Axonal regulation of myelination by 1 Klaus-Armin Nave1,2 and James L Salzer3

Neuregulins comprise a family of epidermal -like In this review, we describe recent progress in elucidating ligands that interact with ErbB receptor kinases to the mechanisms by which motor and sensory axons in the control many aspects of neural development. One of the most peripheral (PNS) regulate the develop- dramatic effects of neuregulin-1 is on glial cell differentiation. ment and differentiation of Schwann cells, most strikingly The membrane-bound neuregulin-1 type III isoform is an axonal during myelination. Unexpectedly, a single growth factor, ligand for glial ErbB receptors that regulates the early Schwann neuregulin-1 (NRG1), has emerged as the pivotal signal cell lineage, including the generation of precursors. Recent that controls Schwann cells at every stage of the lineage. studies have shown that the amount of neuregulin-1 type III expressed on axons also dictates the glial phenotype, with a Neuregulin-1 and ErbB receptors threshold level triggering myelination. The Neuregulin-1 (NRG1) family comprises more than Remarkably, neuregulin-1 type III also regulates Schwann cell 15 membrane-associated and secreted proteins [4,5]. membrane growth to adjust sheath thickness to match These are derived from one of the largest mammalian axon caliber precisely. Whether this signaling system operates (on human 8p22 and mouse chromo- in central nervous system myelination remains an open some 8A3) and are generated by use of multiple tran- question of major importance for human demyelinating scription sites and by extensive alternative RNA splicing diseases. [6]. All NRG1 isoforms share an Addresses (EGF)-like signaling domain that is necessary and suffi- 1 Max Planck Institute of Experimental Medicine, D-37075 Goettingen, cient for activation of their receptors. NRG1 isoforms are Germany subdivided into several subtypes on the basis of their 2 Hertie Institute of Multiple Sclerosis Research, Goettingen, Germany 3 Departments of Cell Biology and Neurology, and the Molecular distinct amino-termini [4,7]. NRG1 type I (also known as Neurobiology Program, Skirball Institute of Biomolecular Medicine, New heregulin, neu differentiation factor, or acetylcholine York University School of Medicine, New York, NY 10016, USA receptor-inducing activity [ARIA]) and NRG1 type II (also known as glial growth factor [GGF]) have N-term- Corresponding author: Nave, Klaus-Armin ([email protected]) inal immunoglobulin-like domains. Transmembrane forms of NRG1 undergo proteolytic cleavage by metal- Current Opinion in Neurobiology 2006, 16:492–500 loproteinases (MP), including TACE (tumor-necrosis factor-a-converting enzyme) [8]. As a consequence, This review comes from a themed issue on NRG1 type I and II are shed from the neuronal cell Neuronal and glial cell biology Edited by Kelsey C Martin and Elior Peles surface and function as paracrine signaling molecules (schematically depicted in Figure 1). NRG1 type III is Available online 7th September 2006 defined by its cysteine-rich domain (CRD), which func- 0959-4388/$ – see front matter tions as a second transmembrane domain. Consequently, # 2006 Elsevier Ltd. All rights reserved. NRG1 type III remains tethered to the cell surface after cleavage and functions as a juxtacrine signal [9]. In DOI 10.1016/j.conb.2006.08.008 addition, exons encoding shorter amino termini of NRG1 have been identified by sequence analysis (referred to as types IV–VI), but these isofoms have Introduction not been further characterized [10]. NRG1 expression Reciprocal interactions between neurons and glia are is not specific to the nervous system, it also has a major crucial for the organization and function of the nervous role in cardiac and mammary tissue development system, from neurogenesis in embryonic development to (reviewed in [11]). Indeed, mice lacking NRG1, or its synaptic plasticity in the adult brain. Glial cells that receptors (ErbB2, ErbB3 and ErbB4), are embryonic synthesize myelin are essential for normal motor and lethal because NRG1–ErbB signaling is essential for cognitive functions, with the fine tuning of myelination cardiac development. contributing to the millisecond precision of the nervous system [1]. Furthermore, myelin-forming glial cells are Within the nervous system, NRG1 types I and III are the also required for the long-term integrity of axons, inde- most abundant forms and have been detected in many pendently of myelin itself [2,3]. Axons, in turn, crucially projection neurons, most notably in spinal motor neurons regulate the behavior of myelinating glia: that is, Schwann and dorsal root ganglia (DRG) neurons, but also in glia cells and . However, the molecular [4,12]. In addition to the axon–glia signaling detailed mechanisms by which neurons and glial cells commu- below, the proposed functions of NRG1 include the devel- nicate remain poorly understood. opment of motor endplates, migration of interneurons, and

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Figure 1

NRG1 isoforms: membrane disposition and signalling. (a) Types I and II are synthesized as single pass transmembrane proteins; Type III has two transmembrane domains. (b) With metalloproteinase (MP) cleavage, types I and II are shed as paracrine signals, type III remains tethered through its cysteine rich domain (CRD) and is a juxtacrine signal; this cleavage is enhanced by released by Schwann cells. The cytoplasmic domain undergoes further cleavage stimulated by binding of ErbB receptors to NRG1, followed by translocation to the nucleus. See [4]for additional details. and synaptic plasticity in the CNS [5]. adaptor molecules and activation of downstream signaling Many NRG1-expressing neurons also express transcripts pathways (see below). Whereas Schwann cells principally for NRG2 and NRG3, two structurally related growth express ErbB2 and ErbB3, cells in the factors with EGF-like signaling domains, the function of lineage express all three ErbB receptors, in a developmen- which in the nervous system remains largely unknown tally regulated manner, in addition to the EGFR (ErbB1), [12,13]. indicating significant complexity of potential ErbB recep- tor heterodimers and downstream signaling events in these ErbB receptors cells [16,17]. NRG1 isoforms mediate their effects by binding to ErbB receptors, members of the EGF receptor superfamily [14]. NRG1 ‘back signaling’ NRG1 binds to either ErbB3, which lacks an active kinase Intriguingly, NRG1 might also signal bidirectionally. Bind- domain, or ErbB4, which has such a kinase domain; each ing of recombinantly produced ‘soluble’ ErbB receptors to receptor, in turn, can heterodimerize with ErbB2, which membrane-bound NRG1 type III of transfected neurons cannot bind NRG1 directly but also has an active kinase results in proteolytic cleavage of NRG1, releasing its domain. ErbB receptors dimerize not by virtue of a brid- cytoplasmic C-terminal domain (CTD) from the mem- ging effect of NRG1, but following a ligand-activated brane [18]. Remarkably, the CTD then rapidly translocates conformational change in the ectodomain of ErbB3 or into the cell nucleus of the cultured neurons where it ErbB4 [15]. Crystallographic data indicate that ErbB2 activates transcription and enhances survival. Whether constitutively exposes a dimerization loop required to NRG1 ‘backsignaling’ occurs in vivo, particularly when form heterodimers with ligand-activated ErbB3 or ErbB4 NRG1 expressed on the axon is engaged by ErbB receptors receptors. Because the ability of ErbB2 to form homodi- of myelinating glia, remains to be established. It will also be mers is poor, and ErbB4 is minimally expressed by important to determine whether loss of the anti-apoptotic Schwann cells, ErbB2–ErbB3 is the relevant NRG1 effect of the CTD is normally responsible for the degen- Schwann cell receptor. NRG1 binding induces ErbB2– eration of dorsal root ganglion (DRG) and motor neurons ErbB3 heterodimer formation, which leads to receptor that is observed in NRG1 null mutant mice. Alternatively, cross-phosphorylation, recruitment of SH3-containing conventional NRG1 (forward) signaling could elicit www.sciencedirect.com Current Opinion in Neurobiology 2006, 16:492–500 494 Neuronal and glial cell biology

reciprocal trophic support of ensheathed axons, such as indicate that final matching of SCPs to axons is mediated through NRG1-stimulated release of neurotrophins by glia through competition for a NRG1-mediated survival sig- [19]. Finally, the cytoplasmic tail of NRG1 has been nal [24–26]. NRG1 type III is the key isoform required reported to interact directly with LIM kinase, a regulator for SCP survival and migration during early embryogen- of the actin cytoskeleton [20]; the biological significance of esis [26]. this interaction is not yet established. Threshold levels of NRG1 type III are an instructive The role of NRG1 in Schwann cell myelination signal for myelination NRG1 has a crucial role at essentially every develop- Once generated, SCPs differentiate into mature Schwann mental stage of Schwann cells, as first indicated by both cells that either ensheath multiple small, unmyelinated culture studies and analysis of knockout mice [21,22]. axons, forming a Remak bundle, or sort larger axons into a These functions include promoting the gliogenic fate of 1:1 relationship that they subsequently myelinate (sche- trunk neural crest cells, the migration of Schwann cell matically summarized in Figure 2). These alternative precursors (SCP) along axons, and their subsequent phenotypes are distinguishable not only by their anatomic proliferation and survival induced by axons. A recent relationship to the axon but by the repertoire of transcrip- study, analyzing zebrafish ErbB mutants, strongly sup- tion factors and proteins that Schwann cells express. The ports the key role of NRG1–ErbB signaling in SCP axon determines this binary choice in Schwann cell proliferation and directed migration along axons phenotypes, as first demonstrated in classic studies per- although, surprisingly, not for survival [23]. This result formed over a century ago in which myelinated and non- contrasts with studies in rodents and chick, which myelinated nerves were cross-anastomosed [27]. As

Figure 2

Axonal NRG1 regulates successive steps of Schwann cell differentiation. (a) Schwann cells (in blue) arise from neural crest precursor cells (in green) and interact with both large and small caliber axons of spinal motor and sensory neurons. During embryogenesis, NRG1 on the axon regulates Schwann cell development by activating ErbB signaling cascades, thereby promoting Schwann cell differentiation and expansion. The amount of NRG1 type III on the axon detected by committed Schwann cells, which is a function of axon size and NRG1 levels, then drives them either into segregating single axons and myelination (top), or into a non-myelinating phenotype and formation of a Remak bundle (bottom). Above threshold levels, NRG1 type III signals axon size to Schwann cells to optimize myelin sheath thickness. (b) In mouse mutants lacking NRG1 (/), in heterozygous NRG1 (+/) mice, and in transgenic NRG1 overexpressing mice, the amount of myelin made by Schwann cells varies directly as a function of axonal NRG1 type III levels (indicated by yellow dots) rather than as a function of axon diameter. See [30,32] for further details.

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myelination in the PNS typically commences around axon surface. Altering this communication impacts on the axons with a diameter of 1 mm or greater, it was initially g-ratio of myelinated axons. Thus, heterozygous NRG1 posited that a critical axonal diameter was the trigger for type III null mutant mice, that display roughly 50% of the Schwann cell myelination [28,29]. NRG1, are hypomyelinated, have commensurate reduc- tion in myelin transcription factors and exhibit reduced Recent studies by Taveggia et al. [30]ontheroleof nerve conduction velocities [30,32]. By contrast, trans- NRG1 in myelination suggest that the level of NRG1 genic mice that overexpress NRG1 in dorsal root ganglia type III on the axon, rather than axon diameter per se,is and motor neurons (under control of the neuronal ThyI the key instructive signal for myelination. Expression of promoter) become hypermyelinated [32]. Axon-to- NRG1 type III on the axon correlates with the Schwann appears specific to NRG1 type ensheathment fate of axons: unmyelinated, autonomic III, as transgenic mice that overexpress the secreted neurons express low levels of NRG1 type III on the NRG1 type I are not hypermyelinated; this specificity axon surface, whereas brain-derived neurotrophic factor might reflect a requirement for juxtacrine signaling char- (BDNF) and -3 (NT-3) dependent dorsal acteristic of the type III isoform [9,30,32]. root ganglion (DRG) neurons, the axons of which are heavily myelinated, express high levels. DRG axons Whereas NRG1 has emerged as the rate-limiting factor of from NRG1 type III null mice are not myelinated by myelin growth control, ErbB2 and ErbB3 are expressed at Schwann cells in cocultures and do not induce myelin- saturating levels [32]. They must be dramatically reduced specific structural proteins or transcription factors, thus to disrupt myelination, such as in a conditional mouse demonstrating that axonal NRG1 type III is essential for mutant carrying the Schwann cell-specific null mutation myelination [30]. Furthermore, mice haploinsufficient of the ErbB2 [33]. Similarly, mice that express a for NRG1 type III have a significantly higher proportion dominant-negative ErbB receptor in Schwann cells under of axons that are persistently unmyelinated [30]. control of the 20,30-cyclic nucleotide phosphodiesterase Accordingly, pharmacologic inhibitors of ErbB receptors (CNPase) promoter are hypomyelinated and have thinner demonstrated a requirement for NRG1 signaling for the myelin sheaths than normal [34]. The length of myelin initiation of myelination in zebrafish [23]. Strikingly, internodes in mice expressing a dominant negative ErbB forced expression of NRG1 type III in the post-gang- receptor is also shorter than normal [34], in contrast to lionic fibers of sympathetic neurons converts these mice with reduced NRG1 dosages that have internodes of normally unmyelinated fibers to myelinated ones normal length [32]. in vitro [30]. Likewise, in transgenic mice, overexpres- sion of NRG1 type III induces earlier onset of myelina- Taken together, these studies support a model in which tion in the PNS than normal, and results in myelination threshold levels of NRG1 type III are required to trigger of very small-caliber C-fiber axons that would normally myelination. Above this threshold, the amount of myelin be unmyelinated (M Schwab and KA Nave, unpub- formed is graded to the amount of NRG1 type III pre- lished). Taken together, these results suggest that sented by the axon to the Schwann cell — an amount likely threshold levels of NRG1 type III provide the long to reflect both the concentration of NRG1 type III at the sought instructive signal that triggers Schwann cell membrane and the axon surface area (a function of axon myelination (Figure 2). diameter). These results also provide a mechanism by which axons, through differing levels of NRG1 type III, NRG1 type III levels regulate myelin thickness coordinate Schwann cell numbers to their alternative phe- NRG1–ErbB signaling has also been recruited for the notypes. Thus, axons that express higher levels of NRG1 important regulatory step associated with the final stage type III generate the additional Schwann cells required to of Schwann cell differentiation: the quantitative control establish the 1:1 relationship characteristic of myelinated of myelin sheath thickness. When aiming for the most fibers and, subsequently, drive axon segregation and rapid impulse propagation, which is a function of axon myelination. caliber and myelination, the optimal myelin thickness is reached when the ‘g-ratio’ (i.e. the numeric ratio between Role of NRG1 in myelin maintenance and the injury the diameter of the axon cylinder and that of the mye- response linated axon) is close to 0.68. For most vertebrates, this Recent studies have addressed whether ongoing NRG1 ratio is remarkably well maintained for peripheral axons signaling is required to maintain the axon-myelinating and their myelinating glia independent of the specific Schwann cell unit after it has formed, and whether axon diameter as first noted by Donaldson and Hoke [31]. NRG1 has a role during . To This requires axon size to be perceived by myelinating address the first question, Atanasoski et al. [35] ablated Schwann cells, to make the correct number of myelin the ErbB2 receptor gene in adult myelinating Schwann wraps. Michailov et al. [32] demonstrated that this axon cells using tamoxifen-inducible Cre recombinase, size information is encoded, at least in part, by the amount expressed under the control of the proteolipid protein of membrane-associated NRG1 type III displayed on the promoter (PLP); they found, surprisingly, that myelin www.sciencedirect.com Current Opinion in Neurobiology 2006, 16:492–500 496 Neuronal and glial cell biology

sheaths were unaffected even after two months. Results of impaired glial-derived neurotrophic factor (GDNF) from this study strongly suggest that ongoing ErbB2 sig- expression by Schwann cells [43]. These observations naling, and by inference NRG1 activity, is dispensable for also reveal that ensheathing glial cells in the PNS, as maintenance of the myelin sheath in the adult. This result in the CNS [3], have a primary axon-protective function, is also consistent with the maintenance of myelin sheaths independent of myelination. in neuron–Schwann cell cocultures even when continu- ously treated with pharmacologic inhibitors of signaling NRG1-activated signaling pathways and myelination pathways activated by NRG1 [36]. The signaling pathways by which axons, through NRG1, promote Schwann cell myelination have also begun to NRG1 has also been considered a candidate to mediate emerge. NRG1 robustly activates mitogen-activated pro- the Schwann cell response to injury as it is persistently tein (MAP) kinase and PtdIns 3-kinase pathways in cul- expressed by adult axons [30,32,37], and could there- tured Schwann cells (reviewed in [5,44]). Axonal contact fore be released during Wallerian degeneration to sti- also robustly activates these signaling pathways in mulate Schwann cell proliferation. In support, ErbB2 is Schwann cells [36] — NRG1 type III is the key neuronal rapidly activated (phosphorylated) within minutes of signal that mediates PtdIns 3-kinase activation, whereas injury [38] followed by a delayed, and sustained (over other signals, distinct from NRG1 type III but not yet days), upregulation of ErbBreceptorsandNRG1iso- identified, activate MAP kinase [30]. Activation of the formsbySchwanncells[37,39]. In addition, several PtdIns 3-kinase pathway, and its downstream effectors, investigators have reported that addition of NRG1 type notably the serine-threonine kinase Akt, are crucial for the II results in demyelination and proliferation in cocul- trophic, proliferative and differentiative responses of tures [38,40,41] or when expressed as a transgene in Schwann cells to axons and NRG1. Pharmacologic inhibi- Schwann cells under the control of the P0 promoter tion of PtdIns 3-kinase blocks the ability of axons to in vivo [42]. These findings suggest that NRG1 isoforms promote Schwann cell proliferation, survival and induction could function as an injury signal, or contribute to nerve of myelination [36]. Expression of dominant negative pathology, when either misexpressed or aberrantly pre- forms of PtdIns 3-kinase or Akt in Schwann cells inhibits sented to the outer (abaxonal) Schwann cell membrane. myelination in vitro, whereas overexpression of PtdIns Important support for a role of NRG1–ErbB signaling in 3-kinase or activated Akt promote myelin protein expres- the injury response was also provided by studies in sion in vitro and enhanced myelin sheath formation during which pharmacologic inhibition of ErbB2 receptors regeneration in vivo [45]. In contrast to its promyelinating blocked Schwann cell proliferation after injury in vitro effects, activation of MAP kinase pathways inhibits mye- and in vivo [38]. Surprisingly, in a second study in lination [45] and causes myelinating Schwann cells to which ErbB2 expression was conditionally ablated dedifferentiate and proliferate [41]. This has led to the in vivo before injury, no effect on Schwann cell prolif- notion that the balance between these two major signaling eration during Wallerian degeneration was seen [35]. pathways determines the differentiative state of Schwann The reasons for the discrepancy between these two cells [45]. studies is not yet clear. Two important signals that also regulate Schwann cell NRG1 regulates axon–Schwann cell interactions in differentiation, laminin in the extracellular matrix Remak fibers (ECM) and cAMP-dependent pathways, were recently Although axonal NRG1 is the primary signal for immature implicated in NRG1-dependent signaling. Mice defi- Schwann cells to adopt the myelin-forming phenotype cient in laminin expression fail to ensheath axons appro- ([30] and Schwab MH, Humml C, Nave K-A, unpub- priately and exhibit markedly reduced ErbB–PtdIns-3 lished), NRG1–ErbB signaling is also important for the kinase signaling associated with increased Schwann cell organization and function of adult non-myelinating apoptosis [46]. Impaired ErbB signaling might result Schwann cells. Adult heterozygous NRG1 Type III null from aberrant physical interactions between Schwann mice exhibit impaired sorting of unmyelinated axons into cells and axons and/or altered co-signaling by ECM separate Schwann cell pockets, which instead frequently components. Laminin, through interaction with persist as large bundles [30]. These results indicate a receptors, has been reported to switch the signaling more general role for NRG1 signals in axon sorting by pathways mediated by soluble NRG1 isoforms in cul- Schwann cells. Overexpression of a truncated (dominant- tured oligodendrocytes [47] and so might also qualitia- negative) ErbB4 receptor under control of the glial fibril- tively affect Schwann cell signaling activated by NRG1. lary acidic protein (GFAP) promoter in Remak Schwann Moreover, addition of cAMP analogs to cultured cells also leads to significant abnormalities. These include Schwann cells mimics the effect of axonal contact and progressive loss of small C-fiber axons, which leads to has synergistic effects with NRG1 [22]. Elevated death of DRG neurons, pain insensitivity and a novel cAMP increases ErbB receptor expression and leads neuropathy phenotype; neuron cell loss might reflect to sustained activation of NRG1-dependent signaling significant reductions of glial support, potentially because pathways [48,49].

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A role for neurotrophins in axonal NRG1 issue is yet to be resolved. However, several studies signaling suggest that NRG1–ErbB signaling might regulate oli- Neurotrophins exert multiple effects on developing glia, godendrocyte development and provide insights into its including Schwann cells and oligodendrocytes. A remark- potential role during differentiation. able ability to stimulate Schwann cell differentiation in vivo was observed by Griffin and co-workers [50], Initial studies in which oligodendrocyte progenitor cell who found that the injection of glial-derived growth factor (OPC) cultures were supplemented with soluble NRG1 (GDNF) into rats caused non-myelinating Schwann cells isoforms suggested that NRG1 has trophic and mitogenic to proliferate and even to myelinate some of the very effects on cells in the oligodendrocyte lineage; their small caliber C-fiber axons. These experiments do not effects on differentiation varied considerably between distinguish between a direct effect on glia and an indirect studies (reviewed in [53]). Analysis of the role of one (e.g. by stimulating axons to express NRG1). Esper NRG1 in a more physiologic context has been con- and Loeb [51] demonstrated that founded by the early embryonic lethality of NRG1 and (NGF) and GDNF, both of which are expressed by ErbB receptor knockouts. As an alterative strategy, the Schwann cells, induce the rapid release of NRG1 from role of NRG1 and ErbB receptors in oligodendrocyte the axons of cultured DRG neurons and motor neurons. development has been analyzed during the ex vivo devel- Although the molecular mechanisms are not fully under- opment of embryonic spinal cord from mice deficient in stood, this neurotrophin-inducible NRG1 release occurs these proteins. These studies suggest a complex role of within minutes, is dose-dependent, and can be mimicked NRG–ErbB signaling in the oligodendrocyte lineage. by protein kinase C activation. It probably involves OPC development was reported to be markedly deficient regulated proteolytic processing of cleavable NRG1 iso- in spinal cord explants from NRG1 null mice [54]. Loss of forms, such as transmembrane type I and II isoforms. ErbB2 likewise impaired OPC differentiation [55]. How- Taken together with evidence that NRG1 promotes ever, loss of ErbB3 had no effect on oligodendrocyte GDNF expression by Schwann cells in Remak fibers differentiation or myelination [56], whereas loss of ErbB4 [43], it appears that a regulatory loop of glial-axon-glial paradoxically enhanced oligodendrocyte differentiation signaling through growth factors determines both neuro- [17]. It is probably the case that altered rather than nal survival and Schwann cell differentiation in the per- completely absent ErbB signaling in these receptor ipheral nervous system. knockouts accounts for these widely divergent effects on oligodendrocyte differentiation. Mice that express a Independently, Chan et al. [52] showed that neurotro- chimeric, dominant negative ErbB receptor protein to phins have strong but opposing effects on myelination by inhibit ErbB signaling broadly in the oligodendrocyte Schwann cells versus oligodendrocytes. In DRG– lineage had increased numbers of progenitor cells, Schwann cell cocultures, adding NGF to the medium reduced numbers of mature oligodendrocytes, and were promoted myelination, whereas in DRG–oligodendro- hypomyelinated [57]. This hypomyelination suggests cyte cocultures, NGF had an inhibitory effect. The that ErbB signaling, and potentially NRG1 or related requirement of (neuronal) TrkA receptors and the results ligands, could regulate CNS myelination. In the future, of experiments with Campenot chambers suggest that analyses of conditional NRG1 and ErbB receptor knock- neurons, not glial cells, are the crucial target cells of NGF outs are expected to more precisely delineate the impor- signaling. Although the idea that neurons are stimulated tant issue of the role of this signaling pathway in the by NGF is in agreement with the data of Esper and Loeb oligodendrocyte lineage. [51], the divergent responses of Schwann cells and oli- godendrocytes to DRG neurons treated with NGF is Clinical implications unexpected. It contradicts the view that axons that pro- Null mutations of the NRG1 gene and its receptors are ject from the CNS into the PNS (and vice versa) are embryonically lethal in mice [7], suggesting that human uniformly myelinated because they provide the same NRG1 loss-of-functions are unlikely to be a primary cause signals to oligodendrocytes and Schwann cells. Modifying of disease. However, the many roles of NRG1 in glial cell this view might help to elucidate temporal differences in development suggest that dysregulated NRG1 expres- the onset of CNS and PNS myelination. It is not yet sion (or abnormal Nrg1-ErbB-PI3K signaling) contributes known whether some of these effects involve NRG1 to disorders of myelin as a disease modifier or a genetic signaling. The relevance of these in vitro observations risk factor. Although this is an interesting possibility, it in normal development remains to be determined, pos- remains speculative as no evidence directly links NRG1 sibly by careful analysis of existing mouse mutants. to myelin disorders as of yet.

The role of NRG1 in oligodendrocyte One potential example is the inherited demyelinating development neuropathies (Charcot-Marie-Tooth [CMT] disease An obvious question is whether NRG1 type III also type 1) of the peripheral nervous system that result regulates oligodendrocyte myelination — this important from the abnormal expression of myelin membrane www.sciencedirect.com Current Opinion in Neurobiology 2006, 16:492–500 498 Neuronal and glial cell biology

proteins [58,59] including P0, PMP22, and connexin32. plausible susceptibility gene to contribute to human Non-specific pathological features of CMT1 include dys- [70]. Interestingly, CNS myelin abnorm- myelination and ‘onion bulb’ formation by supernumerary alities have also been independently associated with Schwann cells. It is intriguing to speculate that perturbed schizophrenia and other neuropsychiatric diseases on physical interactions, for example between axons and the basis of gene array and imaging studies [71,72], adding ensheathing glial membranes, secondary to abnormal credence to a potential glial contribution. expression of myelin proteins or gap junction proteins, alter NRG1-ErbB-PI3K signaling, and contribute to the Outlook and conclusions CMT1 pathology. The identification of NRG1 as the axonal signal that drives the entire Schwann cell lineage, including myelination, is In the adult central nervous system, multiple sclerosis an important milestone that will facilitate elucidation of (MS) lesions often fail to remyelinate, despite the pre- the mechanisms that underlie the morphogenetic and sence of oligodendrocytes and OPC [60]. If remyelination transcriptional events of myelination. In the PNS, impor- occurs at all, the resulting sheaths are abnormally thin tant remaining questions include how NRG1-dependent [61]. This is reminiscent of the thin sheaths of remyeli- activation of PtdIns 3-kinase initially promotes prolifera- nated peripheral nerves [62] and the hypomyelination tion but later drives differentiation of Schwann cells, how seen in the PNS of mice with reduced NRG1 gene dosage NRG1 signaling strength regulates the binary choice of [30,32]. An intriguing possibility is that the steady-state Schwann cell phenotypes, what limits NRG1 signaling levels of NRG1 or related growth factors on myelinated once myelination is complete, and whether perturbations axons in the adult CNS (or PNS) are reduced below the of NRG1 signaling contribute to neuropathology. Future level required for robust remyelination, particularly in studies should also clarify the role of NRG1, and related demyelinated regions [63,64]. These ideas can be experi- genes, in CNS myelination, including the question of mentally tested in mice with a genetically modified NRG1 whether these axonal factors limit the efficacy of remye- expression level, when challenged by either an experi- lination in the adult brain. Finally, the temporal and mental autoimmune encephalomyelitis (EAE) or a toxin- quantitative control of NRG1 expression by neurons induced demyelination to undergo spontaneous remye- remains to be explored. Elucidating these questions pro- lination. Earlier reports that the systemic application of mises to provide important insights into the role of axon– recombinant NRG1 improved the course of EAE in mice, glial signaling in demyelinating disease and myelin repair. with respect to disease onset and severity [63,65], con- trasts with the inability to improve the degree of remye- Acknowledgements lination in toxin-induced rat CNS lesions [66]. This Owing to space limitations, we regret any omissions in citing other relevant publications. We thank C Birchmeier, D Falls, C Lai, J Loeb, M Schwab, suggests that the beneficial effects of systemic NRG1 and C Taveggia for insightful discussions and for comments on the administration have been indirect, for example, through manuscript. Work from the authors’ laboratories cited in this review has immune modulation. It is our experience from experi- been supported by grants from the Duetsche Forschungsgemeinschaft (Center for the Molecular Physiology of the Brain), National Institutes of ments in the peripheral nervous system that functional Health, and the National Multiple Sclerosis Society. NRG1 signaling to myelinating glia must be presented directly by the axon [30,32], whereas ectopic stimula- References and recommended reading tion by soluble NRG1 might even have detrimental Papers of particular interest, published within the annual period of effects on myelination [40]. This might reflect the review, have been highlighted as: requirement for NRG1 to signal in a juxtacrine mode, of special interest or that NRG1 only promotes oligodendrocyte differentia- of outstanding interest tion in the context of laminin–integrin signaling at the 1. Waxman SG: Axon-glia interactions: building a smart nerve axon–glial interface [47]. fiber. Curr Biol 1997, 7:R406-R410. 2. Griffiths I, Klugmann M, Anderson T, Yool D, Thomson C, Finally, there are intriguing links between NRG1 expres- Schwab MH, Schneider A, Zimmermann F, McCulloch M, sion, myelination and neuropsychiatric disorders. Stefans- Nadon N et al.: Axonal swellings and degeneration in mice lacking the major proteolipid of myelin. Science 1998, son and coworkers [67] reported the association of single 280:1610-1613. 0 region of the nucleotide polymorphisms (SNPs) in the 5 3. Lappe-Siefke C, Goebbels S, Gravel M, Nicksch E, Lee J, NRG1 gene with schizophrenia in the Icelandic popula- Braun PE, Griffiths IR, Nave KA: Disruption of Cnp1 uncouples tion, an association now confirmed in other populations oligodendroglial functions in axonal support and myelination. Nat Genet 2003, 33:366-374. [68]. The molecular consequences of the crucial NRG1 4. Falls DL: : functions, forms, and signaling ‘at risk’ are still unclear as the SNPs all corre- strategies. Exp Cell Res 2003, 284:14-30. spond to non-coding variants, but recent evidence sug- 5. Esper RM, Pankonin MS, Loeb JA: Neuregulins: versatile growth gests alterations of expression of NRG1 types I, III and IV and differentiation factors in nervous system development [6,69]. Because NRG1–ErbB signaling in the CNS affects and human disease. Brain Res Brain Res Rev 2006. A comprehensive review, together with Falls [4], of the structure and oligodendrocyte development in addition to neuroblast function of neuregulin 1 in the developing nervous system, including migration and glutamatergic function, NRG1 is a potential implications in neurologic diseases.

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Inhibiting is required for Schwann cell proliferation and migration in time-lapse ErbB2 kinase markedly slowed the progression of Wallerian degeneration studies, but unexpectedly not for survival; pharmacologic inhibitors both in a coculture model and in vivo, providing presumptive evidence for implicated erbB signaling in myelin initiation. a role of NRG–ErbB signaling in the injury response (see also [35]). www.sciencedirect.com Current Opinion in Neurobiology 2006, 16:492–500 500 Neuronal and glial cell biology

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