Mek/ERK1/2-MAPK and PI3K/Akt/mTOR signaling plays both independent and cooperative roles in Schwann cell differentiation, myelination and dysmyelination.

Multiple signals are involved in the regulation of developmental myelination by Schwann cells and in the maintenance of a normal myelin homeostasis throughout adult life, preserving the integrity of the axons in the PNS. Recent studies suggest that Mek/ERK1/2-MAPK and PI3K/Akt/mTOR intracellular signaling pathways play important, often overlapping roles in the regulation of myelination in the PNS. In addition, hyperactivation of these signaling pathways in Schwann cells leads to a late onset of various pathological changes in the sciatic nerves. However, it remains poorly understood whether these pathways function independently or sequentially or converge using a common mechanism to facilitate Schwann cell differentiation and myelin growth during development and in causing pathological changes in the adult animals. To address these questions, we analyzed multiple genetically modified mice using simultaneous loss- and constitutive gain-of-function approaches. We found that during development, the Mek/ERK1/2-MAPK pathway plays a primary role in Schwann cell differentiation, distinct from mTOR. However, during active myelination, ERK1/2 is dependent on mTOR signaling to drive the growth of the myelin sheath and regulate its thickness. Finally, our data suggest that peripheral nerve pathology during adulthood caused by hyperactivation of Mek/ERK1/2-MAPK or PI3K is likely to be independent or dependent on mTOR-signaling in different contexts. Thus, this study highlights the complexities in the roles played by two major intracellular signaling pathways in Schwann cells that affect their differentiation, myelination, and later PNS pathology and predicts that potential therapeutic modulation of these pathways in PNS neuropathies could be a complex process.

Independent and cooperative roles of the Mek/ERK1/2-MAPK and PI3K/Akt/mTOR pathways during developmental myelination and in adulthood.

Multiple extracellular and intracellular signals regulate the functions of oligodendrocytes as they progress through the complex process of developmental myelination and then maintain a functionally intact myelin sheath throughout adult life, preserving the integrity of the axons. Recent studies suggest that Mek/ERK1/2-MAPK and PI3K/Akt/mTOR intracellular signaling pathways play important, often overlapping roles in the regulation of myelination. However, it remains poorly understood whether they function independently, sequentially, or converge using a common mechanism to facilitate oligodendrocyte differentiation, myelin growth, and maintenance. To address these questions, we analyzed multiple genetically modified mice and asked whether the deficits due to the conditional loss-of-function of ERK1/2 or mTOR could be abrogated by simultaneous constitutive activation of PI3K/Akt or Mek, respectively. From these studies, we concluded that while PI3K/Akt, not Mek/ERK1/2, plays a key role in promoting oligodendrocyte differentiation and timely initiation of myelination through mTORC1 signaling, Mek/ERK1/2-MAPK functions largely independently of mTORC1 to preserve the integrity of the myelinated axons during adulthood. However, to promote the efficient growth of the myelin sheath, these two pathways cooperate with each other converging at the level of mTORC1, both in the context of normal developmental myelination or following forced reactivation of the myelination program during adulthood. Thus, Mek/ERK1/2-MAPK and the PI3K/Akt/mTOR signaling pathways work both independently and cooperatively to maintain a finely tuned, temporally regulated balance as oligodendrocytes progress through different phases of developmental myelination into adulthood. Therapeutic strategies aimed at targeting remyelination in demyelinating diseases are expected to benefit from these findings.

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.

Signaling by FGF Receptor 2, Not FGF Receptor 1, Regulates Myelin Thickness through Activation of ERK1/2-MAPK, Which Promotes mTORC1 Activity in an Akt-Independent Manner.

FGF signaling has emerged as a significant "late-stage" regulator of myelin thickness in the CNS, independent of oligodendrocyte differentiation. Therefore, it is critically important to identify the specific FGF receptor type and its downstream signaling molecules in oligodendrocytes to obtain better insights into the regulatory mechanisms of myelin growth. Here, we show that FGF receptor type 2 (FGFR2) is highly enriched at the paranodal loops of myelin. Conditional ablation of this receptor-type, but not FGF receptor type 1 (FGFR1), resulted in attenuation of myelin growth, expression of major myelin genes, key transcription factor Myrf and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) activity. This was rescued by upregulating ERK1/2 activity in these mice, strongly suggesting that ERK1/2 are key transducers of FGFR2 signals for myelin growth. However, given that the PI3K/Akt/mechanistic target of rapamycin (mTOR) pathway is also known to regulate myelin thickness, we examined FGFR2-deficient mice for the expression of key signaling molecules in this pathway. A significant downregulation of p-mTOR, p-Raptor, and p-S6RP was observed, which was restored to normal by elevating ERK1/2 activity in these mice. Similar downregulation of these molecules was observed in ERK1/2 knock-out mice. Interestingly, since p-Akt levels remained largely unchanged in these mice, it suggests a mechanism of mTORC1 activation by ERK1/2 in an Akt-independent manner in oligodendrocytes. Taken together, these data support a model in which FGFs, possibly from axons, activate FGFR2 in the oligodendrocyte/myelin compartment to increase ERK1/2 activation, which ultimately targets Myrf, as well as converges with the PI3K/Akt/mTOR pathway at the level of mTORC1, working together to drive the growth of the myelin sheath, thus increasing myelin thickness.SIGNIFICANCE STATEMENT It is well accepted that myelin is a biologically active membrane in active communication with the axons. However, the axonal signals, the receptors on myelin, and the integration of intracellular signaling pathways emanating downstream from these receptors that drive the growth of the myelin sheath remain poorly understood in the CNS. This study brings up the intriguing possibility that FGF receptor 2, in the oligodendrocyte/myelin compartment, may be one such signal. Importantly, it provides compelling evidence linking FGFR2 with the ERK1/2-MAPK pathway, which converges with the PI3K/Akt/mTOR (mechanistic target of rapamycin) pathway at the level of mTORC1 and also regulates the transcription factor Myrf, together providing a mechanistic framework for regulating both the transcriptional and translational machinery required for the proper growth of the myelin sheath.

Conditional knockout of TOG results in CNS hypomyelination.

The tumor overexpressed gene (TOG) protein is present in RNA granules that transport myelin basic protein (MBP) mRNA in oligodendrocyte processes to the myelin compartment. Its role was investigated by conditionally knocking it out (KO) in myelinating glia in vivo. TOG KO mice have severe motor deficits that are already apparent at the time of weaning. This phenotype correlates with a paucity of myelin in several CNS regions, the most severe being in the spinal cord. In the TOG KO optic nerve <30% of axons are myelinated. The number of oligodendrocytes in the corpus callosum, cerebellum, and cervical spinal cord is normal. In the absence of TOG, the most patent biochemical change is a large reduction in MBP content, yet normal amounts of MBP transcripts are found in the brain of affected animals. MBP transcripts are largely confined to the cell body of the oligodendrocytes in the TOG KO in contrast to the situation in wild type mice where they are found in the processes of the oligodendrocytes and in the myelin compartment. These findings indicate that MBP gene expression involves a post-transcriptional TOG-dependent step. TOG may be necessary for MBP mRNA assembly into translation permissive granules, and/or for transport to preferred sites of translation. GLIA 2017;65:489-501.

Root canal centering ability of rotary cutting nickel titanium instruments: A meta-analysis.

AIM: To systematically review articles on canal centering ability of endodontic rotary cutting Nickel-Titanium (Ni-Ti) instruments and subject results to meta-analysis. MATERIALS AND METHODS: A comprehensive search was initiated on canal centering ability of different rotary cutting Ni-Ti files such as Protaper, Hero Shaper, K3, Mtwo, Race, Wave One by selecting articles published in peer reviewed journals during 1991-2013 using "Pub Med" database. Inclusion and exclusion criteria were established. A data was created by tabulating: Author name, publication year, sample size, number of experimental groups, methods to evaluate canal centering ability, instrument cross section, taper, tip design, rake angle, mean and standard deviation. The data generated was subjected to meta-analysis. RESULTS: Maximum studies were found to be conducted on mesiobuccal canal of mandibular 1(st) molar with curvature ranging from 15-60°. The difference in canal centering ability of different rotary cutting Ni-Ti instruments was not statistically significant. CONCLUSION: All endodontic rotary cutting Ni-Ti instruments are capable of producing centered preparations. Protaper depicted the best centering ability. Computed tomography is an effective method of evaluating canal centering ability.

Role of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the renal 2',3'-cAMP-adenosine pathway.

Energy depletion increases the renal production of 2',3'-cAMP (a positional isomer of 3',5'-cAMP that opens mitochondrial permeability transition pores) and 2',3'-cAMP is converted to 2'-AMP and 3'-AMP, which in turn are metabolized to adenosine. Because the enzymes involved in this "2',3'-cAMP-adenosine pathway" are unknown, we examined whether 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) participates in the renal metabolism of 2',3'-cAMP. Western blotting and real-time PCR demonstrated expression of CNPase in rat glomerular mesangial, preglomerular vascular smooth muscle and endothelial, proximal tubular, thick ascending limb and collecting duct cells. Real-time PCR established the expression of CNPase in human glomerular mesangial, proximal tubular and vascular smooth muscle cells; and the level of expression of CNPase was greater than that for phosphodiesterase 4 (major enzyme for the metabolism of 3',5'-cAMP). Overexpression of CNPase in rat preglomerular vascular smooth muscle cells increased the metabolism of exogenous 2',3'-cAMP to 2'-AMP. Infusions of 2',3'-cAMP into isolated CNPase wild-type (+/+) kidneys increased renal venous 2'-AMP, and this response was diminished by 63% in CNPase knockout (-/-) kidneys, whereas the conversion of 3',5'-cAMP to 5'-AMP was similar in CNPase +/+ vs. -/- kidneys. In CNPase +/+ kidneys, energy depletion (metabolic poisons) increased kidney tissue levels of adenosine and its metabolites (inosine, hypoxanthine, xanthine, and uric acid) without accumulation of 2',3'-cAMP. In contrast, in CNPase -/- kidneys, energy depletion increased kidney tissue levels of 2',3'-cAMP and abolished the increase in adenosine and its metabolites. In conclusion, kidneys express CNPase, and renal CNPase mediates in part the renal 2',3'-cAMP-adenosine pathway.

Role of CNPase in the oligodendrocytic extracellular 2',3'-cAMP-adenosine pathway.

Extracellular adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2',3'-cAMP (positional isomer of 3',5'-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2',3'-cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2',3'-cAMP and their respective adenosine monophosphates (2'-AMP and 3'-AMP). Cells were also isolated from mice deficient in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2',3'-cAMP to 2'-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3'-AMP was minimal in both oligodendrocytes and neurons. The production of 2'-AMP from 2',3'-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2'-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3',5'-cAMP-3'-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2',3'-cAMP to 2'-AMP and inhibition of classic ecto-5'-nucleotidase (CD73) with α,ß-methylene-adenosine-5'-diphosphate did not attenuate the conversion of 2'-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2',3'-cAMP to 2-AMP in CNS cells. By reducing levels of 2',3'-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury.

The brain in vivo expresses the 2',3'-cAMP-adenosine pathway.

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.

Myelin protein composition is altered in mice lacking either sulfated or both sulfated and non-sulfated galactolipids.

Myelin is highly enriched in galactocerebroside (GalCer) and its sulfated form sulfatide. Mice, unable to synthesize GalCer and sulfatide (CGT(null)) or sulfatide alone (CST(null)), exhibit disorganized paranodal structures and progressive dysmyelination. To obtain insights into the molecular mechanisms underlying these defects, we examined myelin composition of these mutants by two-dimensional differential fluorescence intensity gel electrophoresis proteomic approach and immunoblotting. We identified several proteins whose expressions were significantly altered in these mutants. These proteins are known to regulate cytoskeletal dynamics, energy metabolism, vesicular trafficking or adhesion, suggesting a disruption in these physiological processes in the absence of myelin galactolipids. Further analysis of one of these proteins, nucleotide diphosphate kinase (NDK)/Nm23, showed that it was reduced in myelin of CGT(null) and increased in CST(null), but not in whole brain homogenate. Immunostaining showed an increase in its expression in the cell bodies of CGT(null)- and a decrease in CST(null)-oligodenrocytes, together leading to the hypothesis that transport of NDK/Nm23 from oligodenrocyte cell bodies into myelin may be differentially dysregulated in the absence of these galactolipids. This study provides new insights into the changes that occur in the composition/distribution of myelin proteins in mice lacking either unsulfated and/or sulfated galactolipids and reinforces the role of these lipids in intracellular trafficking.

Induction of oligodendrocyte progenitors in dorsal forebrain by intraventricular microinjection of FGF-2.

During embryonic development, oligodendrocyte progenitors (OLPs) originate from the ventral forebrain under the regulation of Sonic hedgehog (Shh). Shh controls the expression of transcription factor Olig2, which is strongly implicated in OLP generation. Studies of mice deficient in Shh expression suggest, however, that an alternative pathway for OLP generation may exist. The generation of OLPs in dorsal forebrain has been suggested since treatment of dorsal-neural progenitor cells in culture with fibroblast growth factor (FGF-2) results in OLP induction. To ask if dorsal induction of OLPs in embryonic forebrain can occur in vivo and if FGF-2 could initiate an alternative pathway of regulation, we used in utero microinjection of FGF-2 into the lateral ventricles of mouse fetal forebrain. A single injection of FGF-2 at E13.5 resulted in the expression of the OLP markers Olig2 and PDGFRalpha mRNA in dorsal forebrain ventricular and intermediate zones. However, FGF-2 did not induce dorsal expression of Shh, Patched1 or Nkx2.1, and co-injection of FGF-2 and a Shh inhibitor did not attenuate the induction of Olig2 and PDGFRalpha, suggesting that Shh signaling was not involved in this FGF-2-mediated dorsal induction. These results demonstrate that the dorsal embryonic forebrain in vivo has the potential to generate OLPs in the presence of normal positional cues and that this can be driven by FGF-2 independent of Shh signaling.

S-phase entry of oligodendrocyte lineage cells is associated with increased levels of p21Cip1.

The mechanisms regulating the number of myelinating cells in the central nervous system are crucial for both normal development and repair in pathological conditions. Among relevant growth factors involved in this process, fibroblast growth factor-2 (FGF2) induces oligodendrocyte progenitors (OLPs) to proliferate and stimulates mature oligodendrocytes (OLs) to reenter the S-phase of the cell cycle. S-phase entry is modulated by the formation of complexes between cyclins and cyclin-dependent kinases (CDKs), on one hand, and by their interactions with cell cycle inhibitors (e.g., p18INK, p27Kip1, p21Cip1), on the other. Although the roles of cyclin E/CDK2 complexes and the inhibitor p27Kip1 have been extensively investigated relative to proliferation and differentiation in the OL lineage, less is known about the regulation of the formation of cyclin D1/CDK4 complexes and the role of p21Cip1 in these events. In this study, we show that the FGF2-mediated increase in bromodeoxyuridine (BrdU) incorporation into OL progenitors and mature OLs occurs concomitantly with increase in the levels of p21Cip1 and the formation of p21Cip1/cyclin D1/CDK4 ternary complexes. These complexes are functionally active is indicated by the ensuing FGF2-dependent hyperphosphorylation of the downstream target Rb. In untreated mature OLs that do not incorporate BrdU, the levels of p21Cip1 are low, and the level of the inhibitor p18INK is high. Furthermore, p18INK sequesters CDK2 into binary complexes, precluding the formation of p21Cip1/cyclin D1/CDK4 ternary complexes in these cells. Therefore, we propose that p21Cip1 is acting as a positive regulator, rather than an inhibitor, of cell cycle entry by favoring the assembly of active cyclin D1/CDK4 complexes.