Activation of mTORC1 and c-Jun by Prohibitin1 loss in Schwann cells may link mitochondrial dysfunction to demyelination.

Schwann cell (SC) mitochondria are quickly emerging as an important regulator of myelin maintenance in the peripheral nervous system (PNS). However, the mechanisms underlying demyelination in the context of mitochondrial dysfunction in the PNS are incompletely understood. We recently showed that conditional ablation of the mitochondrial protein Prohibitin 1 (PHB1) in SCs causes a severe and fast progressing demyelinating peripheral neuropathy in mice, but the mechanism that causes failure of myelin maintenance remained unknown. Here, we report that mTORC1 and c-Jun are continuously activated in the absence of Phb1, likely as part of the SC response to mitochondrial damage. Moreover, we demonstrate that these pathways are involved in the demyelination process, and that inhibition of mTORC1 using rapamycin partially rescues the demyelinating pathology. Therefore, we propose that mTORC1 and c-Jun may play a critical role as executioners of demyelination in the context of perturbations to SC mitochondria.

TXNIP, a novel key factor to cause Schwann cell dysfunction in diabetic peripheral neuropathy, under the regulation of PI3K/Akt pathway inhibition-induced DNMT1 and DNMT3a overexpression.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM) and the dysfunction of Schwann cells plays an important role in the pathogenesis of DPN. Thioredoxin-interacting protein (TXNIP) is known as an inhibitor of thioredoxin and associated with oxidative stress and inflammation. However, whether TXNIP is involved in dysfunction of Schwann cells of DPN and the exact mechanism is still not known. In this study, we first reported that TXNIP expression was significantly increased in the sciatic nerves of diabetic mice, accompanied by abnormal electrophysiological indexes and myelin sheath structure. Similarly, in vitro cultured Schwann cells TXNIP was evidently enhanced by high glucose stimulation. Again, the function experiment found that knockdown of TXNIP in high glucose-treated RSC96 cells led to a 4.12 times increase of LC3-II/LC3-I ratio and a 25.94% decrease of cleaved caspase 3/total caspase 3 ratio. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza has been reported to benefit Schwann cell in DPN, and here 5-Aza treatment reduced TXNIP protein expression, improved autophagy and inhibited apoptosis in high glucose-treated RSC96 cells and the sciatic nerves of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in TXNIP overexpression in high glucose-stimulated RSC96 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-enhanced TXNIP. Moreover, high glucose-inhibited PI3K/Akt pathway led to DNMT1, DNMT3a, and TXNIP upregulation in RSC96 cells. Knockdown of DNMT1 and DNMT3a prevented PI3K/Akt pathway inhibition-caused TXNIP upregulation in RSC96 cells. Finally, in vivo knockout of TXNIP improved nerve conduction function, increased autophagosome and LC3 expression, and decreased cleaved Caspase 3 and Bax expression in diabetic mice. Taken together, PI3K/Akt pathway inhibition mediated high glucose-induced DNMT1 and DNMT3a overexpression, leading to cell autophagy inhibition and apoptosis via TXNIP protein upregulation in Schwann cells of DPN.

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

TGF-ß1 activates RSC96 Schwann cells migration and invasion through MMP-2 and MMP-9 activities.

Following peripheral nerve injury, remnant Schwann cells adopt a migratory phenotype and remodel the extracellular matrix allowing axonal regrowth. Although much evidence has demonstrated that TGF-ß1 promotes glioma cell motility and induces the expression of extracellular matrix proteins, the effects of TGF-ß1 on Schwann cell migration has not yet been studied. We therefore investigated the cellular effects and the signal transduction pathways evoked by TGF-ß1 in rattus norvegicus neuronal Schwann RSC96 cell. TGF-ß1 significantly increased migration and invasion of Schwann cells assessed by the wound-healing assay and by cell invasion assay. TGF-ß1-enhanced migration/invasion was blocked by inhibition of MMP-2 and MMP-9. Consistently, by real-time and western blot analyses, we demonstrated that TGF-ß1 increased MMP-2 and MMP-9 mRNA and protein levels. TGF-ß1 also increased MMPs activities in cell growth medium, as shown by gelatin zymography. The selective TGF-ß Type I receptor inhibitor SB431542 completely abrogated any effects by TGF-ß1. Indeed, TGF-ß1 Type I receptor activation provoked the cytosol-to-nucleus translocation of SMAD2 and SMAD3. SMAD2 knockdown by siRNA blocked MMP-2 induction and cell migration/invasion due to TGF-ß1. TGF-ß1 also provoked phosphorylation of MAPKs extracellular regulated kinase 1/2 and JNK1/2. Both MAPKs were upstream to p65/NF-kB inasmuch as both MAPKs' inhibitors PD98059 and SP600125 or their down-regulation by siRNA significantly blocked the TGF-ß1-induced nuclear translocation of p65/NF-kB. In addition, p65/NF-κB siRNA knockdown inhibited the effects of TGF-ß1 on both MMP-9 and cell migration/invasion. We conclude that TGF-ß1 controls RSC96 Schwann cell migration and invasion through MMP-2 and MMP-9 activities. MMP-2 is controlled by SMAD2 whilst MMP-9 is controlled via an ERK1/2-JNK1/2-NF-κB dependent pathway.

Schwann-Cell-Specific Deletion of Phosphatidylinositol 4-Kinase Alpha Causes Aberrant Myelination.

Active membrane remodeling during myelination relies on phospholipid synthesis and membrane polarization, both of which are known to depend on inositol phospholipids. Here, we show that sciatic nerves of mice lacking phosphatidylinositol 4-kinase alpha (PI4KA) in Schwann cells (SCs) show substantially reduced myelin thickness with grave consequences on nerve conductivity and motor functions. Surprisingly, prolonged inhibition of PI4KA in immortalized mouse SCs failed to decrease plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) levels or PI 3-kinase (PI3K) activation, in spite of large reductions in plasma membrane PI4P levels. Instead, it caused rearrangements of the actin cytoskeleton, which was also observed in sciatic nerves of knockout animals. PI4KA inactivation disproportionally reduced phosphatidylserine, phosphatidylethanolamine, and sphingomyelin content in mutant nerves, with similar changes observed in SCs treated with a PI4KA inhibitor. These studies define a role for PI4KA in myelin formation primarily affecting metabolism of key phospholipids and the actin cytoskeleton.

Hyperglycemia-induced Bcl-2/Bax-mediated apoptosis of Schwann cells via mTORC1/S6K1 inhibition in diabetic peripheral neuropathy.

Schwann cell apoptosis is one of the characteristics of diabetic peripheral neuropathy (DPN). The mammalian target of rapamycin (mTOR) is a multifunctional signaling pathway that regulates cell apoptosis in various types of tissues and cells. To investigate whether the mTOR pathway is involved in cell apoptosis in the Schwann cells of DPN, diabetic mice and rat Schwann cells (RSC96) were chosen to detect phospho-mTOR (Ser 2448), phospho-S6K1 (Thr 389), phospho-4EBP1 (Thr 37/46), Bcl-2, Bax and cleaved caspase-3 by diverse pathological and biological techniques. The results showed that phospho-mTOR (Ser 2448) was decreased in the sciatic nerves of diabetic mice, concomitant with decreased Bcl-2, increased Bax, cleaved caspase-3 and cell apoptosis. In addition, high glucose treatment for 72 h caused a 35.95% decrease in the phospho-mTOR (Ser 2448)/mTOR ratio, a 65.50% decrease in the phospho-S6K1 (Thr 389)/S6K1 ratio, a 3.67-fold increase in the Bax/Bcl-2 ratio and a 1.47-fold increase in the cleaved caspase-3/caspase-3 ratio. Furthermore, mTORC1 inhibition, rather than mTORC2 inhibition, resulted in mitochondrial controlled apoptosis in RSC96 cells by silencing RAPTOR or RICTOR. Again, suppression of the mTORC1 pathway by a chemical inhibitor led to mitochondrial controlled apoptosis in cultured RSC96 cells in vitro. By contrast, activation of the mTORC1 pathway with MHY1485 prevented decreased phospho-S6K1 (Thr 389) levels caused by high glucose and cell apoptosis. Additionally, constitutive activation of S6K1 avoided high glucose-induced cell apoptosis in RSC96 cells. In summary, our findings suggest that activating mTORC1/S6K1 signaling in Schwann cells may be a promising strategy for the prevention and treatment of DPN.

Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells.

Schwann cells respond to cyclic adenosine monophosphate (cAMP) halting proliferation and expressing myelin proteins. Here we show that cAMP signaling induces the nuclear shuttling of the class IIa histone deacetylase (HDAC)-4 in these cells, where it binds to the promoter and blocks the expression of c-Jun, a negative regulator of myelination. To do it, HDAC4 does not interfere with the transcriptional activity of MEF2. Instead, by interacting with NCoR1, it recruits HDAC3 and deacetylates histone 3 in the promoter of c-Jun, blocking gene expression. Importantly, this is enough to up-regulate Krox20 and start Schwann cell differentiation program-inducing myelin gene expression. Using conditional knockout mice, we also show that HDAC4 together with HDAC5 redundantly contribute to activate the myelin transcriptional program and the development of myelin sheath in vivo. We propose a model in which cAMP signaling shuttles class IIa HDACs into the nucleus of Schwann cells to regulate the initial steps of myelination in the peripheral nervous system.

Activation and expression of µ-calpain in dorsal root contributes to RTX-induced mechanical allodynia.

Background Calpain is a calcium-dependent cysteine protease, and inhibition of calpain by pre-treatment with MDL28170 attenuated the rat mechanical allodynia in a variety of pain models. Postherpetic neuralgia (Shingles) is a neuropathic pain conditioned with the presence of profound mechanical allodynia. Systemic injection of resiniferatoxin can reproduce the clinical symptoms of postherpetic neuralgia. In this study, we determined to study whether activation of calpain contributes to cleave the myelin basic protein of dorsal root and is involved in resiniferatoxin-induced mechanical allodynia of postherpetic neuralgia animal model. Results Resiniferatoxin up-regulated the expression and activation of µ-calpain in dorsal root. The expression of µ-calpain was located in Schwann cell of dorsal root, and resiniferatoxin increased the expression of µ-calpain in Schwann cell in L4-L6 dorsal root at six weeks after injection. Resiniferatoxin also induced myelin basic protein degradation in L4-L6 dorsal root at six weeks after injection. Moreover, intraperitoneal injection of calpain inhibitor MDL28170 prevented the degradation of myelin basic protein and then reduced the sprouting of myelinated afferent fibers into spinal lamina II, thus relieving resiniferatoxin-induced mechanical allodynia. Conclusions Up-regulation and activation of µ-calpain located in Schwann cell may be the mechanism underlying resiniferatoxin-mediated proteolysis of myelin basic protein in dorsal root. Calpain inhibitor MDL28170 prevents resiniferatoxin-induced sprouting of myelinated afferent fibers and mechanical allodynia through inhibition of degradation of the myelin basic protein in dorsal root. Our results indicate that inhibition of pathological µ-calpain activation may present an interesting novel drug target in the treatment of postherpetic neuralgia.

Schwann cell-specific deletion of the endosomal PI 3-kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2-ErbB3 tyrosine kinase signaling.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO ) mice show severe hypomyelination in peripheral nerves. Vps34-/- Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34-/- Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34-/- Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.

Sustained activation of ERK1/2 MAPK in Schwann cells causes corneal neurofibroma.

Recent studies have shown that constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Schwann cells (SCs) increases myelin thickness in transgenic mice. In this secondary analysis, we report that these transgenic mice develop a postnatal corneal neurofibroma with the loss of corneal transparency by age six months. We show that expansion of non-myelinating SCs, under the control of activated ERK1/2, also drive myofibroblast differentiation that derives from both SC precursors and resident corneal keratocytes. Further, these mice also harbor activated mast cells in the central cornea, which contributes to pathological corneal neovascularization and fibrosis. This breach of corneal avascularity and immune status is associated with the growth of the tumor pannus, resulting in a corneal stroma that is nearly four times its normal size. In corneas with advanced disease, some axons became ectopically myelinated, and the disruption of Remak bundles is evident. To determine whether myofibroblast differentiation was linked to vimentin, we examined the levels and phosphorylation status of this fibrotic biomarker. Concomitant with the early upregulation of vimentin, a serine 38-phosphorylated isoform of vimentin (pSer38vim) increased in SCs, which was attributed primarily to the soluble fraction of protein-not the cytoskeletal portion. However, the overexpressed pSer38vim became predominantly cytoskeletal with the growth of the corneal tumor. Our findings demonstrate an unrecognized function of ERK1/2 in the maintenance of corneal homeostasis, wherein its over-activation in SCs promotes corneal neurofibromas. This study is also the first report of a genetically engineered mouse that spontaneously develops a corneal tumor.

The tumour suppressor LKB1 regulates myelination through mitochondrial metabolism.

A prerequisite to myelination of peripheral axons by Schwann cells (SCs) is SC differentiation, and recent evidence indicates that reprogramming from a glycolytic to oxidative metabolism occurs during cellular differentiation. Whether this reprogramming is essential for SC differentiation, and the genes that regulate this critical metabolic transition are unknown. Here we show that the tumour suppressor Lkb1 is essential for this metabolic transition and myelination of peripheral axons. Hypomyelination in the Lkb1-mutant nerves and muscle atrophy lead to hindlimb dysfunction and peripheral neuropathy. Lkb1-null SCs failed to optimally activate mitochondrial oxidative metabolism during differentiation. This deficit was caused by Lkb1-regulated diminished production of the mitochondrial Krebs cycle substrate citrate, a precursor to cellular lipids. Consequently, myelin lipids were reduced in Lkb1-mutant mice. Restoring citrate partially rescued Lkb1-mutant SC defects. Thus, Lkb1-mediated metabolic shift during SC differentiation increases mitochondrial metabolism and lipogenesis, necessary for normal myelination.

Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.

Effect of serum containing Jinmaitong Capsule on rats' Schwann cell apoptosis induced by high glucose concentration.

OBJECTIVE: To evaluate the effect of serum containing Jinmaitong Capsule (JMT) on apoptosis of Schwann cells (SCs) that are cultured in high glucose at the cellular and molecular levels. METHODS: SCs were cultured in Dulbecco's modified Eagle's medium (control group), high glucose (50 mmol/L) medium supplemented with 20% rat serum (HG group), and 50 mmol/L glucose medium supplemented with serum containing JMT (JMT group). SC apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling kit. The expression of Bcl-2 and the caspase-3 p20 subunit in SCs were detected by realtime fluorogenic quantitative polymerase chain reaction and confocal laser scanning microscopy, respectively. RESULTS: No apoptosis was detected in SCs that were cultured in the control group. The percentage of apoptosis of SCs cultured in the HG group was much higher than that in the control group. The apoptosis of SCs in the JMT group was lower than that in the HG group. Fluorescence intensity of Bcl-2 and the expression of Bcl-2 mRNA in SCs that were cultured in the HG group were much lower than those in the control group and much higher than those in the JMT group (P<0.01). The fluorescence intensity of caspase-3 p20 and the expression of caspase-3 p20 mRNA in SCs that were cultured in the HG group were much higher than those in the control group (P<0.01), and they were remarkably lower in the JMT group (P<0.01). CONCLUSIONS: JMT effectively prevents SC apoptosis that is induced by high glucose. This effect may be because of increased expression of Bcl-2 mRNA and protein and decreased expression of caspase-3 p20 mRNA and protein.

Lck tyrosine kinase mediates ß1-integrin signalling to regulate Schwann cell migration and myelination.

The interaction between laminin and ß1-integrin on the surface of Schwann cells regulates Schwann cell proliferation, maturation and differentiation. However, the signalling mediators that fine-tune these outcomes are not fully elucidated. Here we show that lymphoid cell kinase is the crucial effector of ß1-integrin signalling in Schwann cells. Lymphoid cell kinase is activated after laminin treatment of Schwann cells, while downregulation of ß1-integrin with short interfering RNAs inhibits lymphoid cell kinase phosphorylation. Treatment of Schwann cells with a selective lymphoid cell kinase inhibitor reveals a pathway that involves paxillin and CrkII, which ultimately elevates Rac-GTP levels to induce radial lamellipodia formation. Inhibition of lymphoid cell kinase in Schwann cell-dorsal root ganglion cocultures and dorsal root ganglions from Lck(-/-) mice show a reduction of Schwann cell longitudinal migration, reduced myelin formation and internode length. Finally, Lck(-/-) mice exhibit delays in myelination, thinner myelin with abnormal g-ratios and aberrant myelin outfoldings. Our data implicate lymphoid cell kinase as a major regulator of cytoskeletal dynamics, migration and myelination in the peripheral nervous system.

ß-1,4-Galactosyltransferase I involved in Schwann cells proliferation and apoptosis induced by tumor necrosis factor-alpha via the activation of MAP kinases signal pathways.

ß-1,4-galactosyltransferase-I (ß-1,4-GalT-I) plays a critical role in the initiation and maintenance of peripheral nervous system inflammatory reaction. However, the exact function of ß-1,4-GalT-I in the regulation of SCs proliferation and apoptosis remains unclear. In this study, we found that low concentration of tumor necrosis factor-alpha (TNF-α) induced SCs proliferation, while high concentration of TNF-α induced SCs apoptosis. Meanwhile, the expressions of ß-1,4-GalT-I, TNFR1, and TNFR2 were changed following. When ß-1,4-GalT I overexpression, low concentration of TNF-α-induced SCs proliferation was partially repressed. Concurrently, the activity of ERK1/2 was decreased. While knocking down ß-1,4-GalT I expression, high concentration of TNF-α-induced SCs apoptosis was partially rescued. Consistent with this, the activity of P38 and JNK were decreased. We also found anti-TNFR2 antibody suppressed low concentration of TNF-α-induced SCs proliferation, while anti-TNFR1 antibody inhibited high concentration of TNF-α-induced SCs apoptosis. Thus, present data show that ß-1,4-GalT I may play an important role in SCs proliferation and apoptosis induced by TNF-α via different signal pathways and TNFR.

Polysialyltransferase overexpression in Schwann cells mediates different effects during peripheral nerve regeneration.

The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) has been shown to support dynamic changes underlying peripheral nerve regeneration. Using transgenic mice expressing polysialyltransferase ST8SiaIV under control of a glial-specific (proteolipid protein, PLP) promoter (PLP-ST8SiaIV-transgenic mice), we tested the hypothesis that permanent synthesis of PSA in Schwann cells impairs functional recovery of lesioned peripheral nerves. After sciatic nerve crush, histomorphometric analyses demonstrated impaired remyelination of regenerated axons at the lesion site and in target tissue of PLP-ST8SiaIV-transgenic mice, though the number and size of regenerating unmyelinated axons were not changed. This was accompanied by slower mechanosensory recovery in PLP-ST8SiaIV-transgenic mice. However, the proportion of successfully mono-(re)innervated motor endplates in the foot pad muscle was significantly increased in PLP-ST8SiaIV-transgenic mice when compared with wild-type littermates, suggesting that long-term increase in PSA levels in regenerating nerves may favor selective motor target reinnervation. The combined negative and positive effects of a continuous polysialyltransferase overexpression observed during peripheral nerve regeneration suggest that an optimized time- and differentiation-dependent control of polysialyltransferase expression in Schwann cells may further improve recovery after peripheral nerves injury.

Expression of ecto-ATPase NTPDase2 in human dental pulp.

Dental pulpal nerve fibers express ionotropic adenosine triphosphate (ATP) receptors, suggesting that ATP signaling participates in the process of dental nociception. In this study, we investigated if the principal enzymes responsible for extracellular ATP hydrolysis, namely, nucleoside triphosphate diphosphohydrolases (NTPDases), are present in human dental pulp. Immunohistochemical and immunofluorescence experiments showed that NTPDase2 was predominantly expressed in pulpal nerve bundles, Raschkow's nerve plexus, and in the odontoblast layer. NTPDase2 was expressed in pulpal Schwann cells, with processes accompanying the nerve fibers and projecting into the odontoblast layer. Odontoblasts expressed the gap junction protein, connexin43, which can form transmembrane hemichannels for ATP release. NTPDase2 was localized close to connexin43 within the odontoblast layer. These findings provide evidence for the existence of an apparatus for ATP release and degradation in human dental pulp, consistent with the involvement of ATP signaling in the process of dentin sensitivity and dental pain.

Genetic interaction between MTMR2 and FIG4 phospholipid phosphatases involved in Charcot-Marie-Tooth neuropathies.

We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P(2), thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P(2) and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P(2) homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P(2) is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.

Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels.

BACKGROUND: In pediatric neuroblastoma (NBL), high anaplastic lymphoma kinase (ALK) levels appear to be correlated with an unfavorable prognosis, regardless of ALK mutation status. This suggests a therapeutic role for ALK inhibitors in NBL patients. We examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated and wild type (WT) NBL cell lines. METHODS: We measured protein levels by western blot and ALK inhibitor sensitivity (TAE684) by viability assays in 19 NBL cell lines of which 6 had a point mutation and 4 an amplification of the ALK gene. RESULTS: ALK 220 kDa (p = 0.01) and ALK 140 kDa (p = 0.03) protein levels were higher in ALK mutant than WT cell lines. Response to ALK inhibition was significantly correlated with ALK protein levels (p < 0.01). ALK mutant cell lines (n = 4) were 14,9 fold (p < 0,01) more sensitive to ALK inhibition than eight WT cell lines. CONCLUSION: NBL cell lines often express ALK at high levels and are responsive to ALK inhibitors. Mutated cell lines express ALK at higher levels, which may define their superior response to ALK inhibition.

A nuclear variant of ErbB3 receptor tyrosine kinase regulates ezrin distribution and Schwann cell myelination.

Reciprocal interactions between glia and neurons are essential for the proper organization and function of the nervous system. Recently, the interaction between ErbB receptors (ErbB2 and ErbB3) on the surface of Schwann cells and neuronal Neuregulin-1 (NRG1) has emerged as the pivotal signal that controls Schwann cell development, association with axons, and myelination. To understand the function of NRG1-ErbB2/3 signaling axis in adult Schwann cell biology, we are studying the specific role of ErbB3 receptor tyrosine kinase (RTK) since it is the receptor for NRG1 on the surface of Schwann cells. Here, we show that alternative transcription initiation results in the formation of a nuclear variant of ErbB3 (nuc-ErbB3) in rat primary Schwann cells. nuc-ErbB3 possesses a functional nuclear localization signal sequence and binds to chromatin. Using chromatin immunoprecipitation (ChIP)-chip arrays, we identified the promoters that associate with nuc-ErbB3 and clustered the active promoters in Schwann cell gene expression. nuc-ErbB3 regulates the transcriptional activity of ezrin and HMGB1 promoters, whereas inhibition of nuc-ErbB3 expression results in reduced myelination and altered distribution of ezrin in the nodes of Ranvier. Finally, we reveal that NRG1 regulates the translation of nuc-ErbB3 in rat Schwann cells. For the first time, to our knowledge, we show that alternative transcription initiation from a gene that encodes a RTK is capable to generate a protein variant of the receptor with a distinct role in molecular and cellular regulation. We propose a new concept for the molecular regulation of myelination through the expression and distinct role of nuc-ErbB3.