Abstract title: Validation of an in vitro a-synuclein aggregation assay in primary cortical cultures for high throughput RNA interference screening

Poster board number (onsite): 658
Poster number: P0738 / #1381

Authors: Susanne Lardell1 , Åke Jägervall1 , Alexander Back1 , Jonna Sjöholm1 , Tracy Bellande2 , Ronald Melki2 , Joachim Täger3 , Peter Heutink3 , Christiane Volbracht4 , George Tofaris5 , Johan Pihl1
Affiliations: 1Cellectricon AB, R&d, Mölndal, Sweden, 2CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory Of Neurodegenerative Diseases, Fontenay-aux-Roses, France, 3German Center for Neurodegenerative Disease DZNE, Genome Biology Of Neurodegeneration, Tübingen, Germany, 4H. Lundbeck A/S, Neuroscience, Valby, Denmark, 5University of Oxford, Nuffield Department Of Clinical Neurosciences, Clinical Neurology,john Radcliffe Hospital, Oxford, United Kingdom

Aims: In Parkinson’s disease (PD), the formation and propagation of α-synuclein insoluble amyloid structures is a process hypothesized to drive the pathology of disease. However, there is currently a lack of understanding with regards to the pathways and proteins responsible for progression of α-syn aggregation, in particular in idiopathic PD, and in vitro genetic screens is one way to increase the knowledge about this.
Methods: We therefore developed a high-capacity in vitro model based on primary embryonic mouse cortical cultures in the 384-well format, where lentiviral shRNA was added at 3 days in vitro (DIV) to induce gene silencing, and endogenous α-synuclein aggregation was induced at 10 DIV using recombinant human α-syn pre-formed fibrils. Endogenous α-syn aggregation and cell health were then quantified at 17 DIV using immunocytochemistry and automated high content imaging and analysis.
Results: The outcome was a robust and sensitive α-synuclein aggregation assay for high throughput RNA interference screening. The throughput was more than sufficient for managing the library of 900 oligos targeting 300 genes with the required number of technical replicates and biological test occasions. Upon completion of the screen, the quality control metrics, e.g. signal to noise ratio, the variability in total cell count, no of neurons & SSMD all confirmed that the screen was a technical success. Furthermore, a number of genes decreasing as well as increasing α-synuclein aggregation were also identified.
Conclusions: We can conclude that the developed assay is well-suited for high throughput RNA interference for the discovery of new genes involved in α-syn aggregation.