SU32 - PATIENT-SPECIFIC AD IN VITRO MODELS FOR THE ANALYSIS OF LATE-ONSET ALZHEIMER'S DISEASE

Abstract

Background Alzheimer´s Disease (AD) is the most common form of dementia. Environmental and genetic factors contribute to the risk for AD, but the underlying disease mechanisms are poorly understood. AD is genetically complex and shows heritability of up to 80%. Glia cells including astrocytes and microglia are affected in AD patients contributing to the tau pathology and the accumulation of neurotoxic amyloid as well. In recent years, Genome-Wide Association Studies (GWAS) allowed the identification of DNA variations associated with an elevated risk for AD. Together with other groups, we identified a number of AD susceptibility genes including CD33, SORL1, ABCA7 and TREM2, which highly recommends that the immune system plays a major role in onset, progression and treatment of AD. Reprogramming of EBV-transformed patient specific cell lines for the generation of induced pluripotent stem cells (iPS) enables the patient-specific differentiation into neurons, astrocytes, and microglia. Patient-specific cells can be used as a model to functionally characterize disease associated variants. Methods Blood was collected from AD patients carrying risk variants in AD susceptibility genes for the generation of iPS cells. SNP variants in those genes were genotyped based on their potential function according to literature and genomic localization (exonic non-synonymous, binding domain, or promoter affecting SNPs). Reprogrammed iPS cells carried the genetic background of a certain AD patient. Pluripotency was characterized by alkaline phosphatase staining, the expression of pluripotency markers, and the differentiation into the three germ layers. Crucial pluripotency markers are OCT4, SOX2, NANOG, KLF4, MYC, and LIN28. AD iPS cells were differentiated into astrocytes and microglia and the differentiation status was characterized by the expression of crucial glia cell markers. Further, AD susceptibility genes recently published in GWAS were analysed. Results The protein expression was successfully induced shown by immunofluorescence and western-blot analysis. Cells were also screened for the most efficient induction of the three germ layers and the induction of neural cell fates including glia cell fates. The established AD-specific iPS cell lines from AD patients represent a powerful tool for the analysis of molecular and cellular disease mechanisms. We established 4-step protocol for the generation of AD-specific microglia enabling the focused analysis of AD-associated risk variants. The protocol was verified by morphology, FACS analysis, immunofluorescence and RNA expression of crucial microglia marker, including hematopoetic lineage markers and the induction of well-described microglia markers including IBA1. The disease-specific analysis of the genetic background of AD patients represents a completely new approach for the understanding of AD genetics and AD-associated risk variants. Discussion Together, combining molecular genetics of AD for the investigation of risk variants and iPS cell technology for the generation of patient- and disease-specific stem cells provides a promising approach to characterize known disease mechanisms, to deepen the understanding of known disease mechanisms, and to discover unknown disease aspects.