Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

Abstract A common hallmark of age‐dependent neurodegenerative diseases is an impairment of adult neurogenesis. Wingless‐type mouse mammary tumor virus integration site (Wnt)/β‐catenin (WβC) signalling is a vital pathway for dopaminergic (DAergic) neurogenesis and an essential signalling system during embryonic development and aging, the most critical risk factor for Parkinson's disease (PD). To date, there is no known cause or cure for PD. Here we focus on the potential to reawaken the impaired neurogenic niches to rejuvenate and repair the aged PD brain. Specifically, we highlight WβC‐signalling in the plasticity of the subventricular zone (SVZ), the largest germinal region in the mature brain innervated by nigrostriatal DAergic terminals, and the mesencephalic aqueduct‐periventricular region (Aq‐PVR) Wnt‐sensitive niche, which is in proximity to the SNpc and harbors neural stem progenitor cells (NSCs) with DAergic potential. The hallmark of the WβC pathway is the cytosolic accumulation of β‐catenin, which enters the nucleus and associates with T cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors, leading to the transcription of Wnt target genes. Here, we underscore the dynamic interplay between DAergic innervation and astroglial‐derived factors regulating WβC‐dependent transcription of key genes orchestrating NSC proliferation, survival, migration and differentiation. Aging, inflammation and oxidative stress synergize with neurotoxin exposure in “turning off” the WβC neurogenic switch via down‐regulation of the nuclear factor erythroid‐2‐related factor 2/Wnt‐regulated signalosome, a key player in the maintenance of antioxidant self‐defense mechanisms and NSC homeostasis. Harnessing WβC‐signalling in the aged PD brain can thus restore neurogenesis, rejuvenate the microenvironment, and promote neurorescue and regeneration.


| INTRODUC TI ON
Aging is the leading risk factor for Parkinson's disease (PD), the second most diagnosed neurodegenerative disorder (ND), affecting almost 1% of the population over age 60 (Blauwendraat et al., 2019). containing ubiquitin and α-synuclein (α-syn), and astroglial activation (Schapira et al., 2014). The cardinal motor signs of PD include a combination of bradykinesia, postural instability, and resting tremor.
Non-motor symptoms including hyposmia, cognitive dysfunction, and sleep and mental health disorders, often precede and/or accompany PD onset and progression, but the underlying pathological alterations in the brain are not fully understood (Reichmann et al., 2016;Schapira, Chaudhuri, & Jenner, 2017).
PD is the fastest growing ND, and because the world's population is aging the number of individuals affected is expected to grow exponentially: the number of people with PD is forecast to double from 6.9 million in 2015 to 14.2 million in 2040 (Dorsey & Bloem, 2018). Currently, most PD symptoms appear when ≥70% of the DAergic terminals are degenerated in the Str and more than half of the DA synthesizing neurons are lost in the SNpc, therefore early detection and intervention is crucial for effective neuroprotective treatment intended to prevent the degeneration of DAergic neurons and, ultimately, PD pathogenesis (Jankovic, 2019). To date, there are no effective treatments that can stop or reverse the neurodegeneration process in PD and current treatments rely on DAergic drugs, including levodopa (L-DOPA) and DAergic agonists, which only temporarily alleviate motor symptoms (Obeso et al., 2017;Olanow, 2019;Olanow & Schapira, 2013).
Evidence is available that quiescent neuroprogenitors reside in the tegmental aqueduct periventricular region (Aq-PVRs), which is close to the SNpc and harbors clonogenic NSCs endowed with DAergic potential (Hermann et al., 2006(Hermann et al., , 2009Hermann & Storch, 2008; L'Episcopo et al., 2011a). Additionally, adult Aq-PVR NSCs F I G U R E 1 The subventricular (SVZ) and the subgranular (SGZ) zones of the adult rodent brain. (a) Sagittal brain section showing the subventricular zone (SVZ) lining the lateral ventricles (LV) and the adjacent striatum (Str), and the hippocampal subgranular zone (SGZ). In blue the trajectory of migrating neuroblasts along the rostral migratory stream (RMS) reaching the olfactory bulb (OB); in red, the CA1 field of the hippocampus and the SGZ in the dentate gyrus (DG) are shown. (b, c) Schematic representation of the neurogenic regions in the adult brain. The SVZ niche (b) composed of SVZ astrocytes (type B1 cells), rapidly proliferating (type C) cells, migrating neuroblast (type A cells), which migrate through the RMS to the OB, and ependymal cells (type E cells) (Doetsch et al., 1999(Doetsch et al., , 1997. In the SGZ, radial glia-like precursors (RGLs) within the SGZ serve as one type of quiescent NSCs (type 1 cells) and continuously give rise to both DG neurons and astrocytes (Bonaguidi et al., 2011). Mature granule neurons then migrate into the granule cell layer (GCL). (d) A sagittal reconstruction of dual immunofluorescent stained images by confocal laser scanning microscopy. SVZ-migrating doublecortin-positive (DCX + ) neuroblasts in red, and dividing, bromodeoxyuridine-positive (BrdU + ) NSCs in green, are seen forming chains traveling along the RMS to the OB where they become granular and periglomerular interneurons involved in odor discrimination. Magnifications of DCX + / BrdU + NSCs are shown in the boxed areas can be activated and induced to differentiate into DAergic neurons, both in vitro and after PD injury in vivo (Hermann et al., 2006(Hermann et al., , 2009Hermann & Stork, 2008;L'Episcopo et al., 2011aL'Episcopo et al., , 2014aL'Episcopo, Tirolo, Peruzzotti-Jametti, et al., 2018a;Xie et al., 2017).
The therapeutic relevance of endogenous neurogenesis for the recovery of the injured brain and, particularly, the aged PD brain, is being actively investigated, yet remains to be elucidated (van den Barker et al., 2018;Kempermann et al., 2018;Le Grand, Gonzalez-Cano, Pavlou, & Schwamborn, 2015;Neves et al., 2017;van den Berge et al., 2013). This avenue of research is particularly significant in light of the dramatic decline of NSC neurogenic potential in PD brain during aging and neurodegeneration, likely underlying the age-dependent cognitive deficits and the failure to replace or repair dysfunctional or dead neurons (Anacker & Hen, 2017;Katsimpardi & Lledo, 2018;Kemperman et al., 2018;Seib & Martin-Villalba, 2015;Takei, 2019).
Herein, after a description of the key Wnt-signalling components and a synopsis of adult neurogenesis in PD, we will focus on the role of WβC-signalling as a common final pathway in mediating NSC regulation, from development to aging and PD degeneration. We aim to survey recent literature in the field supporting the upregulation of WβC as a means to re-activate neurogenesis and incite regeneration in the injured brain, particularly in the context of modalities through which the inherent self-repair capacities of the aged PD brain, can be engaged Kase et al., 2019;Kaur, Saunders, & Tolwinski, 2017;Mishra et al., 2019;Zeng et al., 2019;Zhao, et al., 2018;Zhang et al., 2018aZhang et al., , 2018b and following sections).

| AC TIVATOR S AND INHIB ITOR S OF THE " WNT-SIG NALOSOME " AND THEIR IMPAC T ON ADULT NEUROG ENE S IS
Wnt signalling is transduced via three different pathways, the socalled "canonical" WβC pathway, and the "non-canonical" Wnt/Ca 2+ and Wnt/planar cell polarity (PCP) pathways. Among them, the WβC signalling pathway has received particular attention due to its crucial roles in regulating cell fate, proliferation and survival, whereas Wnt/Ca 2+ and Wnt/PCP signalling are more associated with differentiation, cell polarity and migration (Nusse & Clevers, 2017). Recent studies indicate that two major branches of the Wnt signalling pathway, the WβC and Wnt/PCP pathways, play essential roles in various steps of adult SVZ and SGZ neurogenesis (as reviewed by Varela-Nallar & Inestrosa, 2013;and Hirota et al., 2016).
Amplification of canonical Wnt signalling can be achieved through the participation of another set of receptors, the leucine-rich repeat-containing G-protein coupled receptors (LGR,(4)(5)(6) and their ligands, the R-spondins (Rspos) (Carmon, Gong, Lin, Thomas, & Liu, 2011;de Lau, Peng, Gros, & Clevers, 2014;Raslan & Yoon, 2019) ( Figure 2). LGR-Rspo complexes at the cell membrane decrease the endocytic turnover of Fzd-LRP5/6 by neutralising the ubiquitin ligases ring finger protein 43 (RNF43) and zinc and ring finger 3 (ZNRF3) (Hao et al., 2012). The crosstalk between the canonical and non-canonical pathways is responsible for the coordination and final outcome of Wnt signalling. Several components of the canonical Wnt pathway (Wnt-agonists, Wnt-receptors, and Wnt-inhibitors) have been described in the neurogenic niches of adult mice (see Hirota et al., 2016 for a comprehensive review). Reportedly, a dynamic interplay between endogenous Wnt-agonists and Wnt-antagonists is at play in finely tuning the strength of Wnt signalling (Niehrs, 2012).
Traditionally, Wnt-agonists referred to as the canonical Wnt1-like (including Wnt1-3a, Wnt8, and Wnt8a) and non-canonical Wnt5a-like (including Wnt4-7a and Wnt11) classes act as intercellular growth signals. With the exception of Norrin, an atypical Fzd4/LRP5 agonist, all 19 human Wnts share a highly conserved two-domain structure which enables it to attach to the Fzd receptor cysteine rich domain (CRD) and bind to LRP5/6 (Janda et al., 2012).
Essentially, Wnt ligands are secreted lipid-modified glycoproteins that act as short-range modulators to activate receptor-mediated signalling pathways. The lipid components of Wnts are required for protein secretion and efficient signalling (Nusse & Clevers, 2017).
Wnt palmitoylation is essential for Wnt signalling and is carried out by Porcupine, an endoplasmic reticulum -localized O-acyltransferase (Herr & Basler, 2012;Torres et al., 2019). Additionally, due to their hydrophobic nature, Wnts require extracellular carriers, such as the Wnt-binding proteins Wntless and Secreted wingless-interacting molecule (Swim), that enable secretion of the active Wnt complex by binding to lipidated Wnt (Bänziger et al., 2006).  & Wurst, 2006;Prakash & Wurst, 2014;Zhang et al., 2015). Hence, canonical Wnt signalling is critical for midbrain DAergic progenitor specification, proliferation, and neurogenesis. The involvement of Wnts in regulating NSC activity has been established through the use of Wnt mutant mice whereby loss of Wnt1 resulted in malformation of most of the midbrain and some rostral metencephalon (see Arenas, 2014;Joksimovic & Awatramani, 2014;Prakash & Wurst, 2014). The removal of β-catenin in tyrosine hydroxylase-positive (TH + ) neural progenitor cells in the VM region negatively regulates midbrain DAergic neurogenesis. Here, β-catenin depletion interferes with the ability of committed progenitors to become DAergic neurons, resulting in adult animals with a significant loss of TH + neurons in the adult VM (Tang et al., 2009). Excessive Wnt signalling is also detrimental for DAergic neuron production, adding to the general notion that morphogen dosage must be tightly regulated (Rawal et al., 2009).
Also, a large number of studies have shown a crucial participation of the WβC-pathway at early stages of hippocampal development. Hence, the expression pattern of the LEF1 gene of the TCF/ LEF family of transcription factors, as well as other TCF/LEF proteins, are critical for the regulation of DG granule cell generation and the entire hippocampal maturation, whereas the conditional inactivation of β-catenin in mice results in an impairment of hippocampus development (Galceran, Miyashita-Lin, Devaney, Rubenstein, & F I G U R E 2 The canonical Wnt/β-catenin(WβC) signalling pathway. In WβC pathway, Wnt signal activation is tightly controlled by a dynamic signalling complex, constituted by class Frizzled (Fzd) of the G-protein-coupled receptor (GPCRs) superfamily, the LDL receptorrelated protein (LRP) 5/6 coreceptors and Dishevelled (Dvl) and Axin adapters. (a) In the absence of a Wnt ligand, (Wnt-off) the signalling cascade is inhibited. Cytoplasmic β-catenin is phosphorylated and degraded via proteasome mediated destruction, which is controlled by the "destruction complex", consisting of glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), the scaffold protein AXIN, and the tumor suppressor adenomatous polyposis coli (APC) (Janda et al., 2012). As a result, the translocation into nucleus is inhibited. Interruption of WβC-signalling also occurs in the presence of the Dkk' and secreted FZD-related proteins (sFRPs) families of Wnt-antagonists, or Wnt inhibitory protein, WIF. (b) Conversely, Wnt ligand binding to Fzd receptors at the surface of target cells (Wnt-on) triggers a chain of events aimed at disrupting the degradation complex via Dvl phosphorylation. Then β-catenin is separated from the destruction complex, resulting in its accumulation and stabilization in the cytoplasm (Janda et al., 2012). Subsequently, β-catenin is imported into the nucleus where it can interact with the TCF/LEF family of transcription factors and recruit transcriptional co-activators, p300 and/or CBP (CREB-binding protein), as well as other components to transcribe a panel of downstream target genes. The amplification of canonical WβC-signalling can be achieved through the participation of another set of receptors, the leucine-rich repeat-containing G-protein coupled receptors (LGR4,5,6) and their ligands, the R-Spondins (Rspos) and the atypical FZD4/LRP5 agonist, Norrin Grosschedl, 2000;Lee, Tole, Grove, & McMahon, 2000;reviewed by Ortiz-Matamoros et al., 2013, andInestrosa, 2013).
Remarkably, studies in cultured hippocampal neurons have found that β-catenin regulates dendritic morphogenesis since the overexpression of a stabilized form of β-catenin leads to the development of a more complex dendritic arborization (Ciani & Salinas, 2005;Salinas, 2005aSalinas, ,2005bSalinas, 2012).
Activation of Wnt signalling plays a role in producing regionally homogeneous populations of NSCs and neurons. For instance, pivotal genes whose mutations are linked to PD negatively impact on WβC-signalling Harvey, 2014, Berwick et al., 2017), resulting in an inhibition of the ability of human induced pluripotent cells (iPSCs) to differentiate into DAergic neurons (Awad et al., 2017;Momcilovic et al., 2014;Moya et al., 2014). Specifically, downregulation of WβC-signalling in iPSC-derived NSCs due to a GBA1 mutation Both at the SVZ and SGZ niches, β-catenin is tightly regulated via phosphorylation by the 'destruction complex', consisting of glycogen synthase kinase 3β (GSK-3β), casein kinase 1α (CK1α), the scaffold protein Axin-1, and the tumour suppressor adenomatous polyposis coli (APC) (Janda et al., 2012) (Figure 2). In the absence of a Wnt ligand, the signalling cascade is inhibited. Cytoplasmic β-catenin is phosphorylated and kept at low levels via proteasome-mediated destruction, which is controlled by the destruction complex. As a result, translocation into the nucleus is inhibited. Conversely, binding of Wnt ligands to receptors at the surface of target cells triggers a chain of events resulting in disruption of the destruction complex via Dvl phosphorylation. β-catenin is then separated from the destruction complex, resulting in its accumulation and stabilization in the cytoplasm (Janda et al., 2012). Subsequently, β-catenin is imported into the nucleus where it can interact with the TCF/LEF family of transcription factors and recruit transcriptional co-activators, p300 and/or CREB-binding protein (CBP), as well as other components to transcribe a panel of downstream target genes ( Figure 2).
The enzyme GSK-3β is a crucial inhibitor of canonical Wntsignalling as it leads to the degradation of β-catenin. Inhibition of GSK-3β activity by molecular compounds and various enzymes is an important step in the activation of the canonical Wnt signalling cascade and the downstream gene expression (Figure 2). The critical role of GSK-3β inhibition leading to β-catenin stabilization in VM precursors, with consequent increased differentiation into DAergic neurons, was highlighted by early studies by Arenas and collaborators (Castelo-Branco, Rawal, & Arenas, 2004;Castelo-Branco et al., 2006; reviewed by Arenas, 2014 andToledo et al., 2017). Hence, two chemical inhibitors of GSK-3β, indirubin-3-monoxime and kenpaullone, were found to increase neuronal differentiation in VM precursor cultures. Additionally, kenpaullone was found to increase the size of the DAergic neuron population through conversion of precursors expressing the orphan nuclear receptor-related factor 1 (Nurr1/Nr4A2) into TH + neurons, thereby mimicking the effect of canonical Wnts (Castelo-Branco et al., 2004). These early studies documented a three-to five-fold increase in precursor differentiation into DAergic neurons, paving the way for the use of GSK-3β inhibitors to improve stem/precursor cell therapy approaches in Parkinson's disease (Arenas, 2014;Brodski et al., 2019;Esfandiari et al., 2012;Kirkeby et al., 2012;Kirkeby, Parmar, & Barker, 2017;Kriks et al., 2011;Parish et al., 2008;Parish & Thompson, 2014;Toledo et al., 2017).
Stimulation of NSC proliferation can also be achieved by Wnt-7a up-regulation, as a result of orphan nuclear receptor Tailless (TLX) activation promoting WβC-signalling (Qu et al., 2010).
Owing to its critical role in the regulation of a multiplicity of cellular functions, Wnt-signalling must be kept under a strict control via a panel of endogenous Wnt-antagonists, including proteins of the Dickkopf (Dkk) and the Sclerostin families (Cruciat & Niehrs, 2013).
Adding a further level of complexity, miRNAs (short noncoding RNAs) are increasingly emerging as critical regulators of Wnt-signalling  and, vice versa, Wnt-signalling components can modulate miRNA activity. In a key finding, Anderegg and colleagues (2013) uncovered a regulatory circuit between LIM homeobox transcription factor 1-beta (LMX1B) and miR-135a2 that modulates Wnt1/Wnt signalling which in turn determines the size of the midbrain DAergic progenitor pool. On the basis of bioinformatics and luciferase assay data, the authors suggested that miR-135a2 modulates LMX1B and many genes in the Wnt signalling pathway (Anderegg & Awatramani, 2015;Anderegg et al., 2013). Chmielarz et al., (2017) underscored the crucial role of Dicer, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes, for maintenance of adult DAergic neurons; a reduction of Dicer in the VM and altered miR expression profiles were observed in laser-microdissected DAergic neurons of aged mice (Chmielarz et al., 2017).  (Zhang, Zhang, Deng, et al., 2018a;Zhang, Shi, et al., 2018b;Zhang, Zhang, Yang, et al., 2018c). Thus, both miRNAs and Wnt-signalling pathways form a network (Ashmawy et al., 2017) that is likely to play a significant role in adult neurogenesis.
Taken altogether, a picture emerges of the complexity of the Wnt signalling cascade whereby the outcome of WβC-signalling activation is context-dependent, with β-catenin activating different and sometimes opposing genetic programs depending on tissue/cellular specificity, the availability of receptor/co-receptors and signalling partners, pathological conditions, and the age of the host. Due to the vital action of this signalling pathway in development and systems maintenance, its dysregulation may culminate in a broad range of diseases, including neurodegeneration and cancer (Nusse & Clevers, 2017).
Thus, WβC-signalling is a likely prominent actor in tipping the balance of adult PD brain NSCs due to a concerted action of diverse upstream and downstream modulatory signals impacting within the specialized neurogenic niches.

| A dynamic interplay of positive and negative regulators coordinates adult SVZ and SGZ neurogenesis in PD
As adult neurogenesis occurring at the SVZ and SGZ levels is a vital ongoing process in the adult brain, its disruption may contribute to various disturbances including reduced neuronal plasticity, deficits in olfaction, cognitive dysfunction, and/or mental health disorders, that may precede and/or accompany PD onset and progression. Therefore, the tight regulation of the sequential steps of adult neurogenesis should be finely orchestrated by a wide panel of transcription factors and epigenetic mechanisms coordinating the progression of neurogenesis (Bond et al., 2015;Hsieh, 2012;Ming & Song, 2005). At the SVZ, located along the ependymal cell layer of the lateral ventricles (Figure 3), four major cell types dynamically interact with each other: slowly dividing SVZ-glial fibrillary acidic protein (GFAP)-positive astrocytes (type B cells), rapidly dividing TAPs (type C cells), migrating neuroblasts (type A cells), and ependymal cells (type E cells) (Doetsch, Caillé, Lim, García-Verdugo, & Alvarez-Buylla, 1999;Doetsch, García-Verdugo, & Alvarez-Buylla, 1997) (Figure 3). Type B cells exhibit a radial morphology and extend a basal process to terminate on blood vessels and an apical process with a primary cilium contacting the cerebrospinal fluid (CSF) in the ventricle (Mirzadeh et al., 2008). B cells give rise to TAPs (Doetsch et al., 1999(Doetsch et al., , 1997, which rapidly divide to become neuroblasts (A cells). Neuroblasts then form a chain and migrate following the rostral migratory stream (RMS) to the olfactory bulb, OB, where they become granular and periglomerular interneurons involved in odor discrimination (Lledo & Valley, 2016) (Figures 1 and 3). In the adult mouse DG, radial glia-like precursors within the SGZ serve as one type of quiescent NSCs and continuously give rise to both dentate granule neurons and astrocytes (Bonaguidi et al., 2011; Figure 1).
Here, different populations of progenitors with different properties have been described, enhancing the complexity of the cellular and molecular mechanisms underlying regulation of adult neurogenesis (Bonaguidi et al., 2011;Encinas et al., 2011;Ming & Song, 2005).
Accordingly, lentivirus-mediated blockade of Wnt signalling reduces the number of immature new neurons in the adult dentate gyrus and impairs hippocampus-dependent spatial-and object-recognition memory (Jessberger et al., 2009).
Not surprisingly, different physio-pathological conditions and pharmacological stimuli dynamically modulate both SVZ-and SGZ-NSCs. Among the positive factors, DAergic innervation and DA-agonists, serotonin, exercise, enriched environment, learning, estrogens, and antidepressant drugs have been variously documented to stimulate NSC neurogenic potential (Baker, Baker, & Hagg, 2004;Hoglinger et al., 2004;Chiu et al., 2015;Ehninger et al., 2011;Kempermann, Kuhn, & Gage, 1997;Kempermann, Kuhn, & Gage, 1998;Kodali et al., 2016;Kohl et al., 2016;van Praag et al., 2005;Salvi et al., 2016;Winner et al., 2009aWinner et al., , 2011a. Conversely, DAergic neurotoxins, aging, inflammation, stress and chronic exposure to certain toxins or drugs decrease the generation of new neurons (Chandel et al., 2016;Das, Gangwal, Damre, Sangamwar, & Sharma, 2014;Hain et al., 2018;Iwata et al., 2010;Klein et al., 2016;L'Episcopo et al., 2011cL'Episcopo et al., , 2012L'Episcopo et al., , 2013van Praag et al., 2005;Seib & Martin-Villalba, 2015;Sung, 2015;Veena et al., 2011;Villeda et al., 2011). NSCs are extremely vulnerable to a wide range of insults and toxic exposures, such as the neurotoxicants used in experimental models of basal ganglia injury (Cintha et al., 2018;He et al., 2006;He, Uetsuka, & Nakayama, 2008;L'Episcopo et al., 2012;Shibui et al., 2009). Remarkably, exposure to the herbicide paraquat, which is associated with an increased risk of idiopathic PD, results in a pro-inflammatory senescence-associated secretory phenotype capable of damaging neuronal, glial and NSC cells, and therefore likely contributing to DAergic neurodegeneration (Chinta et al., 2018). Aging, especially, represents a key driver of neurogenic impairment as a result of a reduced proliferation and differentiation of NSCs both at SVZ and SGZ levels (Ahlenius, Visan, Kokaia, Lindvall, & Kokaia, 2009;Daynac, Morizur, Chicheportiche, Mouthon, & Boussin, 2016;Enwere et al., 2004;Luo, Daniels, Lennington, Notti, & Conover, 2006). NSCs still retain their capacity for proliferation and differentiation into functional neurons, despite lower numbers in the aged brain (Ahlenius et al., 2009). Recent evidence also suggests that mitochondrial dysfunction represents an important cause of the age-related decline in neurogenesis, as exposure of the SGZ or SVZ to mitogens or enhancement of mitochondrial function restored neurogenesis . Notably, the mitochondrial transcription factor A (Tfam), a mitochondrial DNA (mtDNA)-binding protein essential for genome maintenance, has recently gained attention, as its putative dysfunction may play an important role in neurogenesis defects in the aging hippocampus  and aging-dependent neurodegeneration (Kang, Chu, & Kaufman, 2018). Tfam has been shown to play a central role in the mtDNA stress-mediated inflammatory response and recent evidence indicates that decreased mtDNA copy number is associated with F I G U R E 3 Location, proliferation and dopaminergic innervation of SVZ-NSCs. (a) Microscopic brain image at the level of the striatal SVZ. In the inset, a schematic drawing of the four SVZ-cell types: 1. slowly dividing SVZ astrocytes (type B cells), 2. rapidly dividing transitamplifying cells (type C cells, TAPs), 3. migrating neuroblasts (type A cells), and 4. ependymal cells (type E cells) (Doetsch et al., 1997(Doetsch et al., , 1999. (b, c) Nigrostriatal DAergic neurons originating in the SN project to the SVZ. Dual immunofluorescent staining with dopamine transporter (DAT, in red) and bromodeoxiuridine (BrdU, green) showing a dense network of DAT expressing neurons innervating the SVZ (b; and higher magnification in c). (d-f) schematic representation of cell counting performed in coronal sections through the SVZ (d); stereological estimations of BrdU and proliferating cell nuclear antigen (PCNA)-positive cells (e), and representative stainings of DCX + , EGF-R + , BrdU + , counterstained with the nuclear marker 4,6-diamidino-2-phenylindole, Dapi (blue) (f), indicating that MPTPinduced basal ganglia injury resulted in a biphasic time-dependent response: a down-regulation of SVZ-NSC proliferation followed by a return to pre-MPTP levels several aging-related pathologies (Kang et al., 2018). Of special mention, besides its crucial role for the development, maintenance and protection of midbrain DAergic neurons, the relevance of the transcription factor Nurr1 for adult hippocampal neurogenesis in PD has been recently highlighthed, and several lines of evidence indicate that pharmacological stimulation of Nurr1 can improve behavioral deficits via an increase in hippocampal neurogenesis (Kim et al., 2015;Kim et al., 2016 and section 6.4.1).
One key compartment that is dramatically affected in PD is represented by DAergic innervation of the SVZ and SGZ. Hence, nigrostriatal DAergic neurons originating in the SN project to the SVZ in mice, primates, and humans (Borta & Hoglinger, 2007;Freundlieb et al., 2006;Höglinger, Arias-Carrión, Ipach, & Oertel, 2014;Hoglinger et al., 2004). Within the SVZ niche, DAergic terminals create a dense network of fibers innervating the SVZ ( Figure 3 and Figure S1). Accordingly, DA receptors are widely expressed in the SVZ region and are actively involved in the modulation of neurogenesis (Van Kampen et al., 2004). In the hippocampal SGZ, DAergic neurites remain in close contact with NSCs and make their synaptic connections with granular cells in the DG, and it is thought that DA provides an environment for proliferation and differentiation of NSCs (Höglinger et al., 2014(Höglinger et al., , 2004. Accordingly, a number of studies have shown that DA depletion in animal models of PD reduced the proliferation of NSCs in the SVZ and SGZ (recently reviewed by Winner & Winkler, 2015) . Additionally, besides DA, a dysfunction of the serotonergic system projecting to the hippocampus has been reported to impact on adult neurogenesis and suggested to contribute to early non-motor symptoms of PD, such as anxiety and depression .
At the mesencephalic level in idiopathic human PD, multipotent NSCs isolated from the SN appeared to lack key factors required for neuronal differentiation as they must be co-cultured with embryonic stem cell-derived neural precursors to obtain neurons (Wang et al., 2012). Within the hippocampal SGZ, neurogenesis is severely impaired, which may be associated with hippocampal Of special importance, the Str, previously defined as a non-neurogenic region, was recently found to generate neuroblasts in response to different types of brain injury (Luzzati et al., 2011;Nato et al., 2015). Striatal neurogenesis has also been observed in adult non-human primates such as the squirrel monkey (Bedard et al., 2002). Recently, Agnihotri and collaborators (2019), reported that PINK1 deficiency is associated with increased deficits of adult hippocampal neurogenesis and lowers the threshold for stress-induced depression in mice.
As a whole, adult neurogenesis is highly susceptible to multiple

| Dysfunctional Wnt/β-catenin signalling as a critical event in MPTP-dependent SVZ impairment: in vivo studies
Using immunohistochemistry to localize β-catenin in the SVZ of saline-and MPTP-treated mice to unravel the potential role of WβCsignalling in SVZ-NSCs during PD, we first identified a significant proportion of β-catenin + cells co-expressing bromodeoxyuridine (BrdU), indicative of proliferation (L'Episcopo et al., 2012). By contrast, MPTP treatment sharply down-regulated the β-catenin immunofluorescence signal and β-catenin + cell proliferation ( Figure   S2). Since β-catenin is expressed in type C cells (Adachi et al., 2007) and given that MPTP reduced the proliferation of type C cells, we next addressed the relevance of the SVZ-WβC-signalling pathway

| WβC-signalling is a key player in SVZ-NSC homeostasis in PD mice: ex vivo and in vitro findings
Looking at Wnt signalling components using western blot analysis we found a decreased β-catenin signal in NSCs isolated from MPTPbut not saline-treated mice, whereas the active GSK-3β signal was sharply increased. We then used quantitative real-time PCR to show that expression levels of Axin-2, a direct Wnt target induced by WβC activation (Jho et al., 2002), were down-regulated in MPTP-NSCs as Together, the "in vivo", "ex vivo" and "in vitro" results clearly established MPTP/MPP + -induced inhibition of WβC-signalling activity in SVZ-NSCs as a crucial step in the neurogenic impairment of PD mice.

| Wnt signalling crosstalk with neuroinflammatory pathways contributes to SVZ plasticity in PD
Within the mechanisms affecting adult neurogenesis in brain diseases, oxidative, and especially nitrosative stress, are likely to play critical roles, given their contribution to the aging process and the development of age-related diseases . In PD especially, glial inflammatory mechanisms have long been recognized to contribute to both nigrostriatal degeneration and self-repair (see

| Depletion of SVZ-NSC starts by middle age
The process of aging is accompanied by a marked decrease in the neurogenic potential of the SVZ, as revealed by sharp decreases in the total number of BrdU + cells, DCX + neuroblasts, and EGFR + cells (Ahlenius et al., 2009;Enwere et al., 2004;L'Episcopo et al., 2012L'Episcopo et al., , 2013Luo et al., 2006). A significant reduction in BrdU + cells and DCX + neuroblasts has already occurred by middle-age, and a further, albeit smaller, decline in BrdU + cells and DCX + neuroblasts is observed in aged mice. The type C cell compartment is particularly affected, since a sharp loss of EGFR + cells is observed from middleage on (L'Episcopo et al., 2013). These findings suggested that by F I G U R E 4 Cross talk dialogue between inflammatory and WβC-signalling pathways in MPTP-induced SVZ plasticity is lost with age. A simplified scheme summarizing MPTP-induced neuroinflammation and SVZ plasticity in young and aged mice via modulation of WβC-signalling ("Wnt on; Wnt off") is shown. In young mice, during the degeneration phase, hyperactivated M1 microglia contributes to the impairment of SVZ neurogenesis at different levels. By increasing oxidative and nitrosative stress and in synergy with MPTP/MPP + direct toxicity, microglial-derived mediators (PHOX-derived ROS, iNOS-derived NO, and peroxynitrite) may act as molecular switch for cell signalling pathways critically involved in the physiological control of NSC homeostasis, with harmful consequences for astrocyte and NSC physiology, at least in part through GSK-3β activation, followed by phosphorylation and consequent degradation of β-catenin. In young mice, after the acute inflammatory and degenerative phase, a regulatory circuit linking microglial activation and proinflammatory cytokine to Nrf2-ARE protective pathway in SVZ, provides an efficient self-adaptive mechanism against inflammatory/neurotoxin-induced oxidative stress, switching the M1 microglial harmful phenotype, thus mitigating inflammation with a return to pre-MPTP conditions. By contrast, the aging process, in synergy with MPTP exposure, negatively impacts on astrocytic Nrf2-driven Hmox1 response within the SVZ niche in vivo. Hence, this process, resulting from an age-dependent dysregulation of astrocyte-microglia interactions, contributes to the exacerbated oxidative and inflammatory SVZ status and the decline of astrocyte Wnt-dependent regulation, finally leading to NSC neurogenic impairment and loss of SVZ plasticity The mutual role of astrocyte-microglial interactions in the plasticity of SVZ response to MPTP is exemplified by the astrocyte's ability to overcome microglial inhibitory effects, also via cross talk with Wnt/β-catenin signalling. Pharmacological mitigation of inflammation and oxidative stress or GSK-β antagonism upregulates β-catenin and successfully rescues NSC proliferation and neuroblast formation, a process associated with striatal DAergic neuroprotection, with further positive modulation of SVZ proliferation via D2 receptor (D2R) activated mechanisms middle-age, the proliferative ability of type A and type C cells is markedly impaired, indicating that the SVZ neurogenic decline is an early event in mice. This impairment of SVZ neurogenic potential was not associated with changes in dopamine transporter (DAT) immunofluorescence in the Str, nor in striatal DA and high affinity synapotosomal DA uptake, or in the number of DAergic cell bodies in the SNpc (L' Episcopo et al., 2013). These findings thus indicated that, besides the nigrostriatal DAergic influence, other factors contributed to SVZ impairment as early as middle-age.
Aging mice showed an especially-impaired recovery from MPTP-

| Microglial modulation of NSCs is age-and inflammation-dependent
By the use of a controlled in vitro environment, we next addressed the distinct roles of young and aged microglia. In these ex vivo/in vitro cellular models, NSCs derived from young and aged SVZs were cocultured with either young or aged glia, with the aging process ostensibly switching microglia from a neurogenesis-promoting to a neurogenesis-inhibitory phenotype. Here, direct coculture of young  Specifically, astrocytes are central players in Nrf2-Hmox1 induction following different types of brain insult, including MPTP exposure (Chen et al., 2009  Together, these results may suggest that the aging-dependent mitochondrial dysfunction in synergy with neurotoxin exposure may negatively impact on the astrocytic Nrf2-driven Hmox1 response within the SVZ niche in vivo. Remarkably, this process, resulting from an age-dependent dysregulation of astrocyte-microglia interactions, can contribute to the exacerbated oxidative and inflammatory SVZ status and the decline of astrocyte Wnt-dependent regulation, ultimately leading to NSC neurogenic impairment and loss of SVZ plasticity. From the bulk of the summarized results, it is tempting to speculate that in addition to governing the redox balance with in the SVZ niche, the Nrf2-induced Hmox1 target gene may simultaneously protect astrocytes, there by upregulating the expression of vital Wnt signalling elements that switch-on key components required for maintaining SVZ cells in a proliferative state, promoting differentiation, and/or exerting neuroprotective effects (Figure 4).

| Harnessing Wnt signalling targeting the inflammatory Nrf2/Hmox1 axis restores SVZ neurogenesis and promotes DAergic neurorestoration in PD
The development of anti-inflammatory drugs targeting inflammatory molecules to preserve adult neurogenesis during PD neurodegen-

Studies of the last few years have identified the Aq-PVR as a novel
Wnt/β-catenin responsive brain region and addressed the proper-

Looking at the factors/mechanisms regulating the behaviour of these
Aq-PVR-NSCs in vitro, we addressed the neurogenesis-promoting and inhibitory conditions during the process of aging and MPTP-induced nigrostriatal injury, investigating the potential to activate these progenitors to rescue DAergic plasticity in aged-MPTP mice. To this end, we also took advantage of transgenic BATGAL mice expressing nuclear beta-galactosidase under the control of the β-catenin-activated transgene (BAT) promoter (Maretto et al., 2003), together with both in vivo experimental PD young and older mice models, coupled to ex vivo/in vitro cell cultures of midbrain-Aq-PVR-NSCs (mNSCs).

| Proliferation and neuron differentiation of mNSCs in vitro depends upon age and MPTP
We first characterized mNSC properties during in vitro clonal expansion supporting their neurogenic potential as indicated by the expression of proliferation, precursor, pro-neural, and astrocyte cell markers, and very low forebrain (distal-less homeobox 2, Dlx2) and hindbrain (homeobox D3, Hoxd3) markers, as opposed to the high ex-

| In vitro studies
By analogy with our studies carried out at the SVZ level, we observed astrocyte-coculture paradigms coupled to Wnt-activation regimens can rescue A-NSCs and promote TH + neuron formation ( Figure 5).

| In vivo studies
The tegmental Aq which is closed to DAergic cell bodies was shown to respond to MPTP-induced DAergic neuron death with an extraor-  developing human mesencephalon show radial glial characteristics (Hebsgaard et al., 2009). The key role of WβC-signalling was demonstrated by Briona, Poulain, Mosimann, and Dorsky (2015), showing its requirement for radial glial neurogenesis following spinal cord injury.

F I G U R E 6
Harnessing WβC-signalling activation in the aged, inflammed PD brain. Schematic representation of the 'Wnt on' neurorestoration instructed by grafted NSCs in the aged PD brain. With age, the inflammed midbrain microenvironment coupled to dysfunctional astrocyte-microglia interactions and environmental toxin exposure (MPTP) inhibit active Wntsignalling in astrocytes ("Wnt-off" condition), resulting in exacerbation of inflammation and inhibition of Wntdependent neuroprotective and proregenerative capacities of astrocytes with harmful consequences for mDA neuron survival and repair from MPTP injury. NSCs, NSC-derived astrocytes and endogenous astrocytes switch the inflammatory/Wnt-genetic cascade via astrocyte-neuron and astrocyte-microglia crosstalk both at the SNpc and Aq-PVR DA niche levels. Reciprocally, astrocyte derived Wnt1 further influence both exogenous and endogenous NSCs, reduce microglia pro-inflammatory status, thus favouring beneficial effects for an overall TH neurorescue ("Wnt on") program. Exogenous pharmacological treatments rescuing the impaired SVZ neurogenis (including GSK-3β antagonists, anti-oxidant and anti-inflammatory drug treatment and DAergic activation promoting SVZ neurogenic rescue) are also illustrated TH + cell bodies with different morphologies and size were seen coursing from the Aq-PVR and along the midline down to the ventral tegmental area (VTA) of NSC grafted mice, mimicking TH + neurons trafficking from the Aq to the SNpc observed in younger MPTP mice during SNpc neurorepair (L'Episcopo et al., 2014a;L'Episcopo, Tirolo, Serapide, et al., 2018b).
Corroborating the role of WβC-signalling in Dkk1-treated NSCgrafted MPTP mice, we observed no TH + neurons in Aq-PVRs. These data, thus, demonstrated that NSC grafts activate β-catenin transcription, inducing a marked increase of TH + cells within the Aq-PVR and VTA, whereas this process is blocked by WβC-antagonism, finally resulting in the inhibition nigrostriatal histopathological and functional repair (L'Episcopo, Tirolo, Serapide, et al., 2018b).
Together, these novel findings supported the potential of nigrostriatal restoration by activating WβC-signalling in Wntresponsive niches, through either pharmacological and cellular approaches aimed at activating/recruiting endogenous progenitors and rescuing the imperiled/diseased DAergic neurons. Moreover, these data suggested that WβC-signalling activation by NSC grafts at the SNpc and Aq-PVR is required for NSC-promoted DAergic functional restoration of aged PD mice. Thus, harnessing WβCsignalling represents a potential means to boost the endogenous self-repair/regenerative capacity of the aged PD brain (Figure 6).

| Up-regulating adult neurogenenesis as a disease modifying strategy for PD: is WβCsignalling the common denominator?
Different conditions, cell therapies and/or pharmacological treatments are being studied for their potential to modulate endogenous neurogenesis to favour neuroprotection, neurorepair and immunomodulation (Wenker & Pitossi, 2019), with an increasing number of manipulations being associated to either a direct or indirect ability to up-regulate the WβC-signalling cascade (Tables 1 and 2, Figure 7).
Owing to its crucial function in stem cell maintenance and tissue ho- Huang , Tang, et al., 2019a;Huang, Yan, et al., 2019b;Janda et al., 2017;Kahn, 2014;Mahmood, Bhatti, Syed, & John, 2016;Maiese, 2015;Narcisi et al., 2016;Nusse & Clevers, 2017). Therefore, indirect WβC modulation appears as an attractive strategy to up-regulate endogenous neurogenesis. Environmental enrichment, physical exercise, the administration of hormones, anti-oxidant and anti-inflammatory molecules, pharmacological, pharmacogenetic and/or epigenetic strategies, optogenetic and neural stimulation, or deep brain stimulation, and stem cell therapies, are all being explored for their potential to reverse neurogenic impairment, incite neurorepair and/or reverse cognitive impairment, apparently through activation of WβC signalling, in experimental models of NDs. Also, a certain number of herbal derivates, primarily from the Traditional Chinese Medicine, endowed with pharmacological properties (including anti-cancer, anti-bacterial, and antioxidant activities) are increasingly being studied for their ability to modulate WβC-signalling (recently reviewed by Liu D et al., 2019), with interesting therapeutic potentials for NDs including PD (Table 1 and 2 and Figure 7).

| Micro-and nanocarriers
Micro/nanoparticulate delivery vehicles may be engineered to release multiple biomolecules with spatio-temporal control are being developed, aimed at mobilizing NSCs efficiently from their niches to promote their engraftment at lesioned areas, or creating a long term anti-inflammatory microenvironment Riabov et al., 2017;Tiwari et al., 2014;Zhang, Zhang, Deng, et al., 2018a;Zhang, Shi, et al., 2018b;Zhang, Zhang, Yang, et al., 2018c;Zhang, Huang, et al., 2016c). Curcumin-loaded nanoparticles powerfully induce adult neurogenesis and reverse cognitive deficits in an Alzheimer's disease model via the activation of WβC-signalling pathway (Tiwari et al., 2015. The ability of curcumin treatment to ameliorate cognitive and mood function was previously associated with increased neurogenesis as well as mitigation of inflammation and mitochondrial dysfunction in the rodent hippocampus . Additionally, loading paclitaxel (PTX)-encapsulated liposomes into a collagen microchannel scaffold, leading to a prolonged sustained release of PTX, was recently shown to provide an instructive microenvironment for neuronal differentiation of NSCs, motor and sensory neuron regeneration, axon extension and beneficial functional outcomes via activation of the WβC-signalling pathway . Conversely, inorganic nanocarriers such as silver nanoparticles have been attributed with negative effects, reportedly disrupting β-catenin signalling and resulting in reduced neurite lengths in differentiating NSCs (Cooper et al., 2019).

| Optogenetics
Optogenetics is a novel approach allowing specific cell stimulation by external illumination which may remotely manipulate intracellular pathways in single cells (Zhang et al., 2016a,b,c), using channelrhodopsin-2 (ChR2) activation to allow cationic currents to depolarize genetically targeted cells. Yang et al. (2014), found that optogenetic activation of VM astrocytes can enhance the DAergic differentiation of stem cells and promote brain repair in PD models in vivo and in vitro, very likely via the mediation of WβC activation. Kaur et al. (2017) addressed the potential of coupling optogenetics and light-sheet microscopy to study Wnt signalling during embryogenesis, showing that WβC-signalling is required not only for Drosophila pattern formation, but also for maintenance later in development. Additionally, Zhang, Huang, et al. (2016c)  Ameliorates cognitive function, enhances neurogenesis, mitigates inflammation and mitochondrial dysfunction in hippocampus in a rodent model of gulf war illeness; WβC-AC involvement to be elucidated Kodali et al. (2018) Exercise, environmental enrichment Physical activity and environmental enrichment regulate the generation of neural precursors in the adult mouse substantia nigra; WβC-AC involvement to be elucidated Klaissle et al. (2012) Endurance exercise promotes neuroprotection against MPTP injury via enhanced neurogenesis, antioxidant capacity and autophagy; WβC-AC involvement to be elucidated Jang, kwon, Song, Cosio-Lima and Lee (2018a), Jang et al. (2018b) Neural activation Neural activity-induced WβC-AC up-regulates expression of BDNF. Zhang, Zhang, Deng, et al., 2018a Neurotrophic factors BDNF promotes growth of neurons and NSCs, possibly through activation of the PI3K/GSK-3β/β-catenin pathway Li et al. (2017) BDNF promotes human neural stem cell growth via GSK-3β-mediated crosstalk with the WβC pathway Yang et al. (2016) Optical depolarization Optogenetic activation of VM astrocytes enhances DAergic differentiation of NSCs and promotes brain repair in PD rodent models; WβC-AC involvement to be elucidated Yang et al. (2014) Optical depolarization of DCX-expressing neuroblasts promotes cognitive recovery and maturation of newborn neurons after traumatic brain injury via WβC-AC Zhang, Huang, et al. (2016c) Coupling of optogenetics and light-sheet microscopy reveals WβC-AC during embryogenesis and post-natal development Kaur et al. (2017) WβC-AC can be controlled in vivo via light responsive capsules. Ambrosone et al. (2016) Optical depolarization promoted the maturation of neural stem cells via WβC-AC Xia et al. (2014) (Continues) activation. WβC-signalling also plays a key role in controlling neuron activity-regulated neurotrophic factor (i.e. Bdnf) expression (Zhang, Zhang, Deng, et al., 2018a;Zhang, Shi, et al., 2018b;Zhang, Zhang, Yang, et al., 2018c). Also, in human NSCs, Yang et al. (2016) showed the ability of BDNF to promote their growth via GSK-3β-mediated crosstalk with the WβC-signalling pathway, and Li et al. (2017) involved the contribution of the PI3K/Akt/GSK-3β/β-catenin pathway in BDNF-induced neuron and NSC growth. Ambrosone et al. (2016) documented the ability of optogenetic stimulation to promote cell proliferation and the migration of SVZ neuroblasts into the peri-infarct cortex, asssociated with increased neuronal differentiation and improvement of long-term functional recovery after stroke. Finally, Mastrodonato et al. (2018) reported enhanced olfactory memory in mice exposed to extremely low frequency electromagnetic fields via WβC-dependent modulation of SVZ neurogenesis.

| Small molecules modulating WβC proteins
In recent years, druggable molecular targets and signalling pathways involved in neurogenic processes have been identified, and as a consequence, different drug types have been developed and tested in neuronal plasticity. The field of small molecules as potential tools to selectively activate or inhibit WβC is increasingly recognized with a number of both established and novel modulators, including Wnt3alike agonists, siRNAs and inhibitors targeting GSK-3β, Axin-LRP5/6 or transcription factor complexes (Table 2, Figure 6). The manipulation of WβC-signalling has become an attractive strategy to ameliorate in vitro differentiation protocols for increasing the fraction of midbrain DAergic neurons (see Kirkeby et al., 2017;Toledo et al., 2017;Brodski et al., 2019).

| Nurr1 activation
An important potential mediator of WβC-signalling activation is Nurr1, a nuclear receptor acting as an intracellular transcription factor recognized to contribute to the proliferation and differentiation of NSCs, both during development and in the adult brain.
A number of studies documented the importance of Nurr1 and WβC/Nurr1 signalling pathways in promoting neurogenesis from DAergic precursors (reviewed in Arenas, 2014;Toledo et al., 2017; Section 2 and section, sectionprevious sections). Several studies have reported the contribution of Nurr1 in the modulation of cognitive functions (see Kim et al., 2015;Kim et al., 2016, and refs therein). Specifically, the pharmacological stimulation of Nurr1 was shown to improve cognitive functions via the enhancement of hippocampal neurogenesis (Kim et al., 2015(Kim et al., , 2016, very likely via up-regulation of WβC-signalling. Hence, Kim et al. (2015) identified two antimalarial drugs, amodiaquine (AQ) and chloroquine that stimulated the transcriptional function of Nurr1. Remarkably, these compounds were able to enhance the Nurr1-dependent transcriptional activation of DAergic-specific genes. Moreover, they further enhanced transrepression of neurotoxic proinflammatory gene expression in microglia (Kim et al., 2015). Of specific interest, pharmacological stimulation of Nurr1 causes both neuroprotection and anti-inflammatory effects in the 6-OHDA lesion model of PD (Kim et al., 2015;Smith et al., 2015). Additionally,
Bold is used to highlight WβC connection TA B L E 1 (Continued) TA B L E 2 Interventions directly targeting WβC-signalling activation (WβC-AC)-targeted in the central nervous system
Wnt3a promoted the proliferation of precursor Nurr1 + cells. Wnt-1 and -5a increased the number of rat midbrain DAergic neurons in E14.5 precursor cultures. Wnt-1 increased the proliferation of Nurr1 + precursors, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 mRNAs Castelo-Branco et al. (2004) Wnt5a increases differentiation of midbrain DAergic cells and phosphorylation of dishevelled Gene KO sFRP-mediated Wnt sequestration represents a potential therapeutic target for Alzheimer's disease. sFRP3 inhibition improves age-related cellular changes in BubR1 progeroid mice CHIR99201 is a substituted aminopyrimidine derivative that potently and selectively inhibits GSK−3 in vitro and in vivo Ring et al. (2003) SB−216763 and SB−415286 are cell-permeable, structurally distinct maleimides that potently and selectively inhibit GSK−3 Coghlan et al. (2000) Midbrain floor plate precursors are derived from hPSCs in 11 days following exposure to small molecule activators of sonic hedgehog and WβC-AC. Enrichment for canonical Wnt signalling upon CHIR99021 treatment. Induction and neurogenic conversion of hESC-derived midbrain floor plate precursors is dependent on CHIR99021 addition Kriks et al. (2011) Establishes a means of obtaining a scalable source of FOXA2 + /TH + neurons for neural transplantation, a major step on the road towards considering a cell-based therapy for PD CHIR99201 counteracts the altered differentiation potential of Gaucher's disease iPSC neuronal progenitors due to Wnt/β-catenin downregulation by WβC-AC these compounds significantly improved behavioral deficits in 6-OHDA lesioned rat model of PD without any detectable signs of dyskinesia-like behavior (Kim et al., 2015), underscoring the potential of small molecules targeting Nurr1 as neuroprotective strategy for PD (Kim et al., 2015). In another study, the anti-malarial AQ powerfully enhanced adult hippocampal neurogenesis, increasing learning and memory processing via a direct neurogenic action of Nurr1 (Kim et al., 2016), as supported by immunocytochemical and immunohistochemical analyses, both in vivo and in vitro (Kim et al., 2016). In addition to its effects on proliferation and differentiation of NSCs, AQ-treated mice showed a significant enhancement of both short-and long-term memory in the Y-maze and the novel object recognition test. Together these data suggest that activation of Nurr1 may enhance cognitive functions by increasing adult hippocampal neurogenesis and also indicate that Nurr1 may be used as a therapeutic target for the treatment of memory disorders and cognitive impairment observed in NDs.  Table 1 Sivrastava and Sivrastava Note: 3xTg, APP/PS1/Nestin-GFP triple transgenic mice; 6-OHDA, 6-hydroxydopamine; APC, adenomatous polyposis coli; Aq-PVR, mesencephalic aqueduct-periventricular region; bFGF, basic fibroblast growth factor; DA, dopamine; EGF, epidermal growth factor; FOXA2, forkhead box A2; Fzd, frizzled; GSK-3β, glycogen synthase kinase 3β; hESC, human embryonic stem cell; hPSC, human pluripotent stem cell; icv, intracerebroventricularly; iPSC, induced pluripotent stem cell; KO, knockout; Lrp, low-density lipoprotein receptor-related protein; MAP2A, microtubule-associated protein 2a; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NSC, neural stem/progenitor cell; Nurr1, nuclear receptor related 1 protein; OTX2, orthodenticle homeobox 2; PD, Parkinson's disease; SAMP8, senescence accelerated mouse-prone 8; sFRP, secreted Frizzled-related proteins; SNpc, substantia nigra pars compacta; SVZ, sub-ventricular zone; TH, thyrosine-hydroxylase; VM, ventral midbrain; WβC-AC, Wnt/β-catenin signalling activation.

TA B L E 2 (Continued)
WβC-signalling transduction. The authors also identified the critical residues on Axin for HLY78 binding and showed that HLY78 may weaken the autoinhibition of Axin (Wang et al., 2013). Further results have been recently gathered by Chen et al. (2019) on the design, synthesis and structure-activity relationship for optimization of phenanthridine derivatives as new WβC-signalling pathway agonists, including evidence for a protective role of the Wnt-agonists in in vivo cytotoxicity models (Huang, Tang, et al., 2019a;Huang, Yan, et al., 2019b). Recently, protein phosphatase-2A (PP2A), a multi-subunit serine/threonine phosphatase that positively regulates the Wnt pathway, has been shown to directly interact with Axin, APC, and β-catenin, and thus identified as a target of the Wnt agonist/IQ and sodium selenate (Jin et al., 2017). Another possible avenue by which to up-regulate WβC is to target sFRP-mediating Wnt sequestration.

| Fzd-LRP5/6 heterodimerizers
A novel class of WβC-agonists are the Fzd-LRP5/6 heterodimerizers (Janda et al., 2017), called "surrogate Wnt-agonists". The water-soluble Fzd-LRP5/6 heterodimerizers, consist of "Fzd5/8specific and Fzd-reactive binding domains", endowed with a WβCsignalling activating potential through ligand-induced receptor heterodimerization, showed to promote a characteristic β-catenin signalling response in a Fzd-selective manner, including the growth of a broad range of primary human organoid cultures, in a fashion comparable to Wnt3a (Janda et al., 2017). The ability of these compounds to be systemically expressed and exhibit Wnt activity in vivo was demonstrated, suggesting a potential "new avenue to facilitate functional studies of WβC-signalling" (Janda et al., 2017).

| GSK-3 β-antagonism
A great number of studies indicates that GSK-3β acts as a key regulator in neural development, including neuroblast generation/migration, neuroprogenitor homeostasis, neural induction, neuronal polarization, axon growth/guidance, and synaptic plasticity (Jope et al., 2017). The activation of GSK-3β has a role in the phosphorylation of microtubuleassociated protein tau (MAPT), triggering cytoskeleton destabilization, Tau aggregation and neuronal dysfunction or death (Beurel, Grieco, & Joper, 2015;Jope et al., 2017;L'Episcopo et al., 2016). As recalled in previous sections, both earlier and more recent reports indicate that GSK-3β inhibitors can promote adult neurogenesis both under normal and injury conditions, either in vivo or in vitro. In vitro protocols that modulate Wnt signalling to improve cell therapies for PD are increasingly being developed (Arenas, 2014;Broski et al., 2019;Joksimovic & Awatrami, 2014;Kirkeby et al., 2012Kirkeby et al., , 2017Kriks et al., 2011;Parish & Thompson, 2014;Prakash & Wurst, 2014;Toledo et al., 2019;Zhang et al., 2015). To date, a number of GSK-3β-antagonists have been described, some of which have been tested for their potential to promote adult neurogenesis (Table 2). Amongs others, CHIR is a small molecule used to promote neuroprogenitor homeostasis and neural induction.
CHIR was shown to restore WβC-signalling and to rescue DAergic differentiation in iPSC-derived NSCs from Gaucher's disease patients, exhibiting developmental defects due to downregulation of canonical with CHIR significantly enhanced neurogenesis, while simultaneously decreasing astrocyte differentiation (Nierode et al., 2019).
The cell permeable selective inhibitor of GSK-3β inhibitor, BIO, is derived from Tyrian purple indirubins, that selectively inhibits the phosphorylation of GSK-3β at Tyr216/276 (Sato et al., 2004) and is widely used to activate Wnt signalling. Recent studies addressing the effect BIO during the sub-acute and chronic phases after ischemic stroke showed its ability to stimulate post-stroke neurogenesis, neuroblast migration to the ischemic cortex, neuronal differentiation and functional recovery after ischemic stroke . Valproic acid has been recently shown to increase β-catenin levels and to induce the expression of NeuroD1, a Wnt target gene involved in neurogenesis in the hippocampus of 3xTgAD mice (Zeng et al., 2019).

| Concluding remarks and future perspectives
Harnessing endogenous pro-neurogenic mechanisms to counteract the decline of adult neurogenesis and promote DAergic plasticity in the aged brain represents a major goal in the PD physiopathological field. We herein discussed the critical role of WβCsignalling in rebuilding a regenerative microenvironment and in promoting neuronal differentiation of endogenous or exogenous NSCs, which is pivotal for the recovery of neurologic functions in the aged PD brain.
There is compelling evidence that WβC-signalling plays a vital role connection" which provides a robust homeostatic regulatory mechanism for NSC survival, proliferation, differentiation and integration.
Herein we provided an overview of the molecular mechanism(s) underlying the crosstalk between Wnt-signalling and NSCs involving astrocyte and microglial mediators, with a crucial role of astrocytic-microglia crosstalk, including modulation of the Nrf2/Hmox1/ WβC-axis, critical for protection against exacerbated inflammation, oxidative stress and mitochondrial dysfunction. As WβC-signalling also plays a prominent role in hippocampal SGZ neurogenesis, and in light of the marked decline of WβC-signalling components in the aged hippocampus, a comparable "WβC-inflammatory (dis)connection" might well be at play in PD-injured SGZ-niche.
Remarkable potential exists to revert some of these age-dependent changes, targeting WβC-signalling components either directly or indirectly. Pharmacological and cellular therapies, in particular NSC-grafts, and immunomodulation were documented to ameliorate the aged microenvironment, promoting endogenous neurogenesis, and ultimately boosting a neurorestoration program in the aged PD brain via the up-regulation of WβC-signalling.
A number of novel molecules and conditions were reviewed for their potential to activate WβC for translational applications in regenerative medicine. Hence, targeting Axin-LRP6 with phenanthridine derivatives, PP2A modulators, sFRP inhibitors, and surrogate Wnt-agonists, showed their ability to promote cell survival/protection and/or immunomodulation. Also, targeting GSK-3β with small inhibitors, such as CHIR or BIO, can promote neuroprogenitor homeostasis and neural induction, and restore WβC-signalling in iPSC-derived neuronal progenitor cells from PD patients. Emerging studies at the interface between NSC biology and tissue engineering are being exploited for innovative therapeutic applications in brain repair/regeneration therapies, including optogenetics, neural stimulation, and micro-and nanocarriers releasing multiple biomolecules involved in WβC activation. These interventions are aimed at mobilizing NSCs efficiently from their niches, and, in combination with sustained release of therapeutic agents, can be envisaged as a promising approach to induce neuronal differentiation of NSCs, down-regulating the exacerbated pro-inflammatory microenvironment and promoting neurorepair in the injured aged PD brain.
In conclusion, the continuous investigations and further deepening of our knowledge on WβC-signalling and its role on endogenous adult stem cell biology, NSC crosstalk within the PD injured microenvironment, the response of NSCs to different pharmacological/cellular strategies, as well as its implication will translate into therapeutic breakthroughs and novel applications. Specifically, harnessing their synergistic interactions may lead to the optimization of cell-based therapies for PD.

ACK N OWLED G M ENTS
This research program has received support from different fund-

CO N FLI C T O F I NTE R E S T
No conflict of interest to declare.

AUTH O R CO NTR I B UTI O N S
Authors contributing to the presented experimental findings and