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. 2022 Jun 13:10:874323.
doi: 10.3389/fcell.2022.874323. eCollection 2022.

Telomere Attrition in Induced Pluripotent Stem Cell-Derived Neurons From ALS/FTD-Related C9ORF72 Repeat Expansion Carriers

Hayley Robinson  1 , Sk Imran Ali  1 , Martha Elena Diaz-Hernandez  2   3 , Rodrigo Lopez-Gonzalez  1
Affiliations

Telomere Attrition in Induced Pluripotent Stem Cell-Derived Neurons From ALS/FTD-Related C9ORF72 Repeat Expansion Carriers

Hayley Robinson et al. Front Cell Dev Biol. .

Abstract

The GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dysregulated DNA damage response and the generation of reactive oxygen species (ROS) have been postulated as major drivers of toxicity in C9ORF72 pathogenesis. Telomeres are tandem-repeated nucleotide sequences that are located at the end of chromosomes and protect them from degradation. Interestingly, it has been established that telomeres are sensitive to ROS. Here, we analyzed telomere length in neurons and neural progenitor cells from several induced pluripotent stem cell (iPSC) lines from control subjects and C9ORF72 repeat expansion carriers. We found an age-dependent decrease in telomere length in two-month-old iPSC-derived motor neurons from C9ORF72 carriers as compared to control subjects and a dysregulation in the protein levels of shelterin complex members TRF2 and POT1.

Keywords: Amyotrophic lateral sclerosis, Frontotemporal dementia; C9orf72; induced pluripotent stem cells; motor neuron differentiation; telomeres.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be interpreted as potential conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Telomere length analysis during the differentiation of iPSCs to MNs from controls and C9ORF72 repeat expansion carriers. Representative images of control and C9ORF72 (A) iPSCs, NEP, and MNP. Telomere length quantification in (B) NEP and (C) MNP, from 3 controls and 3 C9ORF72 iPSC lines, from 3 independent differentiations. Two-tailed t-test with Welch’s correction was applied. ns, not significant. Scale bar = 20 µm.
FIGURE 2
FIGURE 2
Analysis of the expression of telomere maintenance genes during the differentiation of iPSCs to MNs from controls and C9ORF72 repeat expansion carriers. qPCR analysis to compare expression levels (A) TRF1, (B) TRF2 and (C) POT1 transcripts in NEP and levels of (D) TRF1, (E) TRF2 and (F) POT1 in MNP from 3 control subjects and 3 C9ORF72 carriers, from 3 independent differentiations. Western blot analyses to compare protein levels of (G) TRF2 and (H) POT1 in NEP and protein levels of (I) TRF2 and (J) POT1 in MNP, from 3 controls and 3 C9ORF72 iPSC lines, Two-tailed t-test with Welch’s correction was applied. ns, not significant.
FIGURE 3
FIGURE 3
Age-dependent telomere attrition in MNs from C9ORF72 repeat expansion carriers. (A) Representative images from control and C9ORF72 iPCS-derived MN cultures. qPCR analyses of telomere length quantification in postmitotic MNs at (B) 1, (C) 1.5, and (D) 2 months. A one-way ANOVA was applied to compare MNs at 1 month, 1.5 months, and 2 months, from 3 controls and 3 C9ORF72 iPSC lines. ns, not significant. Scale bar = 20 µm.
FIGURE 4
FIGURE 4
mRNA expression of telomere maintenance genes in MNs from C9ORF72 repeat expansion carriers. qPCR analysis of the relative levels of (A) TRF1, (B) TRF2 and (C) POT1, mRNA. A one-way ANOVA was applied to compare iPSC-derived MNs at 1 month, 1.5 months, and 2 months from 3 control subjects or 3 C9ORF72 carriers from 3 independent differentiation experiments. ns, not significant.
FIGURE 5
FIGURE 5
Decreased protein levels of TRF2 and POT1 in 2 month-old-motor neurons. Western blot analyses of (A) TRF2, (B) POT1 in 1-month-old-motor neurons and (C) TRF2, (D) POT1 in 2 month-old-motor neurons from 3 controls and 3 C9ORF72 carriers. Two-tailed t-test with Welch’s correction was applied to western blot data.
FIGURE 6
FIGURE 6
Telomerase activity and hTERT mRNA levels in 2 month-old-motor neurons. qPCR analyses of relative levels of telomerase activity in (A) 2 month-old iPSC-derived MNs from 3 control and 3 C9ORF72 carriers, (B) relative levels of telomerase activity in iPSC lines and 2 month-old iPSC-derived MNs from 3 control and 3 C9ORF72 carriers. qPCR analysis of the relative levels of hTERT in (C) 2-month-old iPSC-derived MNs from 3 control subjects or 3 C9ORF72 carriers, (D) relative levels of hTERT in iPSC lines and 2 month-old iPSC-derived MNs from 3 control and 3 C9ORF72 carriers. Two-tailed t-test with Welch’s correction to compare between controls and C9ORF72 MNs and a one-way ANOVA to compare iPSC to iPSC-derived MNs cultures.
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