YAP and TAZ regulate Schwann cell proliferation and differentiation during peripheral nerve regeneration.

YAP and TAZ are effectors of the Hippo pathway that controls multicellular development by integrating chemical and mechanical signals. Peripheral nervous system development depends on the Hippo pathway. We previously showed that loss of YAP and TAZ impairs the development of peripheral nerve as well as Schwann cell myelination. The role of the Hippo pathway in peripheral nerve regeneration has just started to be explored. After injury, Schwann cells adopt new identities to promote regeneration by converting to a repair-promoting phenotype. While the reprogramming of Schwann cells to repair cells has been well characterized, the maintenance of such repair phenotype cannot be sustained for a very long period, which limits nerve repair in human. First, we show that short or long-term myelin maintenance is not affected by defect in YAP and TAZ expression. Using crush nerve injury and conditional mutagenesis in mice, we also show that YAP and TAZ are regulators of repair Schwann cell proliferation and differentiation. We found that YAP and TAZ are required in repair Schwann cells for their redifferentiation into myelinating Schwann cell following crush injury. In this present study, we describe how the Hippo pathway and YAP and TAZ regulate remyelination over time during peripheral nerve regeneration.

αV integrins in Schwann cells promote attachment to axons, but are dispensable in vivo.

In the developing peripheral nervous system, Schwann cells (SCs) extend their processes to contact, sort, and myelinate axons. The mechanisms that contribute to the interaction between SCs and axons are just beginning to be elucidated. Using a SC-neuron coculture system, we demonstrate that Arg-Gly-Asp (RGD) peptides that inhibit αV -containing integrins delay the extension of SCs elongating on axons. αV integrins in SC localize to sites of contact with axons and are expressed early in development during radial sorting and myelination. Short interfering RNA-mediated knockdown of the αV integrin subunit also delays SC extension along axons in vitro, suggesting that αV -containing integrins participate in axo-glial interactions. However, mice lacking the αV subunit in SCs, alone or in combination with the potentially compensating α5 subunit, or the αV partners ß3 or ß8 , myelinate normally during development and remyelinate normally after nerve crush, indicating that overlapping or compensatory mechanisms may hide the in vivo role of RGD-binding integrins.

Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination.

Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2ß1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin.

Kif13b Regulates PNS and CNS Myelination through the Dlg1 Scaffold.

Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS.

A dual role for Integrin α6ß4 in modulating hereditary neuropathy with liability to pressure palsies.

Peripheral myelin protein 22 (PMP22) is a component of compact myelin in the peripheral nervous system. The amount of PMP22 in myelin is tightly regulated, and PMP22 over or under-expression cause Charcot-Marie-Tooth 1A (CMT1A) and Hereditary Neuropathy with Pressure Palsies (HNPP). Despite the importance of PMP22, its function remains largely unknown. It was reported that PMP22 interacts with the ß4 subunit of the laminin receptor α6ß4 integrin, suggesting that α6ß4 integrin and laminins may contribute to the pathogenesis of CMT1A or HNPP. Here we asked if the lack of α6ß4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP22 and ß4 integrin may not interact directly in myelinating Schwann cells, however, ablating ß4 integrin delays the formation of tomacula, a characteristic feature of HNPP. In contrast, ablation of integrin ß4 worsens nerve conduction velocities and non-compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers.

DDIT4/REDD1/RTP801 is a novel negative regulator of Schwann cell myelination.

Signals that promote myelination must be tightly modulated to adjust myelin thickness to the axonal diameter. In the peripheral nervous system, axonal neuregulin 1 type III promotes myelination by activating erbB2/B3 receptors and the PI3K/AKT/mTOR pathway in Schwann cells. Conversely, PTEN (phosphatase and tensin homolog on chromosome 10) dephosphorylates PtdIns(3,4,5)P3 and negatively regulates the AKT pathway and myelination. Recently, the DLG1/SAP97 scaffolding protein was described to interact with PTEN to enhance PIP3 dephosphorylation. Here we now report that nerves from mice with conditional inactivation of Dlg1 in Schwann cells display only a transient increase in myelin thickness during development, suggesting that DLG1 is a transient negative regulator of myelination. Instead, we identified DDIT4/RTP801/REDD1 as a sustained negative modulator of myelination. We show that DDIT4 is expressed in Schwann cells and its maximum expression level precedes the peak of AKT activation and of DLG1 activity in peripheral nerves. Moreover, loss of DDIT4 expression both in vitro and in vivo in Ddit4-null mice provokes sustained hypermyelination and enhanced mTORC1 activation, thus suggesting that this molecule is a novel negative regulator of PNS myelination.

α6ß1 and α7ß1 integrins are required in Schwann cells to sort axons.

During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the ß1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with ß1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding ß1 integrins in Schwann cells and show that only α6ß1 and α7ß1 integrins are required and that α7ß1 compensates for the absence of α6ß1 during development. The absence of either α7ß1 or α6ß1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all ß1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6ß1 and α7ß1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell ß1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.

Knockout of PA200 improves proteasomal degradation and myelination in a proteotoxic neuropathy.

The cellular response to a decrease in protein degradation by 26S proteasomes in chronic diseases is poorly understood. Pharmacological inhibition of proteasomes increases the expression of proteasome subunits and Proteasome Activator 200 (PA200), an alternative proteasome activator. In the S63del mouse model of the peripheral neuropathy Charcot Marie Tooth 1B (CMT1B), proteasomal protein degradation is decreased and proteasome gene expression is increased. Here, we show an increase in PA200 and PA200-bound proteasomes in the peripheral nerves of S63del mice. To test genetically whether the upregulation of PA200 was compensatory, we generated S63del//PA200-/- mice. Unexpectedly, in the sciatic nerves of these mice, there was greater proteasomal protein degradation than in S63del, less polyubiquitinated proteins and markers of the unfolded protein response, and a greater amount of assembled, active 26S proteasomes. These changes were not seen in PA200-/- controls and were therefore specific to the neuropathy. Furthermore, in S63del//PA200-/- mice, myelin thickness and nerve conduction were restored to WT levels. Thus, the upregulation of PA200 is maladaptive in S63del mice and its genetic ablation prevented neuropathy.

P0S63del impedes the arrival of wild-type P0 glycoprotein to myelin in CMT1B mice.

More than 120 mutations in the Myelin Protein Zero gene (MPZ, P0) cause various forms of hereditary neuropathy. Two human mutations encoding either P0S63C or P0S63del have been shown to cause demyelination in mice through different gain of function pathomechanisms. P0S63del, for example, is retained in the endoplasmic reticulum (ER) and elicits a pathogenetic unfolded protein response (UPR). As P0 likely forms oligomers, another gain of abnormal function could include a dominant-negative interaction between P0S63del and normal P0 (P0wt). To test this idea, we generated a transgenic mouse that expressed a form of P0wt with a myc epitope tag at the C terminus (P0ct-myc). We show that P0ct-myc is trafficked and functions like P0wt, thus providing a new tool to study P0 in vivo. In mice that express both P0ct-myc and P0S63del, P0S63del specifically delays the transit of P0ct-myc through the ER and reduces the level of P0wt in the myelin sheath by half-a level previously shown to cause demyelination in mice and humans. Surprisingly, P0ct-myc does not co-immunoprecipitate with P0S63del, suggesting an indirect interaction. Thus, P0S63del causes not only a UPR-related toxic mechanism, but also a dominant-negative effect on P0wt that probably contributes to demyelinating neuropathy.

A mouse model of Schwartz-Jampel syndrome reveals myelinating Schwann cell dysfunction with persistent axonal depolarization in vitro and distal peripheral nerve hyperexcitability when perlecan is lacking.

Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.

Cholinergic neurons in the basal forebrain are involved in behavioral abnormalities associated with Cul3 deficiency: Role of prefrontal cortex projections in cognitive deficits.

Loss-of-function mutations of the gene Cul3 have been identified as a risk factor for autism-spectrum disorder (ASD), but the pathogenic mechanisms are not well understood. Conditional Cul3 ablation in cholinergic neurons of mice (ChatCRECul3F/+) recapitulated ASD-like social and sensory gating phenotypes and caused significant cognitive impairments, with diminished activity of cholinergic neurons in the basal forebrain (BF). Chemogenetic inhibition of BF cholinergic neurons in healthy mice induced similar social and cognitive deficits. Conversely, chemogenetic stimulation of BF cholinergic neurons in ChatCRECul3F/+ mice reversed abnormalities in sensory gating and cognition. Cortical hypofunction was also found after ChAT-specific Cul3 ablation and stimulation of cholinergic projections from the BF to the prefrontal cortex (PFC) mitigated cognitive deficits. Overall, we demonstrate that cholinergic dysfunction due to Cul3 deficiency is involved in ASD-like behavioral abnormalities, and that BF cholinergic neurons are particularly critical for cognitive component through their projections to the PFC.

Aryl hydrocarbon receptor is a tumor promoter in MYCN-amplified neuroblastoma cells through suppression of differentiation.

Neuroblastoma is the most common extracranial solid tumor in children. MYCN amplification is detected in almost half of high-risk cases and is associated with poorly differentiated tumors, poor patient prognosis and poor response to therapy, including retinoids. We identify the aryl hydrocarbon receptor (AhR) as a transcription factor promoting the growth and suppressing the differentiation of MYCN-amplified neuroblastoma. A neuroblastoma specific AhR transcriptional signature reveals an inverse correlation of AhR activity with patients' outcome, suggesting AhR activity is critical for disease progression. AhR modulates chromatin structures, reducing accessibility to regions responsive to retinoic acid. Genetic and pharmacological inhibition of AhR results in induction of differentiation. Importantly, AhR antagonism with clofazimine synergizes with retinoic acid in inducing differentiation both in vitro and in vivo. Thus, we propose AhR as a target for MYCN-amplified neuroblastoma and that its antagonism, combined with current standard-of-care, may result in a more durable response in patients.

Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4.

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

Regulation of local GTP availability controls RAC1 activity and cell invasion.

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells.

Foot pad skin biopsy in mouse models of hereditary neuropathy.

Numerous transgenic and knockout mouse models of human hereditary neuropathies have become available over the past decade. We describe a simple, reproducible, and safe biopsy of mouse skin for histopathological evaluation of the peripheral nervous system (PNS) in models of hereditary neuropathies. We compared the diagnostic outcome between sciatic nerve and dermal nerves found in skin biopsy (SB) from the hind foot. A total of five animal models of different Charcot-Marie-Tooth neuropathies, and one model of congenital muscular dystrophy associated neuropathy were examined. In wild type mice, dermal nerve fibers were readily identified by immunohistochemistry, light, and electron microscopy and they appeared similar to myelinated fibers in sciatic nerve. In mutant mice, SB manifested myelin abnormalities similar to those observed in sciatic nerves, including hypomyelination, onion bulbs, myelin outfolding, redundant loops, and tomacula. In many strains, however, SB showed additional abnormalities--fiber loss, dense neurofilament packing with lower phosphorylation status, and axonal degeneration-undetected in sciatic nerve, possibly because SB samples distal nerves. SB, a reliable technique to investigate peripheral neuropathies in human beings, is also useful to investigate animal models of hereditary neuropathies. Our data indicate that SB may reveal distal axonal pathology in mouse models and permits sequential follow-up of the neuropathy in an individual mouse, thereby reducing the number of mice necessary to document pathology of the PNS.

Disruption of Mtmr2 produces CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis.

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2-null mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

Human diseases reveal novel roles for neural laminins.

Extracellular matrix molecules such as laminins have a central role in regulating cell behaviour. However, our understanding of their functions in the mammalian nervous system is incomplete. It is important to establish these functions, both for an understanding of normal development and to devise strategies to enhance repair. Here, we review how insights gained from human diseases caused by genetic mutations in laminins or their receptors have revealed significant and sometimes unexpected roles for laminins in neural stem cells, migrating neurons and myelinating glia, in both the PNS and CNS.

Loss of Mtmr2 phosphatase in Schwann cells but not in motor neurons causes Charcot-Marie-Tooth type 4B1 neuropathy with myelin outfoldings.

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth type 4B1 (CMT4B1). This disorder is characterized by childhood onset of weakness and sensory loss, severely decreased nerve conduction velocity, demyelination in the nerve with myelin outfoldings, and severe functional impairment of affected patients, mainly resulting from loss of myelinated fibers in the nerve. We recently generated Mtmr2-null(neo) mice, which show a dysmyelinating neuropathy with myelin outfoldings, thus reproducing human CMT4B1. Mtmr2 is detected in both Schwann cells and neurons, in which it interacts with discs large 1/synapse-associated protein 97 and neurofilament light chain, respectively. Here, we specifically ablated Mtmr2 in either Schwann cells or motor neurons. Disruption of Mtmr2 in Schwann cells produced a dysmyelinating phenotype very similar to that of the Mtmr2-null(neo) mouse. Disruption of Mtmr2 in motor neurons does not provoke myelin outfoldings nor axonal defects. We propose that loss of Mtmr2 in Schwann cells, but not in motor neurons, is both sufficient and necessary to cause CMT4B1 neuropathy. Thus, therapeutical approaches might be designed in the future to specifically deliver the Mtmr2 phospholipid phosphatase to Schwann cells in affected nerves.

Role of integrins in peripheral nerves and hereditary neuropathies.

Interactions between Schwann cells and extracellular matrix on one surface, and axons on the other, are required for correct myelination in the developing peripheral nervous system. Integrins are transmembrane proteins that mediate the former in association with other surface receptors. This review focuses on the role that integrins play in the development of the peripheral nervous system, and in inherited human peripheral neuropathies. Here we describe recent findings on integrin signaling to different intracellular pathways, focusing on cell adhesion, migration, and polarization. Then we use information derived from recent experiments of targeted mutagenesis in mice to show that, consistent with temporally regulated expression, different integrins serve multiple roles in developing nerve.