CRISPR/Cas9 genome editing in human neural progenitor cells demonstrates astrocyte pathology in frontotemporal dementia caused by mutant TAU protein

ABSTRACT: Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the MAPT gene encoding TAU. Here, we applied a stem cell model of FTD to examine if FTD astrocytes carry an intrinsic propensity to degeneration and to determine if they can induce non-cell-autonomous effects in neighboring neurons. We utilized CRISPR/Cas9 technology in human iPS cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU, demonstrated proteasome activation and increased oxidative stress and exhibited disease-associated changes in transcriptome profiles. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study expands the utility of CRISPR/Cas9 genome editing to human NPCs to study the pathogenesis of neurodegenerative diseases such as FTD. For whole genome expression analyses, RNA was isolated from cell lysates using the RNeasy kit (Qiagen) with on-column DNA digestion. 300 ng of total RNA was used as input for cRNA synthesis with the linear amplification protocol (Ambion) comprising the synthesis of T7-linked double-stranded cDNA and in vitro transcription for 12 hr incorporating biotin-labeled nucleotides. Following purification, labelled cRNA samples were hybridized onto HumanHT-12 v4 expression BeadChips (Illumina) for 18 hr following the manufacturer's protocol. The BeadChips were washed as recommended and subsequently stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the associated software. All samples were hybridized as biological replicates. Data were processed by mapping bead intensities to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was achieved by applying the Affymetrix Robust Multiarray Analysis (RMA) background correction model. Variance stabilization was performed using log2 scaling. Gene expression normalization was calculated using the quantile method implemented in the lumi package of R-Bioconductor. In-house developed functions in MATLAB were utilized for data post-processing and the generation of graphical representations. Hierarchical clustering analysis of genes and samples was performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method as previously described.

ORGANISM(S): Homo sapiens

PROVIDER: GSE79557 | GEO | 2017/06/12

SECONDARY ACCESSION(S): PRJNA316159

REPOSITORIES: GEO