Hippo Pathway in Schwann Cells and Regeneration of Peripheral Nervous System

Hippo pathway is an evolutionarily conserved signaling pathway comprising a series of MST/LATS kinase complexes. Its key transcriptional coactivators YAP and TAZ regulate transcription factors such as TEAD family to direct gene expression. The regulation of Hippo pathway, especially the nuclear level change of YAP and TAZ, significantly influences the cell fate switching from proliferation to differentiation, regeneration, and postinjury repair. This review outlines the main findings of Hippo pathway in peripheral nerve development, regeneration, and tumorigenesis, especially the studies in Schwann cells. We also summarize other roles of Hippo pathway in damage repair of the peripheral nerve system and discuss the potential future research which probably contributes to novel therapeutic strategies.

The prior research has illustrated the dominant role of Hippo pathway in the initial development [14][15][16].Though Hippo pathway is a hot spot in mammalian nervous system, no reviews have summarized its function in PNS pathophysiology.Since the development, oncogenesis, and postinjury repair of PNS are mainly contributed by Schwann cells (SCs), we concentrate on the role of Hippo pathway in the development, tumorigenesis, and the repair role of SCs in peripheral nerve injury (PNI).Other roles of Hippo pathway in PNS damage and repair are also summarized in this article.This review provides a deeper insight into the mechanism of Hippo pathway in mammalian PNS, especially in SCs.For this, potential therapeutic strategies of peripheral neuropathy can be anticipated.

Hippo Pathway in PNS Development
Hippo pathway shows the magic of switching between promoting proliferation and facilitating myelination in neural crest cell (NCC) and SC development [15][16][17][18].The brief introduction of the development of SCs and the role of Hippo pathway during the development will be elaborated on below.

Hippo Pathway in the Development of Neural Crest Cells
NCCs arise from the embryonic ectoderm layer after neural tube closure [19].Taking the formation of PNS from the trunk neural crest as an example, NCCs that produce PNS migrate ventromedially between the neural tube and the developing somite.Some NCCs settle down at sites of peripheral ganglia, quit migration, proliferate abundantly, and then enter the state of neurogenesis.Meanwhile, the boundary cap, a transient NCC-derived stem cell niche, begins to populate at the axon entrances of the spinal cord.Following the onset of neurogenesis, the NCCs and boundary cap give rise to satellite glial progenitors located in the ganglia and Schwann cell progenitors (SCPs) that migrate along the axons of the developing nerves.SCPs are pluripotent progenitors that can eventually differentiate into a range of PNS components, including SCs, enteric and parasympathetic neurons, and intraneuronal fibroblasts (Fig. 1) [20][21][22][23].
Since SCPs resemble NCCs with biased multipotency [24], the understanding and research of SCPs are basically obtained through the study of NCCs.The Hippo-YAP/ TAZ pathway promotes cell proliferation and cell migration and impedes cell specification in the development of NCCs [25].YAP and TAZ are expressed in migratory NCCs, dorsal root ganglia progenitors, glial precursors, and satellite glia, but are absent in neuronal precursors and postmitotic neurons [14].Hippo pathway also shows positive bidirectional cross talk between Wnt, Notch, and retinoic acid signaling, which are all key signaling pathways in the regulation of NCC development [15,16,26].Overexpression of YAP and knockdown of Neurofibromin 2 gene (Nf2) which encodes Merlin both lead to immoderate expansion of dorsal root ganglia progenitors, indicating the supporting roles of YAP/TAZ in NCC migration [14].
Hippo Pathway in Schwann Cell Development SCs are myelin-forming cells of peripheral nerves [27].A subset of SCPs differentiate into immature SCs, which subsequently proliferate and give rise to myelinating SCs and nonmyelinating SCs [28].The immature SCs in contact with large bunds of axons set out to differentiate into myelinating SCs.The developing SCs wrap axons with the formation of the basal layer, complete 1:1 radial wrapping of axons (namely radial sorting) under the coordinated progression of the basal layer and cell cycle, and then enter terminal development stage, transfer into mature myelinating SCs, and wrap axons with layers of compressed myelin [29].Nonmyelinating SCs, instead, contact with C-fiber axons with smaller diameters and bundle them close together [20,30].
Hippo pathway serves as a fulcrum balancing immature SC proliferation and lineage progression.In immature SCs, YAP/TAZ are found at high nuclear levels and promote vigorous proliferation [17,31,32].In actively proliferating human Schwann (hSC2λ) cell model, YAP could promote proliferation through inducing the expression of miR-30a, which is a microRNA that could downregulate the growthsuppressive protein PTPN13 [18].In YapTaz dKO immature SCs, genes related to cell growth such as Amotl2, Fgf1, Ect2, and Ddah1 were significantly downregulated; a dramatic drop was also observed in the expression of cell cycle-associated genes Ccnj, Cdk6, Pim3, and Mycn [17].YAP/TAZ mutually interact with Gαs protein, an essential factor that promotes cell cycle exit, to help SCs quit proliferation and set out lineage progression [17].
In promyelinating SCs, YAP and TAZ are significant for radial sorting.YAP/TAZ could bind with TEAD1 to stimulate the expression of the crucial myelin gene Pmp22, the promyelinating regulator Egr2/Krox20, and the major structural component of the myelin sheath Mpz [32][33][34].Double knockout of YAP/TAZ caused a catastrophic defect in SC differentiation and severe tremor and paresis in mice by reducing the expression of myelin-associated factors (e.g., Egr2, Zeb2, Mbp) and crucial regulators of radial sorting (e.g., Dag1, Itgb1, Itga6, Itga8) [32].Despite some degree of functional redundancy, YAP and TAZ may have distinct roles in radial sorting.Silencing YAP resulted in shorter internodal length and reduced myelin thickness, which could not be compensated by TAZ [34].Conditional knockout (cKO) of Taz led to reduced expression of dystroglycan and integrins α6β1 and α7β1, which were all vital for axon sorting, while the Yap cKO sciatic nerves had no significant developmental difference compared to normal nerves [32].Besides, except binding with TEAD1, TAZ was found in mice SCs to be cooccupied with Sox10 at the promoters/enhancers of a series of myelin genes and differentiation regulators, including Pmp22, Mpz, and Mbp, while inhibiting the transcription factors which suppressed SC differentiation like HES1, HES5, and c-Jun (Fig. 2) [17].

Color version available online
Fig. 1.Simple scheme of YAP/TAZ's role in the migration and differentiation of NCCs.NCCs derive from the neural tube.The left part shows NCC migration and the right part illustrates NCC differentiation after the migration.In NCC migration, a high level of nuclear YAP/TAZ stimulates NCCs to proliferate, migrate dorsolaterally and ventromedially, and form ganglia like dorsal root ganglia (DRG).Overexpression of YAP/TAZ will lead to immoderate expansion of NCCs.In NCC differentiation, nuclear YAP/TAZ harbor low level.Some NCCs begin neurogenesis and give rise to sensory neurons and motor neurons.NCCs located at the axon entrances at the spinal cord change into boundary cap cells, which give rise to SCPs.NCCs at the DRGforming site differentiate into satellite glial progenitors.
In mature SCs, Hippo-YAP/TAZ pathway continues to play a role in myelin elongation and maintenance.During individual development, axonal stretching mediates myelin elongation via Hippo pathway.Crb3, a protein located at the tips of the myelin sheath, can target Willin to turn on Hippo pathway and optimize myelin length.The Crb3-Willin-Hippo axis tempers YAP nuclear shift and subsequently inhibits transcriptions of myelin-associated genes, thereby achieving subtle regulation of myelin length [34].YAP/TAZ-TEAD1 was proved to maintain the mature myelin by promoting Krox20 transcription [31].In the short term of Yap/Taz double knockout, it has still been inconclusive that whether YAP/TAZ are necessary for myelin maintenance [17,31].However in mice with a normal lifespan, Jeanette et al. showed that the absence of YAP/TAZ had no effect on the morphology and functions of mature nerves [35].More compelling evidence is needed to further examine the roles of YAP/TAZ in myelin maintenance.
Furthermore, the YAP level in SCs of all the developmental stages can be fine-tuned by Merlin [36].Removal of Merlin in SCs leads to formation anomaly of myelin sheath, including slightly enhanced number of Fig. 2. Overview of the Hippo-YAP/TAZ pathway and the regulation of downstream target genes by YAP/TAZ in SC development.In immature SCs, YAP/TAZ-TEADs promote proliferation by upregulating the cell growth-related genes (Amotl2, Fgf2, Fct2, and Ddah1) and cell cycle-associated genes (Ccnj, Cdk6, Pim3, and Mycn).Under SC proliferation conditions, YAP/TAZ suppress Gnas expression.When SCs start to differentiate, the expression level of Gnas is increased and Gαs negatively regulates YAP/TAZ activity in a feedback loop to facilitate cell cycle exit.In proliferating SC state, YAP/TAZ-TEAD1 not only promote the promyelinating genes and factors like Pmp22, Egr2/Krox20, Mbp, Mag, Rab11, and Laminin γ1 but also upregulate the transcription factor Sox10. TAZ-Sox10 was found to upregulate the gene expression of Pmp22, Mbp, and Mpz and downregulate antimyelinating factors HES1, HES5, and c-Jun.With the completion of SC myelination, the nuclear YAP/TAZ also decrease, and SCs change into myelinating SCs and nonmyelinating SCs according to the diameter of the wrapped axons.

Hippo Pathway in the Peripheral Nervous System
SCs, shortened internodal distance in the adult nerves, and increased numbers of Schmidt-Lanterman incisures in myelinated SCs [37].It is possibly due to the fact that Merlin loss induced the overexpression of YAP, which results in a remarkable downregulation of c-Jun and neurotrophin [38].This indicates that the moderate expression level of the antimyelination regulator c-Jun and its subtle regulation by Hippo-YAP/TAZ pathway are of the essence in appropriate development and myelination of SCs (Table 1).
Mechanical stimuli also play a crucial part in activating YAP in various developmental stages of SCs.It has been well proven that extracellular matrix stiffness and cell geometry could regulate YAP/TAZ activity in a Hippoindependent way [39].In neuronal specification, stiffer substrate could promote the nuclear shift of YAP via βcatenin and therefore suppress neurogenesis [40,41].The combination of physical forces and laminin 211 was revealed to activate YAP in SC radial sorting and myelination [32].On the other hand, using verteporfin to suppress YAP in a hard matrix could stimulate Rac1/ MKK7/JNK signal to restore SC regeneration capacity and attenuate SC differentiation [42].These studies are likely to shed light on novel clinical therapies based on mechanotransduction to better control SC development in diseases like hereditary neuropathy with liability to pressure palsy.

Hippo Pathway in Peripheral Nerve Injury and Repair
According to the classic PNI classification system introduced by Seddon, the axonotmesis is the most classic and common form of PNI due to compressions, tractions, and ischemia [43].Axonotmesis refers to the damage to axons, endoneurium, and even perineurium [44].Wallerian degeneration occurs within 48-72 h after axonotmesis.SCs show excellent plasticity during the recovery.SCs undergo the process of demyelination, phenotype switching, and remyelination [45,46].First, calcium influx activates proteases within 24 h of PNS impairment in injured nerves, which leads to axon degradation in 2 days.Along with this, SCs go on the process of demyelination and phenotype switching [47].SCs suppress the expression of myelin genes including PMP22, MAG, and MPZ and upregulate factors like c-Jun and Sox2, so as to initiate dedifferentiation and convert into repair SCs [48].Repair SCs release cytokines such as IL-1 and TNF-α to recruit macrophages and fibroblasts, promoting debris consumption and tissue repair [49].Then, repair SCs proliferate and migrate to form the regeneration tracks along which the axons regrow, stimulate axonal regrowth, and eventually remyelinate the repaired axons [50].
Hippo Pathway in SC-Led Repair of PNI Accumulating studies have identified a critical role for Hippo pathway in injury and repair of PNS, especially in Wallerian degeneration.YAP and TAZ participate in the whole repair program.In demyelination stage, YAP and TAZ experience a significant loss in SC nuclei, which is in accordance with the repression of myelin genes [51].Then, in SC phenotype switching, YAP and TAZ nuclear levels mediated the SC-type transition efficiency and subsequently determined the remyelination effectiveness [35].As for repair SCs, YAP and TAZ are not involved in repair SC proliferation [51].While the underlying reasons are currently unclear, it is possible to postulate that the robust proliferation driven by YAP/TAZ may exceed the necessary degree of repair SC proliferation required for tissue repair, thereby negating the need for YAP/TAZ activation.Turning into remyelinating state, just as during development, the level of myelination-involved factors (such as Zeb2, Nrg1, Egr2, and Sox10) and myelin proteins in SCs are upregulated [49].Ablation of YAP and TAZ would impair SC remyelination by reducing the expression level of c-Jun and Egr2 [35,51].Merlin also controls the regenerative capacity of SCs [52].Mindos et al. [38] proposed that Merlin-null mice failed in axonal regeneration and remyelination following PNI.Truong et al. [53] tested the effect of Nf2 deletion before and after damage.The absence of Nf2 resulted in decreased axon density, thinner myelin sheaths, and larger endoneurial space.Interestingly, however, the axon conduction velocity and amplitude were almost impervious to Nf2 deletion 90 days after injury.This indicated that Merlin loss may be remedied by the activation of other upstream regulators of Hippo pathway.

Hippo Pathway in PNI-Induced Nerve Scarring
During post-injury demyelination, SC-recruited fibroblasts take an active part in recovery.A fibrotic cascade will be initiated to conserve mechanical stability.However, excessive intraneural fibrosis can produce nerve scarring and hinder axonal regeneration [54].TGF-β signaling plays an important role in scar formation and axon growth inhibition [55,56].The role of YAP/ TAZ in promoting fibrosis in an SECM stiffnessdependent way has been demonstrated in fibroblasts [57,58].The possible link between TGF-β, Hippo-YAP/TAZ signaling pathways, and fibrosis has been implicated in lots of research (reviewed in [59]).Nuclear YAP/TAZ were found to positively impact the expression of TGF-β and Smad in wound healing [60].But, the regulation of Hippo pathway in nerve posttrauma fibrosis is yet to be elucidated.Antifibrotic therapies targeting YAP and TAZ may be beneficial in promoting nerve repair.

Hippo Pathway in PNI-Induced Neuropathic Pain
In response to PNI, other than recovery, maladaptive changes like aberrant stimulus thresholds, enhanced sensitivity, and ectopic transduction may also occur, which can elicit neuropathic pain [61].Hippo pathway has been demonstrated to collaborate with Wnt/β-catenin signaling, a well-known signaling pathway involved in the pathogenesis of neuropathic pain, and serve as a switch for neuropathic pain status [62][63][64].Chronic constriction injury on rodent sciatic nerves dramatically downregulated the phosphorylation levels of MST1/2 and LATS1 in the spinal dorsal horn.The nuclear accumulation of YAP and TAZ showed different changing patterns and distinct functions (Fig. 3).Enhanced accumulation of nuclear YAP/TAZ causes mechanical hypersensitivity similar to that induced by chronic constriction injury in juvenile mice or rats.Adversely, blocking YAP/TAZ-TEAD interaction via verteporfin can "turn off" neuropathic pain [64,65].Xu et al. [64] found a new potent analgesic dCTB, which could significantly relieve neuropathic pain by suppressing the combinations of YAP/TAZ-TEAD and β-catenin-TCF/LEF and blocking both Hippo and Wnt signaling pathways at the same time.Cui et al. [66] discovered the abirritation of an endogenous peptide spinal beta-amyloid 1-42.Spinal beta-amyloid 1-42 could orchestrate Wnt signaling with cytokines and impede the nuclear accumulation of YAP/TAZ to exert an analgesic effect.The above results show the potential of managing the cross talk between Hippo pathway and Wnt signaling in dealing with neuropathic pain.

Hippo Pathway in PNI-Induced Neuronal Death
Damage repair is also accompanied by the death of nerve cells.The PNI resulted from ischemia, morphological abnormalities, and inflammation can lead to metabolic disorders and excessive accumulation of reactive oxygen species, which can induce oxidative stress and neuronal death [67,68].Compelling evidence implicates the crucial role of Hippo pathway in reactive oxygen species-mediated death in nerve cells.MST1/2 are regarded as critical components in this process.Lehtinen et al. [69] revealed that oxidative stress led to MST1 activation and subsequent phosphorylation of Forkhead box O (FOXO) proteins, which motivated the nuclear shift of FOXO and thereby directed neuronal death.Xiao et al. [70] discovered that the upstream protein kinase 385 c-Abl phosphorylated MST1 at Tyr433 and promoted the interaction of MST1 with FOXO3.The regulatory role of the c-Abl-Hippo/MST2 signaling pathway in nerve cell death has also been uncovered.Similarly, c-Abl phosphorylated the conserved site Y81 of MST2, which weakened the combinations between MST2 and Raf-1 protein and enhanced the activity of MST2, leading to the death of nerve cells [71].

Hippo Pathway in Schwann Cell-Derived Tumorigenesis
The dysregulation of Hippo pathway was revealed to exert strong influence on the growth of several kinds of SC-generated tumors.Neurofibromatosis (NF) is a neuro-associated tumor predisposition syndrome, including NF1, NF2, and schwannomatosis [72].The pathogenic mutations of neurofibromin 1 (NF1) and neurofibromin 2 (NF2) lead to NF1 and NF2, respectively, causing the predisposition to develop neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) for NF1 and vestibular schwannomas for NF2 [73,74].The certain cause of schwannomatosis remains unclear [72], but the relationship between schwannomas and defected Hippo pathway has been gradually explored.
Originated from SCs with NF1 mutation, neurofibromas in NF1 patients are benign peripheral nerve sheath tumors comprising a mixture of SCs, perineurial cells, fibroblasts, and mast cells [75].Chen et al. [76] detected 30 sporadic DNA mutations in Hippo pathway in neurofibromas of NF1.YAP and TAZ had enhanced nuclear levels and the downstream regulating genes AXL and CTGF were upregulated [77].Knockdown of LATS1/2 exacerbated tumor growth in NF1, accelerating the death of NF1-muted mice [76].
NF1 gene deficiency is also likely to develop MPNSTs [78].MPNSTs are rare and highly aggressive soft tissue sarcomas consisting of SC-like malignant spindle cells [79].Changes in vital components of Hippo pathway play a part in the formation of MPNSTs.Through wholeexome sequencing, Kim et al. [80] found a nonsense mutation in LATS1 in MPNSTs.Augmented YAP/TAZ expression was detected in human MPNSTs, and the hyperactivation of YAP/TAZ in LATS1/2-ablated SC lineage resulted in high tumorigenicity.Furthermore, TEAD1 could recruit TAZ and occupy the open chromatin loci of cancer-associated genes in NF1 mutant cells, thus initiating tumorigenesis.Antagonizing YAP/TAZ Hippo Pathway in the Peripheral Nervous System together with PDGF pathways could hamper the growth of MPNSTs, which indicated the potential curative targets [81].
The loss-of-function mutation of NF2 accounts for the development of most of the schwannomas in patients [82].Schwannomas are slow-growing benign neoplasms resulting from the aberrant proliferation of neoplastic SCs.Key components of Hippo pathway were found to be mutated in over 70% of schwannomas [83].YAP/TAZ gene expression level is related to the prognosis of schwannomas [84].YAP/TAZ deletion was found to increase oxidative stress-induced cell death and delay schwannoma growth [85].Furthermore, MAPK inhibition sensitized the treatment effect of YAP/TAZ inhibitor JQ1 in schwannomas [84].These studies offer promising pharmaceutical targets for schwannomas.

Prospective
Hippo pathway especially YAP/TAZ is essential in PNS development (Table 2).However, many vital regulators that have been demonstrated to interact with the Hippo-YAP/TAZ pathway in the central nervous system have not been explored in PNS.For instance, bone morphogenetic proteins (BMPs) and their effectors Smad family are expressed in the whole nervous system and BMP-Smad2/4 signaling could promote In the nuclei of injured neurons, YAP rapidly increased as early as in 1 h after injury, peaked at about 3 days (d), fell at 14 d, and returned to normal at 21 d.However, TAZ gradually increased and remained at high levels throughout the examination.Functions of YAP and TAZ also manifest differently: a high nuclear level of YAP contributes to triggering and maintaining neuropathic pain, whereas TAZ is only involved in the later persistence of neuropathic pain.
proliferation exit of NCCs by antagonizing YAP-TEAD combinations [86,87].BMP signaling has also been reported to positively contribute to neurite extension and axonal regrowth in PNS, among which the possible cross talk with Hippo pathway is worth being investigated [88,89].VGLL4, a newly discussed member of the Hippo pathway, mediated the cell activities by negatively regulating the YAP-TEAD complex and connected with some other transcriptional factors such as RUNX2, MyoD, and IRF2BP2 [90][91][92][93].The expression level of VGLL4 is related to some kinds of psychiatric disorders, such as anorexia nervosa, schizophrenia, and neuroticism, which provides a hint for further study of the mechanism of VGLL4 in PNS [94][95][96].Recently, YAP-  MAMLD1 fusion proteins have been reported to promote oncogenic programs and ependymoma tumorigenesis by recruiting tissue factors and coactivators into nuclear condensates [97].We anticipate that more detailed research on the central nervous system (CNS) would provide valuable insights for understanding the roles of the Hippo pathway and its interactions with other signals in PNS (Table 3).
Though the specific mechanism of Hippo pathway in regulating NCC differentiation waits to be further examined, Hippo pathway's regulatory effect in directing other cell lines to differentiate into neuronal-like cells might indicate the direction.SH-SY5Y and NTera2 serve as two commonly used cell models for neuronal development.FAT1 cadherin, an upstream positive regulator of Hippo pathway, contributes to normal neuritogenesis and regulates Hippo pathway in neuronal differentiation of SH-SY5Y and NTera2 cell models [130].The FAT1-Hippo-YAP/TAZ-CTGF/ANKRD1 axis may provide an insightful clue for studying Hippo pathway in NCC specification.
The delicate regulation of Hippo pathway is required for the proper myelination in SC development.Charcot-Marie-Tooth disease type 1A (CMT1A) is an inherited peripheral neuropathy with improperly formed myelin, which is caused by a duplication of PMP22 [131].EGR2 mutation was also observed in CMT [132,133].Given that the Hippo-YAP/TAZ pathway controls the expression of PMP22 and EGR2 in SC myelination, Hippo pathway may manifest medical potential in CMT1A.We look forward to a further investigation into Hippo's role in pathogenesis and its promising application in CMT1A therapy.
Just as in SC development, changes in mechanical force and tissue structures during PNI will affect YAP and subsequent SC repair movement.Nonetheless, the current research only focuses on the loss and gain of functions of YAP/TAZ in SC regeneration and axon recovery.How YAP/TAZ and their position shift are molecularly linked to axonal injury and recovery is insufficiently illustrated.But the physical therapeutic methods still possess a great clinical prospect in accelerating nerve regeneration.In vitro studies of SCs have confirmed the effectiveness of low-intensity pulsed ultrasound in stimulating SC proliferation and viability [134,135].
What's more, the positive effect of low-frequency ultrasound on the Hippo-YAP/TAZ pathway has been discovered [136,137].The possible mechanism of ultrasound to promote SC repair capacity by affecting Hippo pathway is worth exploring.
The repair of PNI, especially the SC movement, partially mimics the process of the development of PNS.It is generally believed that nerve injury repair is equivalent to the processes of dedifferentiation and redifferentiation of SCs.But the differences in the degree of activation and function of Hippo pathway in the two stages of early differentiation and postinjury regeneration in SCs are remained to be elaborated.To sum up, Hippo pathway is of great importance in PNS physiopathology, especially in SCs, making good use of the regulatory role of Hippo pathway may be conducive to treatment and recovery of peripheral neuropathy.

Fig. 3 .
Fig. 3. Summary of Hippo's function in PNI and recovery.Peripheral nerve injury (a); SC demyelination (b); debris consumption (c); proliferation, migration of repair SCs, and axon regrowth (d); and SC remyelination (e).YAP/TAZ show no expression in nuclei of demyelinated SCs.In SC phenotype switching, YAP/TAZ expression determines repair program efficiency and is indispensable for repair SC remyelination.In the nuclei of injured neurons, YAP rapidly increased as early as in 1 h after injury, peaked at about 3 days (d), fell at 14 d, and returned to normal at 21 d.However, TAZ gradually increased and remained at high levels throughout the examination.Functions of YAP and TAZ also manifest differently: a high nuclear level of YAP contributes to triggering and maintaining neuropathic pain, whereas TAZ is only involved in the later persistence of neuropathic pain.

Table 1 .
Mouse PNS phenotypes resulting from gene knockouts or mutations of Hippo pathway

Table 2 .
Essential roles of key members of Hippo pathway in PNS