Dataset Information

Clusterin Is Required for ?-Amyloid Toxicity in Human iPSC-Derived Neurons.

ABSTRACT: Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating ?-amyloid (A?) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in A? processing. Here we investigate how CLU mediates A?-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate A?-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to A? using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with A?25-35 peptides and A?1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following A? treatment. However, in CLU knockout neurons A? exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to A? in neurons with the CLU deletion. Four of the five genes previously identified as downstream to A? and Dickkopf-1 (DKK1) proteins in an A?-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in A? treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to A? compared to the wild type neurons supports the role of clusterin in A?-mediated AD pathogenesis.

SUBMITTER: Robbins JP

PROVIDER: S-EPMC6068261 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Clusterin Is Required for β-Amyloid Toxicity in Human iPSC-Derived Neurons.

Robbins Jacqueline P JP Perfect Leo L Ribe Elena M EM Maresca Marcello M Dangla-Valls Adrià A Foster Evangeline M EM Killick Richard R Nowosiad Paulina P Reid Matthew J MJ Polit Lucia Dutan LD Nevado Alejo J AJ Ebner Daniel D Bohlooly-Y Mohammad M Buckley Noel N Pangalos Menelas N MN Price Jack J Lovestone Simon S

Frontiers in neuroscience 20180725

Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investiga ...[more]

PMID: 30090055

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Project description:Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer's disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU-AD mutations and amyloid-β (Aβ), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aβ toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 -/- edited iPSCs and were incubated with aggregated Aβ peptides. Aβ induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 -/- and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aβ25-35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2 -/- neurons. The latter also displayed partial protection against Aβ-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 -/- neurons, potentially contributing to their reduced sensitivity to Aβ toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aβ toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aβ toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 -/- and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 -/- neurons. This may contribute to the reduced sensitivity of these neurons to Aβ, providing new mechanistic insights into Aβ pathologies and therapeutic targets for AD.

Dataset Information

Clusterin Is Required for ?-Amyloid Toxicity in Human iPSC-Derived Neurons.

Publications

Clusterin Is Required for β-Amyloid Toxicity in Human iPSC-Derived Neurons.

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