Induction of neostriatal neurogenesis slows disease progression in a transgenic murine model of Huntington disease

Abstract

Ependymal overexpression of brain-derived neurotrophic factor (BDNF) stimulates neuronal addition to the adult striatum, from subependymal progenitor cells. Noggin, by suppressing subependymal gliogenesis and increasing progenitor availability, potentiates this process. We asked whether BDNF/Noggin overexpression might be used to recruit new striatal neurons in R6/2 huntingtin transgenic mice. R6/2 mice injected with adenoviral BDNF and adenoviral Noggin (AdBDNF/AdNoggin) recruited BrdU(+)betaIII-tubulin(+) neurons, which developed as DARPP-32(+) and GABAergic medium spiny neurons that expressed either enkephalin or substance P and extended fibers to the globus pallidus. Only AdBDNF/AdNoggin-treated R6/2 mice harbored migrating doublecortin-defined neuroblasts in their striata, and the new neurons expressed p27 as a marker of mitotic quiescence after parenchymal integration. AdBDNF/AdNoggin-treated R6/2 mice sustained their rotarod performance and open-field activity and survived longer than did AdNull-treated and untreated controls. Neither motor performance nor survival improved in R6/2 mice treated only with AdBDNF, and intraventricular infusion of the mitotic inhibitor Ara-C completely blocked the performance and survival effects of AdBDNF/AdNoggin, suggesting that the benefits of AdBDNF/AdNoggin derived from neuronal addition. Thus, BDNF and Noggin induced striatal neuronal regeneration, delayed motor impairment, and extended survival in R6/2 mice, suggesting a new therapeutic strategy in Huntington disease.

Figures

Figure 1. AdBDNF induced striatal neuronal recruitment in both R6/2 and WT mice.

At 1 mo after AdBDNF injection (6 wk), R6/2 striata were double-stained for BrdU (green) and βIII-tubulin (red, A), NeuN (red, B), GAD67 (red, C), or DARPP-32 (red, D). Identically treated WT mice stained for the same markers are shown in E–H. Colabeling of BrdU with each neuronal marker was confirmed by confocal optical sectioning, with orthogonal views in the xz and yz planes (insets). Arrowheads denote double-labeled cells. Scale bars: 10 μm.

Figure 2. AdNoggin potentiated AdBDNF-induced neuronal addition and integration.

(A) Density of BrdU+βIII-tubulin+ cells in the striatum of R6/2 and WT mice injected once at 4 wk of age with AdBDNF/AdNoggin (AdB/N), AdBDNF, AdNoggin, AdNull, or saline. (B–E) Newly generated neurons were recognized in both WT (B) and R6/2 (C–E) mice by confocal imaging of BrdU (green) colabeling with βIII-tubulin (red, B and C), DARPP-32 (red, D), or GAD67 (red, E). (F) Density of BrdU+ cells (green) coexpressing either DARPP-32, GAD67, enkephalin (Enk), or SP in the striatum of AdBDNF/AdNoggin-, AdBDNF-, or AdNull-injected WT and R6/2 mice. (G and H) BrdU-tagged (green) enkephalinergic (red, G) and SP (red, H) neurons in R6/2 mice. (I and J) FG injection of the globus pallidus revealed BrdU-tagged striatal projection neurons in AdBDNF/AdNoggin-injected 11-wk-old WT (I) and R6/2 (J) mice. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by post-hoc Bonferroni t tests. Arrows denote double-labeled cells. Scale bars: 10 μm.

Figure 3. New neurons were the product of antecedent neuronal mitogenesis.

(A) Schematic of a mouse brain section through the striatum (STR) and lateral ventricle (LV) showing the locations of images in B–H (asterisks). CC, corpus callosum. (B) Striatal ventricular wall of an AdBDNF/AdNoggin-treated R6/2 mouse, given BrdU for 3 wk after viral injection at 6 wk of age and sacrificed at 10 wk, immunostained for BrdU (green), βIII-tubulin (red), and Ki67 (blue), which is expressed by mitotically active cells. In the subventricular zone, actively dividing subependymal cells expressed Ki67, whereas BrdU+ daughter cells, the products of earlier divisions, did not. In the neostriatum within the same section, newly generated BrdU+ neurons identified by βIII-tubulin (C) or NeuN (D) did not express Ki67 and thus showed no evidence of either persistent mitotic competence or aberrant cell cycle reentry. To the contrary, newly generated BrdU+βIII-tubulin+ (E) and BrdU+NeuN+ (F) neurons expressed the tumor suppressor p27kip1 (blue, E and F), a marker of mitotic quiescence. A cohort of BrdU+ cells in AdBDNF/AdNoggin-treated mice coexpressed the developmental migratory neuroblastic marker DCX (red, G and H); importantly, these BrdU+DCX+ cells were not found in AdNull-treated R6/2 striata at this age, indicating that the immigration of migrating neuroblasts into the R6/2 striatum was a function of AdBDNF/AdNoggin treatment. Scale bars: 10 μm (B–F); 5 μm (G and H).

Figure 4. Induced neurogenesis was associated with delayed disease progression and prolonged survival.

(A and B) AdBDNF/AdNoggin-cotreated R6/2 mice exhibited slower deterioration of motor performance than did AdBDNF-treated or untreated control R6/2 mice. (A) Time of sustained rotarod performance as a function of time point and treatment, using a 300-s rotarod challenge. (B) Results of the same challenge as in A presented as the percent rotarod impairment (see Methods). (C) Open-field testing revealed that AdBDNF/AdNoggin-treated R6/2 mice sustained volitional explorative behavior, as manifested in spontaneous horizontal locomotion, longer than did mice treated with AdBDNF only or AdNull or left untreated. (D) Kaplan-Meier analysis revealed that survival was significantly extended in R6/2 mice injected with AdBDNF/AdNoggin compared with R6/2 mice injected with AdBDNF only or AdNull or left untreated. Pre, preoperative. *P < 0.05, **P < 0.01 versus AdNull and untreated; ‡P < 0.01 versus AdBDNF.

Figure 5. Ara-C suppressed neuronal mitogenesis.

(A) Number of newly generated striatal neurons in both R6/2 and WT mice after treatment with AdBDNF/AdNoggin or AdNull, both with and without infusion of the mitotic inhibitor Ara-C. Ara-C essentially abrogated AdBDNF/AdNoggin-associated neuronal addition. (B–E) AdBDNF/AdNoggin-treated WT (B) and R6/2 (D) mice exhibited robust incorporation of BrdU (green) by subependymal progenitor cells. In contrast, Ara-C infusion potently suppressed mitotic activity within the subependyma, essentially abrogating neuronal addition to both WT (C) and R6/2 (E) neostriata. **P < 0.001. Scale bar: 100 μm.

Figure 6. Ara-C inhibition of neurogenesis blocked the treatment-associated delay in disease progression.

(A) Ara-C infusion blocked the relative preservation of R6/2 rotarod performance otherwise afforded by AdBDNF/AdNoggin treatment. (B) Rotarod impairment is shown as a function of time point and treatment. (C) Ara-C infusion similarly inhibited the relative maintenance of volitional explorative behavior exhibited by AdBDNF/AdNoggin-treated R6/2 mice. (D) Kaplan-Meier analysis confirmed that survival was significantly extended in R6/2 mice injected with AdBDNF and AdNoggin, compared with AdNull-injected R6/2 mice, and revealed that the Ara-C–mediated inhibition of mitotic neurogenesis completely abrogated that survival benefit. *P < 0.05, **P < 0.01 versus Ara-C–infused AdBDNF/AdNoggin and saline-infused controls.