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Astrocyte pathology in a human neural stem cell model of 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 gene encoding the microtubule-associated protein TAU (MAPT). 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 genome editing in human induced pluripotent stem (iPS) cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs derived from one patient carrying the N279K MAPT mutation, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU isoforms, demonstrated increased vulnerability to oxidative stress and elevated protein ubiquitination and exhibited disease-associated changes in transcriptome profiles when compared to astrocytes derived from one control individual and to the isogenic control. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study highlights the utility of iPS cell-derived NPCs to elucidate the role of astrocytes in the pathogenesis of FTD.

SUBMITTER: Hallmann AL

PROVIDER: S-EPMC5335603 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein.

Hallmann Anna-Lena AL Araúzo-Bravo Marcos J MJ Mavrommatis Lampros L Ehrlich Marc M Röpke Albrecht A Brockhaus Johannes J Missler Markus M Sterneckert Jared J Schöler Hans R HR Kuhlmann Tanja T Zaehres Holm H Hargus Gunnar G

Scientific reports 20170303

Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the gene encoding the microtubule-associated protein TAU (MAPT). 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 genome editing in human induced pluripotent stem (iPS) cell-de ...[more]

PMID: 28256506

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Project description: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.

Project description:BackgroundPallido-ponto-nigral degeneration (PPND), a major subtype of frontotemporal dementia with parkinsonism related to chromosome 17 (FTDP-17), is a progressive and terminal neurodegenerative disease caused by c.837 T > G mutation in the MAPT gene encoding microtubule-associated protein tau (rs63750756; N279K). This MAPT mutation induces alternative splicing of exon 10, resulting in a modification of microtubule-binding region of tau. Although mutations in the MAPT gene have been linked to multiple tauopathies including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, knowledge regarding how tau N279K mutation causes PPND/FTDP-17 is limited.ResultsWe investigated the underlying disease mechanism associated with the N279K tau mutation using PPND/FTDP-17 patient-derived induced pluripotent stem cells (iPSCs) and autopsy brains. In iPSC-derived neural stem cells (NSCs), the N279K tau mutation induced an increased ratio of 4-repeat to 3-repeat tau and accumulation of stress granules indicating elevated cellular stress. More significant, NSCs derived from patients with the N279K tau mutation displayed impaired endocytic trafficking as evidenced by accumulation of endosomes and exosomes, and a reduction of lysosomes. Since there were no significant differences in cellular stress and distribution of subcellular organelles between control and N279K skin fibroblasts, N279K-related vesicle trafficking defects are likely specific to the neuronal lineage. Consistently, the levels of intracellular/luminal vesicle and exosome marker flotillin-1 were significantly increased in frontal and temporal cortices of PPND/FTDP-17 patients with the N279K tau mutation, events that were not seen in the occipital cortex which is the most spared cortical region in the patients.ConclusionTogether, our results demonstrate that alterations of intracellular vesicle trafficking in NSCs/neurons likely contribute to neurodegeneration as an important disease mechanism underlying the N279K tau mutation in PPND/FTDP-17.

Project description:Frontotemporal dementia (FTD) is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein tau. Due to limited availability of neural cells from patients` brains, however, the underlying mechanisms of neurodegeneration in FTD are still only poorly understood. Here, we derived induced pluripotent stem (iPS) cells from individuals with FTD-associated MAPT mutations and differentiated them into neurons for mechanistic studies. Patient-derived neurons demonstrated pronounced tau pathology with increased fragmentation and AT8-immunoreactivity, disturbed neurite extension and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD-neurons showed activation of the unfolded protein response and presented with distinct, disease-associated gene expression profiles in a whole-genome transcriptome analysis. These findings indicate distinct, early neurodegenerative changes in FTD caused by mutant tau and highlight the unique opportunity to use patient-specific iPS cells to identify potential targets for drug screening purposes and therapeutic intervention. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer’s instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The bead intensities were mapped to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model (Irizarry et al., 2003). Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in MATLAB. Hierarchical clusters of genes and samples were performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method.

Dataset Information

Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein.

Publications

Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein.

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