1. Biomarkers of axonal and neuronal injury in CSF of patients with multiple sclerosis Levente Szalárdy, Dénes Zádori, Ágnes Kószó, Krisztina Bencsik, Lászlo Vécsei, Péter Klivényi Department of Neurology, University of Szeged, Szeged, Hungary Two countries, one goal, joint success! Supported by the European Regional Development Fund HURO/0901/021/2.2.3. HURO/0901/021/2.2.3

2. Introduction Chronic progressive CNS disorder InflammationDemyelination Axonal and neuronal degeneration 25 million people worldwide incidence: ~ 1000 prevalence: 20.000 (0.2%) Demography in Hungary ~ 2x 20-40 years 1# CNS symptoms

3. The clinical evolution of multiple sclerosis No remission Continuous progression ‘FCO’ PrimaryProgressiveMS First Clinical Onset Confirmed by MRI and CSF Relapse Remitting Form Form Clinically Isolated Syndrome SecundaryProgressiveMS No more remission Continuous progression No relapse Relapses / remissions alternate ± residual symptoms residual symptoms ↑ ↑ ↑

4. Aims of our study Identifying CSF biomarkers with prognostic value. Structural injury Altered protein processing Tau α-synuclein hyperphosphorlyated Tau β-amyloid 1-42 Hypothesis: Proteins released into the CSF during axonal and/or neuronal injury could be of clinical use.

5. Patients RR RRFCO at the time of LP CIS RR PP n = 9 n = 15 n = 12 n = 35 n = 48 MS patients n = 83 ♀ / ♂ = 1.68; mean age = 35.9 Control subjects n = 37 ♀ / ♂ = 1.17; mean age = 36.9 without evidence of CNS involvement CNS involvement

6. Materials & methods Diagnostic lumbar punctures, 1999-2011 10.000 rpm 15 min Aliquots stored in polypropylene tubes Sandwich ELISA kits Invitrogen Innogenetics ® ; Invitrogen ® SPSS ® Statistics 17.0 5-parameters sigmoid 450 nm O.D.(λ) Sigmaplot ® 10.0 Concentration (pg/ml) Mean Optical densites samples run in duplicates Awareness Technology ® -80 o C

7. Results A sensitivity issue: α-synuclein α-synuclein concentration (ng/ml) mean optical densitiy Reproducibility is very low. Majority of the samples are below detection limit.

8. p = 0.297 Mann-Whitney p = 0.389 Mann-Whitney p = 0.902 Unpaired T-test Results No significant difference between Control (n = 37) and MS (n = 83) groups.

9. p = 0.580 Kruskal-Wallis p = 0.604 Kruskal-Wallis p = 0.809 One-Way ANOVA Results No significant differences between groups divided by the clinical stage at the time of LP.

10. Results No significant differences between disease evolution tendencies of patients with First Clinical Onset within a 5 year follow-up period. p = 0.724p = 0.305 p = 0.669 One-Way ANOVA

11. Results p = 0.051 Mann-Whitney Marginal significance between h-Tau levels during relapse and remission. Independent of total protein content. Spearman’s Rho = 0.069 p = 0.697

12. Discussion Inconclusive published findings hTau: elevated in MS elevated in severe cases elevated only during relapse unchanged [1,2,3,4,5] [6,7,8,9,10,11] [12] [13] References: 1.E. Kapaki, G. P. Paraskevas, M. Michalopoulou, K. Kilidireas, Eur. Neurol. 43, 228 (2000). 2.J. Brettschneider et al., Mult. Scler. 11, 261 (Jun, 2005). 3.M. Terzi, A. Birinci, E. Cetinkaya, M. K. Onar, Acta Neurol. Scand. 115, 325 (May, 2007). 4.H. Bartosik-Psujek, J. J. Archelos, J. Neurol. 251, 414 (Apr, 2004). 5.H. Bartosik-Psujek, M. Psujek, J. Jaworski, Z. Stelmasiak, Acta Neurol. Scand. 123, 252 (Apr, 2011). 6.F. J. Jimenez-Jimenez et al., Acta Neurol. Scand. 106, 351 (Dec, 2002) 7.A. Martinez-Yelamos et al., Neurosci. Lett. 363, 14 (Jun, 2004). 8.I. Guimaraes, M. I. Cardoso, M. J. Sa, Mult. Scler. 12, 354 (Jun, 2006). 9.M. Valis, R. Talab, P. Stourac, C. Andrys, J. Masopust, Neuro. Endocrinol. Lett. 29, 971 (Dec, 2008). 10.K. Hein Nee Maier et al., Neurosci. Lett. 436, 72 (May, 2008). 11.C. E. Teunissen et al., Neurology 72, 1322 (Apr 2009). 12.K. Rostasy et al., J. Child Neurol. 20, 822 (Oct, 2005). 13.S. D. Sussmuth, H. Reiber, H. Tumani, Neurosci. Lett. 300, 95 (Mar, 2001).

13. Discussion Inconclusive published findings pTau: unchanged in MS CSF [9,10] References: 9. M. Valis, R. Talab, P. Stourac, C. Andrys, J. Masopust, Neuro. Endocrinol. Lett. 29, 971 (Dec, 2008). 10. K. Hein Nee Maier et al., Neurosci. Lett. 436, 72 (May, 2008). 14. A. Schneider et al., J Biol Chem 279, 55833 (Dec, 2004). 15. J. M. Anderson et al., Brain 131, 1736 (Jun, 2008). 16. J. M. Anderson et al., Acta Neuropathol 117, 583 (May, 2009). 17. J. M. Anderson et al., Acta Neuropathol 119, 591 (May, 2010). elevated in EAE brain elevated in SPMS brain [14, 15] [15] elevated in PPMS brain [16] elevated in EA-MS brain [17]

14. Discussion Inconclusive published findings Aβ 1-42 : elevated in MS [9] [10] unchanged in MS decreased in MS [18] References: 9. M. Valis, R. Talab, P. Stourac, C. Andrys, J. Masopust, Neuro. Endocrinol. Lett. 29, 971 (Dec, 2008). 10. K. Hein Nee Maier et al., Neurosci. Lett. 436, 72 (May, 2008). F. Mori et al., Neuropsychopharmacology 36, 559 (Feb, 2011). 18. F. Mori et al., Neuropsychopharmacology 36, 559 (Feb, 2011).

15. Discussion Unfortunate insensitivity issue. α-synuclein: upregulated in MS lesions [19, 20] Status in CSF has not yet been demonstrated in MS. References: D. Papadopoulos, L. Ewans, D. Pham-Dinh, J. Knott, R. Reynolds, Mol. Cell. Neurosci. 31, 597 (Apr, 2006). 19. D. Papadopoulos, L. Ewans, D. Pham-Dinh, J. Knott, R. Reynolds, Mol. Cell. Neurosci. 31, 597 (Apr, 2006). 20. J. Q. Lu et al., J. Neuropathol. Exp. Neurol. 68, 179 (Feb, 2009). 21. Z. Hong et al., Brain 133, 713 (Mar, 2010). Similar range by highly sensitive Luminex assay. [21] α-synuclein concentration (ng/ml) mean optical densitiy

16. Conclusions The tested proteins are inappropriate as biomarkers in predicting disease progression and clinical outcome in MS. The observed alterations are of descriptive and not of prospective nature. The correct evaluation of α-synuclein in CSF in MS awaits for technological development.

17. Acknowledgements The project was supported by the European Regional Development Fund HURO/0901/021/2.2.3 and established in the frame-work of Hungary-Romania Cross-Border Co-operation Programme 2007-2013. Two countries, one goal, joint success! HURO/0901/021/2.2.3

18. Thank you for your kind attention!