HUMAN IPSC-DERIVED ALZHEIMER’S DISEASE ASTROCYTES RECAPITULATE DISEASE-RELATED PHENOTYPES

Abstract

The importance of astrocytes in neurodegenerative disorders, including Alzheimer's disease (AD), is well recognized. Yet, their exact role in the disease pathogenesis and progression remains unclear. Interestingly, human astrocytes have been found to be far larger and more complex than those of infraprimate mammals, making it necessary to concentrate research on diseased human astrocytes rather than animal cells. In this study, patient-specific induced pluripotent stem cells (iPSCs) were used as a source for human astrocytes. iPSCs were reprogrammed from skin fibroblasts of familial AD patients with deletion of exon 9 in the PSEN1gene (n=3) and from healthy age-matched controls (n=3). The deletion of exon 9 was corrected with CRISPR/Cas9 genome editing, enabling generation of isogenic control lines for the diseased lines. iPSCs were then further differentiated to GFAP and S100b-positive forebrain astrocytes according to our optimized protocol. iPSC-derived astrocytes are functional cells that are able to propagate calcium waves, uptake glucose, produce cytokines and glutathione, and release gliotransmitters, such as GABA and ATP. In addition, AD astrocytes show calcium leakage from ER and reduced presenilin endoproteolysis, both well-characterized defects linked to PSEN1mutations. Interestingly, inflammatory stimulation of AD astrocytes resulted in increased release of proinflammatory cytokines when compared to control cells. AD astrocytes also have alterations in mitochondrial mass, cellular metabolism and glutathione levels. Most importantly, astrocytes contribute to amyloid pathology and are able to both produce and uptake beta-amyloid. AD astrocytes secrete significantly higher amounts of the 1–42 form and show an elevated 1–42/1–40 ratio as compared to controls, but respond well to treatment with gamma-secretase inhibitor DAPT. Our data show that pathological findings commonly seen in AD patients are recapitulated in the iPSC-derived astrocytes. Thus, these cells offer a valuable tool for studying the disease mechanisms and for drug screening and testing novel therapeutic approaches in a cell-type specific manner.