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CRISPR/Cas9 genome editing of CLU to examine clusterin’s contribution to neurodegeneration and Alzheimer’s disease
Author(s) -
Foster Evangeline M.,
Ribe Elena M.,
Robbins Jacqueline,
Hublitz Philip,
Pangalos Menelas,
Buckley Noel,
Lovestone Simon,
Neuroscience Translational,
Research Dementia
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.040292
Subject(s) - clusterin , exon , biology , neurodegeneration , induced pluripotent stem cell , microbiology and biotechnology , gene , genetics , medicine , apoptosis , disease , embryonic stem cell
Abstract Background Traditionally, clusterin is referred to as a secreted protein however, more recent evidence indicates clusterin may exist intracellularly where it may have a pro‐apoptotic role. Previously, our lab observed altered clusterin trafficking in rodent neurons in response to amyloid‐β suggesting it may be a key factor in neurodegeneration. We aim to use CRISPR/Cas9 in human iPSCs to explore the contribution that clusterin plays in cellular vulnerability to amyloid‐β. Method We aim to remove exon 2 from CLU since this exon contains the ER‐signal peptide thought to be required for secretion of clusterin protein. So far, we have generated heterozygous exon 2 knockout iPSCs (exon 2+/‐) which have been differentiated into cortical neurons and astrocytes. Clusterin gene and protein expression has been characterised in these iPSCs compared to control iPSCs. Response of control and exon 2+/‐ neurons and astrocytes to amyloid‐β treatment has also been explored. Result Surprisingly, exon 2+/‐ iPSCs express significantly less intracellular clusterin than control iPSCs. Secreted clusterin expression is unaltered by the edit achieved but glycosylation state is altered. We build on previous findings in rodent neurons, showing that clusterin trafficking is also altered by amyloid‐β in both control iPSC‐neurons and iPSC‐astrocytes. This response is observed in exon 2+/‐ neurons but is absent in exon 2+/‐ astrocytes. Divergence in gene expression changes of DKK1‐signature genes is observed. Here, we describe differences in both gene and protein expression changes in response to amyloid‐β treatment in control and exon 2+/‐ cells. Conclusion Currently, we are exploring whether differences observed in neuronal and astrocytic responses of exon 2+/‐ cells convey protection or vulnerability to amyloid‐β induced toxicity. To do so, we are using imaging techniques to quantify changes in cell death and neurite projections for both genotypes in response to treatment.