Fig 2: Schematic illustration of the central action of novel generation hormone therapy targeting the androgen signaling axis, abiraterone acetate+prednisone (AAP) and enzalutamide (ENZ) in aged castrated mice. Note. (A) ENZ which is able to cross the blood–brain barrier (BBB) can bind and inhibit ARs expressed in peripheral and brain organs. More specifically, CYP17A1 and AR are localized in both SNpc and VTA neurons in aged castrated mouse brains. ENZ leads to reduced P-DARPP-32 activated signaling in mature neurons expressing DR1 in the striatum through the nigrostriatal pathway and in the vHP via part of the mesolimbocortical pathway. This inhibition of DA-expressing afferents likely accounts for diminished locomotor activity and exploration behavior. ENZ also depressed mature neuron activity evidenced by c-fos expression in dHP potentially responsible for an altered spatial cognitive performance. (B) AAP is a prodrug, generating abiraterone selective for CYP17A1 enzyme activity, thus inhibiting androgen biosynthesis in the testis and the adrenal gland, but potentially in the VTA and SNpc. AA may have the ability to cross the BBB. In order to avoid cortisol deficiency, the treatment is combined with chronic use of prednisone (P) sufficient to activate the HPA-related feedback loop and to reduce the plasma ACTH level allowing physiological cortisol production in patients. CYP17A1 and AR are expressed in the dHP, and here the AAP effect on proliferation of precursor cells and inhibition of BDNF production without modification of mature neurons would hypothetically involve glucocorticoids receptors. In the vHP, the AAP-evoked reduction of c-Fos in mature neurons can sustain the delay in efficiency in spatial learning acquisition. AA, abiraterone acetate; AAP, abiraterone acetate-prednisone; ACTH, adrenocorticotropic hormone; AR, androgen receptor; BBB, blood–brain barrier; BDNF, Brain-derived neurotrophic factor; c-fos, cellular oncogene c-fos; CYP17A1, Cytochrome P450 Family 17 Subfamily A Member 1; DA, dopamine; dHP, dorsal hippocampus; ENZ, enzalutamide; HPA, hypothalamic–pituitary–adrenal; P, prednisone; P-DARPP-32, Phosphorylated form of Dopamine cAMP-Regulated Neuronal Phosphoprotein; DR1, DA receptor 1; SNpc, substantia nigra pars compacta; vHP, ventral hippocampus; VTA, ventral tegmental area.

Fig 3: AR and CYP17A1 are expressed in the substantia nigra pars compacta and ventral tegmental area and ENZ inhibits dopaminergic activity in both midbrain nuclei. Note. (A) Comparative Radar Chart of performance index scores across emotional and exploratory behavioral domains after ENZ (left panel) or AAP (right panel) treatments compared with Vehicles as presented in Figure 2A and Table S3. Based on the behavioral modifications, schematic representation (Middle panel) of nigrostriatal and corticolimbic circuits potentially involved in ENZ-altered locomotor activity and exploration. Based on previous studies, AR may be expressed in ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) dopaminergic neurons. SNpc projects dopamine (DA) axons to the striatum (nigrostriatal pathway) while VTA projects DA axons to ventral hippocampus (vHp) (corticolimbic pathway), also expressing AR. Statistical analysis was done on performances of vehicle (n = 14) and treated (n = 16) mice using unpaired t-test or Mann–Whitney test; Data are mean ± SEM; * p = 0.05. (B) Schematic drawing of SNpc and VTA dopaminergic neurons expressing AR. Tyrosine hydroxylase (TH) (green) and AR (red)-immunoreactivity in the SNpc and VTA of ENZ-, AAP- and Vehicle-treated mice. The boxed areas showed magnification of clusters of TH+/AR+ cells. Scale bar: 250 µm and 100 µm. (C) Schematic drawing of Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17A1) expression in SNpc and VTA and potential binding of local androgens on AR expressed by dopaminergic neurons. Representative example of CYP17A1 (green), NeuN (red) and GFAP (cyan) immunoreactivities in the SNpc and VTA of ENZ-, AAP- and Vehicle-treated mice. Scale bar: 250 µm. (D) Whisker boxes represent the number of TH+ cells and AR+ cells in VTA (left panel) and in SNpc (right panel) of ENZ- and AAP- treated mice compared with respective vehicles. Statistical quantification was performed by using Mann–Whitney test. Data are represented as box and whiskers and mean ± SEM (n = 4 mice), * p < 0.05. AAP, abiraterone acetate-prednisone; AR, androgen receptor; BBB, brain blood barrier; CYP17A1, cytochrome P450 family 17 subfamily A member 17; DA, dopamine; ENZ, enzalutamide; EPM, elevated plus maze; FST, forced swim test; GFAP, Glial Fibrillary Acidic Protein; LDB, light-dark box; NeuN, Neuronal Nuclei Antigen; OFT, open-field test; SNpc, substantia nigra pars compacta; TH, Tyrosine hydroxylase; TST, tail suspension test; vHp, ventral hippocampus; VTA, ventral tegmental area.

Fig 4: AAP and ENZ treatment impairs adult neurogenesis and neuronal activity in dorsal hippocampus respectively. Note. (A) Immunofluorescence of TH+-DA projections to dHP of ENZ-, AAP- and vehicle-treated mice (seen on magnification of squared areas). Whisker boxes represent the number of TH+ fibers in dHP. Statistical quantifications were performed using Mann–Whitney test. Bars are mean ± SEM (n = 4 mice). Scale bars: 100 µm. (B) Representative immunofluorescence of CYP17A1 (green), NeuN (red) and GFAP (cyan) in the DG of dHP of ENZ-, AAP- and vehicles-treated mice showing specific localization of CYP17A1 in neuronal and not glial cells. (C) Upper panel, schematic of mouse dorsal hippocampal (dHp) CYP17A1-mediating production of testosterone and DHT, potentially responsible for spatial learning deficits. Lower panel, hypothetical illustration of the impact of testosterone and DHT or prednisolone on neurogenesis. CYP17A1 expression by mature hippocampal neurons (neuron in light blue; astrocyte in red) of granule cell layer (GCL) of DG; AR is not expressed in neural progenitors cells stained by an anti-BrdU (orange) in the SGZ and by immature hippocampal neurons classically revealed by staining of an anti-DCX (grey) in the DG of vHP. AR expressed by mature hippocampal neurons revealed by anti-NeuN (light blue). AR (green) activation by T or DHT would control mature neuron (blue) activity as illustrated by c-fos immunolabeling. (D) Upper panel, representative immunolabeling of BrdU (green) and AR (red) immunofluorescence in dHP of ENZ-, AAP- and vehicles-treated mice. The boxed areas showed a magnification of lack of BrdU+/AR+ co-stained progenitor cells. Middle panel, immunolabeling of DCX (grey) and AR (red) immunofluorescence in dHp of ENZ-, AAP- and vehicle-treated mice. The boxed areas showed a magnification of lack of DCX+/AR+ co-stained immature migratory neurons in the SGZ. Lower panel, immunolabeling of NeuN (grey) and AR (red) in dHp of ENZ-, AAP- and vehicle-treated mice. The boxed areas showed a magnification of NeuN/AR co-expression in mature neurons. Scale bar: 100 µm and 50 µm. AAP, abiraterone acetate-prednisone; AR, androgen receptor; BrdU, Bromodeoxyuridine; c-fos, cellular oncogene c-fos; CYP17A1, Cytochrome P450 Family 17 Subfamily A Member 1; DA, dopamine; DCX, doublecortin; DG, dentate gyrus; dHP, dorsal hippocampus; DHT, dihydrotestosterone; ENZ, enzalutamide; GCL, granule cell layer; GFAP, Glial Fibrillary Acidic Protein; NeuN, Neuronal Nuclei Antigen; SGZ, sub-granular zone; T, testosterone; TH, Tyrosine hydroxylase; vHP, ventral hippocampus.