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Journal of Clinical Medicine

Review Can the Fact That Are Old and Break down Explain the Origin of in Some People?

Roger J. W. Truscott * and Michael G. Friedrich

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-2-4298-3503; Fax: +61-2-4221-8130

 Received: 11 July 2018; Accepted: 12 September 2018; Published: 14 September 2018 

Abstract: Recent discoveries may change the way that multiple sclerosis (MS) is viewed, particularly with regard to the reasons for the untoward immune response. The fact that myelin proteins are long-lived, and that by the time we are adults, they are extensively degraded, alters our perspective on the reasons for the onset of autoimmunity and the origin of MS. For example, myelin basic (MBP) from every past the age of 20 years, is so greatly modified, that it is effectively a different protein from the one that was laid down in childhood. Since only a subset of people with such degraded MBP develop MS, a focus on understanding the mechanism of immune responses to central (CNS) antigens and cerebral immune tolerance appear to be worthwhile avenues to explore. In accord with this, it will be productive to examine why all people, whose brains contain large quantities of a “foreign antigen”, do not develop MS. Importantly for the potential causation of MS, MBP from MS patients breaks down differently from the MBP in aged controls. If the novel structures formed in these MS-specific regions are particularly antigenic, it could help explain the origin of MS. If verified, these findings could provide an avenue for the rational synthesis of drugs to prevent and treat MS.

Keywords: proteomics; age-related; isoaspartate; posttranslational modification; protein decomposition;

1. Introduction Despite many years of research, the initiating events that lead to multiple sclerosis (MS) remain a mystery. Several have been linked to a higher likelihood of developing MS, in particular, those of the human leukocyte antigen (HLA) system. A consistent finding is the association between MS and DR15 and DQ6 alleles of the major histocompatibility complex. Genome-wide association studies have uncovered over 200 other genes outside of the HLA that increase the probability of MS to a small extent [1,2]. Environmental contributors, such as infections, have been difficult to link directly with the onset of MS. The role of microbiota is becoming increasingly of interest following the discovery that microbes can trigger chronic immune disorders to dietary antigens, e.g., celiac disease [3], in addition to affecting the outcome of neurological diseases [4]. In a remarkable linkage between cellular and microbiological strands, it has been shown recently that metabolites derived from gut bacteria can enter the CNS where they act on transcription factors present in microglia and astrocytes [5]. In this way, the gut can modulate inflammation in the CNS and affect the outcome of experimental autoimmune encephalomyelitis (EAE), a common animal model of MS.

J. Clin. Med. 2018, 7, 281; doi:10.3390/jcm7090281 www.mdpi.com/journal/jcm J. Clin. Med. 2018, 7, 281 2 of 12

2. Long-Lived Proteins and Autoimmunity J. Clin. Med. 2018, 7, x FOR PEER REVIEW 2 of 12 The reasons for developing autoimmunity in general, and MS in particular, are not well understood.2. Long-Lived One Proteins feature and that Autoimmunity has been largely overlooked is the role of endogenous antigen modificationThe reasons in triggering for developing autoimmunity. autoimmunity In the in general, bodies and of MS adults, in particular, long-lived are proteinsnot well are ubiquitousunderstood.[6–8] . One In early feature life, that such has long-lived been largely proteins overlooked are recognised is the role as of “self” endogenous however, antigen over time, they breakdownmodification in and triggering this decomposition autoimmunity. effectively In the bodies creates of adults, new long proteins-lived thatproteins may are be ubiquitous recognised by [6–8]. In early life, such long-lived proteins are recognised as “self” however, over time, they the body as being “foreign”. The subject of this review are the structural changes to long-lived proteins breakdown and this decomposition effectively creates new proteins that may be recognised by the that occur inevitably with age. If these alterations are significant, new antigens are effectively created body as being “foreign”. The subject of this review are the structural changes to long-lived proteins withinthat the occur body. inevitably Such processes with age. mayIf these have alterations particular are significant, relevance new for the antigens induction are effectively of MS, since created myelin proteinswithin are the long-lived body. Such proteins. processes may have particular relevance for the induction of MS, since myelin Overproteins time, are long long-lived-lived proteins. proteins undergo a plethora of modifications, such as racemisation, truncation,Over , time, long-lived deimination, proteins undergo and cross-linking a plethora of (reviewed modifications, in Ref. such [ 9 as]) with racemisation, each single modificationtruncation, potentially deamidation, being deimination sufficient, and to break cross-linking immune (reviewed tolerance. in Ref. Age-related [9]) with modification each single of proteinsmodification at a number potentially of sites beingin the sufficient body may to underlie break immune the observation tolerance. that Age- therelated levels modification of autoantibodies of increaseproteins as a at function a number of age of sites [10]. in In the the body case ofmay another underlie autoimmune the observation condition, that the systemic levels of lupus autoantibodies increase as a function of age [10]. In the case of another autoimmune condition, erythematosus, [11], Mamula et al. have proposed a role for isoaspartate (isoAsp), an isomer of Asp systemic lupus erythematosus, [11], Mamula et al. have proposed a role for isoaspartate (isoAsp), an that is formed spontaneously in old proteins, [12] (see Figure1) in generating the autoantibodies to isomer of Asp that is formed spontaneously in old proteins, [12] (see Figure 1) in generating the histones;autoantibodie anothers family to histones; of long-lived another family proteins. of long-lived proteins.

FigureFigure 1. When 1. When old old proteins proteins in in the the body body degrade, degrade, novel structures structures are are formed. formed. L-AspL-Asp and andL-AsnL -Asn residuesresidues can spontaneously can spontaneously convert convert over over time timeto 4 isomeric to 4 isomeric forms, forms, via a cyclic via a intermediate cyclic intermediate as illustrated. as An unchargedillustrated. AsnAn uncharged becomes Asn a mixture becomes of a four mixture negatively of four negatively charged Asp charged residues. Asp residues. Probably Probably of greatest of greatest impact as potential immune inducers are L-isoAsp and D-isoAsp. In the case of D-isoAsp impact as potential immune inducers are L-isoAsp and D-isoAsp. In the case of D-isoAsp the α-carbon the α-carbon is racemised and the polypeptide chain is also extended by the insertion of a CH2 group, is racemised and the polypeptide chain is also extended by the insertion of a CH2 group, as highlighted as highlighted by the red bonds. In adult MBP, many positively charged Arg residues have been by the red bonds. In adult MBP, many positively charged Arg residues have been converted to neutral converted to neutral citrulline residues. Together these abundant post translational modifications citrulline(PTMs residues.) lead to protein Together unfolding these abundantand the generation post translational of novel antigens. modifications (PTMs) lead to protein unfolding and the generation of novel antigens. J. Clin. Med. 2018, 7, 281 3 of 12

3. Long-Lived Proteins and Multiple Sclerosis Amongst the list of long-lived proteins are the structural proteins of myelin [7,9]. Indeed, these may not turn over at all. Evidence for myelin proteins being long-lived comes from two main sources. Firstly, stable-isotope labelling revealed little or no turnover [6]. Secondly, the degree of racemisation of human MBP as a function of age [12] matched closely that of crystallin proteins from the lens that are well-known not to be replaced during our lifetime [7]. Once the myelin sheath is laid down in childhood, the major proteins within it, such as myelin basic protein (MBP), may be with us for all of our lives. Although this is, in many ways, a remarkable finding, why does this matter, and how could this play a role in the onset of human MS? It turns out that proteins are not stable in a biological environment. Over time they break down. To a large degree it seems that decomposition is largely non-enzymatic and that old proteins decay due to the intrinsic instability of certain amino acid residues. Some reactions have been known for years. For example, and racemise i.e., they change from the L-form to the D-isomer, as well as converting to a different isomeric form, isoAsp (Figure1)[13]. Other spontaneous reactions of old proteins include crosslinking [14,15], deamidation [16], and bond cleavage [17]. It should be noted that not all modifications to long-lived proteins are spontaneous, for example, deimination of residues has been attributed to the action of protein arginine deiminase (Figure1). The many novel modifications of old proteins, and their time courses as a function of age [16,18], have been documented in the human lens, where it is well-established that proteins do not turn over [8]. It is of interest that another age-related disease, cataract, can be traced to a greater degree of protein modification at specific sites in lens crystallins [19,20]. If these reactions are extensive, the structure and properties of the affected long-lived proteins change dramatically. This may have significant consequences, for example, it is possible that the body may no longer recognise the altered protein as being “self”. If this occurs, an immune response could be mounted against the modified old protein. These subjects have been summarised in a recent review [9].

4. Myelin Basic Protein and Age Proteomic analysis of the human cerebellum showed that MBP is highly modified in adults [9]. The extent of posttranslational modification is large, such that every region of the protein, showed some form of covalent alteration. In addition, these changes are quantitatively major (Figure2). The covalent changes included deamidation of Asn and Gln residues, isomerisation of Asp, as well as deimination of Arg residues to citrulline. Some of these changes increased with age. Most were abundant even at age 18, which was the youngest sample available from the brain bank. That extensive MBP modification has taken place by such a young age may at first seem surprising, however the same phenomenon has been observed in the human lens. In the lens, the great majority of posttranslational modifications occur by age 20 [18]. Twenty years is, after all, equivalent to incubating a life-long protein at 37 ◦C for more than 175,000 h! It should be noted that the PTMs of adult myelin discussed in this article take place on MBP that was significantly modified during development [21,22]. In accord with other long-lived proteins, one of the major changes characterised in adult human MBP was the formation of isoAsp residues. This form of Asp is quite different from the normal L-Asp that was present originally in the protein, and is due to the spontaneous formation of a cyclic intermediate [13]. At each site of isoAsp formation from L-Asp (or L-Asn), the polypeptide backbone has been extended by the insertion of a CH2 group (Figure1). In reality, this particular modification leads to an even greater alteration in protein structure because a common product is D-isoAsp. In this case, the original L-amino acid site has been both extended and racemised. As well as creating novel antigens, protein unfolding will accompany these chemical transformations. J. Clin. Med. 2018, 7, 281 4 of 12 J. Clin. Med. 2018, 7, x FOR PEER REVIEW 4 of 12

FigureFigure 2. 2.MyelinMyelin basic basic protein protein isis highlyhighly modified modified in in the the adult adul humant human brain. brain. Histogram Histogram showing showing the the relativerelative abundances abundances of of some some of ofthe the major major sites of sites modification of modification in MBP from in MBP the cerebellum from the in cerebellum controls in controlsand patients and patients with MS. with Three MS. main Three types main of modifications types of modifications were observed: were deimination observed: of deimination Arg (to form of Arg citrulline), racemisation and isomerisation of Asp, and deamidation of Asn and Gln. It should be (to form citrulline), racemisation and isomerisation of Asp, and deamidation of Asn and Gln. It noted that each site of Asn/Gln deamidation contains four Asp/Glu isomers due to the process of should be noted that each site of Asn/Gln deamidation contains four Asp/Glu isomers due to the spontaneous racemisation and isomerisation (see Figure1). The average ages of the two groups were processcontrols of 46spontaneous± 22 years (nracemisation= 10) and MS and patients isomerisation 60 ± 12 years (see (n Figure= 8). Shown 1). The are average mean values ages± ofSD. the two groupsDetailed were subject controls data from46 ± Friedrich22 years ( etn al.,= 10) 2016 and [9]. MS patients 60 ± 12 years (n = 8). Shown are mean values ± SD. Detailed subject data from Friedrich et al., 2016 [9]. 5. Myelin Basic Protein and MS It shouldMBP from be noted an adult, that is the highly PTMs heterogeneous of adult myelin and is discussed a different in protein this article from take the MBP place that on wasMBP that wasdeposited significantly originally modified in childhood. during development In one sense, an[21,22] abundant. new protein has been created in our brain.In accord Every with MBP polypeptideother long-lived in cerebellar proteins, myelin one containsof the major isoAsp changes residues, characterised as well as a numberin adult of human MBPcitrulline was the residues formation [9] and of each isoAsp of these residues. amino acids This is novel.form of These Asp structures is quite are different not found from in normal the normal L-Aspproteins, that was including present the originally original translated in the protein, version ofand MBP. is due to the spontaneous formation of a cyclic It is not hard to imagine that in some cases the body may sense this ‘new’ form of MBP as being a intermediate [13]. At each site of isoAsp formation from L-Asp (or L-Asn), the polypeptide backbone foreign antigen and could commence an immune response. The precise cellular location of MBP is has been extended by the insertion of a CH2 group (Figure 1). In reality, this particular modification not yet clear. If it is partly extracellular then cellular degradation may not be required for immune leads to an even greater alteration in protein structure because a common product is D-isoAsp. In priming. If MBP is an intracellular protein [23] then some cellular damage may be necessary. Not only thisdoes case, this the provide origina anl underlyingL-amino acid mechanism site has forbeen the both autoimmune extended response, and racemised. it also may As explain well as why creating novelmany antigens, cases of protein MS become unfolding evident will in the accompany fourth decade these of chemical life [24]. Eventransformations. by the 30s, MBP has been almost totally transformed into a new protein. Although data on isoAsp formation and other PTMs in 5. Myelinthe MBP Basic from childrenProtein isand lacking, MS it is likely that significant levels are present even in pre-teen years. This is because in other long-lived proteins (LLPs), such as lens proteins, racemisation occurs most MBP from an adult, is highly heterogeneous and is a different protein from the MBP that was rapidly in the first decade of life [18]. Therefore, the age-related changes to MBP and other myelin deposited originally in childhood. In one sense, an abundant new protein has been created in our proteins could even play a role in the genesis of pediatric MS. brain. Every MBP polypeptide in cerebellar myelin contains isoAsp residues, as well as a number of citrulline6. Myelin residues Basic Protein [9] and from each MS of Patients these Isamino Different acids is novel. These structures are not found in normalIf proteins, this ‘old including protein’ hypothesis the original for translated the origin forversion MS is of correct, MBP. why doesn’t everyone suffer fromIt is the not consequences hard to imagine of such that an in untoward some cases immune the body attack, may since sense the brains this ‘new’ of all form adults of are MBP full as of being a foreigndegraded antigen MBP? and One could possibility commence is that thean MBPimmune from response. MS patients The could precise be modified cellular location in a different of MBP is not yet clear. If it is partly extracellular then cellular degradation may not be required for immune priming. If MBP is an intracellular protein [23] then some cellular damage may be necessary. Not only does this provide an underlying mechanism for the autoimmune response, it also may explain why many cases of MS become evident in the fourth decade of life [24]. Even by the 30s, MBP has been almost totally transformed into a new protein. Although data on isoAsp formation and other PTMs in the MBP from children is lacking, it is likely that significant levels are present even in

J. Clin. Med. 2018, 7, x FOR PEER REVIEW 5 of 12 pre-teen years. This is because in other long-lived proteins (LLPs), such as lens proteins, racemisation occurs most rapidly in the first decade of life [18]. Therefore, the age-related changes to MBP and other myelin proteins could even play a role in the genesis of pediatric MS.

6. Myelin Basic Protein from MS Patients Is Different If this ‘old protein’ hypothesis for the origin for MS is correct, why doesn’t everyone suffer from the consequences of such an untoward immune attack, since the brains of all adults are full of degradedJ. Clin. MBP? Med. 2018 One, 7, 281possibility is that the MBP from MS patients could be modified 5in of 12a different way from the MBP in non-MS patients. Proteomic analysis showed that this was indeed the case. Althoughway cerebellum from the MBP samples in non-MS from patients. both normal Proteomic and analysis MS patients showed thatcontained this was highly indeed themodified case. MBP, the MBPAlthough from MS cerebellum patients samples was consistently from both normal different and MS from patients the containednormal MBP highly at modified several MBP,sites (Figure the MBP from MS patients was consistently different from the normal MBP at several sites (Figure2). 2). At eachAt eachsite, site, the thedegree degree of ofchange change was was greater greater inin MSMS patients; patients; the the only only exception exception being being Arg 31. Arg 31. In addition,In once addition, these once MS these-specific MS-specific sites sites of ofmodification modification were incorporated incorporated into into a structure a structure of MBP, of MBP, they appearedthey appeared to be tolocalised be localised into into clusters clusters ( (FigureFigure3 ).3).

Figure 3.Figure MBP 3. inMBP MS in patients MS patients is different is different from from MBPMBP in in controls. controls. Highlighted Highlighted in red in (Arg) red and (Arg) blue and blue (Asp) are(Asp) 5 amino are 5 amino acid acidresidues residues that that differ differ significantly significantly within within a 19 a amino 19 amino acid sequence. acid sequence. This cluster This cluster includes Arg 31, Asp 34, Arg 43, Asp 48, and Arg 49. For each site, the degree of change is greater includes Arg 31, Asp 34, Arg 43, Asp 48, and Arg 49. For each site, the degree of change is greater in in the MS patients; the only exception being Arg 31. Statistical data are shown in Figure2 and are the MS summarised patients; the from only Friedrich exception et al. [9]. being The model Arg of 31. MBP Statistical was generated data using are I-Tasser shown [25 in] and Figure PyMOL 2 and are summarisedMolecular from Graphics Friedrich System et was al. used[9]. toThe render model the 3D of structure. MBP was generated using I-Tasser [25] and PyMOL Molecular Graphics System was used to render the 3D structure. One such cluster is a 19-residue sequence encompassing Arg 31 to Arg 49 (Figures2 and3). This region includes Arg 31, Asp 34, Asp 48, Arg 43, and Arg 49 where in each instance, the extent of One such cluster is a 19-residue sequence encompassing Arg 31 to Arg 49 (Figures 2 and 3). This modification is significantly greater in MS patients. The single exception is Arg 31, where it is lower. region includesOur hypothesis Arg 31, is that Asp regions 34, Asp such as48, these Arg may 43, beand particularly Arg 49 antigenicwhere in and each could instance, precipitate the an extent of modificationimmune is responsesignificantly that leads greater ultimately in MS to MS.patients. There are The precedents single exception for each of the is majorArg 31, modifications where it is lower. Our hypothesis(i.e., isoAsp is [that9] and regions citrulline such [26 ])as found these to may be more be particula abundantrly within antigenic the MBP and clusters could from precipitate MS an immunepatients, response acting that as potent leads antigens. ultimately In MBP to from MS. MS There patients are both precedents of these immune for promoters each of are the major found close together in space [9] so there could well be a synergistic interaction of these novel amino modifications (i.e., isoAsp [9] and citrulline [26]) found to be more abundant within the MBP clusters acids. In effect, aged MBP from MS patients may be ‘more antigenic’ than aged MBP of unaffected from MSindividuals. patients, It acting should be as noted potent that antigens. other proteomic In MBP investigations from MS have patients also detected both differences of these immune promoters(e.g., are in deamidation)found close between together the in MBP space of control [9] so and there MS patientscould well [26]. Thesebe a synergistic proteomic data interaction are of these novelbolstered amino by studies acids. that In haveeffect, reported aged that MBP myelin from from MS MS patients patients differsmay frombe ‘more controls antigenic’ e.g., in being than aged MBP of‘developmentally unaffected individuals. immature’ [ 27 It]. should be noted that other proteomic investigations have also detected differences (e.g., in deamidation) between the MBP of control and MS patients [26]. These proteomic data are bolstered by studies that have reported that myelin from MS patients differs from controls e.g., in being ‘developmentally immature’ [27].

J. Clin. Med. 2018, 7, 281 6 of 12

7. Considerations If MS Results from Age-Related Changes to MBP There are a number of consequences that flow from the discovery that MBP is highly modified with age and that some changes to MBP are characteristic of MS. Firstly, if indeed human MS is an autoimmune disease provoked by an endogenously-formed antigen, what, if any, biological processes exist to modulate the response? Can some protein modifications be tolerated? After all, despite MBP being highly modified in all adults, only a small fraction of people suffer disease symptoms. To what extent does ongoing (in the adult) immune tolerance play a role in the body where there is, in effect, a failure to respond to the formation of a major antigen that was absent at birth? Foxp3 + T regulatory cells are likely to play an important role in this modulation. In addition, there are several chaperone proteins present in the CNS (e.g., alpha crystallin) that can bind to modified, unfolded proteins and in this way could cover potentially antigenic sites before they are recognised by the immune surveillance machinery. Could this putative protective system be less effective in people who go on to develop MS? As with immunisation, the presence of a novel antigen alone may be insufficient to trigger a full immune response. An adjuvant is necessary. Another immune-promoting event (e.g., an infection) may be necessary for a complete response which ultimately leads to MS. In this regard, it is interesting to note that bacterial and viral proteins contain unnatural amino acids such as D-isoAsp and D-isoGlu, so molecular mimicry could play a role. It is known that there is a link between infection and MS [28] and the presence of predisposing genes is also likely to be implicated. T cells have long been thought to be implicated in MS pathogenesis, in particular the inflammation associated with the condition. Recent studies have also highlighted the important contribution of B cells. Evidence for a major role of B cells in MS is suggested by the clinical benefit derived by peripheral B depletion using drugs such as rituximab, an anti-CD20 agent [29]. A possible way in which modified MBP (and other myelin proteins) may elicit an immune response is depicted in Figure4. It is not yet known how normal individuals are able to maintain immunological tolerance towards a new self-antigen (i.e., modified MBP) that is present in large amounts in the adult brain. As mentioned above, there are some intriguing observations that suggest a role for peripheral B cell depletion in this process. In an elegant study to determine whether B cell tolerance checkpoints are defective in MS patients, it was discovered that most suffer from impaired peripheral B cell tolerance, resulting in the accumulation of large numbers of autoreactive and polyreactive mature naive B cells, which express white matter–reactive [30]. Aspects of B cell biology [31] and interactions with respect to MS have been more extensively reviewed elsewhere [32]. Interestingly, in surgical brain injury, it has been found that immune tolerance can be induced by hepatic portal vein and intrathymic injection of MBP [33]. Possibly, in individuals who are not affected by MS, continuous exposure to low levels of modified MBP via the route depicted in Figure4 may act in a similar manner to therapeutic administration of a causative antigen as outlined by Verhagen et al. [34]. In theory, if such a mechanism were impaired or disrupted, exposure to modified MBP may trigger MS. J. Clin. Med. 2018, 7, 281 7 of 12 J. Clin. Med. 2018, 7, x FOR PEER REVIEW 7 of 12

Figure 4. A simplified simplified scheme illustrating how modifiedmodified MBP in the brain could lead to the induction of MS. One mechanism that couldcould underpinunderpin the onsetonset ofof MSMS isis illustrated.illustrated. MBP in the human CNS is heavily modifiedmodified byby ageage 18,18, withwith somesome modificationmodification sitessites beingbeing MS-specific.MS-specific. ModifiedModified MBPMBP and/orand/or peptidespeptides derived from it byby autolysisautolysis [[14,35],14,35], oror possiblypossibly proteaseprotease action,action, diffuse viavia thethe lymphaticlymphatic systemsystem toto thethe CSF. CSF. Subsequent Subsequent drainage drainage via via the the lymphatic lymphatic vessels vessels to the to deepthe deep cervical cervical lymph lymph node node may permitmay permit priming priming of T cell of responses.T cell responses. Once primed, Once primed, T and B cellsT and become B cells self-reactive. become self In-reactive. ways analogous In ways toanalogous those described to those for described EAE, these for self-reactive EAE, these species self-reactive cause damagespecies tocause the myelindamage and to inflammationthe myelin and of theinflammation CNS, which of manifests the CNS, aswhich MS (seemanifests text for as details). MS (see APC, text for Antigen-presenting details). APC, Antigen cell. -presenting cell.

8. Gender, MS,MS, andand Age-RelatedAge-Related ChangesChanges toto MBPMBP ItIt isis wellwell knownknown thatthat MSMS affectsaffects moremore femalesfemales thanthan males.males. If MS cancan indeedindeed bebe ascribedascribed to thethe body’s response to myelin deteriorationdeterioration in the brain,brain, then it may bebe predictedpredicted thatthat womenwomen couldcould harbour a greatergreater degreedegree ofof proteinprotein modificationmodification thanthan men.men. Remarkably this has been observed for MBP (Figure(Figure5 ).5). The The numbers numbers analysed analysed thus thus far far are are small, small, however however the findingsthe findings are in are agreement in agreemen witht awith separate a separate study of study human of MBP human that MBP showed that higher showed levels higher of Asn levels deamidation, of Asn deamidation, Gln deamidation, Gln anddeamidation Arg deimination, and Arg in deimination women than in men women [35]. than men [35].

J. Clin. Med. 2018, 7, 281 8 of 12 J. J.Clin. Clin. Med. Med. 2018 2018, 7, ,7 x, xFOR FOR PEER PEER REVIEW REVIEW 8 8of of 12 12

FigureFigure 5. 5.5. MBP MBPMBP is is more more more highly highly highly racemized racemized racemized in in females. infemales. females. Racemisation Racemisation Racemisation of of aspartic aspartic of aspartic acid acid + acid +asparagine asparagine + asparagine (Asx) (Asx) in(Asx)in thethe the in MBPMBP MBP the of of MBPof age-matchedage age -ofmatched-matched age-matched male male (( ( )male ) and and ( female female ) and ( (female)) subjects.)subjects. subjects. ( ) Elevatedsubjects. Elevated Elevated levels levels of of Dlevels D-Asx-Asx-Asx wereof were D-Asx found found were in in femalesfoundfemales in byby femalesby comparisoncomparison comparison by comparison toto to malesmales males ( (pto p( p== males = 0.029, 0.029, ( Mann–WhitneyMannp Mann= 0.029,–Whitney–Whitney Mann– UUWhitney U test).test test).). MaleMale Male U test). nn n == = 4, Male4, female female n = n 4, n = female =4, 4, age age rangen range = 4, 51–8751age51–87– range87 years. years. 51 – Mean 87Mean years. ± ±SEM. SEM.SEM. Mean ± SEM. 9. Linkage between the CNS and the Periphery in MS 9.9. Linkage Linkage between between the the CNS CNS and and the the Periphery Periphery in in MS MS One reason for believing that an immune response is involved in the induction of MS comes OneOne reason reason for for believing believing that that an an immune immune response response is is involved involved in in the the induction induction of of MS MS comes comes from animal studies. EAE is a widely studied animal model of human (CNS) fromfrom animal animal studies. studies. EAE EAE is is a a widely widely studied studied animal animal model model of of human human central central nervous nervous system system (CNS) (CNS) demyelinating diseases, such as multiple sclerosis [36]. EAE can be induced in a number of species and demyelinatingdemyelinating diseases, diseases, such such as as multiple multiple sclerosis sclerosis [36][36]. [36]. .EAE EAE can can be be induced induced in in a a number number of of species species the most commonly used antigens for injection are spinal cord homogenates, myelin proteins such as andand the the most most commonly commonly used used antigens antigens for for injection injection are are spinal spinal cord cord homogenates, homogenates, myelin myelin proteins proteins MBP, proteolipid protein (PLP) and myelin glycoprotein (MOG), as well as suchsuch as as MBP, MBP, proteolipid proteolipid protein protein (PLP) (PLP) and and myelin myelin oligodendrocyte oligodendrocyte glycoprotein glycoprotein (MOG), (MOG), as as well well as as derived from these proteins. Each results in a model with slightly different disease characteristics peptidespeptides derived derived from from these these proteins. proteins. Each Each results results in in a a model model with with slightly slightly different different disease disease regarding both immunology and pathology. EAE can also be induced by the passive transfer of T cells characteristicscharacteristics regarding regarding both both immunology immunology and and pathologypathology. pathology. .EAE EAE can can also also be be induced induced by by the the passive passive reactive to these myelin antigens [37]. transfertransfer of of T T cells cells reactive reactive to to these these myelin myelin antigens antigens [37][37]. [37]. . EAE demonstrates that once the body has been primed by exposure to a CNS antigen, that it can EAEEAE demonstrates demonstrates that that once once the the body body has has been been primed primed by by exposure exposure to to a a CNS CNS antigen, antigen, that that it it respond with an attack on the brain. As has been recently reviewed by Louveau et al. [38] the CNS, cancan respond respond with with an an attack attack on on the the brain. brain. As As has has been been recently recently revreviewed reviewediewed by by Louveau Louveau et et al. al. [38] [38] the the which was historically viewed as being immune privileged, is now recognized to be in continuous CNS,CNS, which which was was historically historically viewed viewed as as being being immune immune privileged, privileged, is is now now recognized recognized to to be be in in communication with the rest of the body. One way in which an immune response to degraded continuouscontinuous communication communication with with the the rest rest of of the the body. body. One One way way in in which which an an immune immune response response to to MBP could be initiated, is via the “glymphatic” system involving deep cervical lymph nodes [39]. degradeddegraded MBP MBP could could be be initiated, initiated, is is via via th thee “glymphatic” “glymphatic” system system involving involving deep deep cervical cervical lymph lymph The finding that MBP is long-lived, also provides a mechanism for how modified peptides derived nodesnodes [39][39].[39]. . The The finding finding that that MBP MBP is is longlong-lived, long-lived,-lived, also also provides provides a a mechanism mechanism for for how how modified modified from it might travel from their source in the CNS to sites in the periphery where an immune response peptidespeptides derived derived from from it it might might travel travel from from their their source source in in the the CNS CNS to to sites sites in in the the periphery periphery where where could be initiated. Spontaneous cleavage of long-lived proteins at Ser [40] and Asn [13] could lead to anan immune immune respresponse responseonse could could be be initiated. initiated. Spontaneous Spontaneous cleavage cleavage of of longlong-lived long-lived-lived proteins proteins at at Ser Ser [40] [40] and and the release of modified MBP peptides from myelin into the cerebrospinal fluid (CSF) (Figure4). AsnAsn [13] [13] could could lead lead to to the the release release of of modified modified MBP MBP peptides peptides from from myelin myelin into into the the cerebrospinal cerebrospinal The various T cell responses in the CNS are controlled in a complex manner as summarised fluidfluid ((CSF) CSF(CSF) )((Figure Figure(Figure 4). 4). in a recent comprehensive review [41] and structural organisation of the CNS plays a key role in TheThe various various T T cell cell responses responses in in the the CNS CNS are are controlled controlled in in a a complex complex manner manner as as summarised summarised in in a a determining the details of the overall immune response [36]. Evidence in support of the scheme recentrecent comprehensive comprehensive review review [41][41] andand structural structural organisation organisation of of the the CNS CNS plays plays a a key key role role in in outlined in Figure4 is derived from several sources. For example, induction of apoptosis in determiningdetermining the the details details of of the the overall overall immune immune response response [36] [36].[36]. . Evidence Evidence in in support support of of the the scheme scheme is accompanied by the accumulation of phagocytes containing myelin antigens outlinedoutlined in in FigureFigure 44 is is derived derived from from several several sources. sources. For For example, example, induction induction of of apoptosis apoptosis in in in the lymph nodes that drain the CNS [42]. In addition, oligodendrocyte death can cause CNS oligodendrocytesoligodendrocytes is is accompanied accompanied by by the the accumulation accumulation of of phagocytes phagocytes containing containing myelin myelin antigens antigens in in antigens to drain from the CNS in amounts sufficient to induce a systemic T cell response against thethe lymph lymph nodes nodes that that drain drain the the CNS CNS [42][42].[42]. . IInn In addition, addition, oligodendrocyte oligodendrocyte death death can can cause cause CNS CNS MOG [43]. antigensantigens to to drain drain from from the the CNS CNS in in amounts amounts sufficient sufficient to to induce induce a a systemic systemic T T cell cell response response against against MOG [43]. 10.MOGMOG Autoantibodies [43] [43]. . and MS

10.10. Autoantibodies AutoantibodiesThere is compelling and and MS evidence MS from animal model systems that myelin antigens can act as drivers for the induction of MS, and if this were true, it might be expected that MS patients display higher ThereThere is is compelling compelling evidence evidence from from animal animal model model systems systems that that myelin myelin antigenantigens antigens s can can act act as as levels of anti-myelin antibodies than unaffected individuals. The results of human studies driversdrivers for for the the induction induction of of MS, MS, and and if if this this were were true, true, it it might might be be expected expected that that MS MS patients patients display display in MS patients and controls have often been mixed [44,45]. The proteomic results may provide one higherhigher levels levels of of antianti-myelin anti-myelin-myelin antibodies antibodies than than unaffected unaffected individuals. individuals. The The results results of of human human antibody antibody reason for this lack of consistency. Based on the proteomic data from human cerebellum, there is studiesstudies in in MS MS patients patients and and controls controls have have ooften oftenften been been mixed mixed [44,45] [44,45].[44,45]. . The The proteomic proteomic results results may may provideprovide one one reason reason for for this this lack lack of of consistency. consistency. Based Based on on the the proteomic proteomic data data from from human human

J. Clin. Med. 2018, 7, 281 9 of 12 probably very little unmodified MBP left in normal adult brain. The degree of MBP modification is simply so great. Preliminary data suggest that MBP from other brain regions is also highly modified. Therefore, screening for antibodies to the MBP polypeptide as it was originally translated (and as it once existed in childhood), may not be an appropriate method to assess whether MS patients have clinically-relevant autoantibodies to adult myelin proteins. Given the most recent proteomic data [9,26] that has uncovered brain proteins that are highly altered in adults, a better question may be: are antibodies that recognise highly-modified MBP present in MS patients? Antibodies may cross-react to varying degrees with the original unmodified MBP, or possibly may not recognise it at all. Now that MS-specific sites on MBP have been characterised [9,26], a search for antibodies to these particular sites in MS patients should be a priority. In other words, using relevant antigens for antibody screens may be important. Unmodified (original) MBP may not be the best choice and this factor could be one explanation for variability in the published results of serum screening of MS patients for antibodies to MBP [44,45]. Our preliminary data indicate that the other long-lived proteins in myelin (e.g., PLP and MOG [7]) have also been modified over time and could potentially become new self-antigens, which have the potential to trigger autoimmunity. The same caveats with regard to the screening for MS-relevant autoantibodies (and T cells) to these proteins as outlined for MBP, may apply to them as well.

11. Cellular Aspects of MS B and T cell responses to self-antigens are clearly important and mechanisms of central tolerance for B cells have been reviewed [46]. Significant increases in the number of autoreactive B cells have been observed in MS patients [47]. In general, it appears that B cell responses to single sites of PTM in self-antigens are likely to be promiscuous, in the sense that the antibodies recognise both modified and unmodified forms of the self-protein. Presumably this is because the amino acid sequences on either side of the modification remain the same. On the other hand, T cell responses tend to be more specific to the site of modification [48]. In the context of a B cell response in MS, it is important to recognise that MBP has been modified at many sites [9]. Another hypothesis is that the modified MBP could act as a trigger of the innate immune response by binding to pattern recognition receptors and/or damage-associated molecular pattern receptors to drive inflammatory processes and lower activation thresholds of T and B cells.

12. A New Theory for MS It is a corollary that if the extensive modification of MBP in the CNS, coupled with its drainage into the periphery and the consequent induction of an immune response, (as illustrated in Figure4), is validated, it changes the way in which MS is viewed. For example, it renders largely unnecessary a widely held hypothesis for the induction of MS, based on the sensitisation of autoreactive T cells by non-CNS antigens derived from the periphery (e.g., molecular mimicry). In the hypothesis outlined in this article, MS is triggered primarily by the formation of a novel endogenous antigen rather than by the body’s altered response to an existing antigen.

13. Conclusions With regard to the consequences, perhaps the most important for MS is that the existence of MS-specific sites on a major myelin protein opens up the possibility of targeted drug design to prevent, or treat, MS. Although it is not yet clear why MS presents in several forms, if MS does result from an immune response to particular modified sites on MBP, then compounds that specifically recognise and mask these sites could potentially offer a degree of protection. This may be the dawn of a new era in the treatment of MS. J. Clin. Med. 2018, 7, 281 10 of 12

Author Contributions: Original draft preparation, R.J.W.T.; review & editing, M.G.F. Funding: Some funding for this work was supported by a grant from the National Health and Medical Research Council of Australia (NHMRC #1008667). Acknowledgments: Ron Sluyter is thanked for helpful comments and for carefully reviewing the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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