TY - JOUR

T1 - Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells

AU - Morozova, Ksenia N.

AU - Suldina, Lyubov A.

AU - Malankhanova, Tuyana B.

AU - Grigoreva, Elena V.

AU - Zakian, Suren M.

AU - Kiseleva, Elena

AU - Malakhova, Anastasia A.

PY - 2018/10/17

Y1 - 2018/10/17

N2 - 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.

AB - 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.

KW - CRISPR-Cas Systems

KW - Clone Cells/metabolism

KW - Endoplasmic Reticulum/ultrastructure

KW - Gene Knockout Techniques

KW - HEK293 Cells

KW - Humans

KW - Huntingtin Protein/antagonists & inhibitors

KW - Huntington Disease/genetics

KW - Lysosomes/ultrastructure

KW - Microscopy, Electron, Transmission

KW - Mitochondria/ultrastructure

KW - Mutant Proteins/genetics

KW - Mutation

KW - Trinucleotide Repeat Expansion

KW - MUTANT HUNTINGTIN

KW - MITOPHAGY

KW - AUTOPHAGY

KW - NUCLEAR-MEMBRANE

KW - PATHOGENESIS

KW - PLURIPOTENT STEM-CELLS

KW - MITOCHONDRIAL DYNAMICS

KW - DISEASE

KW - BRAIN

KW - LYSOSOMAL ACTIVITY

UR - http://www.scopus.com/inward/record.url?scp=85055071087&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0204735

DO - 10.1371/journal.pone.0204735

M3 - Article

C2 - 30332437

AN - SCOPUS:85055071087

VL - 13

SP - e0204735

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 10

M1 - 0204735

ER -