These results indicate that the concurrent overexpression of BDNF and Noggin may be used to induce quantitatively substantial and functionally significant neuronal recruitment from endogenous progenitor cells into the R6/2 huntingtin mutant neostriatum. When R6/2 mice were assessed at 10 wk of age, BDNF overexpression beginning at 6 wk was found to have induced the addition of 138 ± 34 new neurons/mm3
. Although significant, this represents just 0.6% of the striatal neuronal population. On this basis, we then coexpressed BDNF together with Noggin so as to suppress non-neurogenic pathways of subependymal cell differentiation and thereby increase the pool of progenitor cells potentially responsive to BDNF. By this means, 268 ± 53 neurons/mm3
were added to the AdBDNF/AdNoggin-treated R6/2 striatum within a month of viral injection, representing an increment of 1.2% of the mean striatal neuronal pool during that month. Whether such numbers can be uniformly recruited over longer periods of time using more sustained expression vectors will depend upon the capacity for sustained neurogenesis by the R6/2 neural stem cell pool, as well as the sustained receptivity of the tissue. Nonetheless, recent reports of expanded endogenous neural stem cell populations in both R6/2 mice (42
) and HD patients (43
) suggest that subependymal progenitor cell populations potentially receptive to BDNF/Noggin-induced mobilization indeed remain abundant and active in the HD brain.
Adenoviral gene expression is transient, so that meaningful BDNF and Noggin expression persists for less than 2 mo after intraventricular viral injection (11
). Nonetheless, a pronounced functional benefit was noted in these AdBDNF/AdNoggin-treated mice that persisted long after their effective period of BDNF and Noggin overexpression: AdBDNF/AdNoggin treatment delayed the onset and slowed the course of motor deterioration of the R6/2 mice, just as it extended their survival. Interestingly, the enhanced performance and prolonged survival of the treated mice was thus associated with neuronal addition that had occurred during the discrete period of BDNF and Noggin overexpression, which likely ceased months before the treated animals actually died. This argues for the sustained functional benefits potentially associated with antecedent neuronal addition and suggests the even greater likely value of more sustained BDNF and Noggin overexpression strategies.
In this regard, it is important to note that the AdBDNF/AdNoggin-associated survival benefit was entirely abolished by concurrent intraventricular infusion of the mitotic inhibitor Ara-C, delivered at low doses that impede central neurogenesis (41
) but otherwise lack known central or systemic toxicity (44
). These results argue that induced restorative neurogenesis can delay symptom progression in a prototypic mouse model of HD. In addition, the inhibition of this effect by the concurrent Ara-C–mediated suppression of neurogenesis indicated that the improved motor performance and survival of AdBDNF/AdNoggin-treated R6/2 mice required neuronal addition; both BDNF and Noggin might still have exercised neuroprotective effects on threatened striatal neurons, but if such effects were operative, they were alone insufficient to significantly improve either motor performance or survival in these mice.
Our evidence strongly points to mitotic neurogenesis as the basis for BrdU incorporation by the BrdU+
neurons that we noted. This point bears scrutiny, given several recent reports of BrdU incorporation in the setting of neuronal injury and degeneration. Kuan and colleagues (32
) noted that neurons dying of hypoxic ischemia may reenter the cell cycle, and as a result express markers consistent with G1/S phase transition, such as Ki67, while incorporating BrdU and losing expression of the CDK proteins p16 and p27. However, no such evidence of aberrant cell cycle reentry was noted in a variety of other insults, which included adrenalectomy, kainite toxicity, and major trauma. Kuan et al. assessed the temporal relationship of ectopic cell cycle reentry to neuronal death in detail and noted that BrdU-incorporating neurons appeared by 5 d after ischemic injury, but were no longer evident by 28 d. When apparent at 5 d, they expressed the mitotic marker Ki67 and had lost expression of the mitotic inhibitor p27, suggesting cell cycle reentry. In addition, Kuan et al. noted that BrdU-incorporating neurons arising after ischemia quickly lost expression of the neuronal skeletal constituent βIII-tubulin, so that the cells had to be identified as such by colabeling with the more stable marker NeuN. As a result, these authors found no evidence of BrdU+
cells after the first week after ischemic injury (32
In contrast to the failure of Kuan et al. to observe BrdU+
neurons at 4 wk after ischemia (32
), we noted abundant BrdU+
neurons in the AdBDNF/AdNoggin-treated neostriata of both R6/2 and WT mice as long as 6 wk after viral injection. These cells expressed DARPP-32 and either enkephalin or SP and maintained projections to the globus pallidus that could be FG backfilled as long as 7 wk after viral treatment. Furthermore, all of our quantitative scoring was done using an BrdU+
end point at 4 wk after viral injection; Kuan et al. noted the complete disappearance of this antigenic phenotype by 4 wk (32
). To emphasize this point, we reliably noted over 300 BrdU+
added to both R6/2 and WT mice after AdBDNF/AdNoggin treatment, a number that vastly surpassed the few BrdU+
striatal neurons (and lack of BrdU+
neurons) noted by Kuan et al. 4 wk after ischemic injury (32
). In this regard, it is also worth noting that we have previously reported the persistence of these cells as long as 56 d after production in rats, with little fall-off from the numbers noted at 21 d in that study (18
). Thus, unlike the transient appearance and rapid clearance of a BrdU-incorporating βIII-tubulin+
neuronal phenotype immediately following hypoxic ischemia, the striatal neurons generated in response to BDNF and Noggin overexpression in the R6/2 brain were long lasting and stably integrating.
In studies that paralleled those of Kuan et al. (32
), Herrup and colleagues reported that neuronal cell death in both Alzheimer disease and ataxia telangiectasia can be preceded by cell cycle reentry (33
). These observations again raise the possibility that neuronal death in some pathologies might be heralded by aborted cell cycle reentry, with attendant neuronal incorporation of BrdU. To assess whether R6/2 HD mice exhibit BrdU incorporation by neurons dying as a result of aborted cell cycle reentry, we performed a number of additional experiments. First, TUNEL staining at 10 wk revealed very few dying neurons, consistent with previous reports that R6/2 mice exhibit little if any apoptotic neuronal death (23
). Second, immunostaining for the mitotic marker Ki67 revealed no evidence of Ki67-expressing βIII-tubulin+
striatal neurons; to the contrary, BrdU+
neurons were noted to invariably express the CDK inhibitor p27, as would be expected of postmitotic neurons that had incorporated BrdU previously. In addition, it is important to note that we observed BrdU+
neurons as a function of BDNF/Noggin treatment, but not as a function of disease; at 10 wk, these cells were present in both AdBDNF/AdNoggin-treated WT and HD striata. Indeed, the similar incidence of BrdU-tagged neurons in the WT and HD striata is hard to reconcile with any disease-associated aberration in neuronal mitotic quiescence. Together, these observations strongly suggest that the BrdU+
neurons we observed in the AdBDNF/AdNoggin-treated R6/2 striata were the products of mitotic neurogenesis.
Although the mechanisms of neurotoxicity in HD remain unclear, BDNF transcription has been reported to be impeded by CAG-expanded huntingtin protein (46
). Because BDNF supports MSNs via its anterograde transport and release from corticostriatal afferents (47
), mutant huntingtin’s suppression of corticostriatal BDNF or loss of WT huntingtin function may deprive MSNs of their trophic support. As a result, BDNF has been investigated as a neuroprotective agent for MSNs in the setting of HD (48
). The improved motor performance of our AdBDNF/AdNoggin-treated mice may derive in part from BDNF’s neurotrophic effects on resident MSNs, over and above its induction of new neurons from resident progenitors (51
). Yet neither rotarod performance nor survival was significantly improved in R6/2 mice treated with AdBDNF alone, save for a minor delay in their rotarod deterioration rate. This contrasted with the robust increments in each outcome measure noted in R6/2 mice treated with AdBDNF and AdNoggin together. Because Noggin itself has no known neurotrophic actions and exhibited no significant induction of neuronal addition relative to AdNull-treated mice, the beneficial effects of Noggin’s addition to BDNF in this study would appear to derive from its potentiation of BDNF-induced striatal neuronal recruitment. In this regard, in pilot studies of the effects of AdNoggin on the motor performance and survival of R6/2 mice, we noted no significant differences from AdNull- or saline-treated mice (our unpublished observations), suggesting that AdNoggin’s suppression of subependymal gliogenesis did not provide observable benefit to R6/2 mice. These data argue that the performance and survival increments of AdBDNF/AdNoggin-treated R6/2 mice were specifically caused by the AdBDNF/AdNoggin-associated addition of new MSNs to the R6/2 striatum and did not derive from BDNF’s neuroprotective effects. That being said, the neurotrophic actions of BDNF delivered to the striatal wall may have potentiated the relative contribution of the new neurons by preserving the functional competence of the network into which they integrated.
Together, these observations suggest that BDNF/Noggin-stimulated neurogenesis may represent a means of both replacing neurons lost to striatal neurodegeneration and conferring therapeutic benefit in HD. By using this fundamentally restorative strategy in parallel with independent neuroprotective approaches, such as minocycline inhibition of caspase activity (53
), histone deacetylase inhibitor support of neuronal transcription (24
), Coenzyme Q and creatine support of neuronal energy reserves (39
), and FGF2 infusion (57
), we might reasonably hope to establish a protocol for combination therapy of HD in affected individuals. In this regard, the 17% net increase in R6/2 survival that we observed following a single intraventricular injection of AdBDNF/AdNoggin is as robust an effect as any previously noted using any of these alternative neuroprotective strategies, alone or in combination. Yet because BDNF and Noggin were likely overexpressed for no longer than 1–2 mo after viral injection (11
), we can reasonably postulate that more sustained BDNF and Noggin delivery, whether afforded by protein delivery or more persistent transgene expression, may yield proportionately greater benefits to the performance and survival of affected subjects. Used in tandem with these other, mechanistically independent, approaches toward the protection of threatened striatal neurons, induced neurogenesis may prove an effective strategy for delaying both extrapyramidal dysfunction and overall disease progression in HD. More broadly, these findings suggest that induced neurogenesis from resident progenitor cells may comprise a feasible strategy reconstituting lost multinuclear circuits in the diseased adult brain.