Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several statesincluding chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia/macrophages, which can be polarized to distinct functional phenotypes: proinflammatory (M1) or anti-inflammatory/immunoregulatory (M2). Here we show that a switch from an M1-to M2-dominant response occurred within microglia and peripherally-derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 conditioned media, and impaired in vivo following intra-lesional M2 depletion. M2 densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger animal, and in MS lesions that normally show remyelination. Blocking M2-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Our results therefore show that M2 polarization is essential for efficient remyelination and identify activin-A as a novel therapeutic target for CNS regeneration.Remyelination, the formation of myelin sheaths around demyelinated axons by newly differentiated oligodendrocytes, can occur efficiently following central nervous system (CNS) demyelination. A major component of this regenerative process is a robust innate immune response consisting of peripherally-derived macrophages and their CNS-resident counterparts, microglia. Although these microglia/macrophages are implicated in CNS autoimmune disease via secretion of toxic molecules 1 and antigen presentation to cytotoxic lymphocytes 2 , they also exhibit regenerative properties through the phagocytosis of myelin debris 3, 4 and secretion of growth/neurotrophic factors 5 . Regenerative properties of Corresponding author: Veronique E. Miron, MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh, EH16 4UU, Tel: +44 (0) 131 651 9570, Fax: +44 (0) 131 651 9501, vmiron@staffmail.ed.ac.uk. Author Contributions: V.M. conceived the project, designed and carried out experiments, performed data acquisition, quantification, and analysis, and wrote the manuscript; A.B. and A.W. assisted in in vivo studies, and A.W. assisted in data interpretation; J.-W.Z. contributed to analysis and quantification of parabiosis lesion tissue; A.B., A.W., T.Y., and P.v.W. performed lesioning experiments to provide lesion tissue and assisted in tissue selection; J.R., J.S., A.J.W, R.J.M.F. provided parabiosis lesion tissue; R.J.M.F. assisted in study design, data interpretation, and manuscript writing; C.ff.-C. supervised the project, assisted in study design, data interpretation, figure preparation, and writing of the manuscript. Europe PMC Funders GroupAuthor Manuscript Nat Neurosci. Author manuscript; available in PMC 2014 April 07. Published in final edited form as:Nat Neurosci. 2013 September ; 16(9): 1211-1218. doi:10.1038/nn.3469. Europe PMC Funders Author Manus...

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.

Microglia are cells of myeloid origin that populate the CNS during early development and form the brain's innate immune cell type. They perform homoeostatic activity in the normal CNS, a function associated with high motility of their ramified processes and their constant phagocytic clearance of cell debris. This debris clearance role is amplified in CNS injury, where there is frank loss of tissue and recruitment of microglia to the injured area. Recent evidence suggests that this phagocytic clearance following injury is more than simply tidying up, but instead plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair. Insufficient clearance by microglia, prevalent in several neurodegenerative diseases and declining with ageing, is associated with an inadequate regenerative response. Thus, understanding the mechanism and functional significance of microglial-mediated clearance of tissue debris following injury may open up exciting new therapeutic avenues.

The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, we performed a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions. We report that ;50 transcription factor-encoding genes show dynamic expression during repair and that expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned-but not normal-adult white matter. We report that b-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, we observed similar regulation of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. We show that dysregulation of Wnt-b-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of b-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC Min mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, our findings indicate that dysregulated Wnt-b-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders.[Keywords: Oligodendrocyte; CNS development; Wnt; multiple sclerosis; remyelination; Olig2] Supplemental material is available at http://www.genesdev.org.

Multiple sclerosis is a common cause of neurological disability in young adults. The disease is complex -- its aetiology is multifactorial and largely unknown; its pathology is heterogeneous; and, clinically, it is difficult to diagnose, manage and treat. However, perhaps its most frustrating aspect is the inadequacy of the healing response of remyelination. This regenerative process generally occurs with great efficiency in experimental models, and sometimes proceeds to completion in multiple sclerosis. But as the disease progresses, the numbers of lesions in which demyelination persists increases, significantly contributing to clinical deterioration. Understanding why remyelination fails is crucial for devising effective methods by which to enhance it.

Demyelination in the adult CNS can be followed by extensive repair. However, in multiple sclerosis, the differentiation of oligodendrocyte lineage cells present in demyelinated lesions is often inhibited by unknown factors. In this study, we test whether myelin debris, a feature of demyelinated lesions and an in vitro inhibitor of oligodendrocyte precursor differentiation, affects remyelination efficiency. Focal demyelinating lesions were created in the adult rat brainstem, and the naturally generated myelin debris was augmented by the addition of purified myelin. After quantification of myelin basic protein mRNA expression from lesion material obtained by laser capture microdissection and supported by histological data, we found a significant impairment of remyelination, attributable to an arrest of the differentiation and not the recruitment of oligodendrocyte precursor cells. These data identify myelin as an inhibitor of remyelination as well as its well documented inhibition of axon regeneration.

SUMMARY Remyelination is a regenerative process in the central nervous system (CNS) that produces new myelin sheaths from adult stem cells. The decline in remyelination that occurs with advancing age poses a significant barrier to therapy in the CNS, particularly for long-term demyelinating diseases such as multiple sclerosis (MS). Here we show that remyelination of experimentally-induced demyelination is enhanced in old mice exposed to a youthful systemic milieu through heterochronic parabiosis. Restored remyelination in old animals involves recruitment to the repairing lesions of blood-derived monocytes from the young parabiotic partner, and preventing this recruitment partially inhibits rejuvenation of remyelination. These data suggest that enhanced remyelinating activity requires both youthful monocytes and other factors, and that remyelination-enhancing therapies targeting endogenous cells can be effective throughout life.