Wild-type Schwann Cells Research Articles

Sarm1-Dependent Metabolic Reprogramming of Schwann Cells Following Nerve Injury.

Schwann cells (SCs) transition into a repair phenotype after peripheral nerve injury, which is crucial for supporting axon regeneration. However, the early SC injury response preceding the repair state remains poorly understood. Here, we demonstrate that Sarm1, a key regulator of axon degeneration, is expressed in SCs and has a critical role in the early SC injury response. Leveraging the fact that Sarm1 deletion impairs the SC transition to the repair state, we used single-nucleus RNA sequencing to compare the transcriptional responses of wild-type and Sarm1 knockout SCs 24 hours after nerve injury. Remarkably, Sarm1-deficient SCs, unlike wild-type SCs, showed increased expression of genes involved in oxidative phosphorylation and the TCA cycle. These findings were functionally validated, revealing that Sarm1 knockout SCs displayed increased mitochondrial respiration in response to injury. Intriguingly, Sarm1 knockout SCs also exhibited enhanced axon protection compared to wild-type SCs in an in vitro model of axon degeneration. We propose that Sarm1 gates the transition of SCs from a protective, oxidative phosphorylation-dependent state (which we term Protection Associated Schwann Cells or PASCs) to a glycolytic, pro-regenerative repair phenotype after injury. Our findings challenge the prevailing view of Sarm1 as an exclusively axon-autonomous regulator of degeneration and reveal a paradigm shift in understanding the role of Sarm1 in the SC injury response, with broad implications for the treatment of peripheral neuropathies and neurodegenerative diseases.

Abstract LB326: Combined inhibition of BRD4 and FAK1 as a novel therapeutic strategy for neurofibromatosis type 2 related schwannomas

Abstract Neurofibromatosis type 2 (NF2) is a rare disorder that is inherited in an autosomal-dominant manner and is attributed to the loss of heterozygosity (LOH) of the NF2 gene, which encodes for the tumor suppressor protein Merlin. Patients affected by the disease develop vestibular schwannomas (VS), meningiomas, and ependymomas resulting in high morbidity and premature mortality. To date, neurofibromatosis type 2 has no FDA-approved drug-based treatment. Merlin plays a central role in mediating cell contact inhibition (CI) of proliferation. Loss of Merlin leads to abnormal activation of multiple signaling pathways, including those regulated by small G-proteins Ras/Rac/cdc42 and the Hippo-YAP pathway. The Hippo-YAP pathway plays a major role in cell growth and organ size control and at its core is comprised of a kinase cascade that regulates the transcriptional regulator YAP. The function of YAP is crucial for VS development, and there are several mechanisms by which YAP regulates transcription. Some of these functions have been shown to involve the Bromodomain and Extra-Terminal domain (BET) proteins acting as co-factors. We have previously shown that the BET inhibitor JQ1 can selectively reduce the growth of the NF2-null schwannoma and Schwann cells in vitro and tumorigenesis in vivo. Additionally, evidence has shown that Merlin loss can lead to increased cell proliferation that it requires the activity of Focal Adhesion Kinase 1 (FAK1). We have previously demonstrated that FAK1 inhibition via Crizotinib has antiproliferative effects in NF2-null Schwann cells, and is currently in phase II clinical trials in NF2-related VS. In this study, wild-type and NF2-null Schwann cells and schwannoma cell lines were used to determine the impact on cell growth by employing the treatment with the BET inhibitor JQ1 and the FAK1 inhibitor Crizotinib. Our analysis in NF2-null Schwann cells and schwannoma cell lines shows that the combined inhibition exerts an antineoplastic effect at nanomolar concentrations. Moreover, this combination resulted in selective inhibition of NF2-null Schwann cell proliferation when compared to wild-type Schwann cells in vitro, when compared to either drug alone. Our preliminary data suggest that combined targeting of BET and FAK1 may offer a potential therapeutic option for the treatment of NF2-related schwannomas. Citation Format: Maria Alejandra Gonzalez, Joseph Kissil, Scott Troutman. Combined inhibition of BRD4 and FAK1 as a novel therapeutic strategy for neurofibromatosis type 2 related schwannomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB326.

Nitration of Hsp90 Affects its Spatial Distribution and Promotes Schwannoma Cell Proliferation

Neurofibromatosis Type 2 is a genetic disorder characterized by the development of schwannomas throughout the nervous system caused by loss of merlin tumor suppressor activity. Solid tumors develop in an oxidative environment due to production of oxidants such as peroxynitrite by tumor and infiltrating immune cells. We discovered peroxynitrite production and subsequent protein tyrosine (Y) nitration support schwannoma cell survival/proliferation. Further, Y nitration induces a metabolic reprogramming characterized by decreased oxidative phosphorylation activity, and increased glycolysis and glutaminolysis. Here, we identified the first nitrated protein supporting schwannoma cell proliferation, the molecular chaperone heat shock protein 90 (Hsp90). Out of five Y residues prone to nitration in Hsp90, we showed nitration at Y33 down‐regulates mitochondrial metabolism, while nitration at Y56 activates the ATP‐gated receptor P2X7, which increases glycolysis in tumor cells. We found schwannomas contain endogenous levels of Hsp90 nitrated at either position. Therefore, we hypothesized that nitrated Hsp90 (Hsp90NY) promotes schwannoma cell proliferation by regulating their metabolism. To investigate the role of Hsp90NY in proliferation, we increased the intracellular concentration of Hsp90NY in schwannoma cells by delivering Hsp90NY intracellularly at concentrations like those endogenously present in these cells (~6.5% of cellular Hsp90, calculated by quantitative dot blot). Doubling the intracellular concentration of Hsp90NYbut not wild‐type Hsp90 significantly increased cell proliferation 24 and 48 h post‐delivery. To establish the metabolic role of Hsp90NY, we delivered wild‐type or Hsp90NY into wild‐type Schwann cells (WT‐SC) and studied the metabolic changes by extracellular flux analysis 24 h post‐delivery. Hsp90NY decreased all parameters of mitochondrial activity, with no effect on the glycolytic rate. To test if Y33 and/or Y56 were responsible for the tumorigenic activity of Hsp90NY, we intracellularly delivered to WT‐SC either wild‐type Hsp90, or different site‐specific nitrated Hsp90 proteins produced by genetic code expansion: Hsp90 nitrated at Y33 (Hsp90NY33), at Y56 (Hsp90NY56), or at Y33 and Y56 simultaneously (Hsp90NY33+56). All three forms of Hsp90NY significantly increased WT‐SC proliferation at 24 and 48 h post‐delivery, suggesting Y33 and Y56 induce schwannoma cell proliferation independently. Using a three‐dimensional schwannoma cell culture model (tumoroids), we discovered Hsp90NY33colocalized with mitochondria and remained in the tumoroid periphery, while Hsp90NY56was also found in nuclei with a homogenous distribution throughout the tumoroid. Collectively, our results show that Hsp90, when nitrated at Y33 or Y56, gains a tumorigenic activity that supports schwannoma cell proliferation. In addition, residue‐specific nitration results in distinct localization, and potentially function, of the protein. This is the first nitrated protein shown to support tumor cell proliferation, and a potential tumor‐directed target for therapeutic development.

Curcumin and Ethanol Effects in Trembler-J Schwann Cell Culture.

Charcot-Marie-Tooth (CMT) syndrome is the most common progressive human motor and sensory peripheral neuropathy. CMT type 1E is a demyelinating neuropathy affecting Schwann cells due to peripheral-myelin-protein-22 (PMP22) mutations, modelized by Trembler-J mice. Curcumin, a natural polyphenol compound obtained from turmeric (Curcuma longa), exhibits dose- and time-varying antitumor, antioxidant and neuroprotective properties, however, the neurotherapeutic actions of curcumin remain elusive. Here, we propose curcumin as a possible natural treatment capable of enhancing cellular detoxification mechanisms, resulting in an improvement of the neurodegenerative Trembler-J phenotype. Using a refined method for obtaining enriched Schwann cell cultures, we evaluated the neurotherapeutic action of low dose curcumin treatment on the PMP22 expression, and on the chaperones and autophagy/mammalian target of rapamycin (mTOR) pathways in Trembler-J and wild-type genotypes. In wild-type Schwann cells, the action of curcumin resulted in strong stimulation of the chaperone and macroautophagy pathway, whereas the modulation of ribophagy showed a mild effect. However, despite the promising neuroprotective effects for the treatment of neurological diseases, we demonstrate that the action of curcumin in Trembler-J Schwann cells could be impaired due to the irreversible impact of ethanol used as a common curcumin vehicle necessary for administration. These results contribute to expanding our still limited understanding of PMP22 biology in neurobiology and expose the intrinsic lability of the neurodegenerative Trembler-J genotype. Furthermore, they unravel interesting physiological mechanisms of cellular resilience relevant to the pharmacological treatment of the neurodegenerative Tremble J phenotype with curcumin and ethanol. We conclude that the analysis of the effects of the vehicle itself is an essential and inescapable step to comprehensibly assess the effects and full potential of curcumin treatment for therapeutic purposes.

Modulation of Small RNA Signatures in Schwann-Cell-Derived Extracellular Vesicles by the p75 Neurotrophin Receptor and Sortilin

Schwann cells (SCs) are the main glial cells of the peripheral nervous system (PNS) and are known to be involved in various pathophysiological processes, such as diabetic neuropathy and nerve regeneration, through neurotrophin signaling. Such glial trophic support to axons, as well as neuronal survival/death signaling, has previously been linked to the p75 neurotrophin receptor (p75NTR) and its co-receptor Sortilin. Recently, SC-derived extracellular vesicles (EVs) were shown to be important for axon growth and nerve regeneration, but cargo of these glial cell-derived EVs has not yet been well-characterized. In this study, we aimed to characterize signatures of small RNAs in EVs derived from wild-type (WT) SCs and define differentially expressed small RNAs in EVs derived from SCs with genetic deletions of p75NTR (Ngfr−/−) or Sortilin (Sort1−/−). Using RNA sequencing, we identified a total of 366 miRNAs in EVs derived from WT SCs of which the most highly expressed are linked to the regulation of axonogenesis, axon guidance and axon extension, suggesting an involvement of SC EVs in axonal homeostasis. Signaling of SC EVs to non-neuronal cells was also suggested by the presence of several miRNAs important for regulation of the endothelial cell apoptotic process. Ablated p75NTR or sortilin expression in SCs translated into a set of differentially regulated tRNAs and miRNAs, with impact in autophagy and several cellular signaling pathways such as the phosphatidylinositol signaling system. With this work, we identified the global expression profile of small RNAs present in SC-derived EVs and provided evidence for a regulatory function of these vesicles on the homeostasis of other cell types of the PNS. Differentially identified miRNAs can pave the way to a better understanding of p75NTR and sortilin roles regarding PNS homeostasis and disease.

Development of 3D culture models of plexiform neurofibroma and initial application for phenotypic characterization and drug screening

Plexiform neurofibromas (PNs), which may be present at birth in up to half of children with type 1 neurofibromatosis (NF1), can cause serious loss of function, such as quadriparesis, and can undergo malignant transformation. Surgery is the first line treatment although the invasive nature of these tumors often prevents complete resection. Recent clinical trials have shown promising success for some drugs, notably selumetinib, an inhibitor of MAP kinase kinase (MEK). We have developed three-dimensional (3D) cell culture models of immortalized cells from NF1 PNs and of control Schwann cells (SCs) that we believe mimic more closely the in vivo condition than conventional two-dimensional (2D) cell culture. Our goal is to facilitate pre-clinical identification of potential targeted therapeutics for these tumors. Three drugs, selumetinib (a MEK inhibitor), picropodophyllin (an IGF-1R inhibitor) and LDN-193189 (a BMP2 inhibitor) were tested with dose-response design in both 2D and 3D cultures for their abilities to block net cell growth. Cell lines grown in 3D conditions showed varying degrees of resistance to the inhibitory actions of all three drugs. For example, control SCs became resistant to growth inhibition by selumetinib in 3D culture. LDN-193189 was the most effective drug in 3D cultures, with only slightly reduced potency compared to the 2D cultures. Characterization of these models also demonstrated increased proteolysis of collagen IV in the matrix by the PN driver cells as compared to wild-type SCs. The proteolytic capacity of the PN cells in the model may be a clinically significant property that can be used for testing the ability of drugs to inhibit their invasive phenotype.

Schwann Cell Expressed Nogo-B Modulates Axonal Branching of Adult Sensory Neurons Through the Nogo-B Receptor NgBR.

In contrast to the central nervous system (CNS) nerve fibers do regenerate in the peripheral nervous system (PNS) although in a clinically unsatisfying manner. A major problem is excessive sprouting of regenerating axons which results in aberrant reinnervation of target tissue and impaired functional recovery. In the CNS, the reticulon protein Nogo-A has been identified as a prominent oligodendrocyte expressed inhibitor of long-distance growth of regenerating axons. We show here that the related isoform Nogo-B is abundantly expressed in Schwann cells in the PNS. Other than Nogo-A in oligodendrocytes, Nogo-B does not localize to the myelin sheath but is detected in the ER and the plasma membrane of Schwann cells. Adult sensory neurons that are cultured on nogo-a/b deficient Schwann cells form significantly fewer axonal branches vs. those on wildtype Schwann cells, while their maximal axonal extension is unaffected. We demonstrate that this effect of Nogo-B on neuronal morphology is restricted to undifferentiated Schwann cells and is mediated by direct physical contact between these two cell types. Moreover, we show that blocking the Nogo-B specific receptor NgBR, which we find expressed on sensory neurons and to interact with Schwann cell expressed Nogo-B, produces the same branching phenotype as observed after deletion of Nogo-B. These data provide evidence for a novel function of the nogo gene that is implemented by the Nogo-B isoform. The remarkably specific effects of Nogo-B/NgBR on axonal branching, while leaving axonal extension unaffected, are of potential clinical relevance in the context of excessive axonal sprouting after peripheral nerve injury.Main PointsNogo-B is prominently expressed in Schwann cells and localizes to the ER and plasma membrane. It distributes to the external cytoplasmic compartment of Schwann cells in vivo, but is absent from the myelin sheath.Genetic deletion of Nogo-B in Schwann cells reduces axonal branching, but not long-distance growth, of co-cultured adult sensory neurons.Schwann cell expressed Nogo-B interacts with neuronal NgBR. Blockade of NgBR mimics the loss-of-nogo branching phenotype.

Schwann cells reposition a peripheral nerve to isolate it from postembryonic remodeling of its targets

Although much is known about the initial construction of the peripheral nervous system (PNS), less well understood are the processes that maintain the position and connections of nerves during postembryonic growth. Here, we show that the posterior lateral line nerve in zebrafish initially grows in the epidermis and then rapidly transitions across the epidermal basement membrane into the subepidermal space. Our experiments indicate that Schwann cells, which myelinate axons in the PNS, are required to reposition the nerve. In mutants lacking Schwann cells, the nerve is mislocalized and the axons remain in the epidermis. Transplanting wild-type Schwann cells into these mutants rescues the position of the nerve. Analysis of chimeric embryos suggests that the process of nerve relocalization involves two discrete steps - the degradation and recreation of the epidermal basement membrane. Although the outgrowth of axons is normal in mutants lacking Schwann cells, the nerve becomes severely disorganized at later stages. In wild-type embryos, exclusion of the nerve from the epidermis isolates axons from migration of their targets (sensory neuromasts) within the epidermis. Without Schwann cells, axons remain within the epidermis and are dragged along with the migrating neuromasts. Our analysis of the posterior lateral line system defines a new process in which Schwann cells relocate a nerve beneath the epidermal basement membrane to insulate axons from the postembryonic remodeling of their targets.

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

Ablation of adhesion molecule L1 in mice favours Schwann cell proliferation and functional recovery after peripheral nerve injury

The adhesion molecule L1 is one of the few adhesion molecules known to be beneficial for repair processes in the adult central nervous system of vertebrates by promoting axonal growth and neuronal survival. In the peripheral nervous system, L1 is up-regulated by myelination-competent Schwann cells and regenerating axons after nerve damage but its functional role has remained unknown. Here we tested the hypothesis that L1 is, as in the central nervous system, beneficial for nerve regeneration in the peripheral nervous system by performing combined functional and histological analyses of adult L1-deficient mice (L1y/-) and wild-type (L1y/+) littermates. Contrary to our hypothesis, quantitative video-based motion analysis revealed better locomotor recovery in L1y/- than in L1y/+ mice at 4-12 weeks after transection and surgical repair of the femoral nerve. Motoneuron regeneration in L1y/- mice was also enhanced as indicated by attenuated post-traumatic loss of motoneurons, enhanced precision of motor reinnervation, larger cell bodies of regenerated motoneurons and diminished loss of inhibitory synaptic input to motoneurons. In search of mechanisms underlying the observed effects, we analysed peripheral nerves at short time-periods (3-14 days) after transection and found that Schwann cell proliferation is strongly augmented in L1y/- versus L1y/+ mice. L1-deficient Schwann cells showed increased proliferation than wild-type Schwann cells, both in vivo and in vitro. These findings suggest a novel role for L1 in nerve regeneration. We propose that L1 negatively regulates Schwann cell proliferation after nerve damage, which in turn restricts functional recovery by limiting the trophic support for regenerating motoneurons.

LPA and NRG‐1b synergistically promote proliferation and migration of wild‐type and neoplastic Schwann cells via an ERRFI1‐dependent mechanism

In some cell types, lysophosphatidic acid (LPA)‐induced proliferation and migration requires transactivation of the EGF receptor (erbB1). This led us to hypothesize that LPA stimulates the proliferation and migration of normal and neoplastic Schwann cells and does so via a mechanism involving the EGFR‐related NRG‐1 receptors erbB2, 3 and/or 4. We have found that LPA stimulates the proliferation and migration of wild‐type Schwann cells and malignant peripheral nerve sheath tumor (MPNST) cells in a concentration‐dependent manner. Co‐stimulation of Schwann cells with NRG‐1β and LPA increases mitogenesis to a greater extent than the combined effects of each factor alone, indicating that LPA and NRG‐1β synergistically enhance proliferation of these glia. Further, LPA‐induced Schwann cell migration is ablated by treatment with the erbB kinase inhibitor PD168393 or GM6001, a MMP inhibitor which blocks the release of soluble NRG‐1. Microarray analyses also show that LPA represses Schwann cell expression of the pan‐erbB inhibitor Erffi1, thereby potentially enhancing erbB action. These findings suggest that LPA stimulates Schwann cell proliferation and migration synergistically with NRG‐1β and does so via a previously unknown mechanism that involves modulation of Erffi1 expression. Funded by R01 NS048353 and CA122804.

Scar Formation

Injured tissue produces fibrin, which makes a provisional extracellular matrix (ECM). Degradation of this fibrin ECM by plasmin is required for minimizing scar formation. Plasmin is produced by proteolytic cleavage of plasminogen by plasminogen activators (tPA and uPA), which are inhibited by plasminogen activator inhibitor-1 (PAI-1). Sachs et al . find that mice deficient in p75 neurotrophin receptor (p75 NTR ) exhibit reduced fibrin deposition after sciatic nerve crush injury or lipopolysaccharide-induced pulmonary fibrosis. The decrease in fibrin associated with sciatic nerve injury was due to increased expression and activity of tPA, whereas the decrease in fibrin associated with pulmonary fibrosis was due to decreased PAI-1 abundance. In wild-type mice after sciatic nerve injury, Schwann cells in which p75 NTR was abundant lacked tPA, which suggests that cells expressing p75 NTR did not express tPA. The increased proteolytic activity toward fibrin (measured by in situ zymography) associated with the nerve injury in p75 NTR −/− mice was prevented by application of a specific inhibitor of tPA or by crossing the mice with tPA −/− mice to produce mice deficient in both tPA and p75 NTR . When cultured on a fibrin gel, Schwann cells from the p75 NTR −/− mice degrade the fibrin, whereas wild-type Schwann cells form a monolayer. Studies with a transfected fibroblast cell line overexpressing p75 NTR showed increased abundance of transcripts for PAI-1 and decreased transcripts and activity of tPA. Transcriptional regulation of tPA required a cAMP (adenosine 3′,5′-monophosphate) pathway. Inhibition of tPA activity in the p75 NTR -overexpressing cells was prevented if cAMP concentrations were increased pharmacologically and cAMP concentrations were decreased in the p75 NTR -overexpressing cells. The injured sciatic nerves also showed increased cAMP in the p75 NTR −/− mice compared with injured nerves from wild-type mice. Coimmunoprecipitation studies and in vitro interaction studies indicated that p75 NTR and phosphodiesterase 4A5 (PDE4A5) interacted. The authors propose that recruitment of PDE4A5 to the membrane inhibited cAMP signaling, a hypothesis supported by decreased activation of a plasma membrane protein kinase A reporter in the p75 NTR -overexpressing cells. Tissue remodeling and scar formation appear to be additional ways that p75 NTR signaling is involved in response to injury. B. D. Sachs, G. S. Baillie, J. R. McCall, M. A. Passino, C. Schachtrup, D. A. Wallace, A. J. Dunlop, K. F. MacKenzie, E. Klussmann, M. J. Lynch, S. L. Sikorski, T. Nuriel, I. Tsigelny, J. Zhang, M. D. Houslay, M. V. Chao, K. Akassoglou, p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway. J. Cell Biol. 177 , 1119-1132 (2007). [Abstract] [Full Text]

Suppression of galactosylceramidase (GALC) expression in the twitcher mouse model of globoid cell leukodystrophy (GLD) is caused by nonsense-mediated mRNA decay (NMD)

The twitcher mouse is a pathologically and enzymatically authentic model of globoid cell leukodystrophy (GLD, Krabbe disease) that has been widely used for the evaluation of potential therapeutic approaches. This naturally occurring mouse model contains a premature stop codon (W339X) in the galactosylceramidase (GALC) gene that abolishes enzymatic activity. Using either immunocytochemical approaches or Western blot methodology, we have been unable to detect the truncated form of GALC expected to be produced in these animals. Nonsense-mediated mRNA decay (NMD) is a cellular protection mechanism that degrades newly synthesized transcripts containing a premature termination codon (PTC). Since the naturally occurring mutation in the twitcher mouse introduces a PTC, we hypothesized that NMD might affect the degradation of GALC mRNA in these animals. Consistent with this hypothesis, we determined that the amount of GALC transcript was inversely proportional to the number of twitcher containing alleles. Similar reductions in GALC mRNA were detected in a twitcher-derived Schwann cell line (TwS1) when compared to wild-type Schwann cells (IMS32). Anisomycin, emetine and puromycin, inhibitors of NMD, effectively increased the level of GALC transcript in the TwS1 cells providing further support for nonsense-mediated mRNA decay being the mechanism by which no GALC protein is detected in these animals. Understanding the mechanistic differences between the lack of enzymatic activity in the twitcher model and that observed with the missense mutations that cause human disease yields not only novel therapeutic insights but also highlights the need for additional animal models.

Analysis of congenital hypomyelinating Egr2 Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination

Egr2 is a transcription factor required for peripheral nerve myelination in rodents, and mutations in Egr2 are associated with congenital hypomyelinating neuropathy (CHN) in humans. To further study its role in myelination, we generated mice harboring a hypomorphic Egr2 allele (Egr2Lo) that survive for up to 3 weeks postnatally, a period of active myelination in rodents. These Egr2Lo/Lo mice provided the opportunity to study the molecular effects of Egr2 deficiency on Schwann cell biology, an analysis that was not possible previously, because of the perinatal lethality of Egr2-null mice. Egr2Lo/Lo mice phenocopy CHN, as evidenced by the severe hypomyelination and increased numbers of proliferating Schwann cells of the peripheral nerves. Comparison of sciatic nerve gene expression profiles during development and after crush injury with those of Egr2Lo/Lo Schwann cells revealed that they are developmentally arrested, with down-regulation of myelination-related genes and up-regulation of genes associated with immature and promyelinating Schwann cells. One of the abnormally elevated genes in Egr2Lo/Lo Schwann cells, Sox2, encodes a transcription factor that is crucial for maintenance of neural stem cell pluripotency. Wild-type Schwann cells infected with Sox2 adenovirus or lentivirus inhibited expression of myelination-associated genes (e.g., myelin protein zero; Mpz), and failed to myelinate axons in vitro, but had an enhanced proliferative response to beta-neuregulin. The characterization of a mouse model of CHN has provided insight into Schwann cell differentiation and allowed the identification of Sox2 as a negative regulator of myelination.

Induction of Postnatal Schwann Cell Death by the Low-Affinity Neurotrophin ReceptorIn Vitroand after Axotomy

Schwann cells express the low-affinity neurotrophin receptor (p75), but no role for either the neurotrophins or their cognate receptors in Schwann cell development has been established. We have found that Schwann cells isolated from postnatal day 1 (P1) or P2 mice that were p75-deficient exhibited potentiated survival compared to wild-type cells after growth factor and serum withdrawal. There was, however, no disparity in the survival of p75-deficient and wild-type Schwann cells isolated at embryonic day 15, suggesting that the death-inducing effects of p75 are developmentally regulated. A comparable degree of cell death was also observed in the sciatic nerves of both wild-type and p75-deficient mice at P1. However, 24 hr after axotomy, there was a 13-fold increase in the percentage of apoptotic nuclei in the distal nerve stumps of the transected sciatic nerves of neonatal wild-type but not p75-deficient mice. The expression of both the p75 and nerve growth factor (NGF) genes was upregulated after axotomy in neonatal wild-type nerves. Collectively, these results suggest that NGF-mediated activation of p75 is likely to be an important mediator of Schwann cell apoptosis in the context of peripheral nerve injury.

Nerve Growth Factor Signaling through p75 Induces Apoptosis in Schwann Cells via a Bcl-2-Independent Pathway

Apoptosis is involved in the regulation of Schwann cell numbers during normal development and after axonal damage, but the molecular regulation of Schwann cell death remains unknown. We have used stably transfected rat Schwann cell lines to study the potential roles of nerve growth factor (NGF), the antiapoptotic protein Bcl-2 and the cytokine response modifier A (CrmA) in modulating Schwann cell death in vitro. Bcl-2 inhibited Schwann cell apoptosis induced by survival factor withdrawal, whereas CrmA did not. In contrast, Bcl-2-transfected Schwann cells were susceptible to apoptosis in response to exogenous NGF, whereas CrmA-expressing cell lines were resistant. Demonstration of high levels of the low-affinity neurotrophin receptor p75 but not the high-affinity TrkA receptor on the Bcl-2-transfected cell lines suggested that the NGF-induced killing was mediated by p75. This was confirmed by resistance of Schwann cells isolated from p75 knockout mice to the NGF-induced cell death. Nerve growth factor also promoted the death of wild-type mouse and rat Schwann cells in the absence of survival factor withdrawal. Endogenous Bcl-2 mRNA was expressed by wild-type Schwann cells in all conditions that promoted survival but was downregulated to undetectable levels after survival factor withdrawal. In conclusion, our results demonstrate the existence of two separate pathways that expedite apoptosis in Schwann cells: a Bcl-2-blockable pathway initiated on loss of trophic support, and a Bcl-2-independent, CrmA-blockable pathway mediated via the p75 receptor.