STUDY ON THE LEVELS OF TAU AND β-AMYLOID IN CSF OF PATIENTS WITH MULTIPLE SCLEROSIS Levente Szalárdy MD Department of Neurology University of Szeged Hungary Hungarian-Romanian Multidisciplinary PhD School Programme in Medicine Two countries, one goal, joint success! HURO/0901/021/2.2.3

Introduction Multiple Sclerosis (MS) Chronic, progressive, autoimmune CNS, white matter, oligodendrocytes Demyelination  conductance ↓, injury ↑ Female Male > Axonal & Neuronal loss 2x 25 million people worldwide incidence: ~ prevalence: years 85% relapse remitting form 1# CNS symptoms in Hungary severe residual symptoms ↑ ↑ ↑ progression permanent disability

Diagnosis Disseminated CNS symptoms in time and space MRI UnidentifiedBrightObject Black Hole CSF Oligoclonal gammopathy (OGP) Inflammation total protein ↑ local IgG synthesis OGP γ T 1 T 2 (FLAIR) active lesion inactive lesion

Clinical course No remission Continuous progression ”CIS” PrimaryProgressiveMS First clinical onset Meets diagnostic criteria Symptoms longer than 24 h Relapse Remitting Form Form Clinically Isolated Syndrome SecundaryProgressiveMS No more remission Continuous progression No relapse Relapses / remissions alternate Relapse = recurrent acute exacerbation  neurological dysfunction s  remission neurological dysfunction s  remission ± residual symptoms residual symptoms ↑ ↑ ↑

Clinical course No remission Continuous progression ”CIS” PrimaryProgressiveMS First clinical onset Meets diagnostic criteria Symptoms longer than 24 h Relapse Remitting Form Form Clinically Isolated Syndrome SecundaryProgressiveMS No more remission Continuous progression No relapse Relapses / remissions alternate ? ? Biomarkers of axonal and neuronal injury? ± residual symptoms residual symptoms ↑ ↑ ↑

Biomarkers hTaupTau β-amyloid 1-42 Stability of axonal microtublesAlzheimer’s pathology Released after axonal and neuronal injury. Kapaki et al. 2000: increased Tau Süssmuth et al, 2001:increased Tau in active compared to resting MS Jiménez-Jiménez et al, 2002: no changes in Tau Bartosik-Psujek et al, 2004:increased Tau Martínez-Yélamos et al, 2004:no changes in Tau(some correlations) Brettschneider et al, 2005:increased Tau Rostasy et al, 2005:increased Tau only in very severe cases Guimarães et al, 2006: no changes in Tau Terzi et al, 2007: elevated Tau Valis et al, 2008: hTau, pTau not altered, β-amyloid 1-42 ↑ Hein Neé Maier et al, 2008: no changes in hTau, pTau and β-amyloid1-42 Teunissen et al, 2009: no changes in Tau Bartosik-Psujek et al, 2011: increased Tau Mori et al, 2011: β-amyloid 1-42 ↓ correlates with cognitive deficit

Materials & methods -80 o C Diagnostic lumbar punctures rpm 10 min Aliquots stored in polypropylene tubes Sandwich ELISA kits INNOGENETICS ® SPSS ® Statistics parameters sigmoid Awareness Technology ® INC 450 nm O.D.(λ) Sigmaplot ® 10.0 Concentration (pg/ml) Mean Optical densites run in duplicates

Sandwich ELISA Precoated Plate Capture Antibody Target Protein Detection Antibody TMB 1-2. Samples / Standards 1-2. Detection AB 3. Incubation 4. Washing 5. HRP-Linked AB 6. Incubation 7. Washing 8. Substrate (TMB) 9. Incubation HRP 10. Stop solution 11. Detection at 450nm 450 nm O.D. duplicates CV < 20 %

Samples n (MS) = 83 (52 ♀ ; 31 ♂ ) age = 35.9 Mann-Whitney U Kruskal-Wallis Mann-Whitney U Kruskal-Wallis ANOVA t-Test non-parametricnon-parametric parametric hTaupTauβ-Amyloid 1-42 n (Co) = 41 (23 ♀ ; 18 ♂ ) age = 37.1

Results Control vs MS Control MS hTau (pg/ml) Control MS pTau (pg/ml) Control MS β-amyloid (pg/ml) p = p = p = 0.855

Results Control vs CIS / RR Control CIS hTau (pg/ml) pTau (pg/ml) β-amyloid (pg/ml) p = p = p = RR Control CISRR Control CISRR

Results Progression CIS  CIS CIS  PP hTau (pg/ml) β-amyloid (pg/ml) p = p = CIS  RR CIS  CIS CIS  PP CIS  RR CIS  PP CIS  CIS pTau (pg/ml) p = n = 12 p = 0.12 p = 0.16

Results No significant differences between groups devided by relapse, gender. No significant correlation between protein concentrations and age hTau:Spearman’s Rho p = 0,973 pTau:Spearman’s Rho p = 0,677 amyloid:Pearson’s Corr.p = 0,594 No significant differences either between control and MS group or between different progression tendencies.

Conclusions More focus should be devoted to primary progressive (PP) MS with more patients involved with well- established diagnosis. These proteins are not appropriate as biomarkers in predicting disease progression.

Thank you for your kind attention! Tutor: Dr. Peter Klivenyi Professor: Prof. Dr. Laszlo Vecsei Assistant: Agnes Koszo