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• W1965571714 abstract "The radical response of peripheral nerves to injury (Wallerian degeneration) is the cornerstone of nerve repair. We show that activation of the transcription factor c-Jun in Schwann cells is a global regulator of Wallerian degeneration. c-Jun governs major aspects of the injury response, determines the expression of trophic factors, adhesion molecules, the formation of regeneration tracks and myelin clearance and controls the distinctive regenerative potential of peripheral nerves. A key function of c-Jun is the activation of a repair program in Schwann cells and the creation of a cell specialized to support regeneration. We show that absence of c-Jun results in the formation of a dysfunctional repair cell, striking failure of functional recovery, and neuronal death. We conclude that a single glial transcription factor is essential for restoration of damaged nerves, acting to control the transdifferentiation of myelin and Remak Schwann cells to dedicated repair cells in damaged tissue. The radical response of peripheral nerves to injury (Wallerian degeneration) is the cornerstone of nerve repair. We show that activation of the transcription factor c-Jun in Schwann cells is a global regulator of Wallerian degeneration. c-Jun governs major aspects of the injury response, determines the expression of trophic factors, adhesion molecules, the formation of regeneration tracks and myelin clearance and controls the distinctive regenerative potential of peripheral nerves. A key function of c-Jun is the activation of a repair program in Schwann cells and the creation of a cell specialized to support regeneration. We show that absence of c-Jun results in the formation of a dysfunctional repair cell, striking failure of functional recovery, and neuronal death. We conclude that a single glial transcription factor is essential for restoration of damaged nerves, acting to control the transdifferentiation of myelin and Remak Schwann cells to dedicated repair cells in damaged tissue. Schwann cell c-Jun is a master regulator of the PNS injury response c-Jun activates a defined repair program in Schwann cells of damaged nerves c-Jun controls transdifferentiation of differentiated Schwann cells to repair cells Schwann cell c-Jun is essential for neuronal survival and functional recovery How transcription factors control cellular plasticity and maintain differentiation is currently of great interest, inspired by the success of experimental reprogramming, where remarkable phenotypic transitions can be induced by enforced expression of fate determining factors (Zhou and Melton, 2008Zhou Q. Melton D.A. Extreme makeover: converting one cell into another.Cell Stem Cell. 2008; 3: 382-388Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). These findings raise a key question: to what extent are natural transitions in the state of differentiated cells also governed by specific transcription factors? Such phenotypic transitions are seen in tumorigenesis, dedifferentiation and transdifferentiation. They are also fundamental to tissue repair and regeneration, and in regenerative systems, a major focus of work is identification of gene programs that are selectively activated after injury and which impact the repair process. The striking regenerative capacity of the PNS rests on the surprising plasticity of Schwann cells, and the ability of these cells to switch between differentiation states, a feature that is highly unusual in mammals (Jessen and Mirsky, 2005Jessen K.R. Mirsky R. The origin and development of glial cells in peripheral nerves.Nat. Rev. Neurosci. 2005; 6: 671-682Crossref PubMed Scopus (981) Google Scholar, Jessen and Mirsky, 2008Jessen K.R. Mirsky R. Negative regulation of myelination: relevance for development, injury, and demyelinating disease.Glia. 2008; 56: 1552-1565Crossref PubMed Scopus (366) Google Scholar; Jopling et al., 2011Jopling C. Boue S. Izpisua Belmonte J.C. Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration.Nat. Rev. Mol. Cell Biol. 2011; 12: 79-89Crossref PubMed Scopus (458) Google Scholar). In a process reminiscent of the radical injury responses of zebrafish cardiomyocytes or pigment cells of the newt iris, nerve injury, and loss of axonal contact causes mammalian Schwann cells to lose their differentiated morphology, downregulate myelin genes, upregulate markers of immature Schwann cells, and re-enter the cell cycle. This radical process of natural dedifferentiation has few if any parallels in mammalian systems. At the same time as Schwann cells dedifferentiate, they upregulate genes implicated in promoting axon growth, neuronal survival, and macrophage invasion, and activate mechanisms to break down their myelin sheaths and transform morphologically into cells with long, parallel processes. This allows them to form uninterrupted regeneration tracks (Bands of Bungner) that guide axons back to their targets (Chen et al., 2007Chen Z.L. Yu W.M. Strickland S. Peripheral regeneration.Annu. Rev. Neurosci. 2007; 30: 209-233Crossref PubMed Scopus (596) Google Scholar; Vargas and Barres, 2007Vargas M.E. Barres B.A. Why is Wallerian degeneration in the CNS so slow?.Annu. Rev. Neurosci. 2007; 30: 153-179Crossref PubMed Scopus (354) Google Scholar; Gordon et al., 2009Gordon T. Chan K.M. Sulaiman O.A. Udina E. Amirjani N. Brushart T.M. Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury.Neurosurgery. 2009; 65: A132-A144Crossref PubMed Scopus (108) Google Scholar). Collectively, these events together with the axonal death that triggers them are called Wallerian degeneration. This response transforms the normally growth-hostile environment of intact nerves to a growth supportive terrain, and endows the PNS with its remarkable and characteristic regenerative potential. To complete the repair process, Schwann cells envelop the regenerated axons and transform again to generate myelin and nonmyelinating (Remak) cells. Little is known about the transcriptional control of changes in adult differentiation states, including natural dedifferentiation and transdifferentiation, in any system (Jopling et al., 2011Jopling C. Boue S. Izpisua Belmonte J.C. Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration.Nat. Rev. Mol. Cell Biol. 2011; 12: 79-89Crossref PubMed Scopus (458) Google Scholar). In line with this, although Wallerian degeneration including the Schwann cell injury response are key to repair, the molecular mechanisms that control these processes are not understood (Chen et al., 2007Chen Z.L. Yu W.M. Strickland S. Peripheral regeneration.Annu. Rev. Neurosci. 2007; 30: 209-233Crossref PubMed Scopus (596) Google Scholar; Jessen and Mirsky, 2008Jessen K.R. Mirsky R. Negative regulation of myelination: relevance for development, injury, and demyelinating disease.Glia. 2008; 56: 1552-1565Crossref PubMed Scopus (366) Google Scholar). Conceptually also, the nature of the Schwann cell injury response has remained uncertain, since the generation of the denervated Schwann cell is commonly referred to either as dedifferentiation or as activation. These terms highlight two distinct aspects of the process, namely loss of the differentiated Schwann cell phenotypes of normal nerves and gain of the regeneration promoting phenotype, respectively, without providing a framework for analysis and comparison with other regenerative models. Here, we use mice with selective inactivation of the transcription factor c-Jun in Schwann cells to show that c-Jun is a global regulator of the Schwann cell injury response that specifies the characteristic gene expression, structure, and function of the denervated Schwann cell, a cell that is essential for nerve repair. Consequently, axonal regeneration and functional repair are strikingly compromised or absent when Schwann cell c-Jun is inactivated. Notably, the effects of c-Jun are injury specific, since c-Jun inactivation has no significant effects on nerve development or adult nerve function. These observations provide a molecular basis for understanding Schwann cell plasticity, show that c-Jun is a key regulator of Wallerian degeneration, and offer conclusive support for the notion that glial cells control repair in the PNS. They also show that the Schwann cell injury response has much in common with transdifferentiation, since it represents the generation, by dedicated transcriptional controls, of a distinct Schwann cell repair phenotype, specialized for supporting axon growth and neuronal survival in injured nerves. Because these cells form the regeneration tracks called Bungner’s bands, we will refer to them as Bungner cells. Earlier, we found that neonatal mice with conditional deletion of c-Jun in Schwann cells (c-Jun mutant mice) show delayed loss of myelin proteins and mRNA after nerve injury (Parkinson et al., 2008Parkinson D.B. Bhaskaran A. Arthur-Farraj P. Noon L.A. Woodhoo A. Lloyd A.C. Feltri M.L. Wrabetz L. Behrens A. Mirsky R. Jessen K.R. c-Jun is a negative regulator of myelination.J. Cell Biol. 2008; 181: 625-637Crossref PubMed Scopus (285) Google Scholar). This suggested that Schwann cell c-Jun might play an important role in specifying the phenotype of denervated Schwann cells. To test this comprehensively, we used Affymetrix whole-genome microarray to examine gene expression in the sciatic nerve of adult c-Jun mutant mice and control (WT) littermates and compared this with gene expression in denervated cells in the distal stump of transected nerves without regenerating axons, to avoid the complicating effects of axon-induced redifferentiation (Figure 1). We chose 7 days after injury since in regenerating mouse nerves this is near the mid-point of active axonal regrowth. Seven day denervated cells therefore represent the terrain that confronts regenerating axons in WT and mutant nerves. Before injury, the nerves of adult c-Jun mutant mice were normal on the basis of a number of criteria. Thus, the numbers of myelinated and unmyelinated axons (see Figures 4E and 4F), myelinating Schwann cells and Remak bundles (see Table S1 available online), g-ratios (Figure S1), sciatic functional index (SFI) (see Figure 7E), motor performance in a rotarod test (unpublished), and responses to heat and light touch (see Figures 7B and 7C) were similar to WT controls. While c-Jun was excised from almost all Schwann cells (Parkinson et al., 2008Parkinson D.B. Bhaskaran A. Arthur-Farraj P. Noon L.A. Woodhoo A. Lloyd A.C. Feltri M.L. Wrabetz L. Behrens A. Mirsky R. Jessen K.R. c-Jun is a negative regulator of myelination.J. Cell Biol. 2008; 181: 625-637Crossref PubMed Scopus (285) Google Scholar), c-Jun expression in neurons, macrophages, and fibroblasts was normal, and the rate of axonal disintegration after cut was similar in WT and mutants (Figures S2 and S3). The close similarity between WT and mutant nerves was confirmed by the Affymetrix screen (Figure 1), since only two genes (keratin 8 and desmoplakin) were differentially expressed. Furthermore, following injury, a comparable number of genes changed expression in WT and c-Jun mutants (Figure 1A). Importantly, however, comparison of the distal stumps of WT and c-Jun mutants revealed 172 significant differences in gene expression (Figure 1 and Tables S2 and S3). The differentially regulated genes included genes which have been implicated in regeneration and trophic support such as BDNF, GDNF, Artn, Shh, and GAP-43 that failed to upregulate after injury, together with genes that failed to downregulate normally after injury such as the myelin genes Mpz, Mbp, and Cdh1 (also known as E-cadherin). Gene ontology analysis indicated that known functions of these 172 genes were particularly related to neuronal growth and regeneration (Figure 1C). We selected 32 of the 172 disregulated genes for further analysis by RT-QPCR. In every case this confirmed the disregulation shown by the microarray data (Figures 1D–1F and Table S3). Six of the thirty-two genes were then analyzed in purified Schwann cell cultures. Comparison of c-Jun mutant and WT cells confirmed the regulation seen in the distal stumps. Furthermore, as predicted, enforced c-Jun expression in c-Jun mutant cells, by adenoviral gene transfer, activated c-Jun, BDNF, and GDNF expression but suppressed Chd1, Mpz, and Mbp expression (Figure 2A). These results show directly that c-Jun regulates these genes in Schwann cells, demonstrates that this control is independent of the nerve environment, and confirms results obtained by microarray and RT-QPCR. Lastly, we found that three proteins implicated in regeneration, N-cadherin, p75NTR, and NCAM, were disregulated in cut mutant nerves, although their mRNAs were normally expressed. Injured mutant nerves expressed strongly reduced N-cadherin and p75NTR but elevated levels of NCAM (Figures 2B and 2C). Sox2 protein, which, like c-Jun, is upregulated in WT Schwann cells of injured nerves (Parkinson et al., 2008Parkinson D.B. Bhaskaran A. Arthur-Farraj P. Noon L.A. Woodhoo A. Lloyd A.C. Feltri M.L. Wrabetz L. Behrens A. Mirsky R. Jessen K.R. c-Jun is a negative regulator of myelination.J. Cell Biol. 2008; 181: 625-637Crossref PubMed Scopus (285) Google Scholar), remained normally upregulated in injured nerves of c-Jun mutants (Figure S4). Denervated Schwann cells in injured adult nerves are often considered similar to immature Schwann cells in developing nerves. However, the immature cells for instance do not share the axon guidance, myelin breakdown and macrophage recruitment functions of denervated cells, and these cells differ in molecular expression (Jessen and Mirsky, 2008Jessen K.R. Mirsky R. Negative regulation of myelination: relevance for development, injury, and demyelinating disease.Glia. 2008; 56: 1552-1565Crossref PubMed Scopus (366) Google Scholar). To explore the idea that the denervated cell represents a distinct Schwann cell phenotype regulated by c-Jun, we examined three genes, Olig1, Shh, and GDNF, which showed strong, c-Jun-dependent activation in denervated cells (Figure 1D). Using RT-QPCR and in situ hybririsization we confirmed strong expression of these genes in WT adult denervated cells, but found that they were not (Olig1 and Shh) or borderline (GDNF) detectable in immature Schwann cells (from WT embryo day 18 nerve). They were also essentially absent from uncut nerves (Figures 2D and 2E and Table S4). This supports the notion that denervated adult Schwann cells and immature Schwann cells in perinatal nerves represent distinct cell types. It shows also that c-Jun takes part in controlling the distinctive molecular profile of the adult denervated cell. The response of neonatal cells to injury remains to be determined. Together these results show that c-Jun controls the molecular reprogramming that transforms mature Schwann cells to the denervated cell phenotype following injury. This includes the regulation of genes that differentiate denervated from immature cells and extends to the posttranscriptional control of protein expression. Denervated Schwann cells form cellular columns that replace the axon-Schwann cell units of intact nerves and serve as substrate for growing axons. We examined these structures by electron microscopy in the distal stump 4 weeks after cut. Because these cells have been without axonal contact for 4 weeks they are comparable to the cells encountered by growing axons in distal parts of crushed nerves in the c-Jun mutant where regeneration is delayed beyond the normal 3–4 week period, while at this time WT nerves have just reached their targets. We found that the structure of these regeneration tracks is strikingly abnormal in c-Jun mutants (Figure 3A). There are many fewer cell profiles per column, indicating reduced process formation (Figure 3B) and the cells have flattened as confirmed by reduction in the roundness index of cell profiles in vivo (Figure 3C). Flattening and paucity of processes are also seen even in c-Jun−/− cells from neonatal nerves in vitro (Figures 3D and 3E). Therefore, this is a robust phenotype that does not depend on long term denervation in vivo. Thus, c-Jun is an cell-intrinsic determinant of Schwann cell morphology that controls the structure of the essential regeneration tracks that guide growing axons back to correct targets. c-Jun specification of gene expression and morphology of denervated cells suggested that Schwann cell c-Jun might exert a decisive control over nerve repair. Because survival of injured neurons is the basis for repair, we measured the survival of small and large dorsal root sensory (DRG) neurons following sciatic nerve crush at the sciatic notch. We counted axons in L4 dorsal roots (Coggeshall et al., 1997Coggeshall R.E. Lekan H.A. Doubell T.P. Allchorne A. Woolf C.J. Central changes in primary afferent fibers following peripheral nerve lesions.Neuroscience. 1997; 77: 1115-1122Crossref PubMed Scopus (65) Google Scholar) and the tibial nerve, and neuronal somas and nucleoli in DRGs. Comparable results were obtained using all methods. Axon counts in WT dorsal roots showed that 20%–25% of the unmyelinated axons were lost following crush, as expected (Coggeshall et al., 1997Coggeshall R.E. Lekan H.A. Doubell T.P. Allchorne A. Woolf C.J. Central changes in primary afferent fibers following peripheral nerve lesions.Neuroscience. 1997; 77: 1115-1122Crossref PubMed Scopus (65) Google Scholar). In contrast, 55%–60% of these axons were lost in c-Jun mutants, showing increased death of small DRG neurons in the mutant. This was confirmed by corrected (Abercrombie, 1946Abercrombie M. Estimation of nuclear population from microtome sections.Anat. Rec. 1946; 94: 239-247Crossref PubMed Scopus (3912) Google Scholar) counts of B neuron profiles in DRGs, showing 25%–30% loss in WT but 45%–65% loss in the mutants (Figures 4A and 4B ). The number of myelinated axons in dorsal roots remained unchanged in injured WT mice as expected (Coggeshall et al., 1997Coggeshall R.E. Lekan H.A. Doubell T.P. Allchorne A. Woolf C.J. Central changes in primary afferent fibers following peripheral nerve lesions.Neuroscience. 1997; 77: 1115-1122Crossref PubMed Scopus (65) Google Scholar). But surprisingly, in the mutants the number of myelinated axons was reduced by 30%–35%, indicating death of large DRG cells. In confirmation, the corrected number of large A cell profiles in DRGs was reduced by about 40% in the mutants. The number of these profiles did not change significantly in injured WT (Figures 4C and 4D). We also carried out counts on DRG sections using nucleoli as the counted entity, an approach that theoretically provides increased accuracy. Nucleoli in A type DRG neurons from uncut WT (n = 3), 10 week cut WT (n = 3), and 10 week cut c-Jun mutant (n = 3) mice were counted, corrected (Abercrombie, 1946Abercrombie M. Estimation of nuclear population from microtome sections.Anat. Rec. 1946; 94: 239-247Crossref PubMed Scopus (3912) Google Scholar), and expressed as percentage of uncut WT. This showed a 12% reduction in cut WT, (not significant; p > 0.40) but a 50% reduction in the c-Jun mutant (highly significant; p < 0.017). This provides a third line of evidence (in addition to counts of myelinated axons in dorsal roots and cell profiles) for the notion that nerve injury results in the loss of A type DRG neurons in mice that selectively lack c-Jun in Schwann cells. Consistent with neuronal death, the number of myelinated axons in the mutant tibial nerve 10 weeks after crush was reduced by about 35% and unmyelinated axons were reduced by about 65%, both compared to crushed WT controls (Figures 4E and 4F). These experiments show that without Schwann cell c-Jun, small, unmyelinated DRG neurons are about twice as likely to die following axonal damage. Significantly, about a third of the large, myelinated DRG neurons also die in crushed c-Jun mutants, although none die in injured WT controls, and in other studies these cells are resistant to death following axonal damage (Tandrup et al., 2000Tandrup T. Woolf C.J. Coggeshall R.E. Delayed loss of small dorsal root ganglion cells after transection of the rat sciatic nerve.J. Comp. Neurol. 2000; 422: 172-180Crossref PubMed Scopus (189) Google Scholar). These experiments establish that a key function of denervated Schwann cells is to prevent the death of injured neurons and that this rescue depends on c-Jun activation. The number of myelinated axons in ventral roots of both WT and mutant mice remained unchanged following sciatic nerve crush (Table S6). Therefore, unlike DRG neurons, survival of injured ventral horn motoneurons is independent of Schwann cell c-Jun. Nevertheless, the corrected (Abercrombie, 1946Abercrombie M. Estimation of nuclear population from microtome sections.Anat. Rec. 1946; 94: 239-247Crossref PubMed Scopus (3912) Google Scholar) counts of motoneurons that reconnected with the target muscle showed a large reduction in the mutant, even as late as 10 weeks after injury, reaching only about 55% of that in controls, judged by motoneuron backfilling (Figures 5A and 5B ). This indicates that in the mutants, axonal regeneration by surviving neurons is severely and permanently compromised. To analyze regeneration, we examined sciatic nerves 4 days after crush, using the nerve pinch test and by quantifying the number and length of axons in longitudinal sections immunolabeled by CGRP or galanin antibodies to label regenerating DRG and motoneurons. This showed a strong decrease in axon outgrowth in the mutants compared to WT (Figures 5C–5H). Regeneration in the mouse sciatic nerve is independent of Schwann cell proliferation (Kim et al., 2000Kim H.A. Pomeroy S.L. Whoriskey W. Pawlitzky I. Benowitz L.I. Sicinski P. Stiles C.D. Roberts T.M. A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1.Neuron. 2000; 26: 405-416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar; Yang et al., 2008Yang D.P. Zhang D.P. Mak K.S. Bonder D.E. Pomeroy S.L. Kim H.A. Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis.Mol. Cell. Neurosci. 2008; 38: 80-88Crossref PubMed Scopus (75) Google Scholar). Nevertheless, because c-Jun contributes to proliferation in vitro (Parkinson et al., 2008Parkinson D.B. Bhaskaran A. Arthur-Farraj P. Noon L.A. Woodhoo A. Lloyd A.C. Feltri M.L. Wrabetz L. Behrens A. Mirsky R. Jessen K.R. c-Jun is a negative regulator of myelination.J. Cell Biol. 2008; 181: 625-637Crossref PubMed Scopus (285) Google Scholar), we counted Schwann cells in WT and mutant distal stumps (Table S7). In crushed, actively regenerating nerves (14 days after injury) Schwann cell numbers were not significantly different between WT and mutants; both were elevated 5- to 6-fold compared to uncut nerves. Four days after crush, cell numbers were higher in WT nerves, while 7 days after cut, again the difference between mutants and WT was not significant. The tendency toward lower Schwann cell numbers in the mutants is in line with the involvement of c-Jun in proliferation (Parkinson et al., 2004Parkinson D.B. Bhaskaran A. Droggiti A. Dickinson S. D’Antonio M. Mirsky R. Jessen K.R. Krox-20 inhibits Jun-NH2-terminal kinase/c-Jun to control Schwann cell proliferation and death.J. Cell Biol. 2004; 164: 385-394Crossref PubMed Scopus (184) Google Scholar). Together this shows that in the absence of Schwann cell c-Jun, the regeneration of axons from surviving neurons is severely reduced, leading to a permanent deficit in the number of neurons that reconnect with denervated targets. The observation that that Schwann cell numbers in regenerating mutant nerves are elevated up to 5-fold compared to uninjured nerves, together with the independence of regeneration from elevated Schwann cell numbers (Kim et al., 2000Kim H.A. Pomeroy S.L. Whoriskey W. Pawlitzky I. Benowitz L.I. Sicinski P. Stiles C.D. Roberts T.M. A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1.Neuron. 2000; 26: 405-416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar; Yang et al., 2008Yang D.P. Zhang D.P. Mak K.S. Bonder D.E. Pomeroy S.L. Kim H.A. Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis.Mol. Cell. Neurosci. 2008; 38: 80-88Crossref PubMed Scopus (75) Google Scholar) and analysis using microfluidic chambers (following section), shows that that regeneration failure in c-Jun mutants is not caused by lack of Schwann cells. A nerve is a complex cell community. We therefore used microfluidic chambers containing neurons and purified Schwann cells to test whether the poor axon growth in mutants was caused by disturbance of direct axon-Schwann cell interactions or whether the effect depended on other cells. Axon regeneration by axotomized, adult WT DRG neurons was strongly stimulated by control Schwann cells relative to laminin substrate alone, as expected (Figures 5I–5K). The c-Jun mutant cells, however, were ineffective, the number and area of axons extending on their surface falling to only 40%–50% of that seen on WT cells. Importantly, reactivation of c-Jun in mutant cells by adenoviral gene transfer, fully restored axon number and length to WT levels. These experiments show that injury-activated Schwann cell c-Jun controls direct communication between Schwann cells and growing neurites. We have shown that c-Jun controls three important functions of denervated Schwann cells, formation of regeneration tracks, support of neuronal survival, and promotion of axon regrowth. A fourth major role classically ascribed to these cells is removal of myelin and associated growth inhibitors, a task they accomplish by breaking down myelin early after injury and indirectly by instructing macrophages to complete myelin clearance (Hirata and Kawabuchi, 2002Hirata K. Kawabuchi M. Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration.Microsc. Res. Tech. 2002; 57: 541-547Crossref PubMed Scopus (148) Google Scholar). We found that myelin clearance was substantially delayed in mutants. Four weeks after sciatic nerve transection (without regeneration), the distal stump of WT nerves was translucent, while mutant nerves remained gray/white (Figure 6A). Osmium stained lipid debris occupied about 10-fold larger area in the mutant than WT nerves (Figure 6B). Electron microscopy revealed that although transected mutant nerves did not contain intact myelin, many Schwann cells contained lipid droplets, a late product of myelin breakdown (Figure 6C). This was not seen in 4 week transected WT controls. We therefore tested whether myelin breakdown was impaired in mutant Schwann cells. First, in cut adult nerves, the loss of myelin sheaths was delayed in the mutants (Figure 6D). This was not due to infiltrating macrophages, because the difference between WT and mutants was fully maintained when the cut nerves were maintained in vitro (Figure 6E). Second, this delay was confirmed by slower breakdown of myelin basic protein (MBP) in vivo (Figures S5A and S5B). Third, when myelinating cells from postnatal day 8 nerves were cultured, myelin proteins were broken down slowly by c-Jun mutant cells compared to WT, and mutant cultures contained many Schwann cells bloated with myelin debris (Figures 6F–6H). Both types of culture contained similar numbers of F4/80+ macrophages (5.6+/−1.8% and 5.8+/−1.7%, n = 4, in WT and mutants, respectively, at 3 days in vitro), and no F4/80+ cells containing myelin proteins were seen, suggesting macrophages are not significantly involved in myelin breakdown in these experiments. These findings show that c-Jun mutant Schwann cells are deficient in their ability to break down myelin. Surprisingly, abnormalities in myelin breakdown extended to the macrophage compartment, although the macrophages are genetically normal (Figure S2). Four weeks after cut, macrophages in the mutants contained large amounts of myelin debris, and counts of lipid droplets per macrophage showed that they were about 7 times more numerous than in WT (Figures 6I and 6J). Furthermore, the number of very large (>150 μm2) foamy macrophages bloated with debris was elevated 3-fold in mutant nerves at this time point. Macrophage numbers in the distal stump were strongly elevated in both WT and c-Jun mutant nerves after injury. Three days after cut, their number close to the injury site was significantly higher in WT mice, and a migration assay using Boyden chambers showed that WT nerves attracted more macrophages than mutant nerves (Figures S5C and S5D). But at 1 and 6 weeks after cut, the number of macrophages was similar in WT and mutants, and at 4 and 14 days after crush, macrophage numbers were not significantly different (Table S7). RT-QPCR of cytokines in the distal stumps 36 hr after cut did not reveal significant differences between WT and mutants (Figure S5E). This indicates that c-Jun mutants do not suffer from a major failure in macrophage recruitment. Reduced numbers shortly after injury in the mutant might relate to lower Schwann cell numbers rather than to significant disturbance in the expression of macrophage attractants by individual cells. These results show that injured c-Jun mutant nerves develop substantial problems with myelin clearance. This is evident not only in Schwann cells but also in macrophages, an observation that suggests a role for Schwann cells in the control of macrophage activation and myelin degradation. Previous sections show that injury-activated Schwann cell c-Jun controls the generation of the dener" @default.
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• W1965571714 title "c-Jun Reprograms Schwann Cells of Injured Nerves to Generate a Repair Cell Essential for Regeneration" @default.
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• W1965571714 doi "https://doi.org/10.1016/j.neuron.2012.06.021" @default.
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