Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells

Ksenia N. Morozova, Lyubov A. Suldina, Tuyana B. Malankhanova, Elena V. Grigoreva, Suren M. Zakian, Elena Kiseleva, Anastasia A. Malakhova

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

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.

Original languageEnglish
Article number0204735
Pages (from-to)e0204735
Number of pages23
JournalPLoS ONE
Volume13
Issue number10
DOIs
Publication statusPublished - 17 Oct 2018

Keywords

  • CRISPR-Cas Systems
  • Clone Cells/metabolism
  • Endoplasmic Reticulum/ultrastructure
  • Gene Knockout Techniques
  • HEK293 Cells
  • Humans
  • Huntingtin Protein/antagonists & inhibitors
  • Huntington Disease/genetics
  • Lysosomes/ultrastructure
  • Microscopy, Electron, Transmission
  • Mitochondria/ultrastructure
  • Mutant Proteins/genetics
  • Mutation
  • Trinucleotide Repeat Expansion
  • MUTANT HUNTINGTIN
  • MITOPHAGY
  • AUTOPHAGY
  • NUCLEAR-MEMBRANE
  • PATHOGENESIS
  • PLURIPOTENT STEM-CELLS
  • MITOCHONDRIAL DYNAMICS
  • DISEASE
  • BRAIN
  • LYSOSOMAL ACTIVITY

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