| Summary: | <p>Clusterin’s role in neurodegeneration and Alzheimer’s disease (AD) remains unclear. Clusterin (CLU) protein expression and trafficking are altered in response to stress; the treatment of rodent neurons with beta-amyloid (Aβ) increases intracellular retention and reduces secretion of clusterin proteins. CLU knockdown in rodent neurons provides protection against Aβ-induced toxicity including cell death indicating clusterin may mediate Aβ toxicity.
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<p>Although traditionally referred to as a highly glycosylated and secreted protein, clusterin can also be found intracellularly. However, the biogenesis, structure, and functional importance of these intracellular proteins remains debated. </p>
<p>In this thesis, CRISPR/Cas9 genome engineering was used to remove exon 2 from CLU, since this exon contains the endoplasmic reticulum (ER) signal peptide required for the generation of glycosylated, secreted clusterin proteins. This generated heterozygous and homozygous exon 2 knockout (KO) iPSC lines. These were then differentiated into neurons and treated with Aβ to compare cellular and genetic responses of KO and control cells.</p>
<p>The data demonstrated that heterozygous KO neurons responded similarly to control neurons when treated with Aβ, displaying both cell death and neurite retraction indicating no altered sensitivity. Furthermore, Aβ treatment, and not neuronal genotype, was identified as the largest contributor to gene expression changes measured by RNA-Seq. In contrast, homozygous exon 2 KO was sufficient to generate intracellular, non-glycosylated clusterin proteins, and prevent secretion. This manifested as reduced sensitivity to Aβ: neurons displayed cell death but no evidence of neurite damage indicating exon 2 KO provides partial protection to iPSC-neurons.</p>
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