Site-specific modulation of brain glucocorticoid receptor and corticotropin-releasing hormone expression using lentiviral vectors

Abstract

The glucocorticoid receptor (GR) and corticotropin-releasing hormone (CRH) are important molecular regulators of an individual's ability to respond to stressful stimuli in an adaptive manner. Impaired signaling of both GR and CRH often leads to dysfunction of the hypothalamic-pituitary-adrenal axis, which underlies the etiology of many affective disorders such as anxiety and depression. Studies focusing on how GR and CRH influence the stress response are limited as they generalize to broad brain regions, thus hindering identification of how specific CNS nuclei contribute to maladaptive stress responses. Our objective is to distinguish the site-specific involvement of GR and CRH in limbic regions involved in the stress response. With that intent, we use lentiviral (LV) vectors in combination with transgenic mouse lines, enabling us to modify expression of GR or CRH in a very localized manner. This paper describes the generation of several distinct LV vectors and transgenic mice models that will help further elucidate the site-specific actions of GR and CRH.

Figure 1. Constructs of lentiviral vectors packaged into replication deficient virions

Each LV vector contains an HIV-based system including 3' and 5' long terminal repeats (LTR), a 5' self-inactivating (SIN) element to prevent replication and a woodchuck post-transcriptional regulatory element to enhance protein expression (a.) LV-Cre (b.) LV-GFP (c.) LV-GR (d.) LV-rtTA (e.) LV-tetopCRH. ψ = packaging signal; RRE = Rev response element; cPPT = central polyurine tract; gag = group-specific antigen; EF1α, MNDU3, and tetop = promoters; nls = nuclear localization signal; eGFP = enhanced green fluorescent protein; GR = glucocorticoid receptor; rtTA = reverse tetracycline transactivator; CRH = corticotropin-releasing hormone.

Figure 2. GR protein levels after LV-GR and LV-GFP infection of CHO-K1 cells

Lysates were separated by SDS PAGE and immunoblots were probed with an anti-GR antibody recognizing the N-terminus. LV-GR virus stably expresses full-length mGR protein representing efficient transduction in CHO-K1 cells. Control samples represent CHO-K1 cells without LV infection. B-actin antibody served as an internal control to ensure equal protein loading.

Figure 3. In vitro analysis of pLV-rtTA

CHO cells were co-transfected with pTetop-luc and pLV-rtTA in the presence and absence of doxy to test the efficiency of the pLV-rtTA plasmid. Luciferase expression was then measured. pLV-rtTA displays robust expression in the presence of doxy with low levels of expression in the absence of doxy. RLU = relative light units, doxy = doxycycline

Figure 4. CRH immunoreactivity and hemotoxylin & eosin (H&E) staining in tetopCRH mice injected with LV-rtTA

(a.) CRH protein (dark brown pigment) is present when mice are on doxy chow. (b.) H & E staining displays inflammatory cell infiltration within the injected region.

Figure 5. CRH immunoreactivity and hemotoxylin & eosin (H & E) staining in CamKIIα-tTA mice injected with LV-tetopCRH

(a.) CRH protein (dark brown pigment) is present when mice are off doxy chow. (b. & c.) CRH protein is absent when mice are on doxy chow or in non-injected controls. H & E staining shows normal cells in the (d.) absence or (e.) presence of doxy as well as in (f.) non-injected controls.