Analysing Alzheimer's disease risk variants in CD33 and TREM2 using microglia-like cells derived from Alzheimer-specific induced pluripotent stem cells

Introduction:

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Accumulation of intercellular β-amyloid plaques and intracellular neurofibrillary tangles are two hallmarks of AD that may drive neuronal death and the corresponding dramatic loss of cognitive abilities. A complex interaction between genetic and environmental factors contributes to molecular processes that drive AD. Microglial-mediated processes are key determinants to the accumulation of ß-amyloid deposits in AD, playing roles in amyloid degradation, and initiation and growth of plaques. Environmental and genetic factors contribute to the risk for AD, but the underlying disease mechanisms are poorly understood. In recent years, genome-wide association studies (GWAS) allowed the identification of DNA variations associated with an elevated risk for AD. A number of AD susceptibility genes including CD33, SORL1, ABCA7 and TREM2 point towards the immune system as a player in onset, progression and treatment of AD. The generation of patient specific induced pluripotent stem cell (iPS) lines enables differentiation into microglia. Patient-specific cells can be used as a model to functionally characterize disease associated variants. Potentially functional SNP variants in CD33, SORL1, ABCA7 and TREM2 were tested for association with AD. iPS cells were generated from patients carrying risk variants in these genes.

Methods:

Pluripotency was characterized by alkaline phosphatase staining, the expression of pluripotency markers, and the differentiation into the three germ layers. AD iPS cells were differentiated into microglia characterized by the expression of crucial glia cell markers. Motility, phagocytosis, and behaviour of processes were examined to determine functionality. The protein expression of pluripotency marker genes was successfully induced as shown by IF and WB analyses. Cells were also screened for the most efficient induction of neural cell fates including glia cell fates and the capability to generate derivatives of the three germ layers.

Results:

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, IF analysis, and RNA expression of hematopoietic lineage markers and crucial microglia markers. The established AD-specific iPS cell lines from late-onset AD patients represent a powerful tool for the analysis of molecular and cellular disease mechanisms.

Conclusion:

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.