not used as alteratnive start sites (Chen et al., 1997). The position of the BZIP domain present in all FosB gene products is shown. B) Western blotting of FosB gene products in NAc after repeated administration of cocaine (C, 15 mg/kg i.p., n=4–5) or saline (S) for 7 days, with mice analyzed 2 hr after the last injection. C) Western blotting of FosB gene products in NAc in bitransgenic mice that overexpress ΔFosB (Kelz et al., 1999), and to a much lesser extent Δ2ΔFosB, in the absence of doxycycline (DOX) (n=3–6). Histone H3 or GAPDH (glyceraldehyde 3-phosphate dehydrogenase), which are not regulated under the experimental conditions used, were utilized as a loading control. *p<0.05 by t-tests.

Figure 2. Selective expression of FosB gene products with AAV vectors in Neuro2A cells

Neuro2A cells were transfected with 0.25 μg of each AAV vector, or mock transfected with no vector, and cell extracts were analyzed by Western blotting. Arrowhead denotes full length FosB, two black arrows ΔFosB, and open arrow Δ2ΔFosB. Note the much lower levels of Δ2ΔFosB expressed by wildtype ΔFosB mRNA but not by mΔFosB mRNA which is not subject to alternative translation.

Figure 3. Effect of FosB gene products in NAc on behavioral responses to cocaine or social stress

AAV vectors described in Figure 2 were injected bilaterally into the NAc and separate groups of animals were studied in a battery of behavioral assays. A) Shows the targeting of the medial NAc by the viral vectors. AC, anterior commissure. B) Shows results of social interaction test one day after 10 days of social defeat stress (n=22–40). **p<0.01, ***p<0.001 by t-tests. C) Shows conditioned place preference for a threshold dose of cocaine (7.5 mg/kg i.p.) (F(3,107)=4.890, p < 0.001). *p<0.05, **p<0.01, ***p<0.001 in post-hoc test after ANOVA. D) Percent time spent in the open arms of an elevated plus maze. FosB and Δ2ΔFosB increased time in the open arms compared to Venus controls (*p<0.05 by t-tests), an effect not seen for ΔFosB (n=22–40). E) General locomotor activity in the animals’ home cage during the dark phase. No FosB gene product was different from Venus controls (n=22–40).