Predetermining the fate of transplanted NPCs and directing their maturation will form a cornerstone for enhancing the success of therapeutic transplantation. Our results demonstrate that in vitro priming of adult NPCs with lithium chloride before transplantation leads to a reduction in gliogenesis and enhanced occurrence of DARPP-32-positive neurons 12 weeks after transplantation into the QA-lesioned striatum. In addition, lithium chloride priming increased the formation of anatomically appropriate efferent projections and accelerated improvement in sensorimotor-function performance when compared with that of nonprimed adult NPC transplants.
Regional environmental cues present in the adult brain play a strong role in determining the lineage potential of adult NPCs. Previous studies have demonstrated that transplantation of adult NPCs into homotrophic or heterotrophic neurogenic regions of the normal brain results in site-specific neuronal differentiation [14, 29–31], whereas transplantation into non-neurogenic sites, such as the striatum, results in the majority of transplanted cells differentiating into glia, with approximately 0.1% to 9% of transplanted NPCs forming neurons in either the normal brain, or in lesion models of Parkinson disease or stroke [10, 12, 13, 15]. We previously demonstrated that SVZ-derived adult NPCs differentiate into GABAergic striatal neurons 8 weeks after transplantation into the QA-lesioned striatum . Nonetheless, the majority of transplanted adult NPCs coexpressed the astrocytic marker GFAP. This indicates that microenvironmental changes induced by a CNS lesion in nonneurogenic brain regions are not sufficient to induce extensive neuronal differentiation from transplanted adult NPCs.
This led us to develop a novel mechanism by which to predirect the neuronal fate of adult NPCs before transplantation to enhance therapeutic efficiency . We demonstrated that transient priming with lithium chloride during expansion of adult SVZ-derived NPCs in culture increased the proportion of cells expressing neuronal markers, while concurrently reducing glial progeny in vitro. Extending these observations, we now demonstrate that in vitro priming of adult SVZ-derived NPCs with lithium chloride before transplantation reduces glial differentiation of transplanted NPCs in the QA-lesioned striatum by nearly 60% compared with nonprimed transplants. Reducing glial differentiation from transplanted NPCs is important for optimizing therapeutic efficacy, as this reduces the potential for transplanted NPCs to contribute to gliosis. Preferential differentiation of transplanted cells into astrocytes may compound the neurologic consequences of glial scarring and impede the development and functional integration of transplant-derived neurons [32, 33]; this is of particular concern for transplantation strategies targeted toward HD due to preexisting glial scarring manifested during the disease process [34–36].
In strong contrast with our previous in vitro observations , lithium chloride priming did not alter the proportion of transplanted adult NPCs expressing the mature neuronal marker, NeuN, when compared with nonprimed transplants. However, we did observe an increase in the proportion of transplanted adult NPCs expressing the striatal phenotypic marker DARPP-32 in rats that received lithium chloride-primed transplants compared with nonprimed transplants. These results suggest that although lithium chloride priming of adult NPCs may not be sufficient to enhance the proportion of newly generated neurons 12 weeks after transplantation, it may provide a mechanism by which to enhance phenotypic differentiation or maturation or both of new neurons in the lesioned striatum, possibly through enhanced expression of the neurogenic factor BDNF [37–41]. This is further supported by the observation that lithium chloride priming augmented the formation of anatomically appropriate efferent projections from the damaged host striatum to the globus pallidus.
In accordance with the effect of lithium chloride priming on the formation of efferent projections from transplanted adult NPCs, rats that received lithium chloride-primed transplants exhibited accelerated sensorimotor function recovery compared with those with nonprimed transplants. This was most prominent in the corridor task, in which rats receiving lithium chloride-primed NPCs exhibited an accelerated onset of sensorimotor-function improvement compared with rats with nonprimed transplants. Most surprising, a significant preference for contralateral retrievals was observed at 5 weeks after transplant in rats receiving lithium chloride-primed NPC transplants compared with both sham-transplanted animals and animals receiving nonprimed adult NPC transplants. Although the mechanism resulting in this contralateral recovery bias is unclear, it may reflect the effect of lithium chloride priming on efferent fiber growth and anatomic integration of transplanted cells with the host.
The molecular mechanisms by which lithium chloride priming directs the differentiation, maturation, survival (or a combination of these) of adult SVZ-derived NPCs remains to be specifically clarified. Previous studies have demonstrated that lithium chloride activates a number of cell-survival factors including the PI3-kinase/Akt signaling pathway, and neurotrophic/neuroprotective proteins such as BDNF, heat-shock protein, and Bcl-2 , as well as promoting neurogenesis through activation of the Wnt/MAPK, ERK-, and CREB-dependent signaling pathways [17, 18, 43, 44].
Alternatively, lithium chloride has been shown to increase the active form of the Notch receptor and upregulate its target genes in SVZ-derived NPCs independent of GSK-3β or inositol signaling . Lithium chloride is well known to act as a direct inhibitor of glycogen synthase kinase-3 (GSK-3), a component of the canonic Wnt signaling pathway. Wexler and colleagues  demonstrated that treatment of adult hippocampal NPCs with lithium chloride in vitro increased proliferation and enhanced neuronal differentiation through inhibition of GSK-3a/β, suggesting that much of lithium chlorides effect on neurogenesis may be attributable to activation of the canonic Wnt pathway. Supporting this hypothesis, Kim and colleagues  demonstrated that treatment of adult rats with lithium chloride increased the proportion of BrdU-labeled hippocampal NPCs cells expressing p-CREB. In addition, Su and colleagues  demonstrated that treatment of spinal cord-derived NPCs with lithium chloride increased proliferation, BDNF production and neuronal differentiation of NPCs in vitro. Inhibition of GSK-3 by lithium chloride in rat cortical neurons increases BDNF by activating BDNF promoter IV [43, 44]. This suggests that the CREB-BDNF pathway is involved in the neurogenic effect observed both in vitro and in vivo after priming of adult NPCs with lithium chloride.
Based on these previous findings, we propose that inhibition of GSK-3 in adult SVZ-derived NPCs before transplantation may facilitate CREB activation and increase the expression and production of BDNF, thereby enhancing NPC differentiation or maturation or both. This is in agreement with our previous findings in which we demonstrated that ectopic expression of BDNF in the striatum promoted neuronal differentiation of transplanted adult SVZ-derived NPCs .