http://www.pnas.org/content/102/50/18189/suppl/DC1.
The striatal cell lines were grown at 33°C in DMEM supplemented with 10% FBS, 1% nonessential amino acids, 2 mM l-glutamine, and 400 mg/ml G418 (Geneticin, Invitrogen). Primary cultures of mouse striatum were prepared as described (1). The cells were resuspended at a density of 4 × 106 cells per 100 ml in Amaxa nucleofector solution (Amaxa, Gaithersburg, MD) and electroporated according to the manufacturer’s specifications with Htt15Q-GFP, Htt138Q-GFP, myc-Htt23Q(1-110) and myc-Htt143Q(1-110). Immunofluorescence was measured as described (2). Cells were diluted with DMEM containing 10% FBS and seeded onto polylysine-coated glass chamber slides (Becton Dickinson) at 2.5-3.5 × 105 cells per cm2. After 30 min, the medium was replaced with neurobasal A medium containing 1 mM glutaMax-1, 24.5 mM KCl and 2% B-27 (Invitrogen). The cultures were incubated at 37°C in 95% air/5% carbon dioxide at 95% humidity. Efficiency of transfection was between 75% and 85% by day 4 as estimated by GFP fluorescence.
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.