Rnf43 is “lord of the ring” finger proteins in remyelination

Oligodendrocyte precursor cell differentiation into myelinating oligodendrocytes is critical for remyelination in the central nervous system after injury. In this issue of Neuron, Niu et al., 2021Niu J. Yu G. Wang X. Xia W. Wang Y. Hoi K.K. Mei F. Xiao L. Chan J.R. Fancy S.P.J. Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation.Neuron. 2021; 109: 3104-3118Abstract Full Text Full Text PDF Scopus (6) Google Scholar detail a novel role for ring finger protein Rnf43, which is expressed in response to injury and is essential to promote remyelination in vivo. Oligodendrocyte precursor cell differentiation into myelinating oligodendrocytes is critical for remyelination in the central nervous system after injury. In this issue of Neuron, Niu et al., 2021Niu J. Yu G. Wang X. Xia W. Wang Y. Hoi K.K. Mei F. Xiao L. Chan J.R. Fancy S.P.J. Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation.Neuron. 2021; 109: 3104-3118Abstract Full Text Full Text PDF Scopus (6) Google Scholar detail a novel role for ring finger protein Rnf43, which is expressed in response to injury and is essential to promote remyelination in vivo. Oligodendrocytes (OLs) are glial cells that produce and assemble myelin, providing support and insulation to axons in the central nervous system (CNS). Loss of myelinating OLs can lead to several neurological disorders, such as multiple sclerosis (MS) or cerebral palsy (CP). In MS, loss of myelin sheaths occurs through injury or autoimmune damage to mature OLs, whereas in CP, neurological injuries to the developing brain such as hypoxic ischemic encephalopathy (HIE) lead to inadequate myelination. These diseases are characterized by the inability of oligodendrocyte precursors (OPCs) to proliferate and differentiate into mature myelinating OLs. Therefore, stimulation of endogenous OPCs to regenerate myelinating OLs provides an attractive route for endogenous myelin repair after human white matter injury. Although this subject has been extensively studied, the identification of oligodendroglial mechanisms that are utilized for remyelination specifically in the context of injury has remained elusive. In this pioneering study, Niu et al. characterize an injury-specific oligodendroglial ring finger protein, Rnf43, which is a critical regulator of remyelination after injury (Figure 1; Niu et al., 2021Niu J. Yu G. Wang X. Xia W. Wang Y. Hoi K.K. Mei F. Xiao L. Chan J.R. Fancy S.P.J. Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation.Neuron. 2021; 109: 3104-3118Abstract Full Text Full Text PDF Scopus (6) Google Scholar). Although the CNS has limited capacity for regeneration, injury to OLs results in a robust regenerative response leading to remyelination. A fundamental principle of regenerative biology is the “recapitulation hypothesis,” wherein restorative processes are analogous to developmental processes. In the context of remyelination, oligodendroglial mechanisms promoting remyelination after injury are a recapitulation of myelination programs that occur during brain development (Fancy et al., 2011Fancy S.P.J. Harrington E.P. Yuen T.J. Silbereis J.C. Zhao C. Baranzini S.E. Bruce C.C. Otero J.J. Huang E.J. Nusse R. et al.Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination.Nat. Neurosci. 2011; 14: 1009-1016Crossref PubMed Scopus (266) Google Scholar; Gallo and Deneen, 2014Gallo V. Deneen B. Glial development: the crossroads of regeneration and repair in the CNS.Neuron. 2014; 83: 283-308Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). While developmental remyelination is a robust process, the regenerative capacity of OPCs leading to remyelination ultimately deteriorates, contributing to the neuronal degeneration that characterizes late stages of demyelinating diseases (Franklin and Ffrench-Constant, 2017Franklin R.J.M. Ffrench-Constant C. Regenerating CNS myelin - from mechanisms to experimental medicines.Nat. Rev. Neurosci. 2017; 18: 753-769Crossref PubMed Scopus (263) Google Scholar). This suggests fundamental differences between remyelination after injury and remyelination during brain development. Here, the authors have identified this fundamental difference through Rnf43, which regulates remyelination specifically after injury, providing an alternative therapeutic target to promote remyelination. Using brain tissue of human MS and HIE samples, the authors demonstrate that Rnf43 is expressed in an injury-specific pattern in active lesions, but not in normal-appearing zones surrounding lesions. Furthermore, colocalization with cell-type-specific markers revealed that Rnf43 expression is restricted to OPCs and is absent in astrocytes, microglia, and even mature OLs. Similarly, using lysolecithin-induced demyelination in mouse corpus callosum (CC), Niu et al. revealed that Rnf43 expression is dramatically elevated in CC lesions alone, in contrast to its complete absence in uninjured CC. These observations provided Rnf43 as a powerful entry point to uncover injury-specific mechanisms of remyelination. Since remyelination programs parallel myelination during brain development, how does the role of Rnf43 differ in developing and injured brain? To interrogate how loss of Rnf43 in OPCs in the developing brain affects myelination, Olig1/2 Cre mice were crossed with the Rnf43 and Znrf43 floxed lines to create a double knockout (Olig1/2cre:RZfl/fl). Znrf3 (zinc and ring finger protein 3) was included because it can functionally compensate for the related Rnf43. Interestingly, OPC development, OL differentiation, and subsequent axon myelination were unaffected in this Rnf43-Znrf3 double-knockout mouse. To examine how loss of OPC Rnf43-Znrf3 in the adult brain affects remyelination after injury, the authors crossed RZfl/fl with PDGFR⍺-CreER to allow temporal, conditional deletion in OPCs in adult mice. Following lysolecithin-induced demyelination in the CC of these mice, significant reductions in myelin basic protein (MBP), myelin-associated glycoprotein (MAG), number of remyelinated axons, and myelin thickness were observed. In addition, this injury-specific role for Rnf43-Znrf3 in the adult brain also extends to neonatal white matter injury; chronic neonatal hypoxia in Olig1Cre:RZfl/fl blocked OL maturation after Rnf43-Znrf3 deletion. Comparative analysis between the effects of Rnf43 and Znrf3 revealed that remyelination phenotypes of RZfl/fl mice can be attributed solely to loss of Rnf43. Taken together, these compelling results showed that Rnf43 is completely dispensable for myelination in the healthy developing brain, but in both injured adult and developing brain, Rnf43 is not dispensable and is critical for remyelination after injury. This highly specific role for Rnf43 in regenerative myelination raises the question of how it fits into the existing framework of OPC regeneration. In seminal papers, Fancy and colleagues established that the Wnt pathway is a critical regulator of remyelination and that high levels of Wnt activity are associated with remyelinating failures in human disease (Fancy et al., 2009Fancy S.P.J. Baranzini S.E. Zhao C. Yuk D.I. Irvine K.A. Kaing S. Sanai N. Franklin R.J. Rowitch D.H. Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS.Genes Dev. 2009; 23: 1571-1585Crossref PubMed Scopus (456) Google Scholar, Fancy et al., 2014Fancy S.P.J. Harrington E.P. Baranzini S.E. Silbereis J.C. Shiow L.R. Yuen T.J. Huang E.J. Lomvardas S. Rowitch D.H. Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer.Nat. Neurosci. 2014; 17: 506-512Crossref PubMed Scopus (80) Google Scholar). Interestingly, prior research demonstrated that Rnf43 is a target of Wnt signaling and a negative feedback repressor of the Wnt pathway. Putting these pieces together led the authors to first examine the relationship between Rnf43 and the Wnt pathway in the context of myelination. The use of Olig2-cre:APCfl/fl mice, wherein conditional loss of Wnt pathway repressor APC leads to excessive Wnt activation in OPCs, showed increased Rnf43 in OPCs, indicating that Rnf43 is a target of the Wnt pathway in OPCs. Next, the authors asked if Rnf43 is also a negative feedback repressor of Wnt. Exposure of the Wnt ligand to OPCs lacking Rnf43 reduced OPC differentiation into MBP-positive cells, revealing that loss of Rnf43 makes OPCs hypersensitive to Wnt. Conversely, gain-of-function studies were performed both in vitro and in vivo in lesions of PDGFR⍺-CreER:APCfl/fl mice. Increased Rnf43 showed reduced Wnt pathway activation and promoted MBP expression by blocking the effect of Wnt ligand exposure. Together, these results provide a mechanism for how Rnf43 promotes remyelination, in which Rnf43 represses the Wnt pathway to promote differentiation of OPCs into myelinating OLs after injury. Next, the authors sought to use this mechanism as a means of stimulating remyelination. Previously, Rnf43 had been shown to repress the Wnt pathway by targeting Frizzled (Fzd) receptors for degradation, and here they found that Rnf43 physically interacts with Fzd1 and alters the sub-cellular localization of Fzd1 from cell surface to cytoplasm in OPCs. This led the authors to focus on compound UM206, which had previously been shown to disrupt the interaction between Wnt ligands and Fzd1/2. In OPC in vitro cultures, UM206 promoted OPC differentiation and reversed Wnt-induced suppression of OPC differentiation. In vivo, UM206 rescued loss-of-function phenotypes of Rnf43 in OPCs, as evaluated by reduction in OPC hypersensitivity to Wnt ligands and differentiation into MBP-positive OLs. Remarkably, using lysolecithin-induced demyelination in mouse CC coupled with osmotic pump delivery of UM206, Niu et al. showed that UM206 significantly increases the rate of OPC differentiation. Electron microscopy of remyelination and quantification of myelinated axons in lesions clearly showed that UM206 promotes remyelination in wild-type mice and rescues the loss of remyelination caused by Rnf43 deletion. These exciting results demonstrate the potential of UM206 for therapeutic targeting of the Wnt pathway in remyelination. A central tenet of oligodendroglial biology is that cell-intrinsic factors that control OPC differentiation into myelinating OLs function in both developmental myelination and remyelination after injury. However, injury-specific cell-intrinsic factors that function solely in remyelination were not thought to exist. This work provides a critical entity in Rnf43, which serves as an injury-specific OPC marker that effectively controls remyelination after injury in both the developing and adult brain. Rnf43 is targeted by Wnt signaling, which is activated in the adult brain only in response to injury. This raises an interesting question regarding how Wnt-dependent gene transcription control Rnf43 injury-dependent activation that would provide molecular insights into Rnf43 regulation. Furthermore, Rnf43 provides an excellent entry point for future studies to interrogate how loss of Rnf43 affects molecular properties of OPCs within lesioned injury sites in comparison to healthy OPCs in uninjured tissue. These studies will reveal undiscovered injury-specific molecular mechanisms in OPCs, expanding the repertoire for therapeutic targeting of regulators crucial for remyelination. Existing therapeutics for patients with demyelinating diseases like MS rely on immune-modulatory mechanisms that do not directly repair the damage to myelin (Plemel et al., 2017Plemel J.R. Liu W.Q. Yong V.W. Remyelination therapies: a new direction and challenge in multiple sclerosis.Nat. Rev. Drug Discov. 2017; 16: 617-634Crossref PubMed Scopus (148) Google Scholar). Therefore, more recent research has focused on targeting OPC-dependent remyelination in the CNS (Najm et al., 2015Najm F.J. Madhavan M. Zaremba A. Shick E. Karl R.T. Factor D.C. Miller T.E. Nevin Z.S. Kantor C. Sargent A. et al.Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.Nature. 2015; 522: 216-220Crossref PubMed Scopus (268) Google Scholar), with the M1 muscarinic receptor and clemastine serving as archetypes for this approach (Mei et al., 2016Mei F. Lehmann-Horn K. Shen Y.A. Rankin K.A. Stebbins K.J. Lorrain D.S. Pekarek K. A Sagan S. Xiao L. Teuscher C. et al.Accelerated remyelination during inflammatory demyelination prevents axonal loss and improves functional recovery.eLife. 2016; 5: 1-12Crossref Scopus (134) Google Scholar). The Wnt pathway, which regulates OPC differentiation into myelinating OLs, has manifested as an important therapeutic node for remyelination. Axin2, which is a scaffolding protein that suppresses Wnt to promote remyelination, has been pharmacologically targeted (Fancy et al., 2011Fancy S.P.J. Harrington E.P. Yuen T.J. Silbereis J.C. Zhao C. Baranzini S.E. Bruce C.C. Otero J.J. Huang E.J. Nusse R. et al.Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination.Nat. Neurosci. 2011; 14: 1009-1016Crossref PubMed Scopus (266) Google Scholar). Daam2, another regulator of the Wnt pathway, had been shown to interact with PIP5K to promote clustering of the Wnt receptor complex, amplifying Wnt signal transduction. Pharmacological targeting of Daam2-PIP5K interaction had therefore enabled disruption of Wnt receptor complex formation to promote remyelination (Lee et al., 2015Lee H.K. Chaboub L.S. Zhu W. Zollinger D. Rasband M.N. Fancy S.P. Deneen B. Daam2-PIP5K is a regulatory pathway for Wnt signaling and therapeutic target for remyelination in the CNS.Neuron. 2015; 85: 1227-1243Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Here, Niu et al., 2021Niu J. Yu G. Wang X. Xia W. Wang Y. Hoi K.K. Mei F. Xiao L. Chan J.R. Fancy S.P.J. Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturation.Neuron. 2021; 109: 3104-3118Abstract Full Text Full Text PDF Scopus (6) Google Scholar provide yet another pharmacological strategy to directly suppress Wnt receptor Fzd1 expression, facilitating OPC differentiation and remyelination. The authors were led to this target via discovery of an injury-specific protein, Rnf43, that regulates Fzd1 cell surface expression in OPCs. This mechanism is specifically activated after injury and is therefore superior to existing pharmacological strategies described above, wherein both Axin2 and Daam2 are not injury specific and are required for myelination in the developing brain. In summary, this work identifies a novel protein, Rnf43, which is activated in OPCs after CNS injury and functions in a negative feedback mechanism to suppress Wnt signaling via Fzd1 receptor internalization to promote OPC differentiation and remyelination. This significant discovery allows direct pharmacological targeting of Fzd1 to promote remyelination in vivo, paving the path forward to decipher disease mechanisms associated with remyelination failures. Oligodendroglial ring finger protein Rnf43 is an essential injury-specific regulator of oligodendrocyte maturationNiu et al.NeuronAugust 13, 2021In BriefNiu et al. identify a factor, Rnf43, that marks activated oligodendrocyte progenitor cells (OPCs) in human white matter injury, which is required in an injury-specific context for their maturation and successful remyelination, and acts by regulating Wnt signal strength at the level of OPC surface presentation of the Wnt receptor Fzd1. Full-Text PDF Open Access