Clusterin Is Required for β-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 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 investigate how CLU mediates Aβ-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 Aβ-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aβ 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 Aβ25-35 peptides and Aβ1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment. However, in CLU knockout neurons Aβ 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 Aβ in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aβ and Dickkopf-1 (DKK1) proteins in an Aβ-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 Aβ treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aβ compared to the wild type neurons supports the role of clusterin in Aβ-mediated AD pathogenesis.

Modeling Alzheimer’s disease with iPSC-derived brain cells

“…APOE4 GABAergic interneurons experienced degeneration in culture (though glutamatergic and dopaminergic neurons did not), while cholinergic neurons exhibited elevated sensitivity and altered Ca 2+ signaling in response to glutamate toxicity [39,110]. Intriguingly, iPSC-derived excitatory neurons lacking the SAD risk factor CLU were less sensitive to Aβ-induced toxicity, consistent with CLU single nucleotide polymorphisms being associated with reduced AD risk [31,115]. Sullivan and colleagues took a broader approach, performing a shRNA knockdown screen of more than 50 AD candidate genes in iPSC-derived neurons, examining effects on Aβ secretion and tau phosphorylation [116].…”

The multiplex model of the genetics of Alzheimer’s disease

“…Common variant stem cell models have generally lagged behind with only a small number of target models developed, most notably APOE 144,145 . Only three loci identified exclusively by GWAS, PICALM 146 , CLU 147…”

Secreted Chaperones in Neurodegeneration

“…In this mouse model, Abeta deposits are found as increased CAA deposits, and overall Abeta load is decreased within the brain ( Wojtas et al, 2017 ). These paradoxical results were corroborated by another group who showed that clusterin expression is required for Abeta toxicity in human induced pluripotent stem cell (iPSC)-derived neurons ( Robbins et al, 2018 ). In agreement, in primary neurons, siRNA silencing of clusterin expression provides protection against Abeta toxicity ( Killick et al, 2014 ).…”

“…In agreement, in primary neurons, siRNA silencing of clusterin expression provides protection against Abeta toxicity ( Killick et al, 2014 ). However, as shown by Robbins et al (2018) , the loss of clusterin expression results in a change in the expression of a variety of genes; thus, it is currently unclear if the paradoxical effect is directly related to clusterin loss or is due to pleiotropic effects. Adding to this complexity, in the 5xFAD (familial Alzheimer’s disease) model mouse background, clusterin null mice showed fewer Abeta plaques and cognitive performance deficits compared to clusterin-expressing mice at 5 months of age, though the differences disappeared by 10 months, suggesting that clusterin expression is only required for early toxicity ( Oh et al, 2019 ).…”

“…We speculate that the variation in results when using different mouse models may be due at least in part to differences in the clusterin: Abeta ratio inherent in the different experimental models. Alternative explanations for the positive effects of clusterin null mutations on neurodegenerative pathology include altered expression of other genes, as observed in Robbins et al (2018) , and thus this paradox remains unexplained.…”