Testosterone-induced matrix metalloproteinase activation is a checkpoint for neuronal addition to the adult songbird brain

Abstract

Testosterone-induced neuronal addition to the adult songbird vocal control center, HVC, requires the androgenic induction of vascular endothelial growth factor (VEGF), followed by VEGF-stimulated angiogenesis. The expanded vasculature acts as a source of BDNF, which supports the immigration of new neurons from the overlying ventricular zone. In tumorigenesis, a similar process of adult angiogenesis is regulated by matrix metalloproteinase (MMP) activity, in particular that of the gelatinases. We therefore investigated the role of the gelatinases in neuronal addition to the HVC of adult female canaries. In situ zymography of the caudal forebrain revealed that testosterone-induced perivascular gelatinase activity that was most prominent in HVC. High-resolution gels revealed distinct MMP activities that comigrated with MMP2 and MMP9, and PCR cloning yielded MMP2 and MMP9 orthologues of 1465 and 1044 bp, respectively. Quantitative PCR revealed that HVC MMP2 mRNA levels doubled within 8 d of testosterone, whereas MMP9 transcript levels were stable. Moreover, isolated adult canary forebrain endothelial cells secreted MMP2, and VEGF substantially increased endothelial MMP2 gelatinase activity. To assess the importance of androgen-regulated, VEGF-induced MMP2 to adult angiogenesis and neurogenesis, we treated testosterone-implanted females with the gelatinase inhibitor SB-3CT. In situ zymography confirmed that SB-3CT suppressed gelatinase activity in HVC, and histological analysis revealed that SB-3CT-treated birds exhibited a decreased endothelial mitotic index and substantially diminished neuronal recruitment to HVC. These data suggest that the androgenic induction of endothelial MMP2 is a critical regulator of neuronal addition to the adult HVC, and as such comprises an important regulatory step in adult neurogenesis.

Figure 1.

Testosterone induces MMP activity within the HVC microvasculature. A, Nissl-stained HVC section of adult female canary brain, including HVC at the dorsal surface of the mesopallium. For terminology used, see Reiner et al. (2004) and Jarvis et al. (2005). B, Estrogen receptor-α immunoreactivity was used to define the borders of HVC for cell quantification purposes. C, D, In situ zymography using Oregon 488-conjugated gelatin was used to localize gelatinase activity (green), in sections counterstained with DAPI (blue). C, A low-magnification image of HVC and its surround in an unimplanted adult female canary. D, A matched adult female experimental given testosterone 9 d before being killed. High signal in the dorsocaudal mesopallium is evident, and essentially restricted to HVC. E, F, Higher-magnification images, without counterstaining, of zymography-defined HVC MMP activity in an unimplanted control (E) and in a testosterone-implanted bird (F). Scale bars, 100 μm.

Figure 2.

VEGF triggers MMP2 secretion by canary forebrain endothelial cells. A, Both pro-MMP2 and pro-MMP9 gelatinase activities were noted by gel zymography of adult female HVC tissue extracts, taken from birds given testosterone and killed 8 d after testosterone treatment. These gels were developed briefly, highlighting the gelatinase activities associated with the MMP2 and MMP9 pro- forms, which are both more abundant than their active products, and enzymatically active in denaturing gels like these. B, Media from 2 d serum-free cultures of isolated canary forebrain endothelial cells were analyzed by gel zymogram; 4 μg of sample were loaded per well. VEGF exposure induced a significant increment in gelatinase activity; testosterone treatment did not. C, D, Pixel analyses of gel zymograms for active and pro-MMP2. Pixel intensity from different gels was normalized against a common control. E, RT-PCR showed that CBECs exhibited increased MMP2 transcript levels in response to VEGF. MMP9 mRNA was also expressed, but exhibited no evident response to VEGF or testosterone. **p < 0.01 by ANOVA with Bonferroni t tests.

Figure 3.

Testosterone treatment stimulated MMP2 transcription in vivo. Quantitative RT-PCR of HVC mRNA derived from testosterone-treated birds revealed that MMP2 levels rose by 4 d (T4) after testosterone and had at least doubled by 8 d; in contrast, MMP9 levels were not affected by testosterone treatment. *p < 0.05; **p < 0.01; ***p < 0.001 by ANOVA with Bonferroni t tests.

Figure 4.

Testosterone-induced HVC MMP activity is blocked by the MMP2/9 inhibitor SB-3CT. A–C, For 9 d after testosterone implantation, birds were given vehicle (A), the MMP2/9 inhibitor SB-3CT at 50 mg/kg (B), or SB-3CT at 100 mg/kg (C). On the ninth day (T9), the birds were killed, and their degree of HVC gelatinase inhibition was assessed by in situ zymography. D–F, Surface plot analysis of signal intensity using ImageJ; D–F individually correspond to A–C. G–I, Distribution histogram for pixel intensity; G–I correspond to A–C. Scale bars, 100 μm.

Figure 5.

MMP2/9 blockade inhibits androgen-induced angiogenesis and neuronal addition. A, The experimental timeline for assessing the effects of SB-3CT-mediated MMP inhibition on testosterone-induced angiogenesis and neuronal addition. B, BrdU⁺/laminin⁺ newly generated endothelial cells, in an adult female HVC microvasculature 9 d after testosterone administration (11 d after vehicle injection), with daily BrdU administration from throughout. C, Confocal validation, with orthogonal side views, of laminin immunoreactivity (red) by the BrdU⁺ (green) cells. D, The incidence of newly generated endothelial cells (BrdU⁺/laminin⁺), expressed as a percentage of all laminin⁺/DAPI⁺ endothelial cells within the border of HVC. E, F, Examples of scored BrdU⁺/Hu⁺ cells. Confocal validation of the double labeling of BrdU⁺ (green)/Hu⁺ (red). G, The incidence of newly recruited BrdU⁺/Hu⁺ neurons, expressed as a percentage of all Hu⁺ neurons in HVC. Scale bars: B, E, 50 μm; C, F, 10 μm. ***p < 0.001 by ANOVA with Bonferroni t tests. T, Testosterone; NS, null (empty) SILASTIC.

Figure 6.

An orchestrated set of paracrine interactions mediates adult HVC neurogenesis. This schematic highlights the role of HVC MMP2 during testosterone-induced neuronal recruitment by the adult HVC. Together with the gonadal steroid-mediated induction of both VEGF and its receptor VEGFR2/flk1, which leads in turn to angiogenesis and BDNF production by the activated microvascular bed, the androgen-triggered vascular production of MMP2 appears to comprise a critical gate-keeping step, that establishes the permissiveness of the adult brain parenchyma to accept new neuronal immigrants (adapted in part from Louissaint et al., 2002).