In this webcast, Paul Karila, Head of Drug Discovery, and Johan Pihl, Chief Operating Officer, discuss the development and validation of Cellectricon’s α-synuclein aggregation assay. The assay, based on RNA interference in primary cortical cultures, can be used to identify and validate novel targets relevant for Parkinson’s disease.
The webcast covers three parts:
- Introduction to Cellectricon’s in vitro platform for neurodegenerative disease research.
- Development and validation of the α-synuclein aggregation assay in primary cortical cultures.
- Applying the assay for high throughput genetic screening for the discovery of genes involved in α-synuclein aggregation.
In Parkinson’s disease (PD), the formation and propagation of α-synuclein (α-syn) insoluble amyloid structures is a process hypothesized to drive the pathology of disease. As part of this process, impaired protein clearance and protein aggregation within neurons occur. From a drug discovery perspective, preventing protein aggregation or promoting protein clearance provide interesting opportunities as pharmacological intervention points.
However, there is currently a lack of understanding of 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.
Therefore, Cellectricon 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-syn 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.
In order to validate the assay, Cellectricon together with the IMPRiND consortium (an EU funded consortium that aims to map and target critical steps in the propagation of misfolded tau and α-synuclein) carried out an in vitro genetic screen targeting 300 individual genes with the aim of finding new targets and pathways involved in α-syn aggregation. Here, the assay was adapted to accommodate IMPRiNDs pre-formed fibrils and lentiviral shRNA library.
The outcome was a robust and sensitive α-syn 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. We can therefore conclude that the developed assay is well-suited for high throughput RNA interference screening for the discovery of novel genes involved in α-syn aggregation.
Paul Karila, PhD
Head of Drug Discovery at Cellectricon AB
Paul joined the company in 2012 as head of Cellectricon’s Discovery Services. He previously worked at AstraZeneca (AZ) where he held leadership positions at the Departments of Molecular Pharmacology and Neuroscience. At AZ, Paul led teams responsible for target identification/target validation and ion channel and GPCR profiling in LI-LO phase, mainly on analgesia targets. Prior to joining AZ, Paul was a Postdoctoral Fellow at School of Medicine, University of Pittsburgh, USA, studying neurobiology using electrophysiological methods. He has a PhD in animal physiology from University of Gothenburg, Sweden.
Johan Pihl, PhD
Chief Operating Officer at Cellectricon AB
Johan joined Cellectricon in 2005 and has been in R&D and commercial positions at the company. He was involved in the development of Cellectricon’s microfluidics-based platforms and managed the development of Cellectricon’s optical electrophysiology platform. In addition to the COO position, Johan is responsible for developing Cellectricon’s internal technology platform for discovery services. He has a PhD in Chemistry from Chalmers University of Technology, Gothenburg, Sweden, and is the author and co-author of a number of scientific publications and patents.