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Kronimus Y, Dodel R, Neumann S. A quantitative view on naturally occurring autoantibodies Neuromedicine in neurodegenerative diseases. J Neurol Neuromedicine (2018) 3(4): 5-11 www.jneurology.com www.jneurology.com Journal of Neurology && NeuromedicineNeuromedicine

Mini Review Open Access

A Quantitative View on Naturally Occurring Autoantibodies in Neuro- degenerative Diseases Yannick Kronimus, Richard Dodel*, Sascha Neumann Department of Geriatrics, University Duisburg-Essen, Germaniastrasse 1-3, 45356 Essen, Germany

Article Info ABSTRACT

Article Notes Accumulation and aggregation of Beta- (Aβ) and Alpha-Synuclein (α-Syn) are Received: May 28, 2018 considered as central or even causative for the development of Alzheimer’s (AD) and Accepted: July 05, 2018 Parkinson’s disease (PD). Therefore, the regulation of these proteins seems to be an *Correspondence: essential aspect for prevention and is of central interest in current research aiming to Dr. Richard Dodel, MD, Department of Geriatrics, University find therapeutic approaches. The human immunological repertoire already contains Duisburg-Essen, Germaniastrasse 1-3, 45356 Essen, Germany; such a regulatory system. Naturally occurring autoantibodies (nAbs) against the Telephone No: +49 (0)201-89760; Fax No: +49 (0)201 8976229; proteins Aβ (nAbs-Aβ) and α-Syn (nAbs-α-Syn) are part of the innate immune system E-mail: [email protected]. and modulate the metabolism of their specific antigens including protein clearance © 2018 Dodel R. This article is distributed under the terms of and inhibition of aggregation. Thus, many researchers hypothesize that in the course the Creative Commons Attribution 4.0 International License of AD and PD, quantitative alterations of nAbs-Aβ and nAbs-α-Syn arise resulting in impaired proteastasis. Such alterations would represent promising, reliable biomarkers and indicate potential approaches for therapeutic strategies. Hence, it is not surprising that many studies dealing with nAbs-Aβ and nAbs-α-Syn titers in AD Keywords: and PD patients in comparison to control participants are available in the literature. Naturally occurring autoantibodies In this mini review, we summarize the current evidence. Furthermore, we critically Alzheimer’s disease discuss problems and future requirements for nAbs quantification when a clinical Parkinson’s disease application is the overriding goal. Serum titers CSF titers Beta-Amyloid; Alpha-Synuclein ELISA; Introduction The human antibody repertoire can be subdivided into conventional and naturally occurring antibodies, based on their originating B cell type. While conventional antibodies derive during life from plasma and memory B cells differentiated from B2 or marginal zone B cells after antigenic activation, naturally occurring antibodies arise in fetogenetic periods and thus are present from birth1-4. They are produced from B1 cells without extrinsic stimuli and T cell assistance. A special

recombined immunoglobulin genes resulting in less hypermutated and characteristic of B1 cells is the germline-closed5. Although configuration conventional of the antibodies are usually the main subject in textbooks, naturally occurring affinityantibodies matured account antibodies for the withgreatest lower portion affinities in humans6. Among them, a fraction shows autoreactivity and is termed naturally occurring autoantibodies (nAbs), which are mostly from the IgM and IgA, fewer from the IgG type7 physiological , support the clearance of distinct secreted proteins and apoptotic. They cells,circulate and through protect humanfrom pathologically body fluids, maintain altered structures like oxidatively damaged, aggregated, and non-functional lipids and proteins8,9. In the following sections we want to focus on

especially for therapeutics and diagnostics in neurodegenerative dis­ ortheders. beneficial aspects of nAbs and their potential clinical applications,

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Clinical Potential of AD and PD Associated nAbs factors for the development of AD and PD. Especially quantitative alterations, namely changed titers, may act as The two most common neurodegenerative disorders biomarkers in clinical applications and illustrate restoring – Alzheimer’s (AD) and Parkinson’s disease (PD) – are among others histopathologically characterized by 42 therapeutic strategy. It is not surprising that a number extra- and intracytoplasmic protein deposits primarily of Aβ and α-Syn specific antibodies as a promising 42 already been published in the past and will be following 10,11. ofdiscussed. studies investigating titers of nAbs-Aβ /α-Syn have Albeitconsisting the ofaccurate the proteins pathomechanism beta-amyloid has (Aβ) not inbeen the fully case of AD or alpha-synuclein (α-Syn) in the case of PD nAbs-Aβ titers that the metabolic dysregulation of the proteins has A large body of literature exists harboring inconsistent, clarified, neither for AD12 nor for PD, it is widely accepted titers in AD 42 etiological significance . For Aβ, especially the isoform patients. In a comprehensive study by Britschgi et al. minatory as it exhibits heightened hydrophobicity. The even contrasting data regarding nAbs-Aβ containing 42 amino acids (Aβ ) has been identified as

patients(2009), nAbs-Aβ and nondemented recognizing controlvarious naturessubjects of of its different antigen accumulation and aggregation of both, Aβ and α-Syn into to many (in)direct neuro- and cytotoxic effects indicating agescould33 be detected within serum and CSF samples of AD small soluble oligomers and fibrils have been connected13-16 their participation in disease onset and progression . the highest reactivity was uncovered for aggregated and . Using ELISA technique and antigen forms. microarrays Between pathological mechanism is highly hypothesized within 40 42 literatureFurthermore, encompassing especially the forspreading α-Syn, of aggregates a transmitting from posttranslationally modified Aβ and Aβ 42 plasma titers against oligomeric or monomeric cell to cell17. AD patients and controls, no significant differences of Although AD- and PD-associated protein deposition tonAbs-Aβ moderately and severely affected AD patients, the latter and the resulting detrimental effects are more obvious protein forms were identified. However, comparing mildly

42 plasma titers suggesting a role in disease group showed in one sample set significantly decreased dysregulation also occurs in the periphery. In particular, this titers in AD patients were nAbs-Aβ in the central nervous system (CNS), Aβ as well as α-Syn 40 and progression.26,28 Unchanged nAbs-Aβ - 42 . Marcello and coworkers (2011) revealed similar also verified in further studies examining nAbs-Aβ andis indicated nerve cells by ofvascular the enteric Aβ deposition system in PD– very patients common18-20 . in results even the experimental setup was limited to IgM AD – as well as α-Syn pathology detectable in erythrocytes autoantibodiesAβ 34. Thus, since many years and considering all aspects, researchers follow the idea of an early and preventive Although they were not able to detect altered nAbs- titers, plasma level of nAbs recognizing N-truncated intervention counteracting protein dysregulation, 42 aggregation, and propagation as it is believed to modify Aβ disease progression in AD, PD, and other proteinopathies. pGlu 3-7 Aβ consisting of the sequence three to seven 42 and a modified pyroglutamate ( Aβ ) was significantly could represent a promising therapeutic strategy due to have often been detected in the of AD patients and decreased in patients suffering from AD. Such peptides35 aHere, targeted Aβ modulation and/or α-Syn of the specific metabolism antibody of their treatment antigen. were shown to strengthen the aggregation process . Three

and experi­ments and revealed positive in-vitro in-vivo further studies identified significantly changed blood titers effectsSuch approaches including reduced have already protein beendeposits tested and indecreased diverse Qu et al. (2014) demonstrated reduced serum titers of nAbs of nAbs against distinctbut unchanged Aβ forms using ELISA techniques. , however, failed in phase three at the 1-15 36 latest when tested in the clinical state of AD21-25. and aggregated full-length in AD patients . Moir andrecognizing colleagues Aβ (2005) found decreasednAbs-Aβ nAbs against levels solubleagainst It is an interesting fact that antibodies that maintain 40 40 in tissue homeostasis and proteostasis are already present AD patients37. In contrast, Gruden and coworkers (2007) in humans26,27. In child- and adulthood as well as in health redox-modified Aβ and again unchanged nAbs-Aβ

42 (nAbs- AD patients, namely autoantibodies against oligomeric detected significantly increased nAbs portions38 in female 42 . This andthat become disease, misfolded nAbs recognizing in disease the cases proteins (such as Aβ and fragment – inter alia detectable in AD brains – is often Aβtau protein)) and α-Syn are detectable (nAbs-α-Syn) in the but serum also and further cerebrospinal proteins usedAβ consisting as a model of the system amino as acids it maintains 25 to 35 the neurotoxic mechanisms2,27-32. Quantitative or qualitative alterations controlling the aggregation process39. Elevated titers of fluidof nAbs (CSF) could suggesting impair their the presencenormal functions, of innate worsen protective the propertiesunbound autoantibodies of Aβ with the recognizing simultaneous these option eleven of amino better protection system, and thus represent causal or supporting acids containing and most toxic sequence may be a hint

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for decreased but necessary antigen binding and thus 42 immune complexes.

25-35 what may result in disease onset or progression. The exclusive investigation of such antibodies sufferingwas applied from to AD analyze was detected nAbs/Aβ44. Although the studies hasregulation the ability of Aβ to be a critical step and advantage as all investigatedHere, a significantly nAbs targeting increased different portion antigenic in the CSF structures, of people similar effects can underlie both observations as decreased further Aβ targeting nAbs with various epitopes could nAbs (immune complexes). overlapAs mentioned a specific effect above, of thethere indeed are crucialalso contrary sequence. data free nAbs titers can be the result of increased Aβ bound available including studies that revealed increased or nAbs-α-Syn titers decreased nAbs portions recognizing the full-length peptide

42 immuno-precipitation based experiments have uncovered Conflicting results of nAbs-α-Syn titers are also available Aβ . On the one hand,serum some titersELISA inas wellAD aspatients radiolabeled when 42 in literature about PD. Two exemplary studies using ELISA compared to healthy or cognitively unremarkable technique and dealing with serum45,46 and CSF nAbs-α-Syn lowered 40,41 nAbs-Aβ in patients suffering from PD . While Horvath and subjects . On the other hand, Nath and colleagues (2003) demonstrate significantly elevated autoantibody titers determined elevated nAbs serum titers against soluble and colleagues (2017) found differences between PD patients 42 42 in AD patients samples have also been investigated, however, only three coworkers (2018) detected only increased nAbs levels in and control subjects in both, CSF and serum, Akhtar and ofpre-aggregated the AD patients Aβ – but interestingly. In the the severely same study, affected CSF ones – and none of the controls contained detectable a stronger effect size for changed serum titers and the loss the CSF. This is of particular interest as Horvath revealed 42 frequent than serum data –it encompasses a more invasive patient cohort into mild and moderate affected individuals. of significant differences of CSF titers when separating the amountsclinical method of nAbs-Aβ – only. Sincea few CSFmore data studies are generallydealing with less In line with these observations, Gruden et al. (2011) as variable nAbs structures have been published. For example, experiments and surface plasmon resonance spectroscopy 40 well as Yanamandra et al. (2011) performed serum ELISA 43. Within a second Dustudy and published colleagues by (2001)Maftei et revealed al. (2013) decreased the same nAbs-Aβ method titers in CSF of AD patients using ELISA with monomeric and aggregated α-Syn, identified increased nAbs-α-Syn titers against both protein forms in PD Table 1. NAbs Serum/Plasma and CSF titers in AD patients compared to control subjects. Summary of exemplified studies investigating changed serum/plasma and CSF concentration of nAbs-Aβ. For each study and if information were available (n.a. = not available),p -value of the statistical analysis, age- (AM), gender-matching (GM), and Mini-Mental-State-Examination Score of the study participants as well as the used method are shown. *No comparison possible as nAbs-Aβ42 were only detectable in the CSF of severely affected AD patients.

Serum/plasma concentration CSF concentration AM/GM/ AM/GM/ titer nAbs reference p-value method titer nAbs reference p-value method MMSE MMSE only detectable 42 n.a. +/+/17 vs 29 ELISA in severe cases* 42 nAbs-Aβ42 0.005 +/+/17 vs 29 ELISA

38 bound nAbs- 44 nAbs-Aβ25-35 < 0.01 +/+/15 vs 27 ELISA 0.03 +/+/20 vs 29 ELISA Aβ42 in AD in AD

Increased Increased bound nAbs- Increased 44 0.03 +/+/20 vs 29 ELISA Aβ42 26 0.85 +/+/n.a. ELISA 28 0.056 +/+/17 vs 29 ELISA 33 n.a. +/-/23 vs 30 ELISA nAbs-Aβ 42 34 n.a. +/+/17 vs n.a. ELISA ELISA 36 n.a. +/+/20 vs 29 unchanged Dot Blot unchanged 28 0.19 +/+/17 vs 29 ELISA nAbs-Aβ 40 37 n.a. +/+/n.a. ELISA 40 < 0.02 +/n.a./n.a. ELISA nAbs-Aβ 42 41 0.001 -/-/21 vs 29 IP 34 nAbs-pGluAβ3-7 0.021 +/+/17 vs n.a. ELISA 43 nAbs-Aβ40 0.02 +/n.a./n.a. ELISA in AD 0.02 ELISA in AD nAbs-Aβ 36 +/+/20 vs 29 Decreased Decreased 1-15 0.0008 Dot Blot Decreased 37 nAbs-redAβ40 0.018 +/+/n.a. ELISA

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Table 2. NAbs Serum/Plasma and CSF titers in PD patients compared to control subjects. Summary of exemplified studies investigating changed serum/plasma and CSF concentration of nAbs-α-Syn. For each study and if information were available (n.a. = not available),p -value of the statistical analysis, age- (AM), gender-matching (GM), and Hoehn & Yahr Scale (H&Y; median or *mean) of the study participants as well as the used method are shown.

Serum/plasma concentration CSF concentration AM/GM/ AM/GM/ titer nAbs reference p-value method titer nAbs reference p-value method H&Y* H&Y* 46 < 0.05 n.a./+/2 ELISA 45 0.016 +/-/3 ELISA 47 < 0.01 +/+/2.1* ELISA

nAbs-α-Syn 48 < 0.007 ELISA/WB nAbs-α-Syn in PD n.a. in PD < 0.001 SPR 46

Increased Increased Increased < 0.05 n.a./+/2 ELISA 49 < 0.001 +/+/1 ELISA 45 0.19 +/+/3 ELISA 51 0.5 -/-/2 ELISA nAbs-α-Syn nAbs-α-Syn 52 0.69 +/+/1.3* ELISA unchanged 53 n.a. -/-/2.4 ELISA/WB unchanged 30 < 0.05 +/+/2 ELISA nAbs-α-Syn 50 0.005 -/-/2 ELISA nAbs-α-Syn in PD in PD bound nAbs- 50 0.042 -/-/2 ELISA Decreased Decreased Decreased Decreased α-Syn patients, and revealed an attenuated effect with increasing last years including increased, decreased, and unchanged disease duration47,48 nAbs levels in affected persons. Not until this drawback is eliminated, the application of nAbs titers has a chance to serum levels to PD patients,. In a further controls, publication, and AD patientsShalash and againcolleagues detected (2017) an elevatedexpanded portion the comparison in the PD ofcohort nAbs-α-Syn49. receiveA number clinical of significance. reasons might be responsible for the great These studies provide the impression of increased variance and contrary data of altered nAbs titer in AD and PD. In many cases, typical parameters like incorrect diagnoses of the included participants, small sample sizes However,nAbs-α-Syn contrasting titers in especially hints with early both, stages short- of PD suggestingand long- and technical limitations are cited as causes. However, it is termdisease affected duration PD patients as a critical and correspondent factor for such differences findings. in age and disease severity exist as well. Besong-Agbo et al. (2013) as well as Brudek et al. (2017) demonstrated appliedlargely impossible to determine that nAbs the contrasting titers. Within findings the areexperimental a method- setups,based effectdifferences as in almostin the allsample cases preparation ELISA technique are more was plasma and serum of PD patients30,50. Additionally, in the likely responsible for contrary results. The utilization lattersignificantly paper PD decreased patients were nAbs-α-Syn also shown serum to exhibit titers inlower the

of different body fluids including plasma, serum, or CSF titers of α-Syn/nAbs immune complexes resulting in a harbors also risks for fluctuations like a previous antibody As opposed to the already outlined studies, Maetzler and decreased total nAbs-α-Syn fraction. purification. Additionally, a number of further variabilities coworkers published in 2014 the presence of comparable 51 ofmay the account incorporated for inconsistent participants data andon nAbs-Aβ 2) the antigenand nAbs-α- that isSyn used titers. to determineHere, two mainits corresponding factors represent nAbs 1)titer. the Control choice twonAbs-α-Syn further serum publications levels in investigating PD and healthy serum persons or plasma. Such probands often range from younger healthy controls to age- samplesunchanged of nAbs-α-SynPD patients titers and have healthy also controlsbeen confirmed52,53. Here, in matched patients without disease-associated symptoms36,51. Furthermore, patient cohorts often differ in disease severity interest as they additionally summarize and demonstrate especially the publication by Smith et al. (2012)52. is of great and duration. Such variabilities result in different physical conflictingConclusion studies and Future regarding Perspectives α-Syn protein isand of immunologicalutmost importance conditions as directly and age may dependent significantly and For the development of reliable diagnostic markers but indirectlyinfluence nAbsmedication titer analyses. mediated Especially, effects on the antibody latter aspect titers 54-56. This should not be disregarded even though authors including individuals without age- research.also as therapeutic However, as targets, mentioned the determination above, in both casesof nAbs-Aβ many havematching been usually identified argue that their cohorts do not show differentand nAbs-α-Syn and contrasting titers are results of certain have interest been published in AD and in thePD positive correlation between age and antibody titers.

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