Mechanisms of axonal degeneration induced by alpha-synuclein Mariana Cerdeira, Jan C. Koch, and Paul Lingor University of Medicine Göengen Results 50 GAPDH 36 WT EGFP Free/polymerized tubulin ra2o (%) characterized by the loss of dopaminergic neurons in the midbrain. Affected neurons typically exhibit aggregates known as Lewy bodies, which contain the protein alpha-‐ synuclein (aSyn). The physiological func2ons of aSyn as well as the mechanisms through which it contributes to axonal degenera2on remain poorly understood (Bendor et al., 2013). In this study, we aimed at inves2ga2ng the cellular targets of aSyn involved in axonal degenera2on. aSyn impairs axonal transport in midbrain neurons (Chung et al., 2009; internal data not published). Also, the enzyme GAPDH has been shown to be essen2al for the energy supply of axonal transport (Zala et al., 2013). Therefore, we hypothesized that aSyn exerts a depolymerizing effect on tubulin and/or an inhibitory effect on GAPDH ac2vity. Tubulin Effects of aSyn on the tubulin network 180 50 kDa 160 140 120 100 80 60 40 aSyn wild type (WT) and its A30P mutant were overexpressed in primary cultures of rat midbrain dopaminergic neurons (MDN) using adeno-‐associated viral (AAV) vectors. Immunofluorescence, Western Blot and ELISA were employed to examine altera2ons of tubulin stabiliza2on and GAPDH ac2vity. Immunostaining of GAPDH on H4 cells containing aSyn aggregates was performed to examine a possible colocaliza2on of the two proteins. aSyn WT monomers in different concentra2ons (500 nM, 1000 nM, 5000 nM or only water) were briefly incubated with MDN lysates and GAPDH ac2vity of the cells was analyzed. Literature cited Bendor JT, Logan TP, Edwards RH. 2013. The Func2on of α-‐Synuclein. Neuron. 79(6):1044-‐66. Chen L, Jin J, Davis J, Zhou Y, Wang Y, Liu J, Lockhart PJ, Zhang J. 2007. Oligomeric alpha-‐ synuclein inhibits tubulin polymeriza2on. Biochem Biophys Res Commun. 356:548-‐ 553. GAPDH ac2vity in MDN (%) 15000 10000 5000 EGFP WT A30P 20 0 Control EGFP aSyn WT aSyn A30P * 110 Materials and methods 20000 0 120 Effects of aSyn on GAPDH Acetylated Tubulin (50 kDa band) A30P Pixel intensity (a.u.) Parkinson's disease (PD) is a neurodegenera2ve disorder Conclusions Acetylated Tubulin kDa GAPDH ac2vity in MDN (%) Introduc)on 100 *** 90 140 120 100 80 60 40 20 0 Control 500 nM 1000 nM 5000 nM aSyn WT monomers 80 70 One-‐way ANOVA and Dunnep's test p-‐value < 0,05 * p-‐value < 0,001 *** 60 Control EGFP aSyn WT aSyn A30P 87% 91% Chung CY, Koprich JB, Siddiqi H, Isacson O. 2009. Dynamic changes in presynap2c and axonal transport proteins combined with striatal neuroinflamma2on precede dopaminergic neuronal loss in a rat model of AAV alpha-‐synucleinopathy. J Neurosci. 29(11):3365-‐73. Zala D, Hinckelmann MV, Yu H, Lyra da Cunha MM, Liot G, Cordelières FP, Marco S, Saudou F. 2013. Vesicular glycolysis provides on-‐board energy for fast axonal transport. Cell. 152(3):479-‐91. aSyn overexpression in MDN did not result in a sta2s2cally significant increase of the free/polymerized tubulin ra2o or a decrease of the total amount of acetylated tubulin, as it was expected based on the literature (Chen et al., 2007). These findings imply that the destabiliza2on of the tubulin network is not a relevant pathomechanism of aSyn in the MDN model. The glycoly2c ac2vity of GAPDH was significantly decreased by aSyn overexpression in midbrain neurons. This enzyme has been previously shown to be the only source of energy that is essen2al for the opera2on of vesicular axonal transport. Therefore, GAPDH may be an addi2onal intermediary between aSyn and its deleterious effect on axonal transport. Incuba2on of different concentra2ons of aSyn monomers with MDN lysates showed no sta2s2cally significant difference between the groups. Nevertheless, there was a clear trend of decreasing levels of GAPDH ac2vity in the treated cells, and this decrease was propor2onal to the concentra2on of aSyn. If this trend is confirmed, it will further support our finding of a detrimental effect of aSyn on GAPDH ac2vity and argue for a direct interac2on of both proteins. GAPDH strongly colocalizes with aSyn in the cytoplasmic inclusions of H4 cells. This colocaliza2on of over 97% was specific for GAPDH, as two other highly expressed proteins, tubulin and ac2n, were not present in the aSyn aggregates (data not shown). These findings further support the possibility of an interac2on between aSyn and GAPDH. Our results contribute to further understanding the pathogenic func2ons of aSyn on axonal degenera2on and give novel insight to poten2al targets of PD pathology interven2on. Acknowledgments We thank Lisa Barski and Vivian Dambeck for technical assistance and Tiago Outeiro for providing the H4 cells. Funding for this project was provided by the Department of Neurology. Student funding was provided by Neurasmus. aSyn GAPDH ac2vity Impaired axonal transport Axonal degenera2on
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