Growth factor regulation of remyelination: behind the growing interest in endogenous cell repair of the CNS

Abstract

Remyelination facilitates recovery of saltatory conduction along demyelinated axons and may help prevent axon damage in patients with demyelinating diseases, such as multiple sclerosis. The extent of remyelination in multiple sclerosis lesions varies dramatically, indicating a capacity for repair that is not fulfilled in lesions with poor remyelination. In experimental models of demyelinating disease, remyelination is limited by chronic disease that depletes the oligodendrocyte progenitor (OP) population, inhibits OP differentiation into remyelinating oligodendrocytes and/or perturbs cell survival in the lesion environment. Manipulating the activity of growth factor signaling pathways significantly improves the ability of endogenous OP cells to accomplish extensive remyelination. Specifically, growth factors have been identified that can regulate OP proliferation, differentiation and survival in demyelinated lesions. Therefore, growth factors may be key signals for strategies to improve conditions with poor remyelination.

Transition to in vivo analysis during remyelination

Analysis of growth factors to promote repair in demyelinating diseases has undergone an important transition to in vivo studies and in doing so has revealed much more than the expected results. Characterization of the bipotential oligodendrocyte-type 2 astrocyte or oligodendrocyte progenitor (OP) cell, and the regulation of cell fate by serum components [1] opened the door for in vitro analysis of growth factor responses in the oligodendrocyte lineage. Numerous subsequent in vitro studies identified specific growth factors capable of regulating differentiation, proliferation, migration and survival at specific stages within this lineage. In vitro studies continue to offer distinct advantages for isolating molecular and cellular mechanisms of action and for characterizing the potential of defined growth factors to induce a specific cellular response. In this context, several cytokines and chemokines have been identified as having direct effects on oligodendrocyte lineage cells that are similar to growth factor actions and so they will be discussed together under the rubric of growth factors. This review will focus on recent in vivo remyelination studies designed to test potential growth factor responses identified in vitro. These in vivo studies demonstrate important effects of growth factors in the context of demyelinating disease models in rodents. However, not all growth factor analyses have resulted in the predicted responses. Importantly, several studies have demonstrated a remarkable capacity of endogenous cells to repair the adult CNS, even following chronic demyelination, using modulation of growth factor signaling to better support oligodendrocyte replacement and myelin formation.

Necessity for different models of experimental demyelination to reveal mechanisms impacting remyelination

In vivo studies of growth factor effects on remyelination require appropriate animal models of experimental demyelination. A complement of experimental models is needed to exploit advantages for analysis of specific features in simplified models, as well as to test responses in models of increasing complexity and disease relevance. Experimental allergic/autoimmune encephalomyelitis (EAE) models in rodents are widely employed for their relevance to multiple sclerosis (MS) pathogenesis [2]. However, analysis of remyelination can be complicated in EAE models because modulation of the growth factor or cytokine under study can also alter the immune response and disease severity [3,4]. Gliotoxin models, such as injection of lysolecithin or ethidium bromide or ingestion of cuprizone, can accomplish a relatively specific period of demyelination after which the demyelinating agent is no longer active [5–7]. Therefore, gliotoxin models circumvent the autoimmune complexity and provide a reproducible demyelination episode to simplify analysis of cellular responses associated with remyelination. The mechanism of each toxin must be considered since this influences the stage of oligodendrocyte lineage cell involved (see below) and the effect on astrocytes, which contribute to the environmental signaling interactions regulating OP responses [8,9].

In rodent models, a single episode of demyelination typically results in extensive spontaneous remyelination if the demyelinating activity does not persist [6,10,11]. Similarly, initial lesions in acute MS can remyelinate spontaneously [12]. Analysis of models of acute demyelination have been well suited for testing the capacity of growth factors to prevent oligodendrocyte cell death and subsequent demyelination [4,13,14]. During acute demyelination, OP cells proliferate to amplify the OP population several-fold and generate oligodendrocytes to repopulate lesioned areas [15,16]. Since transient episodes of acute demyelination have this typically robust OP response and extensive remyelination, such models are advantageous for testing growth factor manipulations that are predicted to impair spontaneous remyelination but may be less useful for testing modifications to promote remyelination [7,14,15,17].

Animal models of demyelination that exhibit relatively poor remyelination must be examined to identify molecular and cellular features limiting remyelination. Indeed, interest in identifying growth factor strategies to promote remyelination is based on the desire to improve functional recovery of patients with demyelinating disease pathology lacking sufficient remyelination. In MS lesions, the extent of remyelination is likely to depend on a combination of features associated with disease pathogenesis and its time course [18–20]. Strategies to improve conditions of poor remyelination may facilitate recovery of saltatory conduction along demyelinated axons and may help prevent axon damage that could otherwise lead to transection and permanent loss of function. To identify how growth factors can promote remyelination in relatively nonpermissive conditions, animal models need to exhibit features of remyelination failure, which may then be overcome with experimental manipulations. Examples of rodent models of limited remyelination over a chronic disease course include persistent viral infection, recurring autoimmune response or continuous cuprizone administration [2,10,11,21].

Targeting cellular & molecular mechanisms that limit remyelination

To promote remyelination, experimental growth factor strategies target specific features or a combination of features predicted to limit remyelination in chronic lesions (Box 1). This review will not focus on the potential of growth factors to modulate ongoing demyelinating disease activity, which is critical for remyelination but involves mechanisms of demyelination pathogenesis rather than remyelination processes. Similarly, remyelination will not be a focus in demyelinating conditions associated with early-stage axonal transection, since enhancing axonal outgrowth is then a primary concern. In chronic conditions in which demyelination is no longer ongoing, modifications of the lesion environment have revealed a significant capacity of endogenous cells to remyelinate viable axons in chronic lesions. The intention in this review is not to imply that inflammation and axonal damage can be separated from demyelination and remyelination in MS pathology, but rather to simplify the context sufficiently to reveal the mechanisms of potential beneficial effects that may focus future directions.

Box 1. Cellular and molecular mechanisms limiting remyelination

• Active demyelination that outpaces remyelination

• Loss of axons or axonal signals capable of supporting remyelination

• Depletion of oligodendrocyte progenitor (OP) cells in lesions

• Poor recruitment of OP cells into lesions

• Improper balance of inhibition/induction of OP differentiation

• Insufficient support of survival of newly generated oligodendrocytes

Stimulating proliferation to counter OP depletion in chronically demyelinated lesions

In experimental models of demyelination in rodents, extensive remyelination requires proliferation of immature OP cells followed by differentiation into myelinating oligodendrocytes [15,16,22]. With repeated transient episodes of demyelination and sufficient time between individual episodes, the OP population can repeatedly generate new oligodendrocytes and regenerate the OP pool [6]. However, as shown with chronic cuprizone administration, a prolonged course of demyelination depletes this OP population [10,11].

Growth factor stimulation of OP proliferation is of interest to promote more rapid remyelination, which could potentially minimize vulnerability of denuded axons to further damage, or to enhance remyelination of areas with low OP density. From in vitro OP analyses, PDGF has been predicted to act independently and/or cooperatively with FGF2 as a mitogen for OP cells from the adult rodent and human CNS [23–25]. In areas of experimental demyelination, PDGF-A ligand expression is locally upregulated in reactive astrocytes, and proliferating OP cells express PDGF-α receptor (PDGF-αR) [22,26]. Furthermore, PDG-FαR expression is associated with proliferating cells in MS lesions [27]. These findings led to complementary studies to test the role of PDGF signaling in OP cells responding to demyelination. PDGFαR deficiency in PDG-FαR heterozygous mice resulted in reduced OP proliferation during acute cuprizone demyelination and reduced oligodendrocyte repopulation of lesion areas during the recovery period following removal of cuprizone from the diet [28]. Mice expressing PDGF-A as a transgene controlled by an astrocytic promoter (GFAP–PDGF-A mice) exhibited PDGF-A overexpression with the temporal and spatial correlation of endogenous PDGF-A from reactive astrocytes in areas of demyelination [7,29]. GFAP–PDGF-A mice exhibited increased OP proliferation and cell number during acute demyelination from gliotoxin administration [7]. Although altering PDGF receptor (PDGFαR^+/− mice) and ligand (GFAP–PDGF-A mice) expression had the predicted effect on the OP proliferative response to demyelination, after a transient episode of demyelination the subsequent spontaneous remyelination was not altered significantly [7,28].

By contrast, during the recovery period following prolonged administration of cuprizone to induce chronic demyelination of the corpus callosum, improved remyelination was observed with overexpression of PDGF-A from the same GFAP–PDGF-A mouse line [29]. Interestingly, OP depletion was not prevented during chronic demyelination in these GFAP–PDGF-A mice, possibly because the PDGF-A transgene, although still exhibiting increased expression, may not have been overexpressed sufficiently to continue to stimulate significant OP proliferation [29]. Alternatively, this result may reflect mechanistic differences in repair of chronic lesions. Different subpopulations of OP cells may be relatively quiescent or proliferate robustly in response to demyelination [30]. The potential existence of OP subpopulations raises further questions: after chronic demyelination, are the persisting OP cells responsive to mitogens? If so, is PDGF the optimal mitogen for this OP subpopulation? Importantly, in the chronic lesion environment PDGF may improve remyelination through a different role as a survival factor, since GFAP-PDGF-A mice display significantly reduced apoptosis during remyelination (discussed below).

Surprisingly TNF-α acting through TNF receptor (TNFR)2 appears to be critical in the OP proliferative response, with a significant impact on oligodendrocyte replacement and remyelination [31]. This beneficial role for TNFα signaling was demonstrated using the cuprizone model, which involves microglial–macrophage activation and astrogliosis but does not have an active autoimmune-mediated demyelinating component. TNF-α appears to induce OP proliferation through activation of TNFR2, in contrast with inducing cell death through TNFR1 [31]. This dual role of TNF-α may underlie the poor outcome observed with inhibition of TNF-α signaling in MS clinical trials [31,32]. TNF signaling is an example of the difficulty of predicting effects of growth factors and cytokines associated with inflammation, which can positively and negatively impact remyelination [33,34]. A further example is that induction of localized inflammation promoted remyelination of transplanted cells in the context of chronic demyelination [35].

Interest in the role of FGF2 in OP generation of remyelinating oligodendrocytes has been fostered by the in vitro characterization of FGF2 as a potent mitogen for OP cells from the adult CNS and by the potential interaction of FGF2 with PDGF [23,24]. Areas of acute and chronic demyelination have increased expression of FGF2 ligand and FGF receptors [11,26,36,37]. In contrast with predictions from in vitro studies, OP proliferation in demyelinated lesions was not impaired in FGF2-null mice, indicating that the potential for FGF2 to act as an OP mitogen may not be a significant role of endogenous FGF2 in vivo during demyelination [37]. Studies testing elevated levels of FGF2 indicate a potential mitogenic role of FGF2 signaling, which may be distinct from the response elicited by endogenous FGF2 in lesioned white matter. The number of OP cells and oligodendrocytes increased with FGF2 overexpression from viral transduction early in the course of EAE and this effect was partially abrogated with a second treatment later in EAE disease progression [38]. One possible explanation for the different effects interpreted from FGF2-null mice versus FGF2 overexpression is that supraphysiological elevation of FGF2 levels could result in proliferation that is not stimulated by endogenous FGF2 levels. Another consideration is that differences in the targeted cell populations could also contribute to different responses to FGF2. The viral transduction experiments increased FGF2 concentration in the cerebrospinal fluid, which could possibly allow FGF2 to act on different cell populations relative to the endogenous FGF2 availability. Indeed, FGF2 administration has been reported to increase recruitment of OP cells from the subventricular zone (SVZ) to nearby areas of demyelination in the corpus callosum [5].

Tapping into multiple sources of OP cells

Several variables may determine the extent to which OP cells proliferate within lesion areas and/or are recruited into demyelinated areas, either from the adjacent normal appearing white matter or from germinal zones, such as the SVZ. The method of inducing demyelination is important to consider in analysis of OP responses in animal models. With cuprizone demyelination of the corpus callosum, toxicity is primarily within mature oligodendrocytes, presumably because of the metabolic load of maintaining the extensive myelin membranes [39]. During cuprizone treatment, OP populations proliferate both within the demyelinated areas of white matter and in the adjacent SVZ [15,29,37]. By contrast, demyelination models of lysolecithin or ethidium bromide injection have significant loss of OP cells within lesion areas [40,41]. These models show that OP cells are recruited into lesions from nearby normal appearing white matter [40,41]. In addition, SVZ cells can contribute to the repair of lesions in the adjacent corpus callosum [5,42]. Similarly, in some MS patients, white matter lesion areas can contain substantial populations of immature oligodendrocyte lineage cells and premyelinating oligodendrocytes [27,43]. Furthermore, MS lesions, especially those in the periventricular white matter, show evidence of progenitor cell activation in the SVZ and mobilization to lesion areas [44]. Thus, growth factors that promote recruitment of OP cells to lesions from the SVZ may enhance the repair response from endogenous cell populations, as has been reported from animal studies [5,42]. Proliferation of SVZ cells in the adult CNS can be stimulated by FGF2 and/or EGF [45,46]. Indeed, OP proliferation in the SVZ, mobilization and remyelination of the corpus callosum was enhanced in mice with OP overexpression of the EGF receptor (EGFR) and, conversely, impaired in mice with a hypomorphic EGFR mutation [42]. In chronic lesions, enhancing recruitment from the SVZ may be particularly important to compensate for OP depletion and to promote remyelination. Activation of the SVZ, as indicated by an increase in the density of actively dividing cells, continues throughout the course of chronic cuprizone demyelination [29]. However, areas of chronic demyelination may not be as permissive as acute lesions and so may hinder effective recruitment of cells from the SVZ to generate remyelinating oligodendrocytes. Indeed, in contrast with the expectation from proximity to the SVZ, in MS cases periventricular lesions may have a lesser extent of remyelination than lesions located in subcortical or deep white matter [19].

Modifying the balance of signals regulating OP differentiation

In the face of a depleted OP population in chronic lesions, the efficiency of OP conversion to remyelinating oligodendrocytes is likely to be more critical than in the conditions of OP amplification that are typical during acute episodes of demyelination. Analysis of FGF2-null mice provides a dramatic example that the extent of remyelination from endogenous cells can be improved significantly by altering the chronic lesion environment [11]. FGF2 is present in demyelinating lesions and inhibits OP differentiation into oligodendrocytes during remyelination [11,37]. Following chronic cuprizone demyelination, wildtype mice exhibit limited remyelination of the corpus callosum even when allowed a relatively long recovery period [10,11,29]. Under the same chronic demyelination conditions, almost complete spontaneous remyelination occurs in FGF2-null mice [11]. Given the complexity of the FGF family of ligands and receptors that are expressed in the CNS, further studies will be required to fully explain the potential direct and/or indirect mechanisms of FGF2 action that are responsible for limiting remyelination of chronically demyelinated axons. These findings suggest that blocking the activity of signals in chronic lesions that inhibit OP differentiation may allow more effective generation of remyelinating oligodendrocytes. In MS lesions, persistence of immature oligodendrocyte lineage cells may indicate inhibition of differentiation by local signals, especially those associated with astrocytic scar formation, such as FGF2 [47–50]. Conversely, signals that are beneficial for oligodendrocyte maturation may not be present at sufficient levels, as has been proposed for neuregulin [51]. The inflammatory response may also modulate oligodendrocyte differentiation in lesions. For example, IL-1β may be necessary during acute demyelination for the amplified pool of OP cells to mature into remyelinating oligodendrocytes [52].

Supporting oligodendrocyte lineage cell survival during remyelination

The efficiency of OP conversion to remyelinating oligodendrocytes in chronic lesions is also critically impacted by the probability of survival of newly generated oligodendrocyte lineage cells. During active periods of demyelination, oligodendrocytes die as targets of the autoimmune response and/or from cytokines and excitotoxic molecules associated with active disease progression. Ongoing demyelination that outpaces remyelination will result in chronic lesions. Growth factors, including IGF-1, LIF and CNTF, can act as survival factors that are sufficiently potent to prevent oligodendrocyte loss due to active demyelination in animal models [4,13,14]. These responses to prevent oligodendrocyte loss from active demyelination relate to the complexity of disease pathogenesis mechanisms in the animal models and in MS lesions, and so are beyond the current scope. The preclinical and clinical studies of IGF-1 provide an example of this complexity [53]. Cell death in the absence of active demyelinating insult may also contribute to limited remyelination of chronic lesions. Marked cell death continues during the recovery period following chronic cuprizone demyelination and removal of cuprizone from the diet [29]. Cell death during the recovery period is significantly reduced in GFAP–PDGF-A transgenic mice (see above) and corresponds with enhanced oligodendrocyte repopulation of lesions and improved remyelination of the chronically demyelinated corpus callosum [29]. Therefore, PDGF-A may be an important survival factor during remyelination – a role distinct from the mitogenic effect observed during acute demyelination (see above). Thus, in MS lesions, survival factors for oligodendrocyte lineage cells may help prevent cell loss during active demyelination and subsequently support survival of newly generated cells. IL-11 is an oligodendrocyte survival factor expressed by reactive astrocytes at the border in both active and silent MS plaques, and so may have potential survival effects at different disease stages [54]. When examining MS lesions with oligodendrocyte loss but in the presence of immature cells, it may be important to consider further lesion characteristics to determine the extent to which OP differentiation is inhibited and/or newly generated mature cells cannot survive prior to contacting and remyelinating denuded axons.

Conclusion

Combining the complexity of growth factor and cytokine signaling pathways with the complexity of cellular responses in the context of pathology has been a difficult but rewarding challenge for studies of remyelination. Aside from the specifics of the signaling pathways involved, animal studies have uncovered a much greater potential for endogenous cycling cells in the white matter and SVZ to contribute to the generation of remyelinating oligodendrocytes. Chronically demyelinated areas of white matter that would normally exhibit only limited remyelination capacity can undergo extensive spontaneous remyelination when the chronic lesion environment is modified to be more permissive for OP cells to generate remyelinating oligodendrocytes. Understanding the range of the cellular responses contributing to remyelination failure versus success will be important for assessing lesion status and for identifying critical components to target in order to optimize potential remyelination capacity. Each signaling pathway involved in overcoming remyelination failure can be considered for therapeutic potential. The complexity of ligand and receptor-signaling pathways highlights the challenge of modulating levels of a known growth factor or cytokine in MS patients. Therefore, the current findings of specific growth factor actions may not be as important for direct translation of that growth factor as a therapeutic but rather may be important for identifying the specific cellular response to which therapeutics can be targeted.

Future perspective

The heterogeneous pathology of MS lesions and the variable progression of MS disease activity must be understood more fully to address repair potential with modifications of growth factor signaling. As treatments advance to slow or stop active demyelination, then attempts to promote repair move forward. Imaging and other means to evaluate disease status in patients will be critical for tailoring therapeutic interventions to correlate with cellular and molecular changes within lesions. Furthermore, the numerous effects of growth factors on the immune response were not addressed specifically here but would need to be coordinated with the desired effects on oligodendrocyte lineage cells to translate the repair potential to an inflammatory environment. Future studies may benefit from testing combinations of growth factors with complementary mechanisms in order to accomplish more desirable results than individual pathway manipulations, as attempted recently in an animal model [55]. When possible, therapeutics should be designed to block pathways that have a detrimental effect and that are upregulated in lesion areas. For example, FGF2 is upregulated in lesion areas and removing this inhibitor of OP differentiation appeared to allow OP cells to differentiate more appropriately, presumably under regulation of other signals that promote differentiation. This example from FGF2 studies also emphasizes the spatial and temporal specificity of growth factor effects; FGF2 administration stimulated proliferation of early-stage cells from the SVZ, but FGF2 in demyelinated areas inhibited OP differentiation. The lessons learned for optimizing remyelination from endogenous cycling cells may also provide important insights to improve remyelination from transplanted cells, which may also be limited by characteristics of the chronically demyelinated lesion environment [56]. An important future challenge will be to not only promote remyelination of viable denuded axons but also to integrate neurotrophic factors, such as BDNF, in order to enhance regeneration of damaged or transected axons to accomplish more extensive recovery [57–59].

Executive summary

Transition to in vivo analysis during remyelination

• Oligodendrocyte progenitor (OP) cells generate oligodendrocytes.

• In vitro studies have identified growth factors (and cytokines) that regulate OP proliferation, differentiation, migration and survival.

• Growth factor regulation of OP generation of remyelinating oligodendrocytes is now being examined using animal models of demyelinating diseases.

• Repair capacity of endogenous cells can be expanded by manipulating growth factor signals.

Necessity for different models of experimental demyelination to reveal mechanisms that impact remyelination

• Rodent models of demyelinating disease allow manipulation of in vivo conditions to identify growth factor functions during remyelination.

• During an acute episode of demyelination, OP cells proliferate robustly to generate oligodendrocytes and accomplish extensive spontaneous remyelination.

• In multiple sclerosis (MS), the extent of remyelination varies dramatically.

• To learn how to promote repair in MS patients with poor remyelination, animal models must be analyzed for molecular and cellular features that limit remyelination.

Targeting cellular & molecular mechanisms that limit remyelination

• This review focuses on studies that provide insight into mechanisms limiting repair, as compared with the pathology of demyelination or axon damage.

Stimulating proliferation to counter oligodendrocyte progenitor cell depletion in chronically demyelinated lesions

• Growth factor stimulation of OP proliferation is of interest to promote more rapid remyelination, which may protect denuded axons, or to enhance remyelination in areas with low OP density.

• PDGF-A regulates OP proliferation during acute demyelination.

• FGF2 depletion does not alter OP proliferation during acute demyelination, but FGF2 administration may stimulate proliferation.

• Inflammatory signals, such as TNF-α, may be important for OP proliferation in response to demyelination.

Tapping into multiple sources of oligodendrocyte progenitor cells

• OP cells can proliferate within demyelinated areas to repopulate lesions.

• OP cells can be recruited into lesions from adjacent, normal-appearing white matter or germinal regions, such as the subventricular zone (SVZ).

• Diverse sites may contribute to the generation of new cells in MS lesions.

• EGF can regulate OP proliferation in the SVZ, and mobilization and remyelination of a lesioned area in the adjacent corpus callosum.

• The SVZ may be a site for generating new cells throughout the course of chronic demyelination.

Modifying the balance of signals regulating oligodendrocyte progenitor cell differentiation

• Suboptimal OP differentiation in chronic lesions may have a severe effect on the capacity of the depleted OP pool to generate remyelinating oligodendrocytes.

• Genetic deletion of FGF2, which inhibits OP differentiation, dramatically improves remyelination of chronic lesions.

Supporting oligodendrocyte lineage cell-survival during remyelination

• In chronically demyelinated white matter, significant cell death continues even after eliminating the cause of demyelination, indicating a lack of survival factors or the continued presence of proapoptotic factors.

• IGF-1, LIF, CNTF and IL-11 are protective factors for mature oligodendrocytes during active demyelination.

• PDGF-A may support survival during remyelination.

• Poor cell survival in the chronic lesion environment may diminish the capacity of the depleted OP pool to generate remyelinating oligodendrocytes.

Future perspective

• Advances in remyelination must correspond with the understanding and treatment of MS pathogenesis.

• Imaging and other assessment methods will need to tailor a treatment regimen to a patient’s pathology and disease activity.

• The complexity of growth factor signaling actions in a pathological environment must be considered when designing therapeutics.

• Therapeutics may be capable of more targeted actions if they are selected to block pathways that have a detrimental effect on remyelination and that are upregulated in lesion areas.

• Modifying the lesion environment may be important for optimizing remyelination from endogenous cell sources or from cell transplantation.

• To accomplish meaningful recovery in lesions with axon loss, treatments will need to enhance axonal regeneration in addition to promoting remyelination.