iCell Microglia PD (SNCA)
Alpha-synuclein A53T PD Model with an Isogenic Control
Parkinson’s disease (PD) affects ~1% of people over the age of 65 and is the second most common neurodegenerative brain disorder. Αlpha-synuclein (encoded by the SNCA gene) is a 140 amino-acid protein, expressed highly in dopaminergic neurons of the substantia nigra pars compacta and is intracellularly localized in presynaptic terminals. Alpha-synuclein proteins have the capacity to self-assemble, from unfolded monomers to oligomeric species, to heavy aggregates (called amyloid fibrils).
Accumulation of these insoluble fibrils progressively promotes the formation of intracellular inclusions called Lewy bodies within neurons and glial cells. Recent studies suggest that alpha-synuclein oligomers bind to lipids, disrupt cellular membrane integrity, and induce microglial activation leading to the death of dopaminergic neurons.
Engineered SNCA A53T HO Model
A known mutation in the N-terminal amphipathic domain of alpha-synuclein has been linked to familial Parkinson’s disease. FCDI created a homozygous (HO) model of this mutation in the iCell® Microglia genetic background line, 01279. The mutation results in a single amino acid change at aa53 from alanine to threonine (Fig. 1).
Comparative Marker Expression
iCell Microglia were labeled for the presence of cell surface (CD45, CD11b, CD11c, TREM2 and, CD33) and intracellular (P2RY12, TMEM119, CX3CR1, IBA1) antigens by flow cytometry (Fig. 2). The specific staining of the engineered iCell Microglia SNCA A53T HO is compared against matched isotype control.
In Figure 3, a comparison is shown between iCell Microglia and the engineered SNCA A53T HO line. Phagocytosis of S.aureus BioParticles® was measured over time using an IncuCyte® S3 Live-Cell Analysis System. The engineered microglia exhibit phagocytic activity, but at a reduced activity relative to the unengineered line.
The cytokine response of stimulated iCell Microglia and the engineered SNCA line were compared by multiplex assay (Fig. 4). iCell Microglia were plated in microglia maintenance media and allowed to recover for 72 h prior to stimulation with LPS or interferon gamma or a combination of both for 24 h. Supernatants were collected and assayed using a Luminex® multiplex cytokine assay.