FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington's disease

Abstract

There is no satisfactory treatment for Huntington's disease (HD), a hereditary neurodegenerative disorder that produces chorea, dementia, and death. One potential treatment strategy involves the replacement of dead neurons by stimulating the proliferation of endogenous neuronal precursors (neurogenesis) and their migration into damaged regions of the brain. Because growth factors are neuroprotective in some settings and can also stimulate neurogenesis, we treated HD transgenic R6/2 mice from 8 weeks of age until death by s.c. administration of FGF-2. FGF-2 increased the number of proliferating cells in the subventricular zone by approximately 30% in wild-type mice, and by approximately 150% in HD transgenic R6/2 mice. FGF-2 also induced the recruitment of new neurons from the subventricular zone into the neostriatum and cerebral cortex of HD transgenic R6/2 mice. In the striatum, these neurons were DARPP-32-expressing medium spiny neurons, consistent with the phenotype of neurons lost in HD. FGF-2 was neuroprotective as well, because it blocked cell death induced by mutant expanded Htt in primary striatal cultures. FGF-2 also reduced polyglutamine aggregates, improved motor performance, and extended lifespan by approximately 20%. We conclude that FGF-2 improves neurological deficits and longevity in a transgenic mouse model of HD, and that its neuroprotective and neuroproliferative effects may contribute to this improvement.

Figures

Fig. 1.

FGF-2 treatment enhances neurogenesis in HD transgenic R6/2. (a and b) HD transgenic R6/2 and wild-type control mice were given i.p. BrdUrd for 3 days, treated with s.c. vehicle (PBS) or FGF-2 (3 weeks), and killed 24 h later. Immunocytochemistry showed a modest increase in the number of BrdUrd-labeled cells (red) in SVZ of PBS-treated HD transgenic R6/2 compared to wild-type mice. FGF-2 enhanced BrdUrd labeling slightly in wild-type and markedly in HD transgenic R6/2 mice. (Scale bar, 150 μm.) Data are representative fields from at least three experiments per panel or mean ± SEM, n = 3. *, P < 0.05; **, P < 0.01 relative to PBS-treated mice. (c and d) Immunocytochemistry with anti-DCX antibody showed an increase in the number of DCX-expressing (new) neurons in FGF-compared to PBS-treated HD transgenic R6/2 mice. (c Inset) DCX migrating cell. (Scale bars, 200 μmin c and 50 μmin d.)

Fig. 2.

FGF-2 treatment generates DARPP-32-expressing striatal and NeuN-expressing cortical neurons. (a and b) DARPP-32 (green) and DCX (red) were coexpressed in striatal (Str) neurons (a) and NeuN (green) and DCX (red) show co-expression in cortical (Ctx) neurons (b) from FGF-2-treated HD transgenic R6/2 mice. Littermate control mice treated with FGF-2 did not have DCX and DARPP-32 coexpressed. (Scale bar, 10 μm.) (c) Mice received stereotaxic injection (anterior posterior -0.3 mm, lateral 1.7 mm, depth 3.5 mm) into the globus pallidus with retrograde tracer Alexa Fluor 488 (Left). Retrograde tracer Alexa Fluor 488 (Right) could be detected in the caudate via retrograde transport. No diffusion of Alexa Fluor 488 was detected from the globus pallidus. (d) Alexa Fluor 488 (green), DCX (red), and NeuN (blue) were coexpressed in FGF-2-treated mice, demonstrating extending fibers into the pallidal targets. (Scale bar, 10 μm.) (e) Confocal images of Alexa Fluor 488 (green) and BrdUrd (red) in the striata of FGF-2-treated HD transgenic R6/2 mice are shown as both single optical sections and orthogonal views in the xz and yz planes, to confirm that some BrdUrd cells were positive for retrograde tracer (Left). Confocal images of DARPP-32 (green) and BrdUrd (red) in the striata of FGF-2 treated HD transgenic R6/2 mice to confirm that some BrdUrd cells were positive for DARPP-32 (Right).

Fig. 3.

FGF-2 prolongs survival and improves rotarod performance in HD transgenic R6/2 mice. (a) HD transgenic R6/2 mice were given PBS (HD untreated) or FGF-2 (HD + FGF-2) beginning at 59 days of age (arrow), and survival was plotted. FGF-2 increased survival, as described in Results (n = 10; *, P < 0.05). (b) Motor performance was evaluated with a rotarod apparatus in 11- and 13-week-old littermate controls (NonTg) and in HD transgenic R6/2 mice (HD) treated with PBS or FGF-2. The total time spent on the rod during a 5-min period (latency) was recorded. Values are the means ± SEM (n = 10 per group); **, P < 0.01 compared to PBS.

Fig. 4.

FGF-2 is neuroprotective in HD striatal neuron cultures and does not increase BDNF or CNTF levels in HD transgenic R6/2 mice. (a) Immortalized striatal neurons expressing wild-type Htt (STHttQ7/Q7) or a knocked-in HD mutation with 111 polyglutamine repeats (STHttQ111/Q111) were subjected to serum withdrawal, and cellular viability was assessed with WST-1 assay. **, P < 0.01 compared to untreated cultures (ANOVA, n = 3). (b) Electroporation with a GFP-expressing vector resulted in >50% transfection efficiency in primary cultures of striatal neurons, as shown by immunostaining for GFP (Left). Striatal neurons transfected with a mutant Htt147Q(1-111) construct showed extensive cell death (72 h, Center), which was rescued by treatment with FGF-2 (Right). Cultures shown at Center and Right were immunostained with monoclonal anti-Htt 2170 (Chemicon; 1:100). Nuclei were counterstained with DAPI (blue). (c) Striatal neurons were transfected with Htt23Q(1-111), Htt138Q-GFP, or Htt147Q(1-111) and cell viability was assessed by using calcein-AM and ethidium homodimer-1 (LIVE/DEAD kit, Molecular Probes) at 48 h. Data shown are mean values ± SEM (n = 3-5). *, P < 0.05 compared to untreated cultures (ANOVA, n = 3). (d) Western blot analysis of BDNF and CNTF levels of striatal lysates from 11-week-old littermate controls (WT) and HD transgenic R6/2 mice (HD) treated with PBS or FGF-2 for 3 weeks; anti-actin was used as a control for differences in protein loading. It should be noted that the absence of BDNF expression changes by Western blot does not rule out involvement of BDNF in specific cells in response to FGF-2.

Fig. 5.

Histopathological evidence of in vivo neuroprotection by FGF-2 in 11-week old HD transgenic R6/2 mice. (a) Ubiquitin immunohistochemistry (brown) in PBS- and FGF-2-treated HD transgenic R6/2 mice showed ubiquitin immunoreactivity in PBS-treated HD mice, but not in wild-type (not shown) or FGF-2-treated HD mice. Htt immunohistochemistry (brown) in neostriatum (Str, data not shown) and cortex (Ctx) showed Htt-immunoreactive aggregates (arrows) in PBS-treated, but not FGF-2-treated, HD transgenic R6/2 mice (bottom two rows). (b and c) CB1 cannabinoid receptor (b) and DARPP-32 immunoreactivity (c) in PBS- and FGF-2-treated littermate control (WT) and HD transgenic R6/2 (HD) mice showed that both CB1 and DARPP-32 were depleted from the affected striatum and restored by FGF-2 treatment. The counterstain is hematoxylin.