Objective: Optimization of a dopamine neuron based,in vitro platform is beneficial for studying molecular mechanisms of alpha-synuclein aggregation,and screening of inhibitory drugs and deducing their mechanism of actions.

Background: Alpha-synuclein aggregation in neurons is a defining pathology of synucleinopathies,including Parkison’s disease.Pre-formed fibrils (PFFs) of alpha-synuclein provide a great tool to study aggregation of alpha-synuclein in cultured neurons and in model animals.Ability of PFFs to promote native proteins and lipids to aggregate into Lewy-body like inclusions have been shown before,including the long-term neurodegenerative consequences of these aggregations [1,2].However,cell-based PFF-models often use cortical and hippocampal neurons that are not the most vulnerable cell populations in Parkinson’s disease and other synucleinopathies.

Method: We have developed a robust,PFF-based midbrain dopamine neuron model with alpha-synuclein aggregation and a machine learning-based analysis pipeline to quantify the aggregation and survival data [3].Pharmacological agents,toxins,lentivirus vectors and CRISPR-Cas9 systems are used with this model.

Results: This platform allowed us to discover the alpha-synuclein aggregation preventative effect of GDNF and to identify the action of crucial molecules at GDNF’s signaling pathway –Ret, Src and Akt-.We have validated this effect of GDNF also by targeted treatment of midbrain dopamine neurons in the mouse brains [4].In addition by using GDNF as a positive control, we have screen other small molecules that may inhibit alpha-synuclein aggregation and validated some of them with our dopamine neuron model (e.g.,bromodomain inhibitors [5]).More examples will be presented at the congress.

Conclusion: It has become evident that aggregation of alpha-synuclein in Lewy bodies impair cellular protein dynamics and overtime, result in neurodegeneration.For the first time,we have shown that a neurotrophic signaling pathway can also prevent alpha-synuclein aggregation.Alongside,we developed a robust platform in midbrain dopamine neurons,a highly vulnerable cell population in movement,for screening candidate molecules against Lewy body-like aggregation and dissecting their molecular signaling pathways responsible of these molecules’ actions.Therapies against Lewy body formation with well-defined molecular mechanisms are essential to cure synucleinopathies.

References: [1] Volpicelli-Daley, L. A., Luk, K. C., Patel, T. P., Tanik, S. A., Riddle, D. M., Stieber, A., Meaney, D. F., Trojanowski, J. Q., & Lee, V. M. (2011), Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron, 72(1), 57–71, doi:0.1016/j.neuron.2011.08.033.
[2] Mahul-Mellier, A. L., Burtscher, J., Maharjan, N., Weerens, L., Croisier, M., Kuttler, F., Leleu, M., Knott, G. W., & Lashuel, H. A. (2020). The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration. Proceedings of the National Academy of Sciences of the United States of America, 117(9), 4971–4982, doi: 10.1073/pnas.1913904117
[3] Er, S., Hlushchuk, I., Airavaara, M., Chmielarz, P., Domanskyi, A. (2020), Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons. J. Vis. Exp, (162), e61118, doi:10.3791/61118.
[4] Chmielarz, P., Er, Ş., Konovalova, J., Bandres, L., Hlushchuk, I., Albert, K., Panhelainen, A., Luk, K., Airavaara, M. and Domanskyi, A. (2020), GDNF/RET Signaling Pathway Activation Eliminates Lewy Body Pathology in Midbrain Dopamine Neurons. Mov Disord, 35: 2279-2289, doi: 10.1002/mds.28258.
[5] Hlushchuk, I., Ruskoaho, H., Domanskyi, A., Airavaara, M., & Välimäki, M. J. (2021). Domain-Independent Inhibition of CBP/p300 Attenuates α-Synuclein Aggregation. ACS chemical neuroscience, 12(13), 2273–2279, doi:10.1021/acschemneuro.1c00215.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/dopamine-neuron-platform-for-screening-molecular-mechanisms-and-inhibitory-drugs-against-alpha-synuclein-aggregation/