Field of the Invention

The present invention relates to new treatments for demyelinating diseases. More particularly, the present invention relates to the use of a novel regenerative therapy for the promotion of remyelination at a cellular level via remyelination of axons by oligodendrocyte cells.

Background of the Invention

It is known that rapid transduction of electrical impulses, termed action potentials, is required for the efficient function of the vertebrate nervous system. The conduction velocity of these impulses is enhanced by the presence of an insulating layer surrounding the axon referred to as the myelin sheath. In the central nervous system (CNS), the oligodendrocyte is the cell responsible for the production and maintenance of myelin, which consists of plasma membrane sheets wrapped tightly around the axon. The myelin sheath is also needed to sustain the axon: in the absence of the myelin sheath the axon may undergo irreversible degeneration.

Demyelinating diseases are diseases of the nervous system in which the myelin sheath which protects the axons is damaged. In people affected by multiple sclerosis (MS), damage to the myelin sheath may be thinning or loss of the sheath. Over time such damage leads to scar-like plaque, also known as sclerosis or lesions, build-up around the now- damaged (injured) axons. Axonal-injury impairs the process by which electrical signals are transmitted and ultimately this impaired axonal function / poor signalling presents as progressive loss of function (disability) in various brain pathways, including visual, cognitive, and motor functions.

Not only are there no current treatments for the promotion of myelin regeneration in demyelinating diseases such as MS, and particularly for the progressive forms, primary progressive and secondary progressive, PPMS and SPMS, prior to the present invention there has been no evidence to support any possible treatment based on active cellular regeneration in the body for the treatment of PPMS or SPMS. To date the developed therapies, particularly for MS, have focused on either minimizing the effects of the disease, modifying the course of the disease i.e. reducing lesion frequency, or managing particular symptoms of the disease. However, the very nature of demyelinating diseases such as MS l which affect the central nervous system (CNS) is their ability to progressively worsen. Management of symptoms and or effect minimization, or even disease course modification do not target the central issue of treating the progressive nature of the disease.

Thus there is a long-felt medical need for an effective treatment for promotion of myelin regeneration in demyelinating diseases and particularly in the progressive forms of demyelinating diseases such as PPMS and/or SPMS.

There are currently no approved treatments for disorders that are targeted primarily at promoting oligodendrocyte differentiation and myelin formation. Oligodendrocyte differentiation occurs during normal development and is initiated during gestation in humans as well as during myelin regeneration ('remyelination') following demyelination of the CNS in adulthood in subjects with myelin disorders, such as MS, and particularly RRMS, and to a lesser extent MS patients affected by PPMS or SPMS. Current therapies include immuno- modulators aimed at dampening initial injury as well as agents attenuating secondary pathologies. However these are ineffective at directly promoting remyelination.

Thus, there is a long-felt medical need for the development of effective treatments for myelin disorders that can directly promote oligodendrocyte differentiation and/or myelin regeneration, for the prevention of neurological deficits in patients affected by myelin disorders.

Some of the current therapies have secondary functions in promoting oligodendrocyte differentiation and/or myelin regeneration in experimental models (i.e. Glatiramer Acetate (Skihar et al. 2009, Aharoni et al. 2008), fumarate esters (Moharregh- Khiabani et al., 2010 PLoS One), statins (Paintlia et al. 2004), FTY720 (Miron et al., 2010)). However, the use of these agents to promote oligodendrocyte differentiation and/or remyelination is complicated by their effect on the immune system and the inability of some of these to access the CNS.

Thus, there is a current need for the development of effective treatments for CNS- implicated myelin disorders that can directly promote oligodendrocyte differentiation and/or myelin regeneration.

It is an object of at least one aspect of the present invention to provide an effective therapeutic solution to at least one or more of the aforementioned long-felt medical needs. It is an object of at least one aspect of the present invention to provide an effective treatment for myelin disorders that can directly promote oligodendrocyte differentiation and/or myelin regeneration.

It is a further object of at least one aspect of the present invention to provide an effective treatment for promotion of myelin regeneration in demyelinating diseases and particularly in the progressive forms of demyelinating diseases such as PPMS and/or SPMS.

It is an object of at least one aspect of the present invention to provide an effective regenerative treatment for the promotion of remyelination at a cellular level via remyelination of axons by oligodendrocyte cells. The Applicants have now developed a novel renewal therapy for the effective treatment of demyelinating diseases.

Summary of the Invention

The Applicant has developed a novel renewal therapy for the treatment of demyelinating diseases.

According to one aspect the present invention provides a treatment for demyelinating diseases.

According to a further aspect the present invention provides an effective treatment for promotion of myelin regeneration in demyelinating diseases. According to a yet further aspect the present invention provides an effective treatment for promotion of myelin regeneration in progressive demyelinating diseases.

According to an additional aspect the present invention provides an effective treatment for promotion of myelin regeneration in primary progressive multiple sclerosis (PPMS). According to an additional aspect the present invention provides an effective treatment for promotion of myelin regeneration in secondary progressive multiple sclerosis (SPMS).

According to a still further aspect the present invention relates to the use of a novel regenerative therapy for the promotion of remyelination at a cellular level via regeneration of oligodendrocyte cells. According to a still further aspect the present invention relates to the use of a novel regenerative therapy for the promotion of remyelination at a cellular level via the remyelination of axons by oligodendrocyte cells.

According to a still further aspect the present invention provides a method for the promotion of oligodendrocyte differentiation and/or myelin regeneration for effective treatment of myelin disorders. The Applicant has developed a novel renewal therapy for the treatment of diseases where oligodendrocyte differentiation is impaired.

According to an aspect the present invention provides a treatment for the promotion of remyelination at a cellular level via remyelination of axons by oligodendrocyte cells for use in treatment of diseases where such regenerative / remyelination processes are impaired.

According to an aspect the present invention provides a treatment of diseases where oligodendrocyte differentiation is impaired via myelin regeneration.

According to an aspect the present invention provides a treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation. According to an aspect the present invention provides a treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation and myelin regeneration.

According to an aspect the present invention provides a treatment of myelin disorders. According to an aspect the present invention provides a treatment of progressive demyelinating diseases.

The present invention additionally provides an effective treatment for progressive demyelinating diseases via promotion of oligodendrovyte differentiation by treatment with activin or an activin isoform of mammalian origin, including activin-A, activin-B, activin-AB, and particularly activin-A, which is a homodimer of beta-A subunits.

As particularly illustrated in the Examples hereinafter the present invention additionally provides activin-A for the treatment of progressive demyelinating disorders via promotion of oligodendrocyte differentiation and myelin regeneration.

Thus, according to a further aspect the present invention provides activin-A for the treatment of progressive demyelinating disorders via promotion of oligodendrocyte differentiation and myelin regeneration. For the avoidance of doubt, it is hereby stated that the information disclosed earlier in this specification under the heading "Background" is relevant to the invention and is to be read as part of the disclosure of the invention.

Where indicated herein various definitions are provided for explanations of terms and methods to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

Detailed Description

The Applicants have developed novel and inventive cellular renewal therapies for treatment of demyelinating diseases. These are based upon the generation of new oligodendrocyte progenitor cells and the active promotion of remyelination and/or oligodendrocyte differenitaion leading to re-ensheathment of damaged nerves with new cellular myelin material. The Applicants have developed an effective regenerative treatment for the promotion of remyelination at a cellular level via remyelination of axons by oligodendrocyte cells.

Regeneration as defined herein means the replacement of cells and/or cellular structures following their loss as a consequence of injury or disease. This may involve a number of distinct cellular replacement processes including: differentiation or dedifferentiation of cells; reorganization of existing cells and/or cellular structures; and/or compensatory growth or function of surviving cells. To date, patients do not recover from MS, although some people who suffer from particular forms of MS may experience periods where their disease does not progress significantly, ultimately the disease, once diagnosed remains a permanent feature at some or other level. The unprecedented potential of the treatment according to the present invention is that a full or partial recovery of functions which have been negatively impacted by MS, particularly for people with progressive forms including PPMS and SPMS may now be possible.

Regeneration is different to, and is distinct from, protection. For example, in neuroprotection when referring to neurons, existing cellular functions are preserved but there is no replacement of cells and/or cellular structures. Myelination as defined herein means the de novo ensheathment of nerves with myelin.

Remyelination as defined herein means the re-ensheathment of nerves with myelin sheath following previous loss of myelin from these cells. Some limited myelin repair function can be observed in the early stages of MS-sufferers whereby a partial re-build of the cells comprising the myelin sheath may occur to partially restore function for a short while. This limited functionality can vary widely from patient to patient and as part of the normal disease progression. For example in multiple sclerosis (MS) patients affected by primary progressive MS (PPMS) and/or secondary progressive MS (SPMS), such re-building becomes increasingly difficult and ultimately less and less effective.

In complete contrast the novel and inventive cellular renewal therapy provided by the present invention provides re-ensheathment with new cellular material, as opposed to limited repair or already damaged / injured and /or impaired cellular material. Thus the present invention additionally provides a unique and unprecedented method for the treatment of progressive demyelinating diseases by the re-ensheathment of nerves with myelin sheath.

Differentiation as defined herein means the process whereby a cell undergoes a change in its morphology or gene/protein expression profile to carry out a specialized function.

Mature myelinating oligodendrocytes are generated by differentiation of oligodendrocyte progenitor cells (OPCs). Oligodendrocyte differentiation involves cell cycle- arrest and expression of markers such galactocerebroside (GalC/01 ), myelin associated glycoprotein (MAG), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte myelin glycoprotein (OMgp) concomitant with arborizations of processes, extension of myelin membranes, and wrapping around axons. Oligodendrocyte differentiation occurs during normal development (initiated during gestation in humans) and during myelin regeneration ('remyelination') following demyelination of the CNS in adulthood in subjects affected by MS.

Oligodendrocyte lineage cells as defined herein means all cells that are, and have the normal potential to differentiate into, oligodendrocytes. This includes oligodendrocyte progenitor cells, immature non-myelinating oligodendrocytes and mature myelinating oligodendrocytes.

As discussed herein before, failed oligodendrocyte differentiation leads to lack of myelin, resulting in impairments in axonal function and deficits in many functions, including visual, cognitive, and motor functions. This is observed in in secondary (SPMS) and primary progressive (PPMS) forms of multiple sclerosis and in paediatric disorders leading to cerebral palsy (CP) i.e. periventricular leukomalacia (PVL). Whilst some MS patients, particularly those suffering from relapsing remitting MS (RRMS) may retain some remyelination capacity, it is proposed herein that enhancement of this functionality may provide an effective therapy for these patients. PVL is the main underlying pathology for the development of CP, involving cerebral ischaemia/reperfusion injury and inflammation. PVL is incurred at 24-32 weeks of gestation when oligodendrocyte progenitors (04+, 01 +, MBP-) predominate in the cerebral white matter and is characterized by cell death, reactive astrogliosis and microgliosis, axonal swellings, tissue cavitation, and a block in oligodendrocyte differentiation. Examination of post-mortem tissue of acute, sub-acute, and chronic PVL lesions showed that there was an increase in the density of cells expressing the oligodendrocyte lineage marker Olig2+ in necrotic foci relative to controls, indicating an accumulation of immature undifferentiated oligodendrocyte progenitor cells. Olig2 is essential for oligodendrocyte development and is the earliest indicator of oligodendrocyte specification. In PVL lesions abnormal expression of the mature myelin marker MBP have been observed, with expression in tubules and surrounding nuclei.

The Applicant has recognised that there is a common link between the underlying cause of CP and adult demyelinating diseases and has developed novel therapy for treatment of these diseases. Multiple sclerosis is the most frequent demyelinating disease in young adults, characterized by demyelination, inflammation, reactive astrogliosis and microgliosis, and axonal loss (Lucchinetti et al. 1999 Brain). Although limited remyelination has been observed in some lesions of multiple sclerosis post-mortem tissue, the majority of MS patients show limited to no remyelination in the chronic lesions which are central to the distressing and life-debilitating symptoms felt by MS patients (Patani et al., 2007 Neuropathol Appl Neurobiol, Prineas and Connell, 1979 Ann Neurol, Barkhof et al. 2003 Arch Neurol, Patrikios et al. 2006 Brain). A major contributor to this is a block in oligodendrocyte differentiation in chronic lesions (Kuhlmann et al., 2008 Brain), such as those found in patients having progressive forms of MS, such as PPMS or SPMS. This block has been shown in post-mortem tissue by a decrease in the densities of maturing oligodendrocytes identified by high expression of Olig2 and the mature oligodendrocyte marker neurite outgrowth inhibitor A (NogoA) (Kuhlmann et al., 2007 JNEN, 2008 Brain).

There are a limited number of identified factors that can directly promote oligodendrocyte differentiation and/or myelin regeneration in experimental models. These include: immunoglobulin (lg)M antibodies, Miller et al. 1994, J Neurosci; retinoid-X-receptor (RXR) gamma receptor agonists, Huang et al., 201 1 Nat Neurosci; endothelin-2, Yuen et al., 2013 Brain; ; and anti- leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1 (LINGO-1 ) antibodies, Mi et al., Nat Med 2007. To date there has been no successful translation of any of these into an approved therapy for the effective treatment of myelin disorders.

Surprisingly, the Applicant has demonstrated that activin-A is associated with the regulation of oligodendrocyte differentiation. Thus the present invention additionally provides an effective treatment for progressive demyelinating diseases via promotion of oligodendrocyte differentiation by treatment with activin-A. As demonstrated in the Experimental results herein the Applicants have developed new experimental models and have shown that macrophages activated with interleukin 13 (IL-13) ('M2' or 'alternatively activated' phenotypes) promote oligodendrocyte differentiation and myelin regeneration in these experimental models. The Applicants have demonstrated that this is mediated by secretion of the TGF family member, activin-A. Activin-A immunoreactivity was found to be more evident in association with M2 macrophages in regenerating lesions of the adult mouse brain at the time when oligodendrocytes are differentiating and myelin regeneration is initiated, and was reduced upon M2 depletion. Mouse brain explant cultures treated with M2 conditioned media during the initiation of myelin regeneration showed increased oligodendrocyte differentiation which was significantly reduced with anti-activin-A blocking IgG supplementation. Examination of expression of receptors that directly bind activin-A, activin receptor (Acvr)2A and (Acvr)2B, showed that oligodendrocyte lineage cells within regenerating lesions express both subtypes and also express Acvrl B, the receptor which is recruited by ligand-bound Acvr2 and which is required for subsequent functional downstream signalling. Together, these findings indicate the capacity of oligodendrocyte lineage cells within remyelinating lesions to directly bind and respond to activin-A.

Activin signalling as defined herein means alterations of cell surface or intracellular signalling molecules following activation of activin receptors by agonists or activins.

The present invention additionally provides a use of an activin receptor activating agent for the treatment for progressive demyelinating diseases. In particular the present invention provides a use of an activin receptor activating agent for the treatment for progressive demyelinating diseases wherein said activating agent is independently selected from: one or more activin receptor agonists; activin as defined herein; and/or mixtures thereof. Activin as defined herein includes activin and all activin isoforms of mammalian origin, including activin-A, activin-B, activin-AB. Preferred herein is activin-A, which is a homodimer of beta-A subunits.

Activin receptors as defined herein means molecules that can directly bind activins (Acvr2a and Acvr2b) and those which associate with Acvr2 receptors to mediate downstream signalling (Acvrl ).

The present invention additionally provides an effective treatment for progressive demyelinating diseases via promotion of oligodendrovyte differentiation by treatment with activin and all activin isoforms of mammalian origin, including activin-A, activin-B, activin-AB, and particularly activin-A, which is a homodimer of beta-A subunits.

As additionally established by the results hereinafter, activin-A treatment of oligodendrocyte lineage cells was sufficient to enhance oligodendrocyte differentiation. In accordance with these results, signalling pathways downstream of activin receptor activation include Rac/Cell division cycle 42 (Cdc42) guanosine triphosphate(GTP)ases, Akt, and mammalian target of rapamycin (mTOR), all of which have been previously implicated in positively regulating oligodendrocyte differentiation and/or myelination (Liang et al., 2004 J Neurosci, Thurnherr et al., 2006 J Neurosci, Flores et al., 2008 J Neurosci, Guardiola-Diaz et al., 2012 Glia), and it is additionally proposed herein that activin-A treatment in accordance with the present invention will have a positive impact on such signalling pathways and as such provide a solution to the long-felt need for an effective method for the treatment of progressive demyelinating diseases. Additionally, TGF isoforms have been shown to induce myelin protein expression (Diemel et al., 2003 J Neurosci Res, McKinnon et al., 1993 J Cell Biol, Dutta et al., 2014 Development).

The unprecedented results provided hereinafter which demonstrate a role for activin- A in the regulation of oligodendrocyte differentiation together with evidence of the expression of its receptors on oligodendrocyte lineage cells in regenerating lesions, as well as up- regulation of its receptor during remyelination effectively demonstrate the potential of activin- A in particular, and activin in general as a novel therapeutic target for the promotion of oligodendrocyte differentiation and myelin regeneration. According to a yet further aspect the present invention provides an oligodendrocyte differentiation regulating agent for the treatment for progressive demyelinating diseases. In particular the present invention provides a use of an oligodendrocyte differentiation regulating agent for the treatment for progressive demyelinating diseases wherein said agent is activin as defined herein. According to a yet further aspect the present invention provides an agent for the promotion of myelin regeneration and/or oligodendrocyte differentiation regulating for use in the treatment for progressive demyelinating diseases. In particular the present invention provides activin for use as a myelin regeneration and /or oligodendrocyte differentiation agent for the treatment for progressive demyelinating diseases.

The invention concerns amongst other things the treatment of demyelinating diseases such as for example MS, and including RRMS, PPMS and SPMS. The term "treatment", as used in relation to the various therapies encompassed by this invention, include the following and combinations thereof: (1 ) inhibiting, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition ; and/or (3) relieving and/or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms "treatment", "prophylaxis" and "inhibitor" and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.

The term "prophylaxis" includes reference to treatment therapies for the purpose of preserving health or inhibiting or delaying the initiation and/or progression of an event, state, disorder or condition, for example for the purpose of reducing the chance of an event, state, disorder or condition occurring. The outcome of the prophylaxis may be, for example, preservation of health or delaying the initiation and/or progression of an event, state, disorder or condition. It will be recalled that, in any individual patient or even in a particular patient population, a treatment may fail, and this paragraph is to be understood accordingly.

The term "inhibit" includes reference to delaying, stopping, reducing the incidence of, reducing the risk of and/or reducing the severity of an event, state, disorder or condition. Inhibiting an event, state, disorder or condition may therefore include delaying or stopping initiation and/or progression of such, and reducing the risk of such occurring. The products of the disclosure may be used to inhibit the progression of MS in PPMS and SPMS and other demyelinating disorders and other events, disorders and/or conditions which are disclosed herein.

As used herein, by "peptide" and "protein" can be used interchangeably and mean at least two covalently attached amino acids linked by a peptidyl bond. The term protein encompasses purified natural products, or products which may be produced partially or wholly using recombinant or synthetic techniques. The terms peptide and protein may refer to an aggregate of a protein such as a dimer or other multimer, a fusion protein, a protein variant, or derivative thereof. The term also includes modifications of the protein, for example, protein modified by glycosylation, acetylation, phosphorylation, pegylation, ubiquitination, and so forth. A protein may comprise amino acids not encoded by a nucleic acid codon.

By "protein modification" or "protein mutation" is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. The proteins of the invention may include at least one such protein modification.

Conservative substitution: One or more amino acid substitutions (for example of 1 , 2, 5 or 10 residues) for amino acid residues having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, a conservative substitution in a contact phase factor inhibitory peptide may be an amino acid substitution that does not substantially affect the ability of the peptide to inhibit a contact phase factor or combination thereof. In a particular example, a conservative substitution in a contact phase factor inhibitory peptide, such as a conservative substitution in SEQ ID NO:1 , is an amino acid substitution that does not significantly alter the ability of the protein to inhibit FXIIa and FXIa, e.g. does not significantly alter the ability of the protein to inhibit FXIIa, FXIa and kallikrein. Methods that can be used to determine inhibition of FXIIa, FXIa and kallikrein are disclosed herein (reconstitution assay; Decrem et al. Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis. J. Exp. Med. Vol. 206 No. 1 1 , 2381 -2395, 2009; section Material & Methods, Effect of Ir-CPI in a reconstituted system page 2392). Screening of variants of SEQ ID NO:1 can be used to identify which amino acid residues can tolerate an amino acid substitution. In one example, inhibition of each of FXIIa and FXIa, e.g. of FXIIa, FXIa and kallikrein, is not altered by more than 25%, for example not more than 20%, for example not more than 10%, when a conservative amino acid substitution is affected.

In one example, one conservative substitution is included in the peptide, such as a conservative substitution in SEQ ID NO:1 . In another example, 10 or fewer conservative substitutions are included in the peptide, such as five or fewer. A peptide or protein of the invention may therefore include 1 , 2, 3, 4, 5, 6, 7, 8, 9 10 or more conservative substitutions. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Alternatively, a polypeptide can be produced to contain one or more conservative substitutions by using peptide synthesis methods, for example as known in the art.

Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Examples of amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for lie; lie or Val for Leu; Arg or Gin for Lys; Leu or lie for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and lie or Leu for Val.

In one embodiment, the substitutions are among Ala, Val Leu and lie; among Ser and Thr; among Asp and Glu; among Asn and Gin; among Lys and Arg; and/or among Phe and Tyr.

Further information about conservative substitutions can be found in, among other locations, Ben-Bassat et al., (J. Bacteriol. 169:751 -7, 1987), O'Regan et al., (Gene 77:237-51 , 1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321 -5, 1988), WO 00/67796 (Curd et al.) and in standard textbooks of genetics and molecular biology.

The term "modified protein" or "mutated protein" encompasses proteins having at least one substitution, insertion, and/or deletion of an amino acid. A modified or mutated protein may have 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid modifications (selected from substitutions, insertions, deletions and combinations thereof).

Functionally Equivalent: Having an equivalent function. In the context of a contact phase factor inhibitory peptide, functionally equivalent molecules include different molecules that retain the function of inhibiting the same contact phase factor(s). For example, functional equivalents can be provided by sequence alterations in contact phase factor inhibitory peptide, wherein the peptide with one or more sequence alterations retains the ability of the unaltered peptide to inhibit one or more contact phase factors. Examples of sequence alterations include, but are not limited to, conservative substitutions, deletions, mutations, and insertions. In one example, a given polypeptide binds an active, and a functional equivalent is a polypeptide that binds the same active. Thus a functional equivalent includes peptides that have the same binding specificity as a polypeptide, and that can be used in place of the polypeptide. In one example a functional equivalent includes a polypeptide wherein the binding sequence is discontinuous, wherein the active binds a linear epitope. Thus, if the peptide sequence is that of amino acids 1 -1 0 of SEQ ID NO: 1 , a functional equivalent includes discontinuous epitopes, that can appear as follows ( ^** =any number of intervening amino acids): NH ₂ -**-A**N**H**K**G**R**G**R**P**A-COOH. In this example, the polypeptide may be considered functionally equivalent to amino acids 1 -1 0 of SEQ ID NO: 1 if the three dimensional structure of the polypeptide is such that it can bind a monoclonal active that binds amino acids 1 -1 0 of SEQ ID NO: 1 . The polypeptide may be considered functionally equivalent to amino acids 1 - 1 0 of SEQ ID NO: 1 if the three dimensional structure of the polypeptide is such that it can inhibit a contact phase coagulation factor, e.g. has an inhibitory activity against FXIIa and FXIa at least that of that of SEQ ID NO: 1 .

The term "isolated" means a biological component (such as a nucleic acid molecule or protein) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.

In one example, isolated refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

In one example, the term "isolated" as used with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally- occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally- occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

SEQ ID NO: 1 may be an isolated protein. SEQ ID NO: 1 may be for use in therapy and/or prophylaxis.

Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl- terminal or side chain, may be provided in the form of a salt of a pharmaceutically- acceptable cation or esterified, for example to form a C1 -C6 alkyl ester, or converted to an amide, for example of formula CONR ¹ R ² wherein R ¹ and R ² are each independently H or C1 -C6 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1 -C6 alkyl or dialkyl amino or further converted to an amide. Hydroxyl groups of the peptide side chains may be converted to alkoxy or ester groups, for example C1 -C6 alkoxy or C1 -C6 alkyl ester, using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as F, CI, Br or I, or with C1 -C6 alkyl, C1 -C6 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability. Peptide products of the invention may be modified as described in this paragraph. Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its environment within a cell, such that the peptide is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that may accompany it. In another example, a purified peptide preparation is one in which the peptide is substantially-free from contaminants, such as those that might be present following chemical synthesis of the peptide.

In one example, a peptide of the disclosure is purified when at least 50% by weight of a sample is composed of the inhibitor, for example when at least 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more of a sample is composed of the peptide. Examples of methods that can be used to purify a peptide, include, but are not limited to the methods disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 17). Protein purity can be determined by, for example, high-pressure liquid chromatography or other conventional methods.

To compare two amino acid sequences, the options of B12seq can be set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1 .txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq-i c:seq1 .txt-j c:seq2.txt-p blastp-o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1 166 matches when aligned with a test sequence having 1 154 nucleotides is 75.0 percent identical to the test sequence (i.e., 1 166/1554 ^* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.1 1 , 75.12, 75.13, and 75.14 are rounded down to 75.1 , while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 1 1 , and a per residue gap cost of 1 ). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full- length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.

When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap I penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.

Variants, fragments or fusion proteins: The disclosed proteins include variants, fragments, and fusions thereof.

Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal.

Pharmaceutical Formulations and Therapeutic Methods

The invention provides formulations for use in the novel methods of treatment comprising activin, in any of the forms detailed herein, and particularly activin-A, formulated for pharmaceutical use and optionally further comprising a pharmaceutically acceptable diluent, excipient and/or carrier. The invention therefore additionally includes pharmaceutical formulations which may include, in addition to activin, a pharmaceutically acceptable diluent, excipient and/or carrier. Such formulations may be used in the methods of the disclosure. Additionally or alternatively, pharmaceutical formulations may include a buffer, stabiliser and/or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient (activin). The precise nature of the carrier or other material will depend on the route of administration, which may be any suitable route, for example by a parenteral route and particularly by infusion or injection (with or without a needle). The route of administration may be subcutaneous injection. The route of administration may be intravenous injection or infusion. Other routes of administration which may be used include administration by inhalation or intranasal administration

Amounts effective for therapeutic use, which may be a prophylactic use, will depend upon the severity of the disease and the general state of the patient's health. A therapeutically effective amount of the active is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

As a particular advantage of the present methods for promotion of myelin regeneration in the treatment of patients suffering from demyelinating diseases, is the unique and unprecedented potential of the treatments according to the present invention for the treatment of progressive forms of such diseases, including PPMS and SPMS, a preferred effective amount is one which provides subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer for a continuous period. In other words, following treatment in accordance with the invention the beneficial relief or identifiable improvement is maintained.

Activin as defined herein may be administered in conjunction with another active agent, whether simultaneously, separately or sequentially. The other active agent may be another form of activin, or a different active agent falling outside the invention.

Single or multiple administrations of the formulations of the disclosure are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the actives disclosed herein to effectively treat the patient, bearing in mind though that it may not be possible to achieve effective treatment in every instance. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of treatment. The dose may be sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

The novel and inventive treatment in accordance with the present invention may also be provided in combination with any of the pre-existing medicaments which are known to have potential for the amelioration of the symptoms of MS, such as for example currently prescribed medicaments, such as for example immuno-modulators aimed at dampening initial injury, as well as agents for the attenuation of secondary pathologies. This approach may be particularly suited to subjects who have a pre-existing diagnosis of a progressive demyelinating disease, particularly PPMS and/or SPMS and whom are already receiving medication for symptom management.

For newly diagnosed patients, or for patients with pre-existing diagnosis of a progressive demyelinating disease, treatment in accordance with the present invention may obviate the need for any of the currently prescribed medicaments, such as for example immuno- modulators aimed at dampening initial injury, as well as agents attenuating secondary pathologies. Thus the present invention additionally provides a method for the treatment of progressive demyelinating disorders with a sole-medicament based on activin in accordance with the present invention wherein said sole-medicament promotes myelin regeneration and/or oligodendrocyte differentiation.

Description of Figures Figures 1 (a) to (c) illustrate a switch from an M1 to an M2-dominant microglia/macrophage response occurs at the initiation of remyelination.

(a) Oligodendroglial lineage cell responses in the mouse corpus callosum at 3, 10, and 21 days post lesion (dpi) induction by stereotaxic injection of lysolecithin (LPC).

(b) Six representative images of lesions immunostained against iNOS, Arg-1 (left), and CD68 (right) at 3, 10 and 21 dpi. Inset: isotype controls for iNOS and Arg-1 antibodies.

Examples of iNOS+ cells (top left-hand image) and Arg-1 + cells (middle and bottom left- hand images) are indicated by arrows. Scale bar, 25 μηι.

(c) Percentage of iNOS+ M1 cells, Arg-1 + M2 cells, and iNOS-Arg1 - (unpolarized) cells per field in lesion of the mouse corpus callosum ± s.e.m. One way ANOVA and Newman-Keuls post-hoc test, ^*** p<0.001 (n=5).

Figures 2(a) to (d) illustrate additional characterization of polarization in corpus callosum lesions. (a) Two representative images of sham PBS-injected lesions of the mouse corpus callosum at 3 and 10 dpi immunostained for iNOS, Arg-1 , and CD68.

(b) Eight representative images showing co-localization of iNOS and Arg-1 in microglia/ macrophage cell bodies (CD68; arrows; top row of images) and processes (F4/80; bottom row of images), (c) Six representative images showing 3 and 10 dpi lesions immunostained for additional M1 markers TNF , CD16/32. Colocalization is observed with M1 marker iNOS.

(d) Six representative images showing 3 and 10 dpi lesions immunostained for additional M2 markers MR, IGF-1 . Colocalization is observed with microglia/ macrophage pan marker CD68. All scale bars in Figure 1 (a) to (d) represent 25 μηι.

Figures 3 (a) to (i) illustrate the switch from M1 - to M2-dominant response in remyelinating lesions of the rat caudal cerebellar peduncle.

(a) Oligodendroglial lineage cell responses following induction of demyelination of the caudal cerebellar peduncle by stereotaxic injection of ethidium bromide (EtBr).

(b) Injection of demyelinating toxin induces accumulation of microglia (CD68+) at 5dpl. In the representative image for 5dpl, the accumulation of microglia (CD68+) is indicated by the area within the dotted line

(c) These are the central and right hand images beside Figure 3(b). In the central image for 10dpi recovery of myelin protein expression is observed with re-expression of MAG. The right hand image illustrates re-expression of MBP and MAG at 21 dpi.

(d) Microglia macrophages (CD68+) are indicated by white arrows. Figure 3(d) illustrates that these are present at 5 dpi, with an increase in abundance at 10 dpi, and a decrease in number by 21 dpi. (e) Three representative images of lesions immunostained for iNOS and Arg-1 . Examples of iNOS-i- cells at 5dpl (left-hand image) and Arg-1 + cells at 10 dpi (middle image) are indicated by white arrows. No iNOS-i- cells or Arg-1 + cells were found in this example image at 21 dpi (right-hand image).

(f) A bar chart illustrating the percentage of iNOS-i- M1 , Arg-1 + M2, and unpolarized (iNOS- Arg1 -) cells per field ± s.e.m. at 5, 10, and 21 dpi. One-way ANOVA and Newman-Keuls post test, ^*** p<0.01 , (n=3). (g) A bar chart illustrating the mean number of iNOS+ M1 , Arg-1 + M2, and unpolarized (iNOS- Arg1 -) cells per field ± s.e.m. at 5, 10, and 21 dpi. One-way ANOVA and Newman- Keuls post test, ^* p<0.05, ^*** p<0.001 . (n=3).

(h) 5 and 10 dpi sections of remyelinating lesions of the rat caudal cerebellar peduncle immunostained for additional M1 (TNF , CD16/32)

(i) 5 and 10 dpi sections of remyelinating lesions of the rat caudal cerebellar peduncle immunostained for additional M2 (IGF-1 , MR) markers.

All scale bars in (b) to (i) represent 25 μηι.

Figures 4(a) to (f) illustrate microglia and peripherally-derived macrophages contribute to both M1 and M2 polarized populations during remyelination.

(a) Illustration of how CCR2 ^_/" mice were lesioned in the corpus callosum to examine polarization in microglia only.

(b) Four representative images of CCR2 ^_/" lesions at 3 and 10 dpi immunostained against iNOS and Arg-1 and CD68. Examples of 1NOS+ cells at 3 dpi, on the top left hand image and Arg-1 + cells at 10 dpi, in the bottom left hand image are indicated by arrows. Scale bar, 25 μηι.

(c) A bar chart illustrating the percentage of iNOS+, Arg-1 +, or unpolarized (iNOS-, Arg-1 -) cells in CCR2 ^_/" lesions of the mouse corpus callosum ± s.e.m. One way ANOVA and Newman-Keuls post-hoc test, ^*** p<0.001 (n=3). (d) Illustration of how CCR2 -/- mice were lesioned in the corpus callosum and injected with GFP-expressing wildtype bone marrow derived cells to examine polarization in peripherally- derived macrophages only.

(e) Images of GFP+ macrophages, indicated by stars, and GFP- microglia, indicated by hash symbols, were iNOS-i- or Arg1 +. Scale bar, 5 μηι. (f) A bar chart illustrating numbers of GFP+ (macrophages; black bars) and GFP- (microglia; white bars) iNOS+ M1 and Arg-1 + M2 cells per field ± s.e.m. (n=3).

Figures 5 (a) to (f) illustrate polarization of cultured microglia to M1 and M2 phenotypes.

(a) The top set of 9 images are representative images of microglia treated with interferon- gamma and lipopolysaccharide (M1 ; left-hand column), or IL-13 (M2a; middle column), or IL- 10 (M2c; right-hand column) immunostained against M1 markers iNOS, CD86, CD16/32. The bottom set of 9 images are representative images of microglia treated with interferon- gamma (IFN- ) and lipopolysaccharide (LPS) (M1 ; left-hand column), or IL-13 (M2a; middle column), or IL-10 (M2c; right-hand column) immunostained against M2 markers MR, Arg-1 , and IL1 Ra. Scale bar, 25 μηι.

(b) A bar chart illustrating the mean percentage of iNOS+ or MR+ cells of total CD68+ cells ± s.e.m., relating to Fig. 5(a). Kruskal-Wallis test and Dunn's multiple comparison post-hoc test, ^* p<0.05, ^** p<0.01 , ^*** p<0.001 ,(n=4). Information from the top row of first 9 set of images and top row of bottom set of 9 images was included into the data-set from which this chart was prepared.

(c) Western blots showing expression of iNOS with IFN /LPS treatment and increase in MR expression with IL-13 or IL-10 treatment, with loading control GAPDH.

(d) to (f) ELISAs used to assay conditioned media from microglia treated with interferon- gamma and lipopolysaccharide, or IL-13, or IL-10 to induce M1 , M2a and M2c phenotypes, respectively, for levels of TNF (d) P=0.0004, IGF-1 (e) P=0.0330, and IL-10 (f) P=0.0051 , presented as mean fold over M0 control ± s.e.m.

A small yet significant increase in IL-10 secretion was observed with IFN / LPS treatment (P =0.0081 ). Polarizing factors alone were included in the assay as a control and did not show detectable levels relative to those measured in conditioned media (n=3-5) (d-f), 2-tailed Student's f-test.

Figure 6 (a) to (i) illustrates M1 and M2 associated gene expression in cultured microglia.

(a) Figure 6(a) illustrates M1 - and M2-associated genes which were identified from a previously performed microarray as being significantly upregulated during remyelination of the rat caudal cerebellar peduncles (CCP). These genes were selected for a custom qPCR array to assess gene expression profiles of polarized microglia in vitro.

(b) Representative images showing confirmation of expression of additional polarization markers CD86, CCL2, CXCL1 1 , in iNOS+ cells and TGF 2 in Arg-1 + cells in the rat CCP, where the presence of these polarization markers are indicated by stars. Scale bar, 5 μηι.

Figures 6(c) to (i) are bar charts illustrating gene expression levels in microglia treated with IFN /LPS (M1 ), IL13 (M2a), and IL10 (M2c), analyzed for gene expression levels of: (c) Cd86; (d) CxcM 1 ; (e) Ccl2; (f) Fcgr2a (Cd32); (g) Mrc7(mannose receptor); (h) Tgfb2; and (i) Arg1, values are represented as 2 ^{" Cp} ± s.e.m. Figure 7(a) to (e) illustrates that M1 and M2 microglia conditioned media increase OPC proliferation and migration.

OPCs were treated with microglia conditioned media for 3d.

(a) Representative images of OPCs treated with microglia unconditioned media alone ('control') or conditioned media from microglia that were untreated (MO) or treated with IFN-

/LPS (M1 ), or IL-13 (M2a), or IL-10 (M2c) immunostained against NG2 and the proliferative marker Ki67. Double-positive cells are highlighted by arrows in each image. Scale bar, 25 μηι.

(b) Bar chart illustrating the increased number of NG2+ cells per field following treatment of OPCs with conditioned media from microglia that were MO, M1 , M2a, or M2c, (black bars) when compared to OPCs treated with unconditioned microglia media (control; white bar). Polarizing factors present in the conditioned media (IFN /LPS, IL-13, or IL-10) were directly applied to OPCs as a control, these are shown as grey bars, and as illustrated IL-10 increased number of NG2+ cells (P=0.0029). ^* p<0.05, ^** p<0.01 . (c) Bar charts illustrating the increased number of Ki67+/NG2+ cells following treatment of OPCs with conditioned media from microglia that were MO (P=0.0084), M1 (P=0.0362), M2a (P=0.0194), M2c (P=0.0192) when compared to OPCs treated with unconditioned microglia media (control; white bar). Polarizing factors present in the conditioned media (IFN /LPS, IL-13, or IL-10) were directly applied to OPCs as a control, these are shown as grey bars. Values were normalized to those from control conditions.

(d) Bar charts illustrating the increased number of BrdU+ cells following treatment of OPCs with conditioned media from microglia that were untreated (M0), M1 (P=0.042), M2a (P=0.0083) and M2c (P=0.0423) when compared to OPCs treated with unconditioned microglia media (control; white bar). Polarizing factors present in the conditioned media (IFN /LPS, IL-13, or IL-10) were directly applied to OPCs as a control, these are shown as grey bars.

Values from conditioned media-treated conditions (black bars) and polarizing factors alone (grey bars) were normalized to control (unconditioned media, white bars) in (c) and (d). Values from (b) to (d) are represented as mean ± s.e.m., 2-tailed Student's f-test. (e) Bar charts illustrating the number of OPCs ± s.e.m. migrated towards microglia conditioned media from M0, M1 , M2a, and M2c microglia (black bars), or polarizing factors alone (grey bars; IFN- /LPS, IL-13, and IL-10), normalized to values in control (unconditioned media, white bar) showing chemotactic properties of both M1 and M2 conditioned media. Kruskal-Wallis test and Dunn's multiple comparison post-test, ^** p<0.01 , ^*** p<0.001 . (n=3).

Figures 8(a) and (b) illustrates M2 polarized microglia conditioned media promotes the survival of OPCs in a death-inducing environment. OPCs were treated with microglia conditioned media for 3d in media devoid of serum and growth factors. Polarizing factors present in the conditioned media (IFN /LPS, IL-13, or IL- 10) were directly applied to OPCs as a control.

(a) Representative images of OPCs under basal growth supplemented conditions, grown in deprivation media alone, or supplemented with microglia conditioned media. OPCs were immunostained against NG2 and apoptotic OPCs were visualized by TUNEL assay. In these images, examples of TUNEL positive cells are indicated by white arrows. Scale bar, 50 μηι.

(b) Bar chart illustrating the mean percentage of TUNEL+ NG2+ OPCs in conditions of deprivation media alone or supplemented with microglia conditioned media or polarizing factors alone normalized to values from OPCs grown under basal growth supplemented conditions (GF control) ± s.e.m. DNase and ethanol treatment were positive controls for TUNEL positivity. Information from the images in (a) was included into the data-set from which this chart was prepared.

Application of microglia CM to OPCs under normal growth conditions did not induce apoptosis (data not shown). Kruskal-Wallis test and Dunn's multiple comparison post-test, ^** p<0.01 . (n=6).

Figures 9(a) to (c) illustrate M2 conditioned media promotes oligodendrocyte differentiation.

(a) Images of OPCs treated with unconditioned microglia unconditioned media (control) or M0, M1 , M2a, or M2c microglia conditioned media immunostained against NG2 and MBP. In these images examples of MBP positive cells are indicated by white arrows. Scale bar, 50 μηι.

(b) Bar chart illustrating the percentage of MBP+ cells following treatment of OPCs with unconditioned microglia media (control) or M0, M1 , M2a, or M2c conditioned media, or polarizing factors alone (IFN /LPS, IL-13, orlL-10), Kruskal-Wallis test and Dunn's multiple comparison post-test, ^*** p<0.001 (n=3). Information from the images in (a) was included into the data-set from which this chart was prepared. (c) Bar chart illustrating the number of MOG+ cells per field ± s.e.m. following treatment of OPCs with unconditioned microglia media (control) or MO, M1 , M2a, or M2c conditioned media. Kruskal-Wallis test and Dunn's multiple comparison post-test, ^** p<0.01 (n=3).

Figures 10(a) to (h) illustrate the selective depletion of M1 polarized microglia/ macrophages using gadolinium chloride.

(a) Representative images of M1 (iNOS+), the left-hand image, and M2a (MR+), the right- hand image, microglia treated with 270 μηι GdCI3 and apoptosis assessed by TUNEL assay. In these images apoptotic cells are indicated by white arrowheads, and were iNOS+ and MR-. Scale bar, 25 μηι.

(b) A bar chart illustrating the percentage of TUNEL+ M1 microglia treated with vehicle (control), or GdCI3 (0.27-270 μηι) from the test illustrated in Fig. 10(a). Mann-Whitney test, P=0.0286. (n=4).

(c) A bar chart illustrating the mean percentage of TUNEL+ cells ± s.e.m. in M0, M1 , M2a, and M2c polarized microglia treated with vehicle or 270 μηι GdCI3 from the tests discussed hereinbefore. One-way ANOVA and Newman-Keuls post-hoc test, ^** p<0.01 (n=6).

(d) Representative images of astrocytes (GFAP+) in control and GdCI3-injected remyelinating lesions of the mouse corpus callosum at 3 dpi show no difference in astrocyte reactivity. This indicates that the GdCI3 did not cause a loss of astrocytes or increase reactivity of astrocytes following its injection.

(e) Bar charts illustrating the percentage of iNOS+ (black bars; first bar) or Arg1 + cells (grey bars; second bar) or unpolarised microglia/ macrophages (iNOS- Arg-1 -) (white bars; third bar) in control or GdCI3-injected lesions of the mouse corpus callosum, P=0.0135 (iNOS), 0.0319 (Arg-1 ), 2-tailed Student's f-test (n=5). (f) Representative images of GdCI3-injected lesions of the mouse corpus callosum immunostained for M1 markers (iNOS, TNF , CD16/32) and M2 markers (MR, IGF-1 ) at 3 dpi for tests as discussed herein. Scale bar, 25 μηι. This indicates that there was little immunoreactivity for other M1 or M2 markers following GdCI3 injection, supporting the depletion of M1 microglia/ macrophages and indicating no increase in M2 microglia/ macrophages in the lesion.

(g) Bar charts illustrating the NG2+ (n=4) and Nkx2.2 (n=5) cells per field in control and GdCI3-treated lesions of the mouse corpus callosum at 3 dpi. 2-tailed Student's f-test, p>0.05. This indicates that GdCI3 injection did not decrease the total number of OPCs in the lesion.

(h) Bar charts illustrating the mean area of MBP and MOG co-localization, fold over control ± s.e.m. in GdCI3-treated lesions of the mouse corpus callosum at 21 dpi. ^** p<0.01 , 2-tailed Student's f-test (n=6).

Figures 11 (a) to (d) illustrate that selective depletion of M1 microglia/ macrophages in a demyelinated lesion in the CNS impairs OPC proliferation.

(a) Illustration of how gadolinium chloride (GdCI3) was injected into corpus callosum lesions at the onset of demyelination (0 dpi) prior to the peak in M1 polarization at 3 dpi.

(b) Three representative images of control or GdCI3-injected lesions of the mouse corpus callosum at 3 dpi with immunostaining against iNOS, Arg-1 , and CD68. Examples of iNOS+ cells (middle image) and Arg-1 + cells (right-hand image) are indicated by white arrowheads. Scale bar, 25 μηι. (c) Bar charts illustrating the numbers of iNOS+ M1 , Arg-1 + M2, or unpolarized (iNOS-, Arg- 1 -) cells per field ± s.e.m. in control and GdCI3-injected lesions of the mouse corpus callosum at 3 dpi. One way ANOVA and Newman-Keuls post-test, ^** p<0.01 (n=5).

(d) Bar chart illustrating the density of PCNA+ Nkx2.2+ proliferating OPCs per mm ² in control and GdCI3-injected lesions of the mouse corpus callosum ± s.e.m. at 3 dpi. P=0.0496, 2- tailed Student's f-test (n=5).

Figures 12(a) to (h) illustrate that selective depletion of M2 microglia/ macrophages in a demyelinated lesion in the CNS impairs oligodendrocyte differentiation.

(a) An illustration of mannosylated clodronate liposomes which were used to induce apoptosis selectively in M2 polarized microglia/ macrophages due to upregulation of mannose receptor, and induce clodronate-mediated apoptosis following phagocytosis.

(b) An illustration of corpus callosum lesions which were injected with MCLs at 8 dpi prior to the peak in M2 polarization at 10 dpi.

(c) Six representative images at 10 dpi of control and MCL-injected lesions of the mouse corpus callosum immunostained for iNOS, Arg-1 , and CD68. Examples of iNOS-i- cells (middle two images) and Arg-1 + cells (right-hand images) are indicated by white arrowheads. Scale bar, 25 μηι.

(d) Bar charts illustrating the number of iNOS+ M1 , Arg-1 + M2, or unpolarized (iNOS-, Arg-1 - ) cells per field ± s.e.m. in control and MCL-injected lesions of the mouse corpus callosum at 10 dpi. One way ANOVA and Newman-Keuls post-test, ^** p<0.01 , ^*** p<0.001 (n=5).

(e) Bar charts illustrating the mean lesion pixel counts ± s.e.m. for MAG and MBP at 10 dpi in mouse corpus callosum lesions. 2-tailed Student's f-test versus control, P=0.0361 and 0.0106, respectively (n=5).

(f) Four representative images of lesions, indicated within the dotted outlines at 10 dpi in control and MCL-injected lesions of the mouse corpus callosum immunostained against

MBP(right-hand column) and MAG (left-hand column). Scale bar, 100 μηι.

(g) A chart illustrating the quantification of nodes of Ranvier (paranodal Caspr expression flanking nodal Ankyrin-G expression) ± s.e.m. in control and MCL-treated lesions of the mouse corpus callosum at 21 dpi. 2-tailed Student's f-test versus control, P=0.0068. (h) Two representative images of control and MCL-treated lesions of the mouse corpus callosum at 21 dpi immunostained against Caspr and Ankyrin-G. In these images, examples of Nodes of Ranvier are indicated by white arrowheads. Scale bar, 25 μηι.

Figures 13(a) to (I) illustrate the selective depletion of M2 polarized microglia/ macrophages using mannosylated clodronate liposomes.

(a) Two representative images of iNOS+ M1 and MR+ M2a microglia treated with MCLs (1 :5) and assessed for apoptosis by TUNEL assay. In M1 polarizing conditions (left-hand image), iNOS+ cells were TUNEL-, as indicated by the white arrows; TUNEL+ cells were iNOS-, as indicated by the white arrowheads. In M2a polarizing conditions (right-hand image), TUNEL+ cells were MR+, as indicated by the white arrowheads, whereas MR- cells were TUNEL-, as indicated by the white arrows.

(b) Bar chart illustrating the mean percentage of TUNEL+ cells ± s.e.m. in M0, M1 , M2a, and M2c polarized microglia treated MCLs (1 :5 dilution), normalized to values from M0 conditions. P=0.027 (M1 vs. M2a), P=0.013 (M1 vs. M2c), 2-tailed Student's f-test (n=6). (c) Bar chart illustrating the mean percentage of TUNEL+ cells ± s.e.m. in M2a polarised microglia treated with MCLs compared to control (white bars) (n=4) across a range of dilutions (1 :200-1 :5; black bars). 1 :5 dilution significantly increased percentage TUNEL + cells compared to control (P=0.015). 2-tailed Student's f-test (n=4).

(d) Bar chart illustrating the mean percentage of TUNEL+ cells ± s.e.m. in M2c polarised microglia treated with MCLs across a range of dilutions (1 :200-1 :5; black bars) compared to control (white bars) (P=0.0251 , 0.0016, 0.03 for 1 :50, 1 :10, 1 :5, respectively) . 2-tailed Student's f-test (n=4).

(e) Bar chart illustrating the mean numbers of microglia per field ± s.e.m. for M2a polarized cells treated with MCLs across a range of dilutions (1 :200-1 :5; black bars) compared to control (white bars), (P=0.02, 0.03, 0.009, 0.007 for 1 :100, 1 :50, 1 :10, 1 :5, respectively) 2- tailed Student's f-test (n=4).

(f) Bar chart illustrating the mean numbers of microglia per field ± s.e.m. for M2c polarized cells treated with MCLs across a range of dilutions (1 :200-1 :5; black bars) compared to control (white bars), (P=0.0003, <0.0001 , 0.0005, 0.0005 for 1 :100, 1 :50, 1 :10, 1 :5, respectively) 2-tailed Student's f-test (n=4). (g) Bar chart illustrating the percentage of iNOS+ M1 , Arg-1 + M2, or unpolarized (iNOS-, Arg-1 -) cells per field ± s.e.m. in control and MCL-injected lesions of the mouse corpus callosum at 10 dpi. P<0.0001 , 2-tailed Student's f-test (n=5). This demonstrates the effectiveness of MCLs in depleting M2 microglia and macrophages in vivo following demyelination. (h) Two representative images of MCL-injected lesions of the mouse corpus callosum, immunostained for additional M1 (iNOS, TNF , CD16/32) and M2 markers (MR, IGF-1 ). Scale bar, 25 μηι.

(i) Bar chart illustrating the mean area of MBP and MOG colocalization fold over control ± s.e.m. in lesions of the mouse corpus callosum at 21 dpi, in control lesions and those injected with MCLs. 2-tailed Student's f-test, P=0.0002 (n=6).

(j) Two representative images of axons (NF+; right-hand image) detectable, running through the lesion (MBP negative; left-hand image) at 10 dpi following MCL injection.

(k) Two representative images showing that MCL injection into lesions of the mouse corpus callosum did not influence astrocytes (GFAP+) within the lesion by 10 dpi compared to control lesions. Scale bar, 25 μηι.

(I) Bar chart illustrating total numbers of Nkx2.2+ OPCs ± s.e.m. in control and MCL injected lesions. 2-tailed Student's f-test, p>0.05 (n=4). Figures 14(a) to (f) illustrate restored remyelination efficiency in aged mice via heterochronic parabiosis is associated with increased densities of M2 polarized cells.

(a) Illustrated that lesion induction in the ventral spinal cord was by lysolecithin injection. Figure (a) further illustrates the parabiotic pairings between young (Y-Y) or old (O-O) animals which were compared to lesions in old animals following heterochronic pairing (Y- O), with the results from previous studies to show restored remyelination efficiency in old partners of Y-0 pairings.

(b) Six representative images of lesions immunostained at 7 dpi against Arg-1 and CD68 in Y/Y, Y/O and 0/0 pairings. Scale bar, 25 μηι.

(c) Bar chart illustrating the MR+ IB4+ M2 cells/ 0.1 mm ² at 7 dpi. 2-tailed Student's f-test: Y/Y vs. 0/0, P=0.048; Y/O vs. 0/0, P=0.005 [n=4 (0/0), 6(Y/0), 4 (Y/Y)]. This is complementary to the images shown in (b) in supporting the finding that M2 microglia/ macrophages are increased in Y/O pairings vs. 0/0 pairings. (d) Bar chart illustrating the CD16/32+ IB4+ cells/0.1 mm ² at 7 dpi. 2-tailed Student's f-test: Y/Y vs. 0/0, P=0.0027; Y/Y vs. Y/O, P=0.014. [n=4 (0/0), 6(Y/0), 4 (Y/Y)]. This shows that M1 microglia/ macrophages were not increased in Y/O pairings vs. 0/0 pairings.

(e) Illustration of the parabiosis between a GFP expressing young mouse and wildtype old mouse with a spinal cord lesion. (f) Six representative images of GFP expressing iNOS+ and CD68+, M1 macrophages (top row), and Arg-1 + and CD68+, M2 macrophages (bottom row). Scale bar, 5 μηι.

Figures 15(a) to (g) illustrate M2 microglia/ macrophage densities are increased in acute active and the rim of chronic active multiple sclerosis lesions.

(a) Graph illustrating that the total number of CD68+ microglia / macrophages / mm ² were significantly increased in acute active (P=0.001 ), chronic active rim (P=0.0156) and centre (P=0.0156), chronic inactive (P=0.001 ) and remyelinated lesions (P=0.002) versus control.

(b) Graph illustrating that the total number of iNOS+ M1 microglia macrophages/ mm ² were increased in acute active (P=0.001 ), chronic active rim (P=0.0156) and centre (P=0.0313), chronic inactive (P=0.0005), and remyelinated lesions (P<0.0001 ) versus control. (c) Graph illustrating that the total number of MR+ M2 microglia/ macrophages / mm ² were increased in acute active lesions (P=0.0068) and the rim of chronic active lesions (P=0.0156) versus control. Mann-Whitney test, n for each lesion type indicated in Table 1 hereinafter. (d) Representative image of MS lesion with iNOS immuno labelling. Scale bar, 100 μηι.

(e) Representative image of MS lesion with MR immuno labelling. Scale bar, 100 μηι.

(f) Four representative images of co-localization of MR (top two images) and iNOS (bottom two images) with microglia macrophage marker CD68. In each of these images the co- localisation with CD68 is indicated by the white arrowheads. (g) Two representative images showing that MR+ (top image) and CD68+ M2 cells (bottom image) were sometimes associated with blood vessels. In each of these images the association with the blood vessels is indicated by the white arrows. Scale bar, 25 μηι.

Figure 16(a) to (h) illustrates that Activin-A is an M2-derived factor that drives oligodendrocyte differentiation in culture and during remyelination, and activin-A receptors are expressed by oligodendrocyte progenitors during remyelination.

(a) Bar chart illustrating the percentage of activin-A immunopositive cells per field in M1 , M2a, and M2c microglia in culture after overnight polarization to M1 and M2 phenotypes, ± s.e.m.

(b) Six representative images of control mouse corpus callosum lesions at 3 and 10 dpi (top two and middle two images, respectively) and MCL-injected lesion at 10 dpi (bottom two images) immunostained for activin-A (left-hand column) and iNOS or MR (right-hand column). Scale bar, 25 μηι.

(c) Six representative images of NG2+ OPCs in remyelinating lesions of the mouse corpus callosum expressing activin receptors Acvr2A, Acvr2B, and Acvrl B. Examples of NG2+ cells positive for these receptors in each of these images are indicated by the white arrows. Scale bar, 10 μηι.

(d) Bar chart illustrating the percentage of MBP+ cells ± s.e.m. in OPC cultures treated with activin-A (1 , 10, 100 ng/ml) or vehicle control for 3 days. One way ANOVA and Newman- Keuls post-hoc test, ^* p<0.05 (n=4). (e) Bar chart illustrating the percentage of MBP+ cells ± s.e.m. in OPC cultures treated with combinations of rat microglia M2a conditioned media, goat isotype control IgG, or goat anti- activin-A blocking antibody. The presence of these factors in a treatment condition is indicated by a plus sign. One way ANOVA and Newman-Keuls post-hoc test, ^** p<0.01 (n=4).

(f) An illustration of the 300 μηι sagittal sections of cerebellum and attached hindbrain which were taken from newborn mouse brain to obtain organotypic cultures, then demyelinated with lysolecithin (LPC) at 21 days in vitro and 2 days later subsequently treated with M2 conditioned media and either anti-activin-A IgG or control goat IgG. Anti-activin-A IgG was also added to cultures on its own.

(g) Two representative images of cerebellar and hindbrain slices treated with M2 conditioned media and goat IgG (left-hand image) or anti-activin-A antibody (right-hand image) immunostained for CC1 and MBP in accordance with the methodology herein. CC1 + cells are indicated in each image with white arrowheads. Scale bar, 25 μηι.

(h) A chart illustrating the percentage of CC1 + oligodendrocytes normalized to values obtained from cerebellar and hindbrain slices treated with goat IgG alone. 2-tailed one- sample or Student's f-test (respectively), M2 conditioned media + IgG vs. IgG, P=0.0213; M2 conditioned media + IgG vs. M2 conditioned media + anti-activin-A IgG, P=0.0071 (n=8). Information from the images in (g) was included into the data-set from which this chart was prepared.

Figure 17 illustrates the expression of activin-A receptors by cells in remyelinating lesions of the mouse corpus callosum.

Fourteen representative images of oligodendrocytes (CC1 +), microglia/ macrophages (CD68+), astrocytes (GFAP+), and neurons (axons; NF+) with expression, or lack thereof, of activin-A receptors Acvr2A and Acvr2B illustrated. In each of these images, cells positive for the expression of the receptors are indicated by the white arrowheads. Scale bar, 10 μηι.

Figure 18 is a bar chart which illustrates the ability of activin-A to promote oligodendrocyte differentiation in an explants model of paediatric myelin disorders. Coronal forebrain sections were treated with lipopolysaccharide (LPS) at 7 days in vitro for 2 hours then exposed to hypoxia (3%02) for 3 days. The bar chart illustrates that the explants demonstrated impaired oligodendrocyte differentiation as indicated by low number of mature oligodendrocytes (MBP+) per field in the control sample. Treatment with activin-A at 1 , 10 and 100 ng/ml during injury induction increased the number of MBP+ cells, (shown is average from n=2 forebrain slices per condition).

Figure 19 is a bar chart which illustrates that activin-A increases oligodendrocyte progenitor cell proliferation. The number of proliferating cells (Ki67+) per field in oligodendrocyte progenitor cells treated with vehicle control or activin-A (at 1 , 10 and 100 ng/ml) are shown. Activin-A at 1 ng/ml significantly increased the number of proliferating progenitor cells (Ki67+) compared to vehicle control. Two-tailed Mann-Whitney test, ^* p<0.05 (n=5).

Figure 20 is a bar chart which illustrates that activin-A promotes oligodendrocyte progenitor cell survival. Oligodendrocyte progenitor cells were either maintained in basal growth media ('GF control') or grown in deprivation media to induce cell death, supplemented with vehicle control ('Deprivation media + vehicle') or activin-A (at 1 , 10 and 100 ng/ml). Activin-A at 100ng/ml significantly decreased the number of apoptotic progenitor cells (TUNEL+) compared to deprivation media with vehicle control. Two-tailed Student's f-test, ^* p<0.05 (n=5). Figure 21 illustrates that activin-A activates activin-receptor associated signalling pathways. Oligodendrocyte progenitor cells were treated with vehicle control or activin-A (at 1 , 10, and 100 ng/ml) for 3 days. As illustrated in the heatmap pictorial representations of phosphorylation of signalling proteins (3 heatmaps in top row), 1 ng/ml activin-A was associated with activation of PI3K/AKT, p38/JNK, and Smad2/3 pathways, whereas as illustrated in 2 heatmaps in the bottom row, 10 ng/ml activin-A was associated with activation of Rac/Cdc42 and ERK1/2 pathways. 100 ng/ml activin-A was associated with low activation of all pathways (top and bottom rows).

Abbreviations The following abbreviations are provided for the avoidance of any doubt as to the meaning of any abbreviated terms used herein.

Axon=long process of nerve cell

CNS=central nervous system

MS=multiple sclerosis PVL=periventricular leukomalacia

CP=cerebral palsy SPMS=secondary progressive multiple sclerosis

PPMS=primary progressive multiple sclerosis

lgM=immunoglobulin M

TNF =tumor necrosis factor alpha

IL1 =interleukin 1 beta

LINGO-1 =leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1

RXR=retinoid X receptor

OPC=oligodendrocyte progenitor cell

GalC=galactocerebroside

MAG=myelin associated glycoprotein

MOG=myelin oligodendrocyte glycoprotein

OMgp=oligodendrocyte myelin glycoprotein

NogoA=neurite outgrowth inhibitor A

IL13=interleukin 13

Acvr=Activin receptor

mTor=mammalian target of rapamycin

Cdc42=cell division cycle 42

TGF transforming growth factor beta

PBS=phosphate buffered saline

TUNEL=terminal deoxynucleotidyl transferase dUTP nick end labelling

IGF1 =insulin-like growth factor 1

IL1 Ra=interleukin 1 receptor antagonist

NG2=neuron-glial antigen 2

Nkx2.2=NK2 homeobox 2 DMEM=Dulbecco's modified essential media PDL=poly-D-lysine

GTPase=guanosine triphosphate-ase LPS=lipopolysaccharide Experimental Methods

Microglia polarization. Microglia were either left untreated or treated overnight with IFN (20 ng/ml) and LPS (0127:B8, 100 ng/ml), interleukin-13 (IL-13; 50ng/ml), or IL-10 (10ng/ml) (all from Sigma-Aldrich; <1 EU/ g). Conditioned media was collected and stored at -20°C. Contaminating astrocytes were < 7% of the culture in all polarization states (data not shown). ELISAs for TNF , IGF-1 , and IL-10 were used according to the manufacturer's instructions (R&D Systems). Absorbance was read at 450nm on a spectrophotometer and sample concentrations calculated using an equation generated from a standard curve. 3-5 independent supernatant sets were used. The rapid depolarization of microglia in the absence of polarizing factors (data not shown) required these to be present during conditioning of media; polarizing factors alone were directly applied to OPCs as a control.

In vivo focal CNS white matter demyelinating lesions and parabiosis

Demyelinating lesions were induced in the corpus callosum (or ventral funiculus of the thoracic column of the spinal cord for the parabiosis experiments) of 10-17 week-old male C57BL/6 mice by stereotaxic injection of 1 -2 μΙ of 1 % lysolecithin (v/v) using a Hamilton syringe. Lesions were also induced in the corpus callosum of 10 week-old male CCR2 null mice (B6.129S4-Ccr2 ^im7/fc /J; The Jackson Laboratory). A subset of these underwent tail vein injections of 5x10 ⁶ bone marrow cells derived from GFP mice of the same age/ sex (C57BL/6-Tg(CAG-EGFP)1310sb/LeySopJ;The Jackson Laboratory) 1 day prior to lesioning (for 3 day time points) or 8 days post lesion (for 10 day time points). The animals were intracardially perfused with 4% paraformaldehyde (PFA), and brains were post-fixed overnight in 4% PFA and cryoprotected in 15% and 30% sucrose overnight prior to OCT embedding (Tissue-Tech) and storage at -80 °C. All experiments were performed under the UK Home Office project licences issued under the Animals (Scientific Procedures) Act. Selective depletion of M1 or M2 microglia in focal demyelinated lesions Gadolinium chloride 3 (GdCI3) (Sigma; 0.27-1000 μΜ) was applied to primary rat microglia in vitro during polarization to assess its specificity in inducing apoptosis in M1 phenotypes by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. PBS or GdCI3 (270 μΜ) was stereotaxically co-injected into the corpus callosum of mice with lysolecithin and depletion of M1 microglia/ macrophages was assessed at 3dpl. Mannosylated clodronate (dichloromethylene diphosphonate; CI ₂ MDP)-encapsulated liposomes (Encapsula Nano Sciences) were applied to polarized microglia in vitro diluted in culture media (1 :5 - 1 :200) to assess specificity in promoting M2 apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Phosphate buffered saline (PBS) or undiluted clodronate liposomes were stereotaxically injected into the lesion site at 8 dpi and depletion of M2 microglia macrophages was assessed at 10 dpi.

Immunohistochemistry.

Slides were air-dried, and blocked for 1 hour in 5% normal horse serum and 0.3% triton-100, and primary antibody applied overnight (diluted in block above) at 4 °C in a humid chamber. Fluorescently-conjugated secondary antibodies were applied for 2 hours (diluted in block above) at room temperature in a humid chamber (1 :500, Molecular Probes and Jackson ImmunoResearch). Following counterstaining with Hoechst, slides were coverslipped with Fluoromount-G (Southern Biotech). Antibodies used to detect M1 microglia/ macrophage markers are as follows: mouse anti-iNOS (BD Biosciences, 610329, 1 :100), rat anti-CD16/32 (BD Pharmingen, 553141/2, 1 :500), goat anti-TNF (R&D Systems, AB-410-NA, 1 :500), rabbit anti-CD86 (Abeam, ab53004, 1 :100), rabbit anti-CCL2 (Biorbyt, orb36895, 1 :100), and rabbit anti-CXCL1 1 (Biorbyt, orb33040, 1 :100). Antibodies used to detect M2 markers are as follows: goat anti-Arginase-1 (Santa Cruz Biotechnology, sc-18355, 1 :50), rabbit anti- mannose receptor (Abeam, ab64693, 1 :600), goat anti-insulin like growth factor (IGF)-1 (R&D Systems, AF791 , 1 :100), rabbit anti interleukin 1 receptor antagonist (IL1 Ra) (Santa Cruz Biotechnology, sc-25444, 1 :100), and goat anti-pan TGF (R&D Systems, AB-100- NA,1 :500). Antibodies used against pan microglia/ macrophage markers include rat anti- CD68 (Abeam, ab53444, 1 :100) and rat anti-F4/80 (Abeam, ab6640, 1 :100). Oligodendrocyte and myelin antigens were detected by rabbit or mouse anti-neuron glial antigen 2 (NG2) (Millipore, MAB5384/ 5320, 1 :200, 1 :100, respectively), mouse anti-NK2 homeobox (Nkx2.2) (Developmental Hybridoma Bank, clone 74.5A5-C, University of Iowa, 1 :100), mouse anti-CC1 (Abeam, ab16794, 1 :100), rat anti-MBP (AbD Serotec, MCA409S, 1 :250), rabbit anti-damaged MBP (Millipore, AB5864, 1 :100), and mouse anti-MOG and anti- MAG (Millipore, MAB5680/ 1567, 1 :100). Antibodies against activin-A or its receptors include goat anti-activin-A (AF338), anti-Acvr1 B (AF1477), anti-Acvr2A (AF340), and anti- Acvr2B (AF339) (all from R&D Systems, 1 :40). Other antibodies include chicken anti-NFH (Encor Biotechnology, Inc., CPCA-NF-H, 1 :10,000), rabbit anti-GFAP (DAKO, Z0334, 1 :500), rabbit anti-Ki67 (Abeam, AB9620, 1 :100), rabbit anti-PCNA (Abeam, ab2426-1 , 1 :100), and chicken anti-GFP (Abeam, ab13970, 1 :100), rat anti-F4/80 (Abeam, ab6640, 1 :100).

Microglia and OPC Culture. Cortical mixed glial cultures were generated from Sprague Dawley rat postnatal day 0-2 pups. Microglia were isolated by collecting the floating fraction of 10 day-old mixed glial cultures following 1 hour on a rotary shaker at 37 °C at 250 rpm, and plated in Dulbecco's modified essential media (DMEM) containing 4.5 g/L glucose, L-glutamine, pyruvate, 10% foetal calf serum, and 1 % penicillin/streptomycin ('DMEM 10%'; GIBCO) on poly-D-lysine (PDL)-coated 16-well glass chamber slides (Lab-TEK) at 5x10 ⁴ cells/well, or at 1 x10 ⁶ cells/ well on PDL-coated glass coverslips in a 24 well plate. Microglia were treated overnight with IL-13 (50ng/ml) (Sigma-Aldrich; <1 EU/ g). Conditioned media was collected and stored at - 20°C. OPCs were isolated by collecting the floating fraction of 10 day-old mixed glial cultures following depletion of microglia as above, 16 hours on a rotary shaker at 37^ at 250 rpm, and depletion of astrocytes by differential adhesion. OPCs were plated in DMEM containing 4.5 g/L glucose, L-glutamine, pyruvate, SATO (16 μg/ml putrescine, 400 ng/ml L- thyroxine, 400 ng/ml tri-iodothyroxine, 60 ng/ml progesterone, 5 ng/ml sodium selenite, 100 μg/ml bovine serum albumin fraction V, 10 μg/ml insulin, 5.5 μg/ml halo-transferrin (all from Sigma-Aldrich)), 0.5% foetal calf serum (GIBCO), 1 % penicillin/streptomycin, 10 ng/ml platelet-derived growth factor, and 10 ng/ml fibroblast growth factor-2 at 2x10 ⁴ cells per well in PDL-coated plastic chamberslides (Lab-TEK). Activin-A (1 -100 ng/ml; R&D Systems) or vehicle (0.1 % BSA in PBS) was added to OPCs for 3 days. Goat anti-activin-A ( A subunit) blocking antibody (200 ng/ml; R&D Systems) or goat IgG control (200 ng/ml; Santa Cruz Biotechnology, Inc.) diluted in OPC culture media was added to cultures with M2a conditioned media in a 1 :1 ratio for 3 days.

Organotypic cerebellar and forebrain slice cultures.

Method 1 involves isolation of cerebellum and attached hindbrain from P0-P2 CD1 mouse pups, sectioned sagitally at 300 μηι on Mcllwain tissue chopper, and plated onto Millipore- Millicel-CM mesh inserts (Fisher Scientific) in 6 well culture plates at 6-8 slices per insert. The media was composed of 50% minimal essential media, 25% heat-inactivated horse serum, 25% Earle's balanced salt solution (all from GIBCO), 6.5 mg/ml glucose (Sigma), 1 % penicillin-streptomycin, and 1 % glutamax, and was changed every 2-3 days. Demyelination was induced at 21 days in vitro (DIV) by a 20 hour incubation in 0.5 mg/ml lysolecithin, slices were washed in media for 10 min. After recovery, slices were treated for 6 days with M2a microglia conditioned media and either anti-activin-A goat IgG or isotype control (200 ng/ml). Method 2 involves coating Millipore-Millicel-CM mesh inserts (Fisher Scientific) with poly-D- lysine for 1 hour at room temperature and 10 g/ml laminin-2 at 37^ overnight, then washing in media (described below). Forebrain slices were isolated from P0-P2 CD1 mouse pups, sectioned coronally at 300 μηι on Mcllwain tissue chopper, plated onto mesh inserts in 6 well culture plates at 3 slices per insert and maintained at 37°C and 7.5% C0 ₂ . Media was composed of 25% Earle's balanced salt solution, 67% Basal Medium Eagle, 27 mM glucose, 5% heat inactivated horse serum, 1 % penicillin-streptomycin, and 1 % glutamax (all from GIBCO) and was changed every 2-3 days. Injury was induced in slices at 7 days in vitro by treatment with lipopolysaccharide (LPS) (100 ng/ml; 0127:B8) diluted in culture media above for 2 hours, washed in media once, media replaced, and grown at 3% 0 ₂ and 7.5% C0 ₂ for 3 days. Slices were treated with activin-A (1 , 10, 100 ng/ml) or vehicle control (final concentration 0.002% bovine serum albumin in phosphate buffered saline), both diluted in media above, for the entire course of treatment. Slices were then washed in phosphate buffered saline and fixed in 4% paraformaldehyde for 10 minutes to 1 hour. Slices were blocked in 5% normal horse serum for 1 hour at room temperature, primary antibody (as listed in immunohistochemistry section) was applied for 48 hours at 4^, slices were washed three times, and fluorescently conjugated antibody was applied overnight at 4°C or 2 hours at room temperature. Following counterstaining with Hoechst for 10 minutes at room temperature and 3 washes in phosphate buffered saline, slices were mounted onto glass slides using Fluoromount-G.

Protein extraction & Western blotting

Lysates were generated using RIPA buffer (Thermo Scientific) supplemented with 1 % protease inhibitor cocktail set III EDTA-free (Calbiochem). Protein concentrations were determined using the Pierce BCA Protein Assay Kit according to the manufacturer's instructions. Samples were diluted in Laemmli buffer [200mM Tris-HCI pH6.8 (Sigma- Aldrich), 8% sodium dodecyl sulphate (Sigma-Aldrich), 40% glycerol (BioRad), 0.2% bromophenol blue (Sigma) and 5% -mercaptoethanol (Sigma)], heated at 95 ^e C for 2 min, and 10 μg of protein was loaded onto an acrylamide gel (4-20%; Thermo Scientific). Gel electrophoresis was performed in Tris-HEPES-SDS running buffer (Thermo Scientific) at 150 V for 45 minutes and proteins transferred onto PVDF membranes (Millipore) for 2h at 400 mA in 10% transfer buffer [3% Tris-HCI (Sigma-Aldrich), 15% glycine (Sigma-Aldrich), pH 8.3] and 20% methanol (Fisher Chemical) diluted in H ₂ 0. Membranes were blocked with 5% powdered milk (<1 % fat) in Tris-buffered saline (TBST) [4% sodium chloride (NaCI), 0.1 % potassium chloride (KCI), 1 .5% Tris-HCI, 0.1 % Tween-20 (all from Sigma-Aldrich), pH 7.4] for 1 h at room temperature on an orbital shaker, and incubated overnight at 4 ^e C with mouse anti-iNOS (1 :500; BD Bioscience) or rabbit anti-MR antibody (1 :500; Abeam), washed thrice in TBST for 5 min, and incubated with horseradish peroxidase (HRP)-lgG secondary antibody conjugates (1 :10,000; Calbiochem) for 1 h at room temperature. Chemiluminescent substrate detection reagent RapidStep ECL Reagent (Calbiochem) and autoradiography film processing was performed. For loading control purposes all membranes were re-blotted with anti-mouse (1 :1000, Millipore) or anti-rabbit glyceraldehyde- 3-phosphate dehydrogenase antibody (GAPDH; 1 :1000, Sigma).

Transfilter microchemotaxis assays

OPCs were plated on PDL-coated poly-carbonate transwell culture inserts (Corning) in a 24 well plate at 5x10 ⁴ cells/ well in basal culture media supplemented with FGF2. PDGF was excluded from the media due to its chemoattractant properties. Microglia media, conditioned media, or recombinant polarization factors were added to the bottom chamber in a 1 :1 ratio with OPC culture media. Cells were allowed to migrate for <24 hrs at 37°C to avoid confounding effects of conditioned media on proliferation. Cells were fixed for 1 h with 4% PFA and 0.1 % glutaraldehyde. Unmigrated cells on the upper side of the transwell were scraped off with a cotton swab, and cells that had migrated to the bottom of the transwell were visualized with Hoechst. Each condition was performed in quadruplicate wells and the experiment was repeated with 3 independent OPC samples and 3 conditioned media samples. 4 images were taken per well at 20X magnification for quantification purposes.

Immunocytochemistry. Cells were fixed with 4% paraformaldehyde (Sigma) for 10-15 min and blocked for 30 minutes at RT. Primary antibodies were diluted in blocking solution and applied for 1 h at RT. The primary antibodies used are as listed in the immunohistochemistry section herein. Cells were incubated with fluorescently conjugated secondary antibodies (1 :1000, Invitrogen) for 1 h at RT, counterstained with Hoechst (5 μg/ml), and coverslipped with fluoromount-G. Apoptotic cells were visualized by TUNEL assay (Promega) according to the manufacturer's instructions. Positive controls for apoptosis included addition of 70% ethanol to live cells, and incubation of fixed cells with DNase (10 units/ml) for 10 min. For proliferation studies, BrdU-treated OPCs were fixed with ice-cold 70% ethanol in 50 mM glycine buffer, pH 2.0, for 20 minutes. Permeabilization was performed with 2M HCI for 15 min at 37 °C, which was neutralized by 3 washes in TBE buffer (1 M Tris, 0.9M Boric acid, 0.01 M EDTA; GIBCO).

RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction array

Microglia were washed with PBS, lysed with RLT buffer supplemented with beta- mercaptoethanol, scraped, and homogenized with 21 guage needles. RNA extraction was performed using the Qiagen minikit (with on-column DNase treatment), and reverse transcription performed using the Invitrogen Superscipt First-strand synthesis system for RT- PCR, both according to the manufacturer's instructions. A custom qPCR array plate (SABiosciences) was designed to include primers for M1 and M2-associated genes that were detected as being significantly upregulated in a microarray screen of a remyelinating lesion of the rat caudal cerebellar peduncles in vivo ¹⁶ . qPCR was carried out using the Roche Light Cycler 480. Cp values were obtained using the second derivative maximum method and Cp was calculated by subtracting the average value from 5 housekeeping genes {Actb, B2m, Ldha, Pgk1, Hprtl). Expression is represented as 2 ^Cp . Table 1 illustrates the different sources of human brain tissue samples which were used in the experiments detailed herein.

Table 1 . Human brain tissue samples

SPMS F 57 19 P3C4 0 2 3 0

10 6 11 14

TOTAL

Carcinoma of M 77 P3B4

the lung

metastasized

Cardiac failure M 64 P2D2

Carcinoma of M 35 P3B3

1 the tongue

Ovarian F 60 P3D2

cancer

Myelodysplast M 82 A2C7

-ic syndrome,

Rheumatoid

Arthritis

1 non-neurological controls

Multiple Sclerosis Tissue. Post-mortem tissue from MS patients and controls that died of non-neurological causes were obtained via a UK prospective donor scheme with full ethical approval from the UK Multiple Sclerosis Tissue Bank (MREC/02/2/39). Diagnosis of MS was confirmed by neuropathological means by Dr F. Roncaroli (Consultant Neuropathologist, Imperial College London) and clinical history was provided by Dr R. Nicholas (Consultant Neurologist, Imperial College London). The antibodies used are as listed in immunohistochemistry section herein). The death-to-tissue preservation interval was from 7- 31 h. Brain blocks numbered by the brain bank locate the position of the block in the brain (i.e. coronal plane: anterior (A) and posterior (P) halves at the mamillary bodies, and 1 cm coronal slices numbered sequentially, vertical plane: 2 x2 cm blocks identified by letters (A- E) and by numbers in the horizontal plane). Snap frozen unfixed tissue blocks (2x2x 1 cm) were cut at 10 m and stored at -80 °C. MS lesions were classified according to the International Classification of Neurological Disease using luxol fast blue staining and CD68+ immunoreactivity. We analysed 5 control blocks and 13 tissue blocks from 10 MS patients; in total, we analysed 10 active lesions, 6 chronic active lesions, 1 1 chronic inactive lesions, and 14 remyelinated lesions. Approximately 170 fields of 50 μηι x 50 μηι were counted per lesion and counts were multiplied to determine density of immunopositive cells/ mm ² . Sections were fixed in 4% PFA for 1 h at RT, washed in PBS, and permeabilized in methanol for 10 min at -20 °C. Following washes in 0.3% Triton X-100 in PBS, sections were microwaved in Vector unmasking solution for 10 min, and endogenous peroxidase activity blocked for 5 min (Envision blocking solution, DAKO). Primary antibody was prepared in antibody diluent (AMS Biotechnology (Europe) Ltd) and applied overnight in a humid chamber at 4 ^q C. Following 3 washes in 0.3% Triton X-100 in PBS, anti-mouse or -rabbit peroxidase conjugated secondary antibody was applied for 2h at RT in a humid chamber. Sections were washed in PBS and stains visualized by DAB chromagen. Following washes in water, sections were dehydrated in increasing concentrations of acetone, then xylene, and coverslipped. For co- localization stains, sections were blocked with 10% normal horse serum and 0.1 % Triton-X 100 for 1 h, antibodies applied overnight as above, and fluorescently conjugated secondary antibodies (Invitrogen) applied for 2h with Hoechst counterstain.

Forward phase protein microarray for assessment of signalling pathway activation Primary rat OPCs were grown in 2 ml of DMEM containing 4.5 g/L glucose, L-glutamine, pyruvate, SATO (16 μg/ml putrescine, 400 ng/ml L-thyroxine, 400 ng/ml tri-iodothyroxine, 60 ng/ml progesterone, 5 ng/ml sodium selenite, 100 μg/ml bovine serum albumin fraction V, 10 μg/ml insulin, 5.5 μg/ml halo-transferrin (all from Sigma-Aldrich)), 0.5% fetal calf serum (GIBCO), 1 % penicillin/streptomycin, 10 ng/ml platelet-derived growth factor (Peprotech), and 10 ng/ml fibroblast growth factor-2 (Peprotech) at 1 x10 ⁶ cells per well in a poly D lysine- coated 6 well plate. Cells were treated with activin-A (1 , 10, 100 ng/ml) or vehicle control (final concentration 0.002% bovine serum albumin in phosphate buffered saline), both diluted in media above, for 3 days at 37 °C and 7.5% C0 ₂ . Cells were washed with cold phosphate buffered saline, scraped and centrifuged at l OOOrpm for 5 minutes at 4°C thrice. Cells were lysed, proteins purified and biotinylated using a kit according to the manufacturer's instructions (Full Moon Biosystems). Lysates were supplemented to a transforming growth factor beta (TGF- ) pathway phospho antibody microarray (according to and provided by Full Moon Biosystems) and read on a Axon 4200a scanner. Background values were subtracted and signal from phosphorylated protein normalized to its unphosphorylated form, then normalized to vehicle control. Values were log2 transformed and plotted using QluCore software into heatmaps of activation.

Quantification and statistical analysis

Lesion pixel counts and area quantification for MAG and MBP expression were performed using 10X objective images thresholded using Image J (NIH). The area of MOG and MBP co-localization in 40X objective images was obtained with Image J using the RG2B Colocalization Plugin followed by area quantification. All manual cell counts were performed in a blinded manner. Data are represented as mean ± s.e.m. Data was first tested for normality using the Kolmogorov-Smirnov test. Multiple comparisons within the same data set were analyzed by one-way ANOVA with Newman-Keuls post-hoc test, or Kruskal-Wallis test and Dunn's multiple comparison test. Single comparisons to control were made using 2- tailed Student's f-test or Mann-Whitney test. P values of <0.05 were considered statistically significant. Data handling and statistical processing was performed using Microsoft Excel and Graph Pad Prism Software.

Where any of the following experiments refers to a general experimental method, other than those specifically indicated hereinbefore, and no detailed description of how to perform such method is provided then any of the known-methods of the art may be used. The identification of such suitable methods and the particular selection of any one known method from another would be a routine matter for one skilled in the art.

The following non-limiting examples are provided. Experimental Results

Example 1. Confirmation of a switch from an M1 -dominant to M2-dominant microglia/ macrophage response occurs at the initiation of remyelination.

To examine the timing of microglia/ macrophage polarization during remyelination, focal demyelination was induced by stereotaxic injection of lysolecithin into the mouse corpus callosum. Tissue was analyzed at 3, 10, and 21 days post lesion (dpi), times that correspond to key steps in the remyelination process: oligodendrocyte progenitor cell (OPC) recruitment into the lesion by proliferation and migration, initiation of remyelination by the differentiation of these OPCs into myelin-sheath forming oligodendrocytes, and completion of remyelination. (Fig. 1a). Studies of microglia/ macrophages polarization state-specific markers revealed for the first time a switch from an M1 - to M2-dominant phenotype at the initiation of remyelination. At 3 dpi, significantly more CD68+ microglia/ macrophages expressed the M1 marker inducible nitric oxide synthase (iNOS) than the M2 marker arginase-1 (Arg-1 ) (Fig. 1 b, c).

However, by 10 dpi there were more Arg-1 + CD68+ M2 cells than iNOS+ CD68+ M1 cells (Fig. 1 b, c). At 21 dpi, there was a reduction in Arg-1 + M2 cells compared to 10 dpi (Fig. 1 b, c). Only a low percentage of CD68+ microglia macrophages remained unpolarized (iNOS- and Arg1 -) throughout remyelination (Fig. 1 c). The polarization switch during remyelination was confirmed using additional markers for M1 (TNF and CD16/32) and M2 phenotypes (MR and IGF-1 ) (Fig. 2). In addition the same switch was also observed from an M1 to M2 response during remyelination in another model of demyelination induced by injection of ethidium bromide into the rat caudal cerebellar peduncles (CCP) (Fig. 3).

As the entry of peripherally-derived macrophages to function alongside CNS-resident microglia is an important part of the innate immune response in the CNS, further studies were carried out to determine whether this switch in polarization phenotype was a consequence of a change in the balance of (differentially-polarized) endogenous microglia vs. peripherally-derived macrophages, or of polarization switching in both populations.

To exclude macrophages from lesions and therefore examine polarization only in microglia, lesions created in CCR2 ^Λ mice (commercially purchased from Jackson Laboratories) whose peripheral monocytes (from which macrophages derive) cannot extravasate were examined, and are thus excluded from entering remyelinating lesions (Fig. 4(a)).

These studies confirmed that the transition from M1 to M2 polarization still occurred, as shown hereinbefore, within the microglia population in CCR2 ^_/" mice, with the majority of CD68+ cells being iNOS+ at 3 dpi and Arg-1 + at 10 dpi (Fig. 4(b), (c)).

However, the absence of peripherally-derived macrophages in CCR2 ^Λ lesions did lead to a reduction in total numbers of iNOS+ M1 cells (to 37 ± 10 and 33 ± 7 % of wildtype control at 3 and 10 dpi, respectively) and Arg1 + M2 cells (to 8 ± 8 and 40 ± 13 % of wildtype controls at 3 and 10 dpi, respectively). To examine polarization only in peripherally-derived macrophages within a lesion, GFP- expressing wildtype bone marrow-derived cells were injected into the circulation of lesioned CCR2 ^_/" mice (Fig. 4(d)). Both GFP+ iNOS+ M1 macrophages and GFP+ Arg1 + M2 macrophages were present at 3 and 10 dpi (Fig. 4(e), (f)). The iNOS+ M1 population was composed of 39 ± 3 % (3 dpi) and 46 ± 14 % GFP+ macrophages (10 dpi) whereas the Arg- 1 + M2 population was composed of 63 ± 12 % (3 dpi) and 68 ± 23 % GFP+ macrophages (10 dpi), showing for the first time that macrophages contribute equally to both M1 and M2 populations before and during the switch in polarization.

These studies confirm that both peripherally-derived macrophages and resident microglia contribute to the switch from M1 to M2 phenotypes observed at the initiation of remyelination. This suggests that M2 microglia and macrophages contribute to oligodendrocyte differentiation during remyelination. Without wishing to be bound to any particular theory it is proposed herein that this supports the use of agents associated with M2 microglia and macrophages for the treatment of diseases where oligodendrocyte differentiation is impaired.

More particularly this data supports the following treatments independently selected from: treatment of diseases where oligodendrocyte differentiation is impaired via myelin regeneration; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation and myelin regeneration; treatment of myelin disorders; treatment of MS; PPMS; RRMS; or SPMS; or promotion of remyelination at a cellular level via regeneration of oligodendrocyte cells, via the use of agents associated with M2 microglia and macrophages.

As demonstrated in Example 5 that M2 microglia and macrophages are confirmed to be the source of activin-A during remyelination and also that activin-A promotes oligodendrocyte differentiation. This data supports our proposal for the use of an activin receptor activating agent, as evidenced by the results obtained with activin-A, for use in the treatment of diseases wherein oligodendrocyte differentiation is impaired, and specifically as may be independently selected from the following treatments: treatment of diseases where oligodendrocyte differentiation is impaired via myelin regeneration; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation and myelin regeneration; myelin disorders; treatment of MS; PPMS; RRMS; or SPMS; or promotion of remyelination at a cellular level via regeneration of oligodendrocyte cells.

The antibodies used in this example were: goat anti-Arginase-1 (Fig1 -4; Santa Cruz Biotechnology, sc-18355, 1 :50), rabbit anti-mannose receptor (Fig 2-3; Abeam, ab64693, 1 :600), goat anti-insulin like growth factor (IGF)-1 (Fig. 2-3; R&D Systems, AF791 , 1 :100), anti-CD68 (Fig. 1 -4; Abeam, ab53444, 1 :100), mouse anti-iNOS (Fig. 1 -4; BD Biosciences, 610329, 1 :100), rat anti-CD16/32 (Fig. 2-3; BD Pharmingen, 553141 /2, 1 :500), goat anti- TNF (Fig. 2-3; R&D Systems, AB-410-NA, 1 :500), chicken anti-GFP (Fig. 4; Abeam, ab13970, 1 :100), rat anti-MBP (Fig. 3; AbD Serotec, MCA409S, 1 :250), anti-MAG (Fig. 3; Millipore, MAB5680/ 1567, 1 :100), rat anti-F4/80 (Fig. 2; Abeam, ab6640, 1 :100).

Example 2: M2 polarized microglia promote oligodendrocyte differentiation.

Following the switch observed in Example 1 , from M1 to M2 polarization at 10 dpi, the Applicant hypothesized that the two polarization states may have distinct roles in regulating the oligodendrocyte differentiation essential for the initiation of remyelination occurring at this time. To confirm this, the responses of OPCs to application of M1 and M2 conditioned media (CM) in vitro were assessed. Microglia rather than peripherally-derived macrophages were used, since the former are always a component of the innate immune response regardless of changes to the blood-brain barrier, and the results observed in Example 1 had confirmed above that microglia alone can undergo the switch in polarization in vivo.

Microglia were polarized to M1 or one of two M2 subtypes (M2a (anti-inflammatory) and M2c (immuno-regulatory)) by exposure to IFN / LPS, IL-13, and IL-10, respectively (Fig. 5, 6). Polarization was examined by immunofluorescence (Fig. 5(a), (b)), ELISA (Fig. 5 (d), (e), (f)), and by gene expression profiling, which confirmed that the phenotypes matched those measured in vivo during remyelination (Fig. 6).

The Applicant has demonstrated for the first time that CM derived from both M1 and M2 microglia increased OPC proliferation. This was evidenced by increased total OPC numbers, cell cycle activity, and BrdU incorporation (Fig. 7(a)-(d)) and also increased chemotaxic migration (Fig. 7(e)). In contrast, only M2 CM prevented OPC apoptosis in media deprived of serum and growth factors (Fig. 8), conditions that reveal the physiological target-dependent survival mechanisms of newly-formed oligodendrocytes in vivo. Additionally, the Applicant has also shown for the first time that only M2 CM increased OLG differentiation. This was assessed by expression of myelin basic protein (MBP) (Fig. 9(a), (b)) and myelin oligodendrocyte glycoprotein (MOG) (Fig. 9(c)). These data show that M2 microglia secrete factors that promote oligodendrocyte progenitor responses that are critical for remyelination: proliferation, survival and differentiation into mature oligodendrocytes. In Example 5 it is confirmed that that one of these factors is activin-A, and that activin-A directly promotes all of these OPC responses.

This suggests that activin receptor activating agents, and in particular activin-A for the treatment of diseases where oligodendrocyte differentiation is impaired via: myelin regeneration; promotion of oligodendrocyte differentiation; via promotion of oligodendrocyte differentiation and myelin regeneration; or promotion of remyelination at a cellular level via regeneration of oligodendrocyte cells. This also suggests that activin receptor activating agents, and in particular activin-A for the treatment of myelin disorders, or progressive demyelinating diseases.

The antibodies used in this example are mouse anti-iNOS (Fig. 5-6; BD Biosciences, 610329, 1 :100), rat anti-CD16/32 (Fig. 5; BD Pharmingen, 553141 /2, 1 :500), rabbit anti- CD86 (Fig. 5-6; Abeam, ab53004, 1 :100), rabbit anti-CCL2 (Fig. 6; Biorbyt, orb36895, 1 :100), and rabbit anti-CXCL1 1 (Fig. 6; Biorbyt, orb33040, 1 :100), goat anti-Arginase-1 (Fig. 5-6; Santa Cruz Biotechnology, sc-18355, 1 :50), rabbit anti-mannose receptor (Fig. 5; Abeam, ab64693, 1 :600), rabbit anti interleukin 1 receptor antagonist (IL1 Ra) (Fig. 5; Santa Cruz Biotechnology, sc-25444, 1 :100), and goat anti-pan TGF (Fig.6; R&D Systems, AB- 100-NA,1 :500), rabbit or mouse (Fig. 6) anti-neuron glial antigen 2 (NG2) (Fig.8-9; Millipore, MAB5384/ 5320, 1 :200, 1 :100, respectively), mouse anti-MOG (Fig.9; Millipore, MAB5680, 1 :100), rat anti-MBP (Fig. 9; AbD Serotec, MCA409S, 1 :250), rabbit anti-Ki67 (Fig. 7; Abeam, AB9620, 1 :100), anti-mouse (1 :1000, Millipore) or anti-rabbit glyceraldehyde-3- phosphate dehydrogenase antibody (Fig. 5; GAPDH; 1 :1000, Sigma).

Example 3: Selective depletion of M2 polarized microglia/macrophages within a lesion inhibits oligodendrocyte differentiation.

Having revealed distinct effects of M1 and M2 CM in cell culture, with only M2 CM enhancing oligodendrocyte differentiation, the effect of selective depletion of each polarized macrophage population during remyelination in vivo was determined. M1 cells were depleted using a novel method development by the Applicant, by administration of gadolinium chloride (GdCI ₃ ) within a lesion, which upon phagocytosis induced apoptosis of inflammatory macrophages via competitive inhibition of Ca ²⁺ mobilization and damage to plasma membranes. The ability of GdCI3 (270 μΜ) to selectively deplete M1 cells in vitro (Fig. 10a-c) and within a lesion in vivo by injection at the time of lesioning was confirmed (Fig. 11 a-c, Fig. 10e, f). GdCI3 injection did not significantly affect M2 cell numbers (Fig. 11 c). This data demonstrates for the first time that M1 depletion reduced the density of PCNA+ Nkx2.2+ proliferating OPCs at 3dpl compared to control (Fig. 11d). This data confirms a role for M1 polarization in regulating OPC proliferation. OPC migration (as measured by numbers of NG2+ or Nkx2.2+ OPCs) and remyelination were not impaired (Fig.10g, h). M2 cells were depleted using a novel method developed by the Applicant using mannosylated clodronate liposomes (MCLs) that bind the mannose receptor (Fig. 12a) upregulated following M2 polarization (Fig. 5), and induce apoptosis within 2-3 days via clodronate-mediated depletion of intracellular iron. Selective M2 depletion in vitro (Fig. 13a- f) and in vivo was confirmed when MCLs were injected into a lesion at 8 dpi (Fig. 12b-d, Fig. 13g, h). This M2 depletion caused an increase in the number and proportion of iNOS+ M1 cells (Fig. 12d, Fig. 13g), which supports the previously discussed reciprocal regulation between M1 and M2 polarization and associated cytokines. We show for the first time that M2 depletion decreased expression of differentiation markers MAG and MBP at 10 dpi (Fig. 12e, f). This indicates a delay in oligodendrocyte differentiation. The consequent delay in remyelination in tissue was measured at 21 dpi following M2-depletion in two different ways. Firstly, decreased co-localization of MBP and the late myelin marker MOG was observed (Fig. 13i). Secondly, a decrease in the number of nodes of Ranvier was demonstrated, indicative of compact myelin, by showing reduced paranodal Caspr localization (flanking nodal Ankyrin-G) (Fig. 12g, h). No change was observed in lesion area, numbers of Nkx2.2+ OPCs, presence of neurofilament+ axons, and GFAP+ astrocyte reactivity within the lesion (Fig. 13j-l).

These data show that depletion of M2 microglia and macrophages leads to impaired oligodendrocyte differentiation and remyelination. In conjunction with our demonstration that M2 microglia and macrophages are the main source of activin-A during remyelination and that activin-A promotes oligodendrocyte differentiation, as detailed in Example 5, these results provide further support for the use of activin receptor activating agents for use in the treatment of diseases where oligodendrocyte differentiation is impaired. More particularly this data supports the following treatments independently selected from: treatment of diseases where oligodendrocyte differentiation is impaired via myelin regeneration; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation and myelin regeneration; treatment of myelin disorders; treatment of MS; PPMS; RRMS; or SPMS; or promotion of remyelination at a cellular level via regeneration of oligodendrocyte cells. The antibodies used in this example are mouse anti-iNOS (Fig. 10-13; BD Biosciences, 610329, 1 :100), rat anti-CD16/32 (Fig. 10, 13; BD Pharmingen, 553141/2, 1 :500), goat anti- TNF (Fig. 10, 13; R&D Systems, AB-410-NA, 1 :500), goat anti-Arginase-1 (Fig. 10-12; Santa Cruz Biotechnology, sc-18355, 1 :50), rabbit anti-mannose receptor (Fig. 10, 13; Abeam, ab64693, 1 :600), goat anti-insulin like growth factor (IGF)-1 (Fig. 10, 13; R&D Systems, AF791 , 1 :100), rat anti-CD68 (Fig. 1 1 -12; Abeam, ab53444, 1 :100), mouse anti- NK2 homeobox (Nkx2.2) (Fig. 10. 1 1 . 13; Developmental Hybridoma Bank, clone 74.5A5-C, University of Iowa, 1 :100), rat anti-MBP (Fig. 10, 12, 13; AbD Serotec, MCA409S, 1 :250), mouse anti-MOG and anti-MAG (Fig. 10, 12; Millipore, MAB5680/ 1567, 1 :100), rabbit anti- Caspr (Fig. 12; Abeam, ab34151 , 1 :500), mouse anti-Ankyrin-G (Fig. 12; UC Davis/ NIH NeuroMab Facility, clone N106/36, 1 :200), rabbit anti-PCNA (Fig. 1 1 ; Abeam, ab2426-1 , 1 :100), chicken anti-NFH (Fig. 13; Encor Biotechnology, Inc., CPCA-NF-H, 1 :10,000), rabbit anti-GFAP (Fig. 10, 13; DAKO, Z0334, 1 :500), rabbit anti-neuron glial antigen 2 (NG2) (Fig. 10; Millipore, MAB5320, 1 :200).

Example 4: Increased densities of M2 polarized cells are associated with efficient remyelination in parabiotic mice and multiple sclerosis lesions. The in vivo depletion experiments discussed hereinbefore have demonstrated that a switch to an M2 dominant phenotype in demyelinated lesions is required for efficient remyelination. The Applicants predicted that experimental manipulations leading to increased numbers of M2 macrophages would enhance remyelination. To confirm this, parabiosis experiments were carried out, where the slow remyelination normally observed in old mice was shown to be reversed by sharing a circulation with young mice, an effect mediated in part by recruitment of young circulating monocytes into the demyelinating lesions of the older mice. These experiments use the methodology as detailed in Ruckh,J.M. et al., Rejuvenation of regeneration in the aging central nervous system, Cell Stem Cell 10, 96-103 (2012), the contents of which are incorporated herein by reference.

Analysis of previously-characterized tissue of lysolecithin-demyelinated ventral spinal cord of one of the two mice in various pairings was carried out (Fig. 14a) in accordance with the method of Ruckh, J.M. et. al, as detailed hereinbefore and as incorporated herein by reference. This analysis has shown for the first time that the densities of M2 microglia/ macrophages (MR+ isolectin B4+ and Arg-1 + CD68+) were higher in the lesions of young animals paired to another young animal (Y/Y) than in old animals paired to another old animal (O/O) (Fig. 14b, c). Pairing of an old animal with a young animal (Y/O), a manipulation that restores remyelination efficiency in the older animal ⁴ , led to increased densities of M2 microglia/ macrophages in the lesions in the older animal relative to those seen in the O/O pairings (Fig. 14b, c). In contrast, the densities of CD16/32+ M1 microglia macrophages were significantly higher in O/O pairings than Y/Y pairings and not significantly reduced by Y/O pairing (Fig. 14d). These results demonstrated for the first time that increasing densities of M2 microglia/ macrophages is associated with enhanced remyelination efficiency and provide yet further support for the proposed uses of activin receptor activating agents proposed herein.

Analysis of the results of parabiosis experiments between a GFP-expressing young mouse and a lesioned wild type old mouse so as to assess the contribution of peripherally-derived young macrophages (GFP+) to the M1 and M2 polarized populations within a remyelinating lesion (Fig. 14e), GFP+ iNOS+ CD68+ M1 macrophages and GFP+ Arg1 + CD68+ M2 macrophages present within the lesion (Fig. 14f) only contributed to 3.6 ± 1 .6 % of iNOS+ M1 cells and 12.4 ± 5 % of Arg-1 + M2 cells. Without wishing to be bound to any particular theory is it proposed herein that the great majority of the increased numbers of M2 cells in the older animal of the Y/O pairings are derived from the microglia/ macrophages of the old animal, with the young macrophages that enter the lesion in the old CNS rejuvenating the lesion environment to promote M2 polarization of endogenous populations. A further proposal from our supposition that a switch to an M2-dominant phenotype in demyelinated lesions is required for efficient remyelination, and one critical for translational relevance, is that M2 macrophages will be abundant in areas of multiple sclerosis (MS) lesions associated with ongoing remyelination. To test this, we examined 5 patterns of MS lesion pathology (acute active, rim of chronic active, centre of chronic active, chronic inactive, remyelinated; Table 1 ). All lesion types showed significantly higher densities of total CD68+ microglia/ macrophages and iNOS+ M1 cells compared to controls (Fig. 15a, b). While a few remyelinating lesions showed increased densities of MR+ M2 cells compared to controls, significantly elevated numbers of M2 cells were seen only in acute active lesions and within the rim of chronic active lesions (Fig. 15c), both areas of recent damage where ongoing remyelination would be expected.

These results provide additional support, in conjunction with the results of Example 5, for our proposal that M2 microglia and macrophages are the major source of activin-A during remyelination, and that activin-A promotes oligodendrocyte differentiation.

Thus, the data from this example provides further support for the use of activin receptor activating agents for use in the treatment of diseases where oligodendrocyte differentiation is impaired. More particularly this data supports the following treatments independently selected from: treatment of diseases where oligodendrocyte differentiation is impaired via myelin regeneration; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation; treatment of diseases where oligodendrocyte differentiation is impaired via promotion of oligodendrocyte differentiation and myelin regeneration; treatment of myelin disorders; treatment of MS; PPMS; RRMS; or SPMS; or promotion of remyelination at a cellular level via regeneration of oligodendrocyte cells.

The antibodies used in this example are mouse anti-iNOS (Fig. 14-15; BD Biosciences, 610329, 1 :100), rat anti-CD16/32 (Fig. 14; BD Pharmingen, 553141 /2, 1 :500), goat anti- Arginase-1 (Fig. 14; Santa Cruz Biotechnology, sc-18355, 1 :50), rabbit anti-mannose receptor (Fig. 14-15; Abeam, ab64693, 1 :600), rat anti-CD68 (Fig. 14; Abeam, ab53444, 1 :100), Biotin-conjugated isolectin B4 (Fig. 14; 1 :100, Sigma Aldrich L-2140) with avidin- Alexa 488 (Fig. 15; 1 :500, Molecular Probes), mouse anti-CD68 (Fig. 15; DAKO, clone KP1 M0814, 1 :100). Example 5: M2-derived activin-A drives oligodendrocyte differentiation during remyelination.

As detailed hereinbefore the foregoing experimental data has indicated for the first time that the switch to M2 polarization is an essential part of the regenerative response in the CNS by promoting oligodendrocyte differentiation. To identify an M2-derived regenerative factor, a candidate approach was taken and the TGF superfamily member activin-A was investigated. Activin-A is produced by inflammatory macrophages and is a marker of M2 polarization. In keeping with this, the results of Example 5 confirmed that more MR+ M2a and M2c polarized microglia expressed activin-A in vitro in comparison to iNOS+ M1 microglia (Fig. 16a). To confirm that M2 polarized cells also contribute activin-A to the environment of remyelinating lesions, we examined expression of activin-A in remyelinating lesions for the first time and found that activin-A immunoreactivity was more evident in association with MR+ M2 cells at 10 dpi compared to iNOS+ M1 cells at 3 dpi, and was reduced upon M2 depletion with MCLS (Fig. 16b). In addition examination of expression of receptors that directly bind activin-A, Acvr2A and Acvr2B, in remyelinating lesions showed for the first time that NG2+ OPCs within remyelinating lesions expressed both subtypes (Fig. 16c).

Our experiments also demonstrated that OPCs also expressed Acvrl B, the receptor which is recruited by ligand-bound Acvr2, and which is required for subsequent functional downstream signalling.

Together, these findings indicate the capacity of OPCs within remyelinating lesions to directly bind and respond to M2-derived activin-A.

Assessment of activin-A binding capacity of other cell types within lesions indicates that Acvr2 is expressed by CC1 + oligodendrocytes (Acvr2B+) and CD68+ microglia/ macrophages (Acvr2A+, Acvr2B+), but not GFAP+ astrocytes or NF+ axons (Fig.17).

To test the effect of activin-A on oligodendrocyte differentiation, we exposed cultured OPCs to activin-A and found for the first time that it was sufficient to enhance oligodendrocyte differentiation (Fig. 16d).

To confirm a role for activin-A in the differentiation-promoting effect of M2 microglia, we supplemented cultures with M2 CM and an activin-A blocking antibody and observed for the first time reduced oligodendrocyte differentiation (MBP+ cells) (Fig. 16e). Given that activin-A mice are neonatally lethal, and activin receptor null mutants are either developmentally lethal or have gastrulation and fertility defects, we investigated the role of activin-A in M2-driven oligodendrocyte differentiation during remyelination in intact CNS using ex vivo organotypic cerebellar slice cultures (Fig. 16f). Following lysolecithin-induced demyelination, slices treated with M2 CM during the initiation of remyelination showed increased numbers of CC1 + MBP+ mature oligodendrocytes, which we show for the first time was significantly reduced with anti-activin-A blocking IgG supplementation (Fig. 16g, h). This was due to blocking of activin-A in M2 CM and not endogenous activin-A, as treatment of slices with anti-activin-A antibody alone did not reduce the number of oligodendrocytes relative to IgG control (Fig. 16h). Together, these data provide a molecular mechanism for M2-driven oligodendrocyte differentiation during remyelination via secretion of activin-A.

To determine whether activin-A can cause differentiation of oligodendrocytes when it is impaired in paediatric myelin disorders, we developed a novel system to model perinatal brain injury using ex vivo forebrain explant cultures from newborn mice and supplemented with activin-A during injury induction. Given that inflammation and hypoxia are considered major pathological contributors to paediatric myelin disorders such as PVL and CP, and inflammation increases sensitivity to subsequent hypoxia and consequent brain injury, explants were first exposed to inflammation (lipopolysaccharide; LPS; 100ng/ml) for 2 hours then hypoxia (3% 0 ₂ ) for 3 days at 7 days in vitro. Treatment with activin-A (1 -100 ng/ml) during the whole 3 days increased the number of mature oligodendrocytes (MBP+) compared to vehicle-treated control [Fig.18], indicating that activin-A increased differentiation of oligodendrocyte progenitor cells in this model.

To determine whether activin-A promotes additional oligodendrocyte progenitor responses directly relevant for remyelination, primary rat OPCs were treated with activin-A for 3 days and assessed for effects on proliferation and survival. 1 ng/ml activin-A increased the number of proliferating oligodendrocyte progenitor cells (Ki67+) [Fig.19]. To assess effects of activin-A on survival of oligodendrocyte progenitor cells, cells were grown either in basal culture media (GF control) or deprivation media devoid of serum and growth factors to induce apoptosis, and supplemented with activin-A. 100 ng/ml rescued cells from supplement withdrawal-induced apoptosis [Fig. 20].

These results demonstrate that activin-A, in addition to promoting oligodendrocyte differentiation as discussed hereinbefore, also promotes oligodendrocyte progenitor proliferation and survival, key cellular responses that may contribute to the effectiveness of remyelination by increasing the pool of progenitor cells that can differentiate into oligodendrocytes that mediate remyelination. To confirm that oligodendrocyte progenitor cells can respond to activin receptor binding by ligand, we assessed activation of signalling pathways downstream of activin receptors (PI3kinase/AKT, JNK/p38, Smad2/3, Rac/Cdc42 GTPase and ERK1/2) [Fig. 21 ]. Primary rat oligodendrocyte progenitor cells were exposed to activin-A and lysates supplemented to an antibody microarray against phosphorylated/ unphosphorylated proteins within the 5 pathways. We observed activation of all 5 signalling pathways with activin-A treatment (1 - 100 ng/ml); in particular 1 ng/ml activin-A induced robust activation of P13kinase/AKT, JNK/p38, and Smad2/3 pathways, 10 ng/m activin-A induced robust activation of Rac/Cdc42 GTPase and ERK1/2 pathways, while 100 ng/ml activin-A was associated with low activation of all pathways. This indicates that activin-A directly activates signalling pathways downstream of activin receptors.

The antibodies used in this example are mouse anti-iNOS (Fig. 16; BD Biosciences, 610329, 1 :100), rabbit anti-mannose receptor (Fig. 16; Abeam, ab64693, 1 :600), rat anti-CD68 (Fig. 17; Abeam, ab53444, 1 :100), rat anti-MBP (Fig. 16; AbD Serotec, MCA409S, 1 :250), rabbit anti-neuron glial antigen 2 (NG2) (Fig. 19; Millipore, MAB5320, 1 :200), rabbit anti-GFAP (Fig. 17; DAKO, Z0334, 1 :500), goat anti-activin-A (AF338), anti-Acvr1 B (AF1477), anti- Acvr2A (AF340), and anti-Acvr2B (AF339) (Fig. 16, 17; all from R&D Systems, 1 :40)), rabbit anti-Ki67 (Fig. 18; Abeam, AB9620, 1 :100), chicken anti-NFH (Fig. 17; Encor Biotechnology, Inc., CPCA-NF-H, 1 :10,000), mouse anti-CC1 (Fig.16; Abeam, ab16794, 1 :100).

Whilst specific embodiments of the present invention have been described above, it will be appreciated that departures from the described embodiments may still fall within the scope of the present invention.