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  • Gut Feelings About a-Synuclein in Gastrointestinal Biopsies:Biomarker in the Making?

    Claudio Ruffmann, MD1,2 and Laura Parkkinen, PhD1,2*

    1Oxford Parkinsons Disease Centre, University of Oxford, Oxford, United Kingdom2Nuffield Department of Clinical Neurosciences, Academic Unit of Neuropathology, University of Oxford, Oxford, United Kingdom

    ABSTRACT: In recent years, several studies haveinvestigated the potential of immunohistochemical detec-tion of a-synuclein in the gastrointestinal tract to diag-nose Parkinsons disease (PD). Although methodologicalheterogeneity has hindered comparability between stud-ies, it has become increasingly apparent that the highsensitivity and specificity reported in preliminary studieshas not been sustained in subsequent large-scale stud-ies. What constitutes pathological a-synuclein in the ali-mentary canal that could distinguish between PDpatients and controls and how this can be reliablydetected represent key outstanding questions in thefield. In this review, we will comment on and comparethe variable technical aspects from previous studies, andby highlighting some advantages and shortcomings wehope to delineate a standardized approach to facilitatethe consensus criteria urgently needed in the field. Fur-thermore, we will describe alternative detection techni-ques to conventional immunohistochemistry that have

    recently emerged and may facilitate ease of interpreta-tion and reliability of gastrointestinal a-synuclein detec-tion. Such techniques have the potential to detect thepresence of pathological a-synuclein and include theparaffin-embedded tissue blot, the proximity ligationassay, the protein misfolding cyclic amplification tech-nique, and the real-time quaking-induced conversionassay. Finally, we will review 2 nonsynonymous theoriesthat have driven enteric a-synuclein research, namely, (1)that a-synuclein propagates in a prion-like fashion fromthe peripheral nervous system to the brain via vagal con-nections and (2) that gastrointestinal a-synuclein deposi-tion may be used as a clinically useful biomarker in PD.VC 2016 The Authors. Movement Disorders published byWiley Periodicals, Inc. on behalf of International Parkin-son and Movement Disorder Society.

    Key Words: biomarker; biopsy; enteric nervous sys-tem; Parkinsons disease; a-synuclein

    Idiopathic Parkinsons disease (PD) is a neurodegener-ative condition characterized by typical motor symp-toms of tremor, bradykinesia, and rigidity, which enable

    its diagnosis in the majority of cases. Nonetheless, mis-diagnosis can occur in up to 20% of cases, particularlyin the early phases of disease.1 Thus, the postmortemdetection by immunohistochemistry (IHC) of aggrega-tion of a-synuclein (ASN) in the brain coupled withneuronal loss in the substantia nigra remains the goldstandard for the definite diagnosis of PD.

    The progressive pathological changes, however, startyears before the clinical onset of symptoms, and by thetime of diagnosis, the disease is too advanced for anyneuroprotective therapies to have full impact. In recentyears, several studies have shown that ASN aggregationcan also be detected outside the central nervous system(CNS), particularly in the enteric nervous system (ENS)of the gastrointestinal (GI) tract of PD patients.2-5 Thecontrasting results and varying methodologies appliedhave recently been reviewed in detail by Visanji and col-leagues.6 However, reflecting the fast development in thisfield, a number of studies on ENS ASN detection as a

    ------------------------------------------------------------This is an open access article under the terms of the Creative CommonsAttribution License, which permits use, distribution and reproduction inany medium, provided the original work is properly cited.

    *Correspondence to: Dr. Laura Parkkinen, Nuffield Department ofClinical Neurosciences, Academic Unit of Neuropathology, University ofOxford, West Wing, Level 1, OX3 9DU Oxford, UK,E-mail: [email protected]

    Funding agencies: This study was funded by the Monument TrustDiscovery Award from Parkinsons UK. Dr. Parkkinen receives researchsupport from Parkinsons UK, the Michael J. Fox Foundation, andAlzheimers Research UK.

    Relevant conflicts of interest/funding disclosures: The authors reportno financial disclosures or conflict of interests.

    Received: 13 August 2015; Revised: 12 October 2015; Accepted: 15October 2015

    Published online 22 January 2016 in Wiley Online Library( DOI: 10.1002/mds.26480

    R E V I E W

    Movement Disorders, Vol. 31, No. 2, 2016 193
  • PD biomarker have since then added to the growingbody of literature.7-10 These have provided new evidencethat GI ASN detection may facilitate a presymptomaticdiagnosis of PD, with evidence of ASN accumulation incolonic biopsies up to 8 years prior to the developmentof motor symptoms,7,11 which also suggests that ASNaccumulation may start in the ENS long before it affectsthe brain. Recent studies have also supported the use ofin vivo gastric detection of ASN to confirm a diagnosisof PD in clinically symptomatic subjects,8 whereas othershave identified the appendiceal mucosa as a potentiallyuseful source of tissue for detection of ASN.9 If con-firmed, these findings could meet the current urgent needfor a biomarker in PD, which could complement or per-haps even be superior to directly imaging changes in thenigrostriatal system to predict, confirm, track progres-sion, and phenotypically stratify PD patients.12

    However, a critical step in this process will be todevelop a method that is sufficiently sensitive and specific.To date, both the use of different types of antibodies andmorphological assessment of pathology have fallen shortof accurately distinguishing between the physiologicaland pathological forms of the ASN protein. Given thatASN accumulation has repeatedly been detected in the GItract of neurologically unimpaired individuals,9,13,14 thequestion of what constitutes the pathological species ofASN has increasingly emerged as the key outstandingquestion in the field and will be addressed throughout thisreview. For the purposes of clarity, we will use the termASN immunoreactive staining (ASN-IRS) to describe anykind of ASN-positive staining described in the literature(ie, aggregates, inclusions, fibers, etc.). Based on the mor-phology of staining, we will subdivide this into somatic(ie, round, Lewy bodylike inclusions or diffuse stainingin the perikaryon of ganglionic cells), neuritic (ie, thread-like staining in dendrites and axons), or synaptic (ie, dot-like staining in the neuropil).

    Sensitivity and Specificity of EntericNeuronal ASN-IRS in PD

    Although initial studies with small cohorts showedpromisingly high sensitivity and specificity rates,3,5 thiswas not sustained in subsequent large-scale studies(Table 1; also see review by Visanji et al).7,15 Sensitivityas low as 10% was reported in the largest cohort todate, in which ASN-IRS was detected in biopsies fromvarious levels of the GI tract in only 7 of 62 PDpatients.7 However, case selection was potentially lessrigorous in this study, which relied entirely on retro-spective review of medical records, and analyzed tissuewas derived from biopsies done for reasons unrelated toPD. On the other hand, studies examining ad hocfresh biopsies from patients included after clinicalexamination by a neurologist collectively obtained asensitivity of 86%.2,3,5,8,14 There is also significant dis-

    crepancy in specificity, with some studies reporting anoptimal 100% specificity,2-5,7 whereas others detectedASN-IRS in more than 50%15 or as many as 82%14 ofcontrol subjects. Visanji and colleagues found 9 of 11control individuals to have ASN-IRS in both colonicand rectal biopsies, which could not be distinguishedfrom PD patients by any specific feature such as loca-tion, area, or intensity.14

    To our knowledge, two studies have reported in vivoASN-IRS exclusively in neurologically unimpaired sub-jects.9,13 In the most recent of these, Gray and colleaguesreported somatic, diffuse ASN-IRS in the appendices of20 of 20 neurologically unimpaired subjects but alsoabundant neuritic and synaptic ASN-IRS in the laminapropria with an apicobasal gradient of increasing fiberdensity.9 It is unlikely that prodromal PD underlies theASN-IRS in all neurologically unimpaired subjectsdescribed in these studies; thus, these findings raise con-cerns about the use of colonic biopsies to diagnose PD.

    Biopsy Sampling

    One highly variable parameter between studies is thesite of the GI tract biopsy (Fig. 1). Several differentregions of the GI tract have been harvested includingsubmandibular glands,16 stomach,8 appendix,9 ascend-ing and descending colon,3 sigmoid colon, and rec-tum.5,14 Both postmortem17-19 and in vivo3,4 studieshave reported a higher density of ASN-IRS in the rostralregions of the GI tract compared with the more distalones. However, findings from more recent studieswould seem to question this hypothesis. Hilton and col-leagues reported the absence of ASN-IRS in the esopha-gus, whereas 8% of biopsies from the stomach and13% of both small and large intestine showed ASN-IRstaining,7 suggesting a caudo-rostral gradient, if any.Furthermore, Visanji and colleagues found equally prev-alent ASN-IRS when comparing biopsies taken 5 cm(rectum) and 20 cm (sigmoid colon) from the analverge.14 Finally, Gray and colleagues showed that neu-ritic ASN-IRS was much more abundant in the mucosaof the appendix than in stomach, ileum, or colon, high-lighting appendix as the optimal anatomical locus.9

    Several reports have also demonstrated the impact ofthe amount of tissue analyzed on the likelihood of cap-turing ASN-IRS.8,18,19 Of note, routine 4- to 6-lm-thickparaffin sections of the GI bio