Therapeutic Strategies for Ischemic Stroke: Modulating the Adult Neural Stem Cell Niche through the Wnt/ β -catenin Pathway

Stroke is a prominent contributor to mortality and impairment on a global scale. Ischemic stroke accounts for approximately 80% of stroke cases and is caused by occlusion of cerebral blood vessels. Enhancing neurogenesis through the modulation of the neural stem cell niche in the adult brain is a promising therapeutic strategy for individuals afflicted with ischemic stroke. Neurogenesis results in the generation of newborn neurons that serve as replacements for deceased neural cells within the ischemic core, thereby playing a significant role in the process of neural restoration subsequent to cerebral ischemia. Research has shown that activation of the Wnt/ β -catenin pathway can augment neurogenesis following cerebral ischemia, suggesting that this pathway is a potentially beneficial therapeutic target for managing ischemic stroke. This review provides an extensive analysis of the current knowledge regarding the involvement of the Wnt/ β -catenin pathway in promoting neurogenesis, thereby offering a promising avenue for therapeutic intervention in the context of ischemic stroke or other neurological impairments.


Introduction
Stroke is a prevalent cerebrovascular disorder characterized by significant rates of disability and mortality.The two primary classifications of stroke are hemorrhagic stroke and ischemic stroke.Ischemic strokes account for approximately 80% of all stroke cases, with middle cerebral artery thromboembolism as the principal etiology [1].Patients who experience ischemic stroke frequently exhibit significant neurological impairments.At present, the sole interventions that have demonstrated efficacy during the acute phase of ischemic stroke are the administration of tissue plasminogen activator (tPA) and mechanical thrombectomy [2].Unfortunately, the therapeutic window is narrow, and only a small proportion of patients are eligible [3][4][5].Therefore, potential drugs for ischemic stroke treatment are urgently needed.The pathophysiology of ischemic stroke involves complicated molecular mechanisms, such as oxidative stress, inflammation and apoptosis.Available pharmacological therapies targeting crucial stages in the pathophysiology of ischemic stroke aim to promote optimal poststroke neurological recovery by reducing neuronal apoptosis, inhibiting inflammation, promoting angiogenesis, and removing free radicals [6,7].However, the performance of these therapies is not clinically satisfactory.In recent years, promoting neurogenesis from endogenous neural stem cells (NSCs) has emerged as a promising therapeutic approach for treating ischemic stroke [8].
Josef Altman was the first to present evidence of neurogenesis in the adult brain when he identified newly formed neurons and glial cells through the use of tritiated thymidine to label proliferating cells [9].Since then, accumulating evidence has revealed that neurogenesis persists in the adult mammalian brain.Although research on adult neurogenesis in humans is controversial, the occurrence of neurogenesis in the adult human brain is widely acknowledged [10,11].Presently, two extensively documented neurogenic niches housing adult NSCs exist within the adult brain: the subventricular zone (SVZ) located in the lateral ventricle (LV) and the subgranular zone (SGZ) situated in the dentate gyrus (DG) of the hippocampus.Under normal physiological conditions, SVZ NSCs are responsible for the production of transit-amplifying cells (TACs), subsequently leading to the emergence of neuroblasts.These neuroblasts undergo migration along the rostral migratory stream (RMS) to reach the olfactory bulb (OB), where they differentiate into interneurons that play a crucial role in the sense of smell.NSCs located in the SGZ are responsible for generating excitatory glutamatergic neurons, which subsequently integrate into the granule cell layer of the DG.These neurons play a crucial role in spatial and temporal memory processing.During ischemic stroke, SGZ NSCs proliferate and migrate toward the granule cell layer within the DG, although their migration remains confined within the boundaries of the hippocampus [12].Simultaneously, ischemic injury drastically increases neurogenesis in both rodent and human SVZs, and neuroblasts deviate from the conventional pathway toward the adjacent parenchyma and striatal ischemic penumbra.Within the ischemic penumbra, a limited population of neuroblasts produces neurons, potentially facilitating tissue restoration and the recovery of locomotor function.However, a significant proportion of NSCs undergo differentiation into astrocytes, which subsequently transform into reactive astrocytes that play a crucial role in the process of glial scar formation.This impedes the effective repair of nerve injuries [8,13,14].A prior investigation employed a genetic approach utilizing the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide system to eliminate doublecortin (DCX) + neuroblasts, which led to increased infarct size and exacerbated neurological impairments in mice that underwent middle cerebral artery occlusion (MCAO) [15].These findings indicate that neurogenesis induced by ischemia appears to play a role in alleviating histological and neurobehavioral impairments.
Taken together, these findings indicate that enhancing neurogenesis and augmenting neuronal regeneration within the ischemic penumbra subsequent to cerebral ischemia can potentially enhance the restoration of neurological function.Multiple pathways are believed to be implicated in adult neurogenesis, including the Notch signaling pathway [16], Sonic Hedgehog signaling (SHH) pathway [17,18], and bone morphogenetic protein (BMP) signaling pathway [19,20].This review primarily concentrates on the role of the Wnt/β-catenin pathway in adult neurogenesis and its potential therapeutic implications for the management of cerebral ischemia (Fig. 1).

The Wnt Signaling Pathway
The Wnt signaling pathway primarily consists of Wnt ligands, which are secreted glycoproteins, and cell-surface receptors called Frizzled.The mammalian genome harbors a total of 19 Wnt genes that encode Wnt ligands.Wnt signaling pathways can be categorized into three different types: the Wnt/β-catenin pathway (also referred to as the canonical Wnt signaling pathway), the planar cell polarity pathway (Wnt/PCP), and the Wnt/Ca 2+ signaling pathway.
In the canonical Wnt signaling pathway, Wnt ligands bind to the Frizzled receptor (Fzd) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6).Then, the cytoplasmic protein Dishevelled (Dvl) is activated.Activation of Dvl leads to the degradation of a complex containing glycogen synthase kinase-3β (GSK-3β), adenomatous polyposis coli (APC), the protein kinase casein kinase 1α (CK1α), and Axin.In the absence of Wnt pathway activation, the GSK-3β/APC/CK1α/Axin complex phosphorylates cytosolic β-catenin, leading to its ubiquitination and subsequent degradation by the proteasome.Conversely, upon activation of the Wnt pathway, the degradation machinery of the GSK-3β/APC/CK1α/Axin complex is hindered, resulting in the cytosolic accumulation of β-catenin and its subsequent transportation into the nucleus.Intranuclear β-catenin participates in interactions with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins, thereby promoting the transcription of downstream genes (Fig. 2) [21][22][23].In addition to the canonical pathway, Wnt ligands can initiate various signaling cascades that do not rely on β-catenin, namely, the Wnt/PCP pathway and the Wnt/Ca 2+ signaling pathway.The Wnt/PCP pathway begins with the interaction between a Wnt ligand and Fzd, subsequently leading to the activation of Dvl.The activation of Dvl, in turn, initiates the activation of small G proteins, including Rac and Rho, which subsequently activate Rho-associated kinase (ROCK) and c-Jun N-terminal kinase (JNK).This intricate series of events ultimately contributes to the establishment of cellular polarity and the facilitation of cell migration [24,25].The initiation of the Wnt/Ca 2+ pathway occurs when a Wnt ligand interacts with Fzd, leading to the subsequent activation of phospholipase C (PLC).This activation subsequently modulates the release of calcium from the endoplasmic reticulum, thereby governing the regulation of intracellular calcium concentrations [26].
Research has demonstrated that the Wnt signaling pathway, particularly the Wnt/β-catenin signaling pathway, is important for neurogenesis.This review aimed to consolidate existing studies pertaining to the role of the Wnt/β-catenin signaling pathway in neurogenesis throughout developmental stages and in the adult brain.Additionally, we explored the potential therapeutic implications of the Wnt/β-catenin pathway in the treatment of cerebral ischemia.

The Role of the Wnt/β-catenin Pathway in Neurogenesis during Brain Development
The Wnt/β-catenin signaling pathway has diverse functions throughout various stages of neural development.Overall, stimulation of the Wnt/β-catenin signaling pathway promotes neurogenesis and suppresses gliogenesis [27,28].
Chenn et al. [29] employed transgenic mice to induce excessive activation of β-catenin in neural precursors, resulting in the identification of enlarged brains characterized by augmented cerebral cortical surface area and folds, in addition to enlarged lateral ventricles lined with neuroepithelial precursor cells.This outcome can be attributed to an increased population of proliferative precursor cells [29].Machon et al. [30] employed a D6-Cre mouse strain to conditionally inactivate β-catenin (Ctnnb1) within the murine cerebral cortex and hippocampus after embryonic day (E) 10.5.The results showed that β-catenin is needed for hippocampal progenitor proliferation, cortical neuronal migration, late-embryonic cortical proliferation, and cortical radial glial cell maintenance [30].Additionally, several other studies have demonstrated the indispensability of the Wnt pathway in the development of the hippocampus [31,32].Furthermore, the Wnt/β-catenin pathway plays a crucial role in the early stages of cerebellar development and assumes a pivotal function in governing differentiation within the cerebellar ventricular zone in the subsequent stages of embryonic development [33].However, in the event of ischemic stroke, neuroblasts deviate from their conventional pathway toward the ischemic penumbra.Within the ischemic penumbra, a limited population of neuroblasts produces neurons.Activation of the Wnt/β-catenin pathway has been shown to facilitate neurogenesis and augment neuronal regeneration within the ischemic penumbra following cerebral ischemia, leading to an improvement in nerve function impairment.SVZ, subventricular zone; NSCs, neural stem cells; TACs, transit-amplifying cells; RMS, rostral migratory stream; OB, olfactory bulb; LV, lateral ventricle; Str, Striatum.
In vitro, the introduction of exogenous Wnt3a protein enhanced the proliferation and differentiation of NSCs isolated from the forebrain of E14.5 mice [34].The inhibition of GSK-3β or the overexpression of β-catenin in ventral midbrain (VM) precursors leads to an increase in neuronal differentiation and the quantity of dopaminergic (DA) neurons [35].Furthermore, Wnt1 and Wnt3a promoted the proliferation of VM precursors obtained from E14.5 rats, and Wnt5a induced the differentiation of DA precursors (from either the cortex or VM) into DA neurons [36,37].These findings indicate that Wnts serve as pivotal regulators of neurogenesis in the VM.
As described above, the Wnt/β-catenin signaling pathway is known to play a crucial role in brain development, particularly in neurogenesis.However, the precise mechanism and downstream target genes involved in the promotion of neurogenesis by Wnt/β-catenin remain uncer-tain.Furthermore, the complexity of the mechanism is further compounded by considerations of spatial heterogeneity.A study revealed that β-catenin plays a crucial role in the proliferation and neuronal differentiation of neural progenitor cells.Chromatin immunoprecipitation (ChIP) and luciferase reporter assays revealed that β-catenin binds directly to the promoter or proximal enhancer regions of proneural genes, including neurogenin 1, neurogenin 2, mash1, and myoD, resulting in their activation [38].A separate ChIP investigation revealed that the β-catenin/T-cell factor 1 (TCF1) complex plays a direct role in modulating the expression of Sox1, a marker of neural precursor cells, in the context of neuronal differentiation in embryonic stem cells [39].In addition, some articles suggest that the Wnt/β-catenin signaling pathway facilitates the proliferation of NSCs while inhibiting their differentiation, thereby playing a role in the preservation of stem cell characteris- tics [40,41].This disparity could be attributed to variations in the culture conditions of neural progenitor cells, specifically the presence or absence of Fibroblast growth factor 2 (FGF2) [38].
Taken together, these findings highlight the involvement of Wnt/β-catenin signaling in NSCs during development and imply that this pathway may also facilitate adult neurogenesis and contribute to nerve regeneration following cerebral ischemia.

Inhibition of the Wnt/β-catenin Pathway Blocks Adult Brain Neurogenesis
Wnt/β-catenin signaling is activated in adult NSCs [42,43].Numerous published studies have shown that the ablation of Wnt/β-catenin signaling impedes the prolifera-tion of adult NSCs and their subsequent differentiation into neurons.The overexpression of mutant Wnt1 protein in the DG led to reduced proliferation of adult hippocampal NSCs and impaired generation of new neurons by suppressing Wnt signaling, consequently impacting spatial memory and object recognition memory [44,45].Knockout of Wnt7a led to a substantial decrease in the abundance of NSCs and an increase in the rate of cell cycle termination in neural progenitors located in the DG and SVZ of adult mice.Furthermore, Wnt7a plays a pivotal role in the process of neuronal differentiation and maturation.The absence of Wnt7a expression led to a noteworthy decrease in the number of newly formed neurons in the DG region of the hippocampus [46,47].Another study showed that the suppression of Wnt signaling through the knockdown of the Frizzled-1 receptor in the hippocampus resulted in reduced neuronal differentiation of NSCs and altered migration patterns of newly generated neurons [48].However, Austin et al. [42] reported that conditional deletion of β-catenin in adult Glast-positive NSCs did not have any impact on their maintenance, activation, or differentiation.Interestingly, a separate investigation indicated that conditional knockout of β-catenin in Sox2-positive NSCs in the DG inhibited newborn neuron generation and the survival of neuronal progenitors [49].

Activation of the Wnt/β-catenin Pathway Promotes Adult Brain Neurogenesis
In contrast to knockout of β-catenin, conditional stabilization of β-catenin in mice resulted in the displacement of NSCs from the SGZ.Additionally, in an in vitro model of hippocampal NSCs, the activation of the Wnt/β-catenin signaling pathway facilitated the differentiation of active NSCs into neurons while also inducing the proliferation or differentiation of quiescent NSCs in a dose-dependent manner [42].Another study indicated that the activation of the Wnt/β-catenin signaling pathway within the SVZ through the suppression of GSK-3β resulted in an elevated quantity of nascent neurons within the olfactory bulb.This phenomenon can be attributed to the increased proliferation of Mash1 + progenitor cells within the SVZ [50].
The observed phenotypes resulting from the activation of Wnt/β-catenin signaling in adult NSCs are consistent with other reports in the literature.Activation of the Wnt/βcatenin signaling pathway leads to increased expression of NeuroD1 and long interspersed nuclear element 1 (LINE-1), both of which are pivotal in the process of neuronal differentiation [49].Overexpression of Wnt7a and stabilized β-catenin promote NSC self-renewal in vitro.The introduction of β-catenin through lentiviral transduction resulted in an increase in the population of type B NSCs within the SVZ of adult brains [46].The overexpression of Wnt3 in NSCs in vitro or in the DG in vivo promoted neuroblast proliferation and neuronal differentiation [44].In another study, the overexpression of Wnt3a or Wnt5a was found to enhance the proliferation and neuronal differentiation of neural progenitor cells derived from postnatal and adult mouse brains [51].In addition, the augmentation of neurogenesis in the hippocampus was also observed upon the removal of Wnt inhibitors [52,53].Moreover, the Wnt/βcatenin signaling pathway plays a significant role in the maturation of newborn neurons, as well as in the growth of dendrites and the formation of dendritic spines in adult hippocampal neurons [53][54][55].

The Role of the Wnt/β-catenin Pathway in Neurogenesis after Cerebral Ischemia
The occurrence of cerebral ischemia leads to the stimulation of NSCs in the SVZ, resulting in their increased proliferation and subsequent asymmetric division into migratory neuroblasts.These neuroblasts subsequently migrate toward ischemic regions, where they undergo differentiation into neurons, potentially facilitating functional recovery.Additionally, NSCs in the SGZ can migrate to the granular cell layer and differentiate into new neurons, potentially reversing the learning and memory deficits caused by ischemia.However, it should be noted that this reparative mechanism is insufficient to fully compensate for the extensive damage caused by severe cerebral ischemia [8,13].Studies have indicated that the Wnt/β-catenin pathway within adult NSC niches facilitates neurogenesis following cerebral ischemia and contributes to the restoration of neurological function (Table 2, Ref. [56][57][58][59]).

Activation of the Wnt/β-catenin Pathway Promotes Neurogenesis after Cerebral Ischemia and is Beneficial for Ameliorating Nerve Function Injury following Cerebral Ischemia
The increase in Wnt3a levels within the SVZ or striatum of mice afflicted with focal ischemic injury was found to play a significant role in facilitating functional recovery subsequent to the ischemic event.This was achieved through the promotion of neurogenesis or the enhancement of neuronal viability [58].A separate study demonstrated that the intranasal administration of Wnt3a subsequent to focal ischemic stroke in mice reduced infarct volume, augmented sensorimotor functions, stimulated neurogenesis in the SVZ and SGZ, increased the number of DCX + /BrdU + colocalized cells migrating from the SVZ toward the periinfarct area, and increased the quantity of newly formed neurons (BrdU + /NeuN + cells) in the peri-infarct zone.Conversely, intranasal administration of a Wnt inhibitor hindered neurogenesis and decreased the quantity of newly generated neurons in the peri-infarct area [59].
In addition, studies have revealed that some treatments and mechanisms can further upregulate the Wnt/β-catenin pathway after cerebral ischemia, thereby promoting neurogenesis and contributing to the reinstatement of neurological function [60][61][62][63][64]. Taken together, these findings indicate that the activation of the Wnt/β-catenin pathway subsequent to cerebral ischemia has the potential to stimulate neurogenesis and ameliorate nerve function impairment, making this pathway a viable therapeutic target for the management of cerebral ischemia.

Limitations in the Available Studies on the Role of the Wnt/β-catenin Pathway in Postischemic Neurogenesis
As stated above, considerable advancements have been made in the field of research pertaining to genetic or pharmacological interventions in the Wnt/β-catenin pathway aimed at increasing neurogenesis following cerebral ischemia.However, most studies lack a quality study design.Many published studies utilized immunofluorescence colocalization of BrdU with neuronal markers or neuroblastic markers to evaluate neurogenesis and the de novo generation of neurons.However, it is unclear whether these proliferative cells were derived from NSCs.Previous studies have reported that astrocytes can transdifferentiate into morphologically mature and functional neurons after cerebral ischemia [65].Therefore, it is recommended that researchers employ lineage tracing techniques in future studies to track the fate of NSCs to elucidate their potential contributions to brain neurogenesis and postischemic stroke recovery.Lineage tracing serves as a valuable tool for investigating the origin of new neurons in is-chemic regions, specifically whether they originate from NSCs within the adult NSC niche.Additionally, it enables the examination of the impact of the Wnt/β-catenin signaling pathway on the cellular fate of different NSC populations.Currently, numerous techniques are available for lineage tracing (for review, see [66]), with one of the most prevalent approaches involving the integration of the Cre mouse line in combination with the Rosa26-CAG-reporter mouse line.For adult NSC lineage tracing, researchers can cross inducible NSC-specific Cyclization recombination enzyme (Cre) lines, such as Nestin-CreERT2 mice [67] and Ascll-CreERT2 mice [68], with Rosa26-CAGreporter lines, such as Rosa26-CAG-tdTomato mice [69], to generate Nestin CreERT 2/+ ; Rosa26 tdT omato/+ mice or Ascll CreERT 2/+ ; Rosa26 tdT omato/+ mice.With tamoxifen administration, the NSCs in lineage-traced mice exhibit stable expression of tdTomato, enabling the tracking of the fate of cells originating from the NSC niche (Fig. 3).
There are additional limitations in studies on the role of the Wnt/β-catenin pathway in postischemic neurogenesis.All current studies on the Wnt/β-catenin pathway in postischemic neurogenesis have been conducted using animal models.Given the ongoing debate surrounding human adult neurogenesis, the transition from preclinical research to clinical trials for the treatment of ischemic stroke through the activation of the Wnt/β-catenin pathway presents significant obstacles.In addition, the potential of agonists of Wnt/β-catenin signaling to enhance neurogenesis following cerebral ischemia remains uncertain.The preclinical exploration of Wnt/β-catenin signaling agonists holds significance for their eventual clinical utility.

Conclusion
Ischemic stroke is characterized by increased morbidity, disability, recurrence, and mortality.Intravenous thrombolysis and thrombectomy are approved medical treatments for acute stroke.However, it is crucial to acknowledge the limited timeframe within which these therapies can be administered, as well as the potential for intracerebral hemorrhage and other bleeding complications that may arise from the utilization of intravenous thrombolysis and mechanical thrombectomy.Thus, there is a need for the development of new treatments.Researchers have focused on endogenous NSC-induced neurogenesis following ischemic stroke due to the demonstrated positive impact of NSCs on neural repair.According to existing studies, the modulation of numerous signaling pathways has been found to facilitate NSC proliferation and differentiation while also augmenting angiogenesis and synaptic plasticity.These mechanisms collectively contribute to the process of neural repair subsequent to ischemic brain injury.Based on the existing evidence, the Wnt/β-catenin pathway is a promising therapeutic target for alleviating the consequences of ischemic stroke.The activation of this pathway facilitates neurogenesis, leading to the generation of newly formed neurons that can replace deceased neural cells within the ischemic core.Future studies should further investigate whether the newly generated neurons are functional in vivo and whether they can integrate with existing neural circuits.Furthermore, additional in-depth lineage tracing experiments should be conducted.

Fig. 1 .
Fig. 1.Activation of the Wnt/β-catenin pathway offers a promising therapeutic strategy for treating ischemic stroke by promoting neurogenesis.Under typical physiological circumstances, SVZ NSCs are responsible for producing TACs, which possess the capacity to generate neuroblasts that subsequently migrate through the RMS toward the OB, where they undergo differentiation into interneurons.