**3. Role of Wnt/β-catenin signaling in facilitating DA neuronal development**

ing pathways, the best studied one is the canonical Wnt pathway, which is highlighted by βcatenin-dependent regulation of down-streaming genes. The canonical Wnt ligands, e.g. Wnt1, Wnt2 and Wnt3a, can be secreted by surrounding neuronal and glial cells in the nerv‐ ous system, bind to Frizzled and Lrp5/6 receptors in the target cells. At absence of binding with ligand Wnts, a protein complex in the cytoplasm, namely Axin2/GSK3β/APC complex makes phosphorylation of β-catenin and degradation of phosphorylated β-catenin, and keeps cytoplasmic β-catenin at such a low level that β-catenin can not be translocated into nucleus. Upon Wnt stimulation, the Axin2/GSK3β/APC complex can be deaggregated, the cytoplasmic β-catenin is accumulated and increased β-catenin imported into nucleus. In the nucleus, β-catenin is recruited by transcription factors TCF1-4 to the promoter regions of the target genes for specific biological effects. These TCF-4 downstream targeting genes include c-myc, mmp-7, cyclin D1, CD44 that are actively and mainly involved in cell proliferation, cycling and cell differentiation (Figure 1). On the other hand, the non-canonical Wnt signal‐ ing pathways, i.e. PCP pathway and Ca2+ pathway, Wnt ligands bind to Frizzled receptors, then activate GTPase or increase intracellular Ca2+, transmitting signals by JNK cascade or

**Figure 1.** The canonical Wnt/β-catenin signaling pathway in regulation of downstream target genes of DA neurogen‐

without any nucleus events [4-6].

142 Trends in Cell Signaling Pathways in Neuronal Fate Decision

esis (*From Ref. 48, Ding, et al., 2011*)

The canonical Wnt/β-catenin signaling pathways is critical for generation of DA neurons during development. Differential regulation of midbrain DA neurogenesis by Wnt1, Wnt3a, and Wnt5a was well studied [9]. The β-catenin was detected in DA precursor cells and β-catenin signaling took place in the precursor cells by assessment of TOPgal reporter mice. Wnt3a promoted prolif‐ eration of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of DA neurons. The Wnt1 and Wnt5a increased the number of midbrain DA neurons in E14.5 possibly by two mechanisms. Wnt1 predominantly increased the prolifera‐ tion of Nurr1+ precursors that acquired a neuronal DA phenotype, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 expression. In contrast, Wnt5a increased proportion of Nurr1+ precursors and up-regulated expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and effects of Wnt1 and Wnt5a on proliferation and acquisition of DA phenotype. For the embryonic expres‐ sion, Wnt1 was throughout of midbrain at E8.5, and then restricted to the roof plate, a subset of floor plate cells and isthemus of midbrain at E9.5. From E10 to E12, Wnt2 was observed in the ven‐ tral midbrain, with highest in the intermediate and marginal zone of ventral midbrain. Wnt3a was expressed in dorsal midbrain of rat at E11.5. The Wnt5a appeared at E9.5 and became restrict‐ ed to the floor plate of midbrain from E11.5 to E13.5. Functionally, mutation of Wnt1 led to re‐ duced DA neurons in late embryos. Mechanistic study showed that Wnt1 and its downstream gene Lmx1 formed a loop to regulate the expression of Octx2, Nurr1 and Pitx3, thereby establish‐ ing identity of DA precursors *in vivo* [10]. This Wnt1-lmx1a regulatory loop synergistically con‐ trolled DA differentiation in the midbrain by antagonizing Shh signaling pathway [11]. Wnt 2 mutation resulted in a decrease in proliferation of DA progenitors and subsequently loss of DA neurons, partially by phosphorylation of Lrp5/6 and Dishevelled 2/3. Wnt 3a promoted the pro‐ liferation of Nurr1-positive DA progenitors. The Wnt5a, derived by the astrocytes and radial glial cells, was demonstrated to promote cell fate commitment of precursors into DA neurons and development of A9-A10 DA neurons *in vivo* [12, 13]. The Wnt5a also regulated DA axon growth and guidance in midbrain development [14]. In mouse embryo at E11.5, Wnt5a was abundantly expressed in the ventral midbrain where it promoted DA neurite and axonal growth. By E14.5, when DA axons were approaching their striatal target, Wnt5a caused DA neurite re‐ traction. Co-culture of ventral midbrain explants with Wnt5a-overexpressing cell aggregates re‐ vealed that Wnt5a was capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a were mediated by the Frizzled receptors and by small GTPase, Rac1. More‐ over, this effect was specifically blocked by Wnt5a antibody. Role of Wnt5a in DA neuronal axon morphogenesis was further verified in Wnt5a-/-mice, where fasciculation of the medial fore‐ brain bundle as well as the density of DA neurites and striatal terminals were disrupted. Al‐ though Wnts can function via intracellularβ-catenin, Ca2+, and JNK signaling, the canonical Wnt/β-catenin signaling pathway shows a major position in regulation of DA neurogenesis. It appeared that all Wnt1, Wnt3a and Wnt5a act in proliferation and DA differentiation of precur‐ sor cells with sequence of proliferation stimulating effect of Wnt1≥Wnt3a≥Wnt5a, or differentia‐ tion facilitating effects of Wnt5a≥Wnt1≥Wnt3a. These findings have evidenced that the Wnt/βcatenin signaling is a key regulator of proliferation and differentiation of DA precursors and different Wnts might have specific and unique activity profiles during DA neurogenesis.

[30]. Accumulating evidence indicated that a population of astrocyte was functionally acti‐ vated, named reactive astrocytes with active proliferation, morphological expanding of cell bodies and increasing generation of various neurotrophic factors, and with predominate dis‐ tribution in the nigra and striatum of PD condition. These reactive astrocytes and Wnt/β-cat‐ enin signaling showed a link of nigrostriatal injury to repair in MPTP model of PD. The Wnt signaling components Frizzled-1 and β-catenin were dynamically regulated in response to MPTP insult-induced DA neuronal degeneration and reactive glial activation. Activated or reactive astrocytes in the ventral midbrain were identified as candidate source of Wnt1. Blocking Wnt/Fzd signaling with Dkk1 also counteracted astrocyte-induced neuroprotection against MPP (+) toxicity in primary mesencephalic astrocyte-neuron cultures. Moreover, as‐ trocyte-derived Wnt1 promoted DA neurogenesis from adult midbrain stem cells or progen‐ itor cells. Conversely, lack of Wnt1 transcription in response to MPTP in aged animals and failure of DA neurons to recover could be reversed by activation of Wnt/β-catenin signaling *in vivo*, suggesting MPTP-reactive astrocytes and Wnt1 worked as neuroprotective activity in DA neural plasticity [31]. Obviously, Wnt1 regulated Frizzled-1/β-catenin signaling path‐ way as a candidate regulatory circuit for DA neuron-astrocyte crosstalk in the ventral mid‐ brain, also implying that Wnt signals might act as the critical messages in neuron-glial

Roles of Wnt/β-Catenin Signaling in Controlling the Dopaminergic Neuronal Cell Commitment of Midbrain and

Therapeutic Application for Parkinson's Disease

http://dx.doi.org/10.5772/53282

145

Moreover, The Wnt/β-catenin signaling also involved in Parkin protection of DA neurons [33]. Differential expression of Wnts was observed after spinal cord contusion injury in adult ro‐ dents [34]. Wnt signaling in the activated microglia; cells exhibited proinflammatory effect. Gene-expression profiling revealed that Wnt3A specifically increased expression of proinflam‐ matory immune response genes in microglia and exacerbated release of IL-6, IL-12, and tumor necrosis factor α [35]. Heterotrimeric G protein-dependent Wnt5A signaling to ERK1/2 mediat‐ ed distinct aspects of proinflammatory transformation in microglial cells [36]. While combin‐ ing nitric oxide release with anti-inflammatory activity preserved DA innervation and prevented motor impairment in the MPTP model of PD [37], switching the microglial harmful phenotype promoted lifelong restoration of DA neurons from inflammatory degeneration in the substantia nigra of aged mice [38]. In addition, activation or inhibition of Wnt/β-catenin sig‐ naling could regulate neuronal and glial differentiation in neurospheres, respectively. Inhibi‐ tion of Wnt signaling promoted gliogenesis from neural stem cells. Long-term activation of Wnt signaling pathway by Wnt-7a or GSK3 inhibitors promoted a moderate increase of neuro‐ nal differentiation and blocked gliogenesis. In contrast, Wnt pathway inhibition by Dkk1overexpression robustly increased gliogenesis [39]. Accumulating evidences suggested that the glial cells including reactive astrocytes and microglial cells might present as crucial turning

**5. Prospect on manipulation of Wnt/β-catenin signaling for regeneration**

The studies have indicated that transplantation of Wnt primed neural stem cells might result in improvements of cellular and functional recovery in PD condition. The midbrain neural

intercommunication in the adult mammalian nervous system [32].

points for the therapeutic strategy against PD [40, 41]

**medicine**

Dynamic temporal and cell type-specific expression of Wnt signaling components was found in the developing midbrain [15]. The DA neuronal cluster size was determined dur‐ ing early forebrain patterning [16]. Furthermore, temporally controlling modulation of FGF/ERK signaling directed midbrain DA neural progenitor fate in mouse and human pluri‐ potent stem cells [17]. Dickkopf-1 (Dkk1), a specific Wnt signaling inhibitor, regulated ven‐ tral midbrain DA neuronal differentiation and morphogenesis [18]. Blockade of Wnt/βcatenin signaling pathway promoted neuronal induction and DA-phenotype differentiation in embryonic stem cells [19]. Delayed DA neuron differentiation was seen in the Lrp6 mu‐ tant mice [20]. Signaling interactions between Wnt/β-catenin and sonic hedgehog (Shh) mechanisms functioned to regulate the production of DA neurons. Specific deletion of intra‐ cellular β-catenin in Shh expressing cells resulted in diminished DA progenitors, NgN2, BrdU labeling cells, and subsequently DA neurons. Permanent stabilization of β-catenin in Shh expressing cells led to more DA progenitors and DA neurons accordingly, and inhibi‐ tion of GSK-3β also increases the differentiation of DA precursors [21]. HPRT deficiency co‐ ordinately dysregulated canonical Wnt signaling in neuro-developmental regulatory process [22]. Wnts showed antagonism of Shh signaling pathway and facilitated neurogene‐ sis in the midbrain floor plate [23]. The β-arrestin was also a necessary component of Wnt/ beta-catenin signaling linking Dvl and axin *in vitro* and *in vivo* functions [24]. Wnt5a induced the DA differentiation of midbrain neural stem cells *in vitro*, and the effect was mediated by the phosphorylation of Dishevelled protein and activation of GTPase RAC1. Wnt5A stimu‐ lated the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins [25]. As to Wnt receptors, mutation of Lrp6 might not affect the patterning; proliferation and cell death in the ventral midbrain, but displayed a delay in the onset of DA precursor differen‐ tiation. Lrp6(-/-) mice exhibited 50% reduction in DA neurons and expression of DA mark‐ ers such as Nurr1 and Pitx3 as well as a defect in midbrain morphogenesis in the mutant embryos at E11.5. The extracellular domain of Lrp5/6 inhibited the non-canonical Wnt sig‐ naling *in vivo* condition [26]. The mitogen-activated protein kinases promoted Wnt/β-catenin signaling via phosphorylation of LRP6 [27]. Ectopic Wnt/β-catenin signaling was also in‐ volved in induction of neurogenesis in spinal cord [28]. In addition, Wnt5a was required for the endothelial differentiation of embryonic stem cells and vascularization via both Wnt/βcatenin signaling and protein kinase C pathways [29].
