In vitro modelling of human microglial alterations associated with Alzheimer’s disease and polygenic risk

It is increasingly clear that Alzheimer’s Disease (AD) presents with a strong genetic component. Recent Genome Wide Association Studies (GWAS) uncovered a huge number of polymorphisms associated with the disease which cumulative effect can be captured by the calculation of the polygenic risk score (PRS). PRS allows the identification of those at the highest and lowest risk of disease, opening new avenues for the understanding of the neurodegenerative processes and protective mechanisms. Samples collected from individuals identified at the extremes of the score can be used to generate stem cells models to investigate molecular mechanisms of the disease on the induced pluripotent stem cell (iPSC)-derived models such as microglial-like cells. In the first part of this study, a cohort of cryopreserved peripheral blood mononuclear cells (PBMC) samples collected from individuals originally sampled for GWAS and identified to have with high or low PRS for AD were used. These samples, collected more than 10 years ago resulted to have suboptimal quality, requiring several protocols and attempts to recover viability and cell number. One of these methods resulted in the successful reprogramming of several samples into iPSCs and their extensively characterisation. One of the most interesting discovery of recent GWAS is the novel R522 variant located in the PLCG2 gene which encode for the phospholipase Cγ2 enzyme, mainly expressed in microglia cells. To set the ground for functional studies, two different iPSCs-derived microglia differentiation protocols were tested and characterisations were carried out. Moreover, phenotypic characterization of iPSCs-derived cortical neurons and astrocytes were completed. iPSCs derived cortical neurons were used to established neuronal-microglia co-culture system; iPSCs-derived astrocyte were used to prepare the astrocyte-conditioned medium (ACM) used to feed cells. PLCγ2 R522 variant has been associated with decreased risk of developing late onset AD, and it represents the first classically drug-targetable vi molecule identified in LOAD genetic studies. Results of this study show that a model of iPSCs-derived microglia-like cells harbouring the PLCG2R522 SNP displays an altered neuroinflammatory response upon several different stimuli, such as LPS, oAβ, zymosan and ATP/ADP, in comparison to the common PLCG2P522 variant. Different outputs were analysed , including the secretion of cytokines (IL-6, TNF, GCSF, IL10), the possible involvement of NLRP3 inflammasome (IL1β), the levels of NO and the calcium response. Taken together the results support the hypothesis of a protective role of PLCγ2R522 variant in the context of AD, via reducing the neuro inflammatory burden in response to several stimuli