Abstract
Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate “healthy” control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington’s disease.
Highlights
Huntington’s disease (Huntington’s chorea, HD) is a severe autosomal dominant disease caused by an increase in the number of CAG trinucleotide repeats in the first exon of the huntingtin (HTT) gene
Using the genome-editing method based on the CRISPR/Cas9 system, we obtained a panel of isogenic cell clones with an identical genetic background differing in the number of CAG repeats in the HTT gene
In spite of the abnormal karyotype of HEK293 cells, we Expanded CAG tract in the huntingtin gene causes ultrastructural defects in human cell culture assumed that mutant-HTT expression should affect the cells and that mutant cell clones should recapitulate some molecular mechanisms of mHTT action because the HD-causing mutation is known to be dominant [1]
Summary
Huntington’s disease (Huntington’s chorea, HD) is a severe autosomal dominant disease caused by an increase in the number of CAG (cytosine-adenine-guanine) trinucleotide repeats in the first exon of the huntingtin (HTT) gene. The mutant HTT protein that is expressed from the gene with more than 35 repeats leads to death of brain cells, which causes impairment of motor and cognitive functions. Studies on mutant neurons have revealed significant disturbances in the structure and dynamics of mitochondria and in their contacts with endoplasmic reticulum (ER) membranes; these problems lead to impairment in calcium ion homeostasis as well as in autophagy and mitophagy [10,11,12]. Elucidation of the influence of HTT mutation on the fine organization of cells and intracellular organelles, such as mitochondria, ER cisternae, and components of the autophagic system, remains one of the essential issues in the HD pathology research
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