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.

Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypomyelinating neuropathy.

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.

Developmental stage-specific role of Frs adapters as mediators of FGF receptor signaling in the oligodendrocyte lineage cells.

FGF signaling is important for numerous cellular processes and produces diverse cellular responses. Our recent studies using mice conditionally lacking FGF-Receptor-1 (Fgfr1) or Fgfr2 during different stages of myelinogenesis revealed that Fgfr signaling is first required embryonically for the specification of oligodendrocyte progenitors (OPCs) and then later postnatally for the growth of the myelin sheath during active myelination but not for OPC proliferation, differentiation, or ensheathment of axons. What intracellular signal transduction pathways are recruited immediately downstream of Fgfrs and mediate these distinct developmentally regulated stage-specific responses remain unclear. The adapter protein Fibroblast-Growth-Factor-Receptor-Substrate-2 (Frs2) is considered a key immediate downstream target of Fgfrs. Therefore, here, we investigated the in vivo role of Frs adapters in the oligodendrocyte lineage cells, using a novel genetic approach where mice were engineered to disrupt binding of Frs2 to Fgfr1 or Fgfr2, thus specifically uncoupling Frs2 and Fgfr signaling. In addition, we used conditional mutants with complete ablation of Frs2 and Frs3. We found that Frs2 is required for specification of OPCs in the embryonic telencephalon downstream of Fgfr1. In contrast, Frs2 is largely dispensable for transducing Fgfr2-mediated signals for the growth of the myelin sheath during postnatal myelination, implying the potential involvement of other adapters downstream of Fgfr2 for this function. Together, our data demonstrate a developmental stage-specific function of Frs2 in the oligodendrocyte lineage cells. This contextual requirement of adapter proteins, downstream of Fgfrs, could partly explain the distinct responses elicited by the activation of Fgfrs during different stages of myelinogenesis.

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.

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.

Strength of ERK1/2 MAPK Activation Determines Its Effect on Myelin and Axonal Integrity in the Adult CNS.

UNLABELLED: Myelin growth is a tightly regulated process driven by multiple signals. ERK1/2-MAPK signaling is an important regulator of myelin thickness. Because, in demyelinating diseases, the myelin formed during remyelination fails to achieve normal thickness, increasing ERK1/2 activity in oligodendrocytes is of obvious therapeutic potential for promoting efficient remyelination. However, other studies have suggested that increased levels of ERK1/2 activity could, in fact, have detrimental effects on myelinating cells. Because the strength, duration, or timing of ERK1/2 activation may alter the biological outcomes of cellular responses markedly, here, we investigated the effect of modulating ERK1/2 activity in myelinating cells using transgenic mouse lines in which ERK1/2 activation was upregulated conditionally in a graded manner. We found enhanced myelin gene expression and myelin growth in the adult CNS at both moderate and hyperactivated levels of ERK1/2 when upregulation commenced during developmental myelination or was induced later during adulthood in quiescent preexisting oligodendrocytes, after active myelination is largely terminated. However, a late onset of demyelination and axonal degeneration occurred at hyperelevated, but not moderately elevated, levels regardless of the timing of the upregulation. Similarly, myelin and axonal pathology occurred with elevated ERK1/2 activity in Schwann cells. We conclude that a fine tuning of ERK1/2 signaling strength is critically important for normal oligodendrocyte and Schwann cell function and that disturbance of this balance has negative consequences for myelin and axonal integrity in the long term. Therefore, therapeutic modulation of ERK1/2 activity in demyelinating disease or peripheral neuropathies must be approached with caution. SIGNIFICANCE STATEMENT: ERK1/2-MAPK activation in oligodendrocytes and Schwann cells is an important signal for promoting myelin growth during developmental myelination. Here, we show that, when ERK1/2 are activated in mature quiescent oligodendrocytes during adulthood, new myelin growth is reinitiated even after active myelination is terminated, which has implications for understanding the mechanism underlying plasticity of myelin in adult life. Paradoxically, simply increasing the "strength" of ERK1/2 activation changed the biological outcome from beneficial to detrimental, adversely affecting myelin and axonal integrity in both the CNS and PNS. Therefore, this study highlights the complexity of ERK1/2-MAPK signaling in the context of oligodendrocyte and Schwann cell function in the adult animal and emphasizes the need to approach potential therapeutic modulation of ERK1/2 activity with caution.

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.

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.

Renal 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Is an Important Determinant of AKI Severity after Ischemia-Reperfusion.

A positional isomer of 3',5'-cAMP, 2',3'-cAMP, is produced by kidneys in response to energy depletion, and renal 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) metabolizes 2',3'-cAMP to 2'-AMP; 2',3'-cAMP is a potent opener of mitochondrial permeability transition pores (mPTPs), which can stimulate autophagy. Because autophagy protects against AKI, it is conceivable that inhibition of CNPase protects against ischemia-reperfusion (IR) -induced AKI. Therefore, we investigated renal outcomes, mitochondrial function, number, area, and autophagy in CNPase-knockout (CNPase(-/-)) versus wild-type (WT) mice using a unique two-kidney, hanging-weight model of renal bilateral IR (20 minutes of ischemia followed by 48 hours of reperfusion). Analysis of urinary purines showed attenuated metabolism of 2',3'-cAMP to 2'-AMP in CNPase(-/-) mice. Neither genotype nor IR affected BP, heart rate, urine volume, or albumin excretion. In WT mice, renal IR reduced (14)C-inulin clearance (index of GFR) and increased renal vascular resistance (measured by transit time nanoprobes) and urinary excretion of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. IR did not affect these parameters in CNPase(-/-) mice. Histologic analysis revealed that IR induced severe damage in kidneys from WT mice, whereas histologic changes were minimal after IR in CNPase(-/-) mice. Measurements of renal cardiolipin levels, citrate synthase activity, rotenone-sensitive NADH oxidase activity, and proximal tubular mitochondrial and autophagosome area and number (by transmission electron microscopy) indicted accelerated autophagy/mitophagy in injured CNPase(-/-) mice. We conclude that CNPase deletion attenuates IR-induced AKI, in part by accelerating autophagy with targeted removal of damaged mitochondria.

Role of ERK1/2 MAPK signaling in the maintenance of myelin and axonal integrity in the adult CNS.

Oligodendrocytes form myelin during postnatal development and then maintain a functional myelin sheath throughout adult life. While many regulators of developmental myelination have been identified, the signal transduction mechanisms that regulate oligodendrocyte functions in adulthood are not well understood. The extracellular signal-regulated kinases-1 and -2 (ERK1/2), downstream mediators of mitogen-activated protein kinases (MAPKs), have emerged as prominent regulators of myelin formation. Here, we investigated whether these signaling molecules are also required for myelin maintenance in the adult CNS. Inducible conditional ablation of Erk1/2 in oligodendrocytes of the adult CNS resulted in a downregulation of myelin gene expression. Although myelin thickness was reduced and some axons were demyelinated, the majority of axons were wrapped by intact myelin sheaths that appeared structurally normal. However, late onset of progressive axonal degeneration, accompanied by astrogliosis, microglial activation, partial loss of oligodendrocytes, and functional impairment, occurred in the adult mice lacking ERK1/2 activity. Conditional ablation of Fibroblast Growth Factor receptors-1 and -2 (FGFR1/2) in oligodendrocytes also resulted in downregulation of myelin gene expression and development of axonal degeneration as the mice aged. Further, the level of the key transcription factor myelin gene regulatory factor (Myrf) was downregulated or upregulated in mice with genetic loss or gain of ERK1/2 function, respectively. Together, our studies demonstrate that ERK1/2-MAPK signaling is required for the long-term maintenance of myelin and axonal integrity in the adult CNS and suggest that FGFR1/2 and Myrf may, in part, contribute to signaling upstream and downstream of ERK1/2 in maintaining these oligodendrocyte functions during adulthood.

Fibroblast growth factor signaling in oligodendrocyte-lineage cells facilitates recovery of chronically demyelinated lesions but is redundant in acute lesions.

Remyelination is a potent regenerative process in demyelinating diseases, such as multiple sclerosis, the effective therapeutic promotion of which will fill an unmet clinical need. The development of proregenerative therapies requires the identification of key regulatory targets that are likely to be involved in the integration of multiple signaling mechanisms. Fibroblast growth factor (FGF) signaling system, which comprises multiple ligands and receptors, potentially provides one such target. Since the FGF/FGF receptor (FGFR) interactions are complex and regulate multiple diverse functions of oligodendrocyte lineage cells, it is difficult to predict their overall therapeutic potential in the regeneration of oligodendrocytes and myelin. Therefore, to assess the integrated effects of FGFR signaling on this process, we simultaneously inactivated both FGFR1 and FGFR2 in oligodendrocytes and their precursors using two Cre-driver mouse lines. Acute and chronic cuprizone-induced or lysolecithin-induced demyelination was established in Fgfr1/Fgfr2 double knockout mice (dKO). We found that in the acute cuprizone model, there was normal differentiation of oligodendrocytes and recovery of myelin in the corpus callosum of both control and dKO mice. Similarly, in the spinal cord, lysolecithin-induced demyelinated lesions regenerated similarly in the dKO and control mice. In contrast, in the chronic cuprizone model, fewer differentiated oligodendrocytes and less efficient myelin recovery were observed in the dKO compared to control mice. These data suggest that while cell-autonomous FGF signaling is redundant during recovery of acute demyelinated lesions, it facilitates regenerative processes in chronic demyelination. Thus, FGF-based therapies have potential value in stimulating oligodendrocyte and myelin regeneration in late-stage disease.

Sustained activation of ERK1/2 MAPK in oligodendrocytes and schwann cells enhances myelin growth and stimulates oligodendrocyte progenitor expansion.

Myelin is a biologically active membrane receiving and processing signals from axons. Although much is known about its structure and molecular composition, the intracellular signal transduction pathways, active during specific phases of myelinogenesis for regulating myelin formation, remain poorly understood. Recent genetic loss-of-function studies have suggested a key role of extracelluar signal-regulated kinases-1 and -2 (ERK1/2), downstream mediators of mitogen-activated protein kinases (MAPKs), in promoting CNS and PNS myelination. In contrast, other studies, largely in vitro, have suggested that activation of ERK1/2 pathway can be detrimental for glial cell function and myelination. Given these conflicting reports, we investigated the effects of cell-autonomous activation of ERK1/2 in glial cells during developmental myelination in the intact CNS and PNS. Two lines of transgenic mice with sustained activation of ERK1/2 in oligodendrocyte progenitors (OPCs), oligodendrocytes, and Schwann cells were generated. Consistent with our loss-of-function studies, gain of ERK1/2 function in oligodendrocyte-lineage cells significantly increased myelin thickness, independent of oligodendrocyte differentiation or initiation of myelination. Additionally, increased activation of ERK1/2 in OPCs during early development resulted in transient hyperproliferation and overproduction of OPCs but generation of normal numbers of myelinating oligodendrocytes. Thus, these in vivo studies suggest a beneficial biphasic requirement of ERK1/2 during developmental myelination in the CNS, deployed first during early stages of the oligodendrocyte lineage for promoting OPC expansion and then redeployed later in myelinating oligodendrocytes for promoting myelin growth. Furthermore, Schwann cells with activated ERK1/2 hypermyelinate PNS axons, suggesting that ERK1/2 signaling is a conserved mechanism that promotes both CNS and PNS developmental myelination.

The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS.

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.

Treatment with thyroxine restores myelination and clinical recovery after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) remains a major cause of white matter injury in preterm infants with no viable therapeutic strategy to restore myelination. Maturation of oligodendrocytes and myelination is influenced by thyroid hormone (TH) signaling, which is mediated by TH receptor α (TRα) and TRß. In the brain, cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that IVH would decrease TH signaling via changes in the expression of deiodinases and/or TRs, and normalization of TH signaling would enhance maturation of oligodendrocytes and myelination in preterm infants with IVH. These hypotheses were tested using both autopsy materials from human preterm infants and a rabbit model of IVH. We found that deiodinase-2 levels were reduced, whereas deiodinase-3 levels were increased in brain samples of both humans and rabbits with IVH compared with controls without IVH. TRα expression was also increased in human infants with IVH. Importantly, treatment with TH accelerated the proliferation and maturation of oligodendrocytes, increased transcription of Olig2 and Sox10 genes, augmented myelination, and restored neurological function in pups with IVH. Consistent with these findings, the density of myelinating oligodendrocytes was almost doubled in TH-treated human preterm infants compared with controls. Thus, in infants with IVH the combined elevation in deiodinase-3 and reduction in deiodinase-2 decreases TH signaling that can be worsened by an increase in unliganded TRα. Given that TH promotes neurological recovery in IVH, TH treatment might improve the neurodevelopmental outcome of preterm infants with IVH.

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.

Fibroblast growth factor receptor signaling in oligodendrocytes regulates myelin sheath thickness.

Formation of the CNS white matter is developmentally tightly regulated, but the molecules and mechanisms of myelination control in the postnatal CNS are poorly understood. Here, we show that myelin growth is controlled by fibroblast growth factor (FGF) signaling, originally identified as a proliferative signal for oligodendrocyte precursor cells (OPCs) in vitro. We created two lines of mice lacking both FGF receptor 1 (Fgfr1) and Fgfr2 in oligodendrocyte-lineage cells but found that in these mice OPC proliferation and differentiation were unaffected. In addition, axonal ensheathment and the initiation of myelination were on time. However, the rapid growth of CNS myelin, normally occurring in the second postnatal week, was strongly inhibited. Throughout adulthood, the myelin sheath remained disproportionately thin relative to the axon caliber. In adult mice, mutant oligodendrocytes were normal in number, whereas the transcription of major myelin genes was reduced. This FGF receptor-mediated stimulation of mature oligodendrocytes could also be modeled in vitro, demonstrating that enhanced expansion of oligodendroglial processes requires signaling by extracellular signal regulated kinase-1 and -2 (Erk1/2), downstream mediators of mitogen-activated protein kinase (MAPK). In vivo, Erk1/2-MAPK activity was reduced in the hypomyelinated CNS of Fgfr1/Fgfr2 mutant mice. These studies reveal a previously unrecognized function of FGF receptor signaling in oligodendrocytes that contributes to the regulation of myelin sheath thickness and that uncouples the initiation of ensheathment from the later phase of continued myelin growth.

ERK1/ERK2 MAPK signaling is required to increase myelin thickness independent of oligodendrocyte differentiation and initiation of myelination.

Wrapping of the myelin sheath around axons by oligodendrocytes is critical for the rapid conduction of electrical signals required for the normal functioning of the CNS. Myelination is a multistep process where oligodendrocytes progress through a well coordinated differentiation program regulated by multiple extracellular growth and differentiation signals. The intracellular transduction of the extracellular signals that regulate myelination is poorly understood. Here we demonstrate a critical role for two important signaling molecules, extracelluar signal-regulated protein kinases 1 and 2 (ERK1/ERK2), downstream mediators of mitogen-activated protein kinases, in the control of CNS myelin thickness. We generated and analyzed two lines of mice lacking both ERK1/ERK2 function specifically in oligodendrocyte-lineage cells. In the absence of ERK1/ERK2 signaling NG2⁺ oligodendrocyte progenitor cells proliferated and differentiated on schedule. Mutant oligodendrocytes also ensheathed axons normally and made a few wraps of compact myelin. However, the subsequent increase in myelination that correlated myelin thickness in proportion to the axon caliber failed to occur. Furthermore, although the numbers of differentiated oligodendrocytes in the adult mutants were unchanged, they showed an inability to upregulate the transcription of major myelin genes that normally occurs during active myelination. Similarly, in vitro ERK1/ERK2-deficient oligodendrocytes differentiated normally but failed to form typical myelin-like membrane sheets. None of these effects were observed in single ERK1 or ERK2 mutants. These studies suggest that the predominant role of ERK1/ERK2 signaling in vivo is in promoting rapid myelin growth to increase its thickness, subsequent to oligodendrocyte differentiation and the initiation of myelination.

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.