Mol. Cells 2023; 46(4): 219-230
Published online January 10, 2023
https://doi.org/10.14348/molcells.2023.2095
© The Korean Society for Molecular and Cellular Biology
Correspondence to : yanjb@shchildren.com.cn (JY); fzeng@vip.163.com (FZ)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Down syndrome (DS) is the most common autosomal aneuploidy caused by trisomy of chromosome 21. Previous studies demonstrated that DS affected mitochondrial functions, which may be associated with the abnormal development of the nervous system in patients with DS. Runt-related transcription factor 1 (RUNX1) is an encoding gene located on chromosome 21. It has been reported that RUNX1 may affect cell apoptosis via the mitochondrial pathway. The present study investigated whether RUNX1 plays a critical role in mitochondrial dysfunction in DS and explored the mechanism by which RUNX1 affects mitochondrial functions. Expression of RUNX1 was detected in induced pluripotent stem cells of patients with DS (DS-iPSCs) and normal iPSCs (N-iPSCs), and the mitochondrial functions were investigated in the current study. Subsequently, RUNX1 was overexpressed in N-iPSCs and inhibited in DS-iPSCs. The mitochondrial functions were investigated thoroughly, including reactive oxygen species levels, mitochondrial membrane potential, ATP content and lysosomal activity. Finally, RNA-sequencing was used to explore the global expression pattern. It was observed that the expression levels of RUNX1 in DS-iPSCs were significantly higher than those in normal controls. Impaired mitochondrial functions were observed in DS-iPSCs. Of note, overexpression of RUNX1 in N-iPSCs resulted in mitochondrial dysfunction, while inhibition of RUNX1 expression could improve the mitochondrial function in DS-iPSCs. Global gene expression analysis indicated that overexpression of RUNX1 may promote the induction of apoptosis in DS-iPSCs by activating the PI3K/Akt signaling pathway. The present findings indicate that abnormal expression of RUNX1 may play a critical role in mitochondrial dysfunction in DS-iPSCs.
Keywords Down syndrome, induced pluripotent stem cells, mitochondrial dysfunction, RUNX1
Down syndrome (DS) or trisomy 21 is one of the most common genetic diseases and is often accompanied by intellectual disability. It occurs with an incidence ~1 in 700-1,000 births (El Hajj et al., 2016). DS is a major cause of congenital malformations and mental retardation in humans. Patients with DS typically display associated dysfunctions and a range of cognitive deficits, with important differences in their phenotype and severe side effects in the nervous system (Vacca et al., 2019). Previous studies on the brains of patients with DS have revealed abnormalities in the structure and function of the central nervous system. Recent studies have shown that alterations in mitochondrial function may be associated with the occurrence of DS (Pecze and Szabo, 2021; Zamponi and Helguera, 2019).
Mitochondria play a central role in metabolism and energy conversion in eukaryotic cells, which generate ATP via oxidative phosphorylation (OXPHOS). These organelles provide energy for various cell-based activities (Ohnishi et al., 2018). Dysregulated mitochondria lead to the excessive production of reactive oxygen species (ROS), which are involved in the regulation of programmed cell death (Li et al., 2019; Wong, 2011). Impaired mitochondrial function leads to significant changes in the cell and affects neural cell proliferation, apoptosis, differentiation, regeneration, and other cellular activities (Beckervordersandforth, 2017; Johnson et al., 2021). It is known that mitochondrial dysfunction/oxidative stress is associated with DS. Mitochondrial dysfunction has been observed in the brain of early fetuses with DS and can manifest as abnormalities in OXPHOS complexes (Coskun and Busciglio, 2012; Salemi et al., 2018), accumulation of oxidative stress products and changes in mitochondrial biosynthesis. In addition, previous studies demonstrated that abnormal expression of genes was associated with mitochondrial function in DS (Qiu et al., 2017; Salemi et al., 2020). Mitochondrial dysfunction leads to impaired neuron proliferation, differentiation, and maturation. Previous studies have shown the presence of mitochondrial dysfunction in neurons, glial cells, and peripheral blood cells in patients with DS, and they were associated with mitochondrial fragmentation, impaired OXPHOS, and reduced ATP levels (Valenti et al., 2018; Zamponi and Helguera, 2019). Collectively, these studies indicated that mitochondrial dysfunction was strongly associated with abnormal development of the nervous system in DS. However, the key genes and molecular mechanisms that lead to mitochondrial dysfunction in patients with DS remain unknown.
DS is caused by an extra copy of the human chromosome 21 (Hsa21). Previous studies have proposed the gene dosage effect hypothesis, suggesting that increased expression of a specific set of dosage-sensitive genes on Hsa21 may eventually cause neurodevelopmental or neurocognitive disorders in DS (Delabar et al., 1993). A recent study indicated that trisomy 21 caused neuronal apoptosis, leading to a decrease in the neural population (Hirata et al., 2020). The DS critical region (DSCR), namely 21q22-21q23, is a genomic region on Hsa21 that is strongly linked to DS phenotypes (Pelleri et al., 2016). Aberrant expression of genes, notably transcription factors located in the DSCR, may be associated with mitochondrial dysfunction in DS (Flippo and Strack, 2017; Izzo et al., 2018).
Runt-related transcription factor 1 (RUNX1), which is encoded by the
Normal induced pluripotent stem cells (N-iPSCs) and induced pluripotent stem cells of patients with DS (DS-iPSCs) were purchased from the American Type Culture Collection (Cat. No. ATCC DYR0100 [normal, newborn, male] and Cat. No. ATCC DYP0730 [DS, newborn, male]; ATCC, USA). The clones were cultured in a feeder-free system. The cell culture dishes were coated with Cell Matrix Basement Membrane Gel (Cat. No. ACS-3035; ATCC) and cultured in Complete Pluripotent Stem Cell SFM XF/FF medium (Cat. No. ACS-3002; ATCC) in the presence of 10 µM inhibitor (Cat. No. Y0503; Sigma, USA). The culture medium was changed the day after cell resuscitation, and daily thereafter until the colonies reached 80% confluence. The cells were typically digested, sub-cultured at a 1:3 ratio using the Stem Cell Dissociation Reagent (Cat. No. ACS-3010; ATCC) and incubated overnight at 37°C in the presence of 5% CO2.
DS-iPSCs were then cultured in the medium supplemented with 20 µM LY294002 (Cat. No. S1105; Selleck, USA) for 1 h. Then the cells were collected, the expression level of Akt was identified by western blotting and the analysis of ROS level and cellular ATP content was also performed. The experiment was repeated in triplicate.
The following vectors were purchased from AddGene (USA); RUNX1 expression vector (pCMV5-AML1B; plasmid Cat. No. 12426) and RUNX1 short hairpin RNA (shRNA) vector (pLKO.1 shRUNX1 puro; plasmid Cat. No. 45816).
Single clones were harvested from a 60-mm dish using Stem Cell Dissociation Reagent within 10 min and washed once with 2 ml phosphate-buffered saline (PBS). Subsequently, the cells were resuspended in R-buffer. N-iPSCs and DS-iPSCs were transfected with the Neon Transfection System (Cat. No. MPK1025; Invitrogen, USA) according to the manufacturer’s instructions. The conditions used were as follows: 1,240 V, 20 ms and 2-times pulses. A total volume of 10 µl containing 1 µg plasmid DNA was used. Following transfection, the samples were placed on a 24-well microplate, and subsequent analysis was performed 48 h after transfection.
Total RNA was extracted from PBMCs and iPSCs using TRIzol® reagent. RT was performed using a reverse transcription kit (Cat. No. 18091050; Invitrogen). RT-qPCR was performed with TaqmanTM Real-Time PCR Assay. The primer sequences for RUNX1 and GAPDH were as follows: RUNX1, forward: 5’-TGGCACTCTGGTCACCGTCAT-3’ and reverse: 5’-GAAGCTCTTGCCTCTACCGC-3’, and GAPDH, forward: 5’-AGAGGGCTGTCGGCGCAGTA-3’ and reverse: 5’-GGCTGTGGTCTCGGTTGGGC-3’. The reactions were performed using an ABI7500 Real-Time PCR system (Thermo Fisher Scientific, USA). The transcript levels of RUNX1 were quantified using the 2-ΔΔCq method, with GAPDH as the reference gene used for normalization.
The iPSCs were scraped off in ice-cold PBS, collected into RIPA lysis buffer (Cat. No. sc-24948; Santa Cruz Biotechnology, USA) with a cell scraper, and incubated on ice for 30 min. The cell lysates were centrifuged at 14,000 ×
ROS levels in iPSCs were measured using a ROS Assay Kit (Cat. No. S0033; Beyotime), following the manufacturer’s instructions. Briefly, iPSCs were cultured in 6-well culture plates pretreated with Matrigel, and subsequently, the cell culture medium was removed. The cells were then incubated with 10 µM DCFH-DA, which is a fluorescent probe used for ROS detection. Following subsequent culture at 37°C for 20 min in the dark, the iPSCs were washed three times with serum-free Dulbecco’s modified Eagle’s medium (DMEM), and the fluorescence emission of the samples was detected by fluorescence microscopy (Nikon Eclipse Ti; Nikon, Japan). The fluorescence intensity was analyzed using ImageJ software (ver. No. 1.6; National Institutes of Health, USA).
BODIPY-C11 staining was performed according to the manufacturer’s protocol. The cells were seeded in a 35-mm-diameter dish. Culture medium was replaced with 2 ml medium containing 5 µM of BODIPY-C11 (Cat. No. D3861; Invitrogen) after 48 h and the culture was returned to the cell culture incubator for 30 min. Finally, the cells were fixed with 4% paraformaldehyde for 15 min and cell nuclei were stained with DAPI for 10 min. Fluorescence intensity was examined by fluorescence microscopy (Nikon Eclipse Ti), and images were analyzed with the ImageJ software.
Cells were seeded in culture flasks, and when they were 60%-70% confluent, the culture solution was aspirated. The cells were then washed once with PBS, and 2 ml cell culture medium containing 1 ml JC-1 solution (Cat. No. S2003S; Beyotime) was added for staining. The solution was thoroughly mixed in a cell culture incubator for 20 min at 37°C. Concomitantly, the JC-1 staining buffer (5×) was diluted five times in water (4 ml distilled water and 1 ml JC-1 staining buffer). The samples were placed on ice. Following incubation at 37°C, the supernatant was aspirated and washed with JC-1 buffer (1×) three times. Upon addition of 2 ml medium, the fluorescence emission of the samples was detected using a fluorescence microscope. The experiments were repeated three times.
Cellular ATP content was determined with an ATP Assay Kit according to the manufacturer’s instructions (Cat. No. S0026; Beyotime). Briefly, the cells were lysed with ATP Detection Lysis Buffer 48 h after transfection, and 100 µl ATP detection working solution was added to the wells for 5 min at room temperature. A total of 20 µl sample or standard was added to each well, and the ATP concentration was measured using luminometry. A BCA Protein Assay Kit (Cat. No. P0010; Beyotime) was used to determine the protein concentrations of each sample, and the total ATP levels were evaluated as the ratio of cellular ATP level to protein concentration.
Following transfection of iPSCs with RUNX1-overexpressing plasmids for 48 h, the transfected cells cultured on 35-mm culture dishes were incubated with 100 nM lysosome probes (Cat. No. M7512; Invitrogen) at 37°C for 20 min. Following incubation, the cells were fixed with 4% paraformaldehyde for 15 min and 0.2% Triton X was used for permeabilization for 10 min. This was followed by DAPI staining (Cat. No. C1002; Beyotime) for 8 min to visualize the nuclei. Red fluorescence was determined by fluorescence microscopy, and image analysis was performed with ImageJ software.
Cell apoptosis was evaluated with a Cell Apoptosis Kit (Cat. No. APOAF; Sigma), according to the manufacturer’s instructions. Briefly, iPSCs were collected 24 h following transfection of RUNX1-overexpressing plasmids. Subsequently, 500 µl binding buffer, 5 µl Annexin V-FITC conjugate and 10 µl propidium iodide solution were added successively into the cell suspension, and incubated at room temperature in the dark for 10 min. The fluorescence emission of the cells was immediately detected with a flow cytometer (BD AccuriTM C6 Flow Cytometer; BD Biosciences, USA).
RNA-seq was conducted with the NovaSeq 6000 platform (Illumina, USA). An experimental group transfected with a RUNX1 expression vector and a control group transfected with a GFP expression vector were used. Each group had three replicates.
Following total RNA extraction using TRIzol® reagent (Cat. No. 15596018; Invitrogen), the quantification, qualification, library preparation, and subsequent RNA-seq of the above two groups were performed by Novogene (China). The details of RNA-seq have been previously described (Jia et al., 2020). Raw data were cleaned by removing reads with adaptors and excluding low quality (>50%) or a high proportion of unknown bases (>10%). Subsequently, the data were assembled using Trinity methodology (Grabherr et al., 2011).
For each sequenced library, the read counts were adjusted using the edgeR program package through one scaling normalized factor prior to differential gene expression analysis. Differential expression analysis of two conditions was performed using the edgeR R package.
Differential expression genes and apoptotic biomarkers were confirmed by RT-qPCR. The primers are listed in Supplementary Table S1. To detect activation of the PI3K-Akt signaling pathway, the protein expression and phosphorylation levels of Akt, S6, and 4E-binding protein (4E-BP1) were detected by western blotting as previously described. The antibodies used are listed in Supplementary Table S2.
Our previous study demonstrated that the expression levels of almost all the genes related to mitochondrial function were downregulated in DS-iPSCs, suggesting that mitochondria were dysfunctional in these cells (Qiu et al., 2017). To further demonstrate the impairment of mitochondrial function in DS-iPSCs, specific changes in mitochondrial MMP, ROS and ATP content were investigated to provide further evidence of impaired mitochondrial function. As shown in Figs. 1A and 1B, a, a high green fluorescence intensity was noted in the DS-iPSCs. The quantification of green fluorescence intensity was analyzed by ImageJ (Alafnan et al., 2021), and the results suggested that the contents of ROS were increased 2.24-fold in DS-iPSCs (0.076 ± 0.004 vs 0.034 ± 0.003;
Previous studies have shown that RUNX1 is involved in the pathogenesis of DS (Bourquin et al., 2006; Edwards et al., 2009; Patel et al., 2011). Furthermore, overexpression of RUNX1 was detected in DS-iPSCs and peripheral blood of children with DS (Supplementary Fig. S1), leading to the speculation that RUNX1 may be a key gene regulating mitochondrial function in DS. To determine whether RUNX1 affects mitochondrial function, RUNX1 was overexpressed in N-iPSCs (Supplementary Fig. S2). The results indicated that mitochondrial dysfunction was present in N-iPSCs following RUNX1 overexpression, which was similar to that in DS-iPSCs. JC-1 staining indicated that the green fluorescence intensity was considerably enhanced in RUNX1-overexpressing N-iPSCs compared with that observed in N-iPSCs expressing normal levels of RUNX1 (0.090 ± 0.017 vs 0.037 ± 0.0039;
Accumulation of ROS in mitochondria causes abnormalities in mitochondrial structure and function, including ATP synthesis obstruction, reduced mitochondrial MMP, and impaired respiratory chain function. Mitochondrial respiratory chain deficiencies block OXPHOS and inhibit ROS production, which aggravates mitochondrial dysfunction and creates a vicious cycle of mitochondrial damage (Bhatti et al., 2017; Hu and Liu, 2011). Thus, mitochondrial function was evaluated in iPSCs overexpressing RUNX1. The results indicated that overexpression of RUNX1 led to a 3.17-fold decrease in ATP content compared with that of control cells (4.75 ± 0.36 vs 2.11 ± 0.26;
High levels of mitochondrial ROS can induce oxidative damage in mitochondria, which impairs the function of the mitochondrial respiratory chain, leading to the accumulation of intracellular ROS. The membrane ROS levels of iPSCs were determined using BODIPYTM C11, and the data confirmed that RUNX1 overexpression increased mitochondrial and cell membrane ROS levels compared with those of control cells (0.10 ± 0.0034 vs 0.077 ± 0.0015;
To further determine whether RUNX1 overexpression could affect mitochondrial function, DS-iPSCs were transfected with RUNX1 shRNA vector, and the mitochondrial function was measured. The results indicated that the green fluorescence intensity was markedly decreased when the expression levels of RUNX1 were inhibited in DS-iPSCs (0.068 ± 0.015 vs 0.029 ± 0.0020;
RNA-seq analysis indicated that 51 differentially expressed genes were identified between the experimental and control groups using the edgeR R package (Fig. 4A). This included 39 upregulated and 12 downregulated genes (Fig. 4B). KEGG analysis revealed a set of 39 upregulated genes, and the results indicated that PI3K-Akt was identified as the most significant signaling pathway, in which five genes (
A previous study has shown that the activation of mTOR signaling via the PI3K/Akt signaling pathway is involved in the regulation of mitochondrial ROS production in PC-12 cells, which can ultimately affect cell apoptosis (Li et al., 2020). In the present study, PI3K/Akt inhibitor LY294002 was used to suppress the expression of Akt in DS-iPSCs. The results showed that the expression level of Akt in DS-iPSCs was significantly decreased, accompanied by a significant increase in ATP content and a decrease in ROS level (Supplementary Fig. S3).
Akt is a major downstream effector molecule of the PI3K/Akt signaling pathway, and increased phosphorylation implies activation of the PI3K signaling pathway (Hart and Vogt, 2011; McCubrey et al., 2012). The present results revealed a significant increase in the phosphorylation of Akt, S6 and 4EBP1 in N-iPSCs + RUNX1 cells (Fig. 5A). These results indicated that upregulation of RUNX1 in iPSCs activated the PI3K-Akt signaling pathway by increasing the phosphorylation of Akt, S6 and 4EBP1.
The effects of RUNX1 overexpression on the induction of apoptosis of iPSCs were investigated by flow cytometry. A significant increase in apoptosis was observed in RUNX1-overexpressing N-iPSCs (19.00 ± 1.79 vs 32.46 ± 1.54;
The most apparent clinical features of DS are neurological complications, including early onset of Alzheimer’s disease (AD) and mental retardation. The central nervous system requires high quantities of energy. Redox homeostasis and adequate ATP levels are essential for normal neurodevelopment (Arrázola et al., 2019; Kann and Kovács, 2007). Mitochondria supply virtually all eukaryotic cells with energy through ATP production by OXPHOS. Accordingly, maintenance of the mitochondrial function is fundamentally important to sustain cellular health. Previous studies reported that mitochondrial abnormalities, including aberrant mitochondrial morphology and function, were detected in both patients with DS and in DS cells (Aburawi and Souid, 2012; Shah et al., 2019). In the present study, mitochondrial abnormalities were confirmed in DS-iPSCs. Therefore, it is reasonable to speculate that the aberrant development of the nervous system in DS is closely associated with mitochondrial dysfunction, as iPSCs have the pluripotency to differentiate into various nerve cell types. However, the mechanism leading to mitochondrial dysfunction in DS is still not clear, and the key genes responsible for this process have not been identified thus far.
RUNX1 is an important transcriptional regulator located in DSCR, which is closely associated with the development and regeneration of the nervous system. A recent study indicated that large-scale hypermethylation of RUNX1 (~28 kb) was the strongest signal within the DS differentially methylated regions in peripheral blood (Laufer et al., 2021), which was also confirmed by the current study. In the present study, overexpression of RUNX1 was observed in both DS-iPSCs and peripheral blood samples of children with DS. It was therefore speculated that RUNX1 may be a crucial regulatory gene involved in the pathogenesis of DS. In a recent study,
The present study investigated whether RUNX1 affects the neurological development of DS by regulating mitochondrial function. The results revealed that overexpression of RUNX1 induced impaired mitochondrial functions, characterized by excessive generation of ROS, decreased MMP and reduced overall energy metabolism. It was also observed that RUNX1 could reduce the number of active lysosomes and cause lysosomal dysfunction. Furthermore, the data confirmed that impaired mitochondrial functions, including excessive generation of ROS, and decreased MMP and ATP content, could be reversed when the expression levels of RUNX1 were inhibited in DS-iPSCs. All these results strongly suggested that the abnormal expression levels of RUNX1 may be a critical factor (although not the sole factor) in the occurrence of mitochondrial dysfunction in DS-iPSCs, and indicated that overexpression of RUNX1 led to neurological abnormalities by affecting mitochondrial function in DS.
It is well known that the neuropathology of AD is an important clinical feature of DS, which is almost always present in patients with DS >40 years of age. Perluigi et al. (2014) demonstrated that the PI3K/Akt signaling pathway displayed increased activity in patients with DS and those with DS/AD. Notably, the PI3K/Akt signaling pathway plays a critical role in the regulation of cell proliferation, death, and metastasis (Lim et al., 2015), RUNX1 overexpression increased the phosphorylation levels of PI3K, Akt, and mTOR proteins (Liu et al., 2020).
Akt is a well-known pro-survival kinase and activated by phosphorylation at Ser 473 via the PI3K signaling pathway. Activated Akt plays a critical role in inhibiting neuronal death through further activation or inhibition of its downstream target proteins (Ye et al., 2015). In the present study, it was further found that overexpression of RUNX1 may activate the PI3K/Akt signaling pathway via increasing the phosphorylation of Akt, S6 and 4EBP1 in iPSCs. Certain upregulated genes in the PI3K-Akt signaling pathway, including
Previous reports showed that Akt plays a central role in apoptosis inhibition through its regulatory effects on various downstream targets such as anti-apoptotic Bcl-2 (Yu et al., 2016). Normal mitochondrial function depends on the integrity of the mitochondrial membrane, which is strictly regulated by the Bcl-2 family of proteins (D'Orsi et al., 2017). The balance of cell death and survival signals in the Bcl-2 protein family determines the cell fate. As a pro-apoptotic member of the Bcl-2 family, Bim induces the release of cytochrome
In conclusion, the present study demonstrated overexpression of RUNX1 in DS-iPSCs, and found that RUNX1 could regulate mitochondrial function and mitochondria-mediated apoptosis via the modulation of the PI3K-Akt signaling pathway in iPSCs. However, further investigation is required to assess whether RUNX1 promotes the neural differentiation of DS-iPSCs by regulating mitochondrial function.
This research was supported by the National Key Research and Development Program of China (2019YFA0801402) and the National Natural Science Foundation of China (81971421, 81471485), Shanghai key clinical specialty project (shslczdzk05705), Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212200).
Y.L., J.Y., and F.Z. conceived and performed experiments, wrote the manuscript, and secured funding. Y.L. and Y.Z. performed experiments. J.Y., Z.R., and F.Z. provided expertise and feedback. All authors have read and agreed to the published version of the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2023; 46(4): 219-230
Published online April 30, 2023 https://doi.org/10.14348/molcells.2023.2095
Copyright © The Korean Society for Molecular and Cellular Biology.
Yanna Liu1,4 , Yuehua Zhang1,4
, Zhaorui Ren1,2
, Fanyi Zeng1,2,3,*
, and Jingbin Yan1,2,*
1Shanghai Children’s Hospital, Shanghai Institute of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China, 2NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology, Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China, 3Department of Histoembryology, Genetics & Development, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China, 4These authors contributed equally to this work.
Correspondence to:yanjb@shchildren.com.cn (JY); fzeng@vip.163.com (FZ)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Down syndrome (DS) is the most common autosomal aneuploidy caused by trisomy of chromosome 21. Previous studies demonstrated that DS affected mitochondrial functions, which may be associated with the abnormal development of the nervous system in patients with DS. Runt-related transcription factor 1 (RUNX1) is an encoding gene located on chromosome 21. It has been reported that RUNX1 may affect cell apoptosis via the mitochondrial pathway. The present study investigated whether RUNX1 plays a critical role in mitochondrial dysfunction in DS and explored the mechanism by which RUNX1 affects mitochondrial functions. Expression of RUNX1 was detected in induced pluripotent stem cells of patients with DS (DS-iPSCs) and normal iPSCs (N-iPSCs), and the mitochondrial functions were investigated in the current study. Subsequently, RUNX1 was overexpressed in N-iPSCs and inhibited in DS-iPSCs. The mitochondrial functions were investigated thoroughly, including reactive oxygen species levels, mitochondrial membrane potential, ATP content and lysosomal activity. Finally, RNA-sequencing was used to explore the global expression pattern. It was observed that the expression levels of RUNX1 in DS-iPSCs were significantly higher than those in normal controls. Impaired mitochondrial functions were observed in DS-iPSCs. Of note, overexpression of RUNX1 in N-iPSCs resulted in mitochondrial dysfunction, while inhibition of RUNX1 expression could improve the mitochondrial function in DS-iPSCs. Global gene expression analysis indicated that overexpression of RUNX1 may promote the induction of apoptosis in DS-iPSCs by activating the PI3K/Akt signaling pathway. The present findings indicate that abnormal expression of RUNX1 may play a critical role in mitochondrial dysfunction in DS-iPSCs.
Keywords: Down syndrome, induced pluripotent stem cells, mitochondrial dysfunction, RUNX1
Down syndrome (DS) or trisomy 21 is one of the most common genetic diseases and is often accompanied by intellectual disability. It occurs with an incidence ~1 in 700-1,000 births (El Hajj et al., 2016). DS is a major cause of congenital malformations and mental retardation in humans. Patients with DS typically display associated dysfunctions and a range of cognitive deficits, with important differences in their phenotype and severe side effects in the nervous system (Vacca et al., 2019). Previous studies on the brains of patients with DS have revealed abnormalities in the structure and function of the central nervous system. Recent studies have shown that alterations in mitochondrial function may be associated with the occurrence of DS (Pecze and Szabo, 2021; Zamponi and Helguera, 2019).
Mitochondria play a central role in metabolism and energy conversion in eukaryotic cells, which generate ATP via oxidative phosphorylation (OXPHOS). These organelles provide energy for various cell-based activities (Ohnishi et al., 2018). Dysregulated mitochondria lead to the excessive production of reactive oxygen species (ROS), which are involved in the regulation of programmed cell death (Li et al., 2019; Wong, 2011). Impaired mitochondrial function leads to significant changes in the cell and affects neural cell proliferation, apoptosis, differentiation, regeneration, and other cellular activities (Beckervordersandforth, 2017; Johnson et al., 2021). It is known that mitochondrial dysfunction/oxidative stress is associated with DS. Mitochondrial dysfunction has been observed in the brain of early fetuses with DS and can manifest as abnormalities in OXPHOS complexes (Coskun and Busciglio, 2012; Salemi et al., 2018), accumulation of oxidative stress products and changes in mitochondrial biosynthesis. In addition, previous studies demonstrated that abnormal expression of genes was associated with mitochondrial function in DS (Qiu et al., 2017; Salemi et al., 2020). Mitochondrial dysfunction leads to impaired neuron proliferation, differentiation, and maturation. Previous studies have shown the presence of mitochondrial dysfunction in neurons, glial cells, and peripheral blood cells in patients with DS, and they were associated with mitochondrial fragmentation, impaired OXPHOS, and reduced ATP levels (Valenti et al., 2018; Zamponi and Helguera, 2019). Collectively, these studies indicated that mitochondrial dysfunction was strongly associated with abnormal development of the nervous system in DS. However, the key genes and molecular mechanisms that lead to mitochondrial dysfunction in patients with DS remain unknown.
DS is caused by an extra copy of the human chromosome 21 (Hsa21). Previous studies have proposed the gene dosage effect hypothesis, suggesting that increased expression of a specific set of dosage-sensitive genes on Hsa21 may eventually cause neurodevelopmental or neurocognitive disorders in DS (Delabar et al., 1993). A recent study indicated that trisomy 21 caused neuronal apoptosis, leading to a decrease in the neural population (Hirata et al., 2020). The DS critical region (DSCR), namely 21q22-21q23, is a genomic region on Hsa21 that is strongly linked to DS phenotypes (Pelleri et al., 2016). Aberrant expression of genes, notably transcription factors located in the DSCR, may be associated with mitochondrial dysfunction in DS (Flippo and Strack, 2017; Izzo et al., 2018).
Runt-related transcription factor 1 (RUNX1), which is encoded by the
Normal induced pluripotent stem cells (N-iPSCs) and induced pluripotent stem cells of patients with DS (DS-iPSCs) were purchased from the American Type Culture Collection (Cat. No. ATCC DYR0100 [normal, newborn, male] and Cat. No. ATCC DYP0730 [DS, newborn, male]; ATCC, USA). The clones were cultured in a feeder-free system. The cell culture dishes were coated with Cell Matrix Basement Membrane Gel (Cat. No. ACS-3035; ATCC) and cultured in Complete Pluripotent Stem Cell SFM XF/FF medium (Cat. No. ACS-3002; ATCC) in the presence of 10 µM inhibitor (Cat. No. Y0503; Sigma, USA). The culture medium was changed the day after cell resuscitation, and daily thereafter until the colonies reached 80% confluence. The cells were typically digested, sub-cultured at a 1:3 ratio using the Stem Cell Dissociation Reagent (Cat. No. ACS-3010; ATCC) and incubated overnight at 37°C in the presence of 5% CO2.
DS-iPSCs were then cultured in the medium supplemented with 20 µM LY294002 (Cat. No. S1105; Selleck, USA) for 1 h. Then the cells were collected, the expression level of Akt was identified by western blotting and the analysis of ROS level and cellular ATP content was also performed. The experiment was repeated in triplicate.
The following vectors were purchased from AddGene (USA); RUNX1 expression vector (pCMV5-AML1B; plasmid Cat. No. 12426) and RUNX1 short hairpin RNA (shRNA) vector (pLKO.1 shRUNX1 puro; plasmid Cat. No. 45816).
Single clones were harvested from a 60-mm dish using Stem Cell Dissociation Reagent within 10 min and washed once with 2 ml phosphate-buffered saline (PBS). Subsequently, the cells were resuspended in R-buffer. N-iPSCs and DS-iPSCs were transfected with the Neon Transfection System (Cat. No. MPK1025; Invitrogen, USA) according to the manufacturer’s instructions. The conditions used were as follows: 1,240 V, 20 ms and 2-times pulses. A total volume of 10 µl containing 1 µg plasmid DNA was used. Following transfection, the samples were placed on a 24-well microplate, and subsequent analysis was performed 48 h after transfection.
Total RNA was extracted from PBMCs and iPSCs using TRIzol® reagent. RT was performed using a reverse transcription kit (Cat. No. 18091050; Invitrogen). RT-qPCR was performed with TaqmanTM Real-Time PCR Assay. The primer sequences for RUNX1 and GAPDH were as follows: RUNX1, forward: 5’-TGGCACTCTGGTCACCGTCAT-3’ and reverse: 5’-GAAGCTCTTGCCTCTACCGC-3’, and GAPDH, forward: 5’-AGAGGGCTGTCGGCGCAGTA-3’ and reverse: 5’-GGCTGTGGTCTCGGTTGGGC-3’. The reactions were performed using an ABI7500 Real-Time PCR system (Thermo Fisher Scientific, USA). The transcript levels of RUNX1 were quantified using the 2-ΔΔCq method, with GAPDH as the reference gene used for normalization.
The iPSCs were scraped off in ice-cold PBS, collected into RIPA lysis buffer (Cat. No. sc-24948; Santa Cruz Biotechnology, USA) with a cell scraper, and incubated on ice for 30 min. The cell lysates were centrifuged at 14,000 ×
ROS levels in iPSCs were measured using a ROS Assay Kit (Cat. No. S0033; Beyotime), following the manufacturer’s instructions. Briefly, iPSCs were cultured in 6-well culture plates pretreated with Matrigel, and subsequently, the cell culture medium was removed. The cells were then incubated with 10 µM DCFH-DA, which is a fluorescent probe used for ROS detection. Following subsequent culture at 37°C for 20 min in the dark, the iPSCs were washed three times with serum-free Dulbecco’s modified Eagle’s medium (DMEM), and the fluorescence emission of the samples was detected by fluorescence microscopy (Nikon Eclipse Ti; Nikon, Japan). The fluorescence intensity was analyzed using ImageJ software (ver. No. 1.6; National Institutes of Health, USA).
BODIPY-C11 staining was performed according to the manufacturer’s protocol. The cells were seeded in a 35-mm-diameter dish. Culture medium was replaced with 2 ml medium containing 5 µM of BODIPY-C11 (Cat. No. D3861; Invitrogen) after 48 h and the culture was returned to the cell culture incubator for 30 min. Finally, the cells were fixed with 4% paraformaldehyde for 15 min and cell nuclei were stained with DAPI for 10 min. Fluorescence intensity was examined by fluorescence microscopy (Nikon Eclipse Ti), and images were analyzed with the ImageJ software.
Cells were seeded in culture flasks, and when they were 60%-70% confluent, the culture solution was aspirated. The cells were then washed once with PBS, and 2 ml cell culture medium containing 1 ml JC-1 solution (Cat. No. S2003S; Beyotime) was added for staining. The solution was thoroughly mixed in a cell culture incubator for 20 min at 37°C. Concomitantly, the JC-1 staining buffer (5×) was diluted five times in water (4 ml distilled water and 1 ml JC-1 staining buffer). The samples were placed on ice. Following incubation at 37°C, the supernatant was aspirated and washed with JC-1 buffer (1×) three times. Upon addition of 2 ml medium, the fluorescence emission of the samples was detected using a fluorescence microscope. The experiments were repeated three times.
Cellular ATP content was determined with an ATP Assay Kit according to the manufacturer’s instructions (Cat. No. S0026; Beyotime). Briefly, the cells were lysed with ATP Detection Lysis Buffer 48 h after transfection, and 100 µl ATP detection working solution was added to the wells for 5 min at room temperature. A total of 20 µl sample or standard was added to each well, and the ATP concentration was measured using luminometry. A BCA Protein Assay Kit (Cat. No. P0010; Beyotime) was used to determine the protein concentrations of each sample, and the total ATP levels were evaluated as the ratio of cellular ATP level to protein concentration.
Following transfection of iPSCs with RUNX1-overexpressing plasmids for 48 h, the transfected cells cultured on 35-mm culture dishes were incubated with 100 nM lysosome probes (Cat. No. M7512; Invitrogen) at 37°C for 20 min. Following incubation, the cells were fixed with 4% paraformaldehyde for 15 min and 0.2% Triton X was used for permeabilization for 10 min. This was followed by DAPI staining (Cat. No. C1002; Beyotime) for 8 min to visualize the nuclei. Red fluorescence was determined by fluorescence microscopy, and image analysis was performed with ImageJ software.
Cell apoptosis was evaluated with a Cell Apoptosis Kit (Cat. No. APOAF; Sigma), according to the manufacturer’s instructions. Briefly, iPSCs were collected 24 h following transfection of RUNX1-overexpressing plasmids. Subsequently, 500 µl binding buffer, 5 µl Annexin V-FITC conjugate and 10 µl propidium iodide solution were added successively into the cell suspension, and incubated at room temperature in the dark for 10 min. The fluorescence emission of the cells was immediately detected with a flow cytometer (BD AccuriTM C6 Flow Cytometer; BD Biosciences, USA).
RNA-seq was conducted with the NovaSeq 6000 platform (Illumina, USA). An experimental group transfected with a RUNX1 expression vector and a control group transfected with a GFP expression vector were used. Each group had three replicates.
Following total RNA extraction using TRIzol® reagent (Cat. No. 15596018; Invitrogen), the quantification, qualification, library preparation, and subsequent RNA-seq of the above two groups were performed by Novogene (China). The details of RNA-seq have been previously described (Jia et al., 2020). Raw data were cleaned by removing reads with adaptors and excluding low quality (>50%) or a high proportion of unknown bases (>10%). Subsequently, the data were assembled using Trinity methodology (Grabherr et al., 2011).
For each sequenced library, the read counts were adjusted using the edgeR program package through one scaling normalized factor prior to differential gene expression analysis. Differential expression analysis of two conditions was performed using the edgeR R package.
Differential expression genes and apoptotic biomarkers were confirmed by RT-qPCR. The primers are listed in Supplementary Table S1. To detect activation of the PI3K-Akt signaling pathway, the protein expression and phosphorylation levels of Akt, S6, and 4E-binding protein (4E-BP1) were detected by western blotting as previously described. The antibodies used are listed in Supplementary Table S2.
Our previous study demonstrated that the expression levels of almost all the genes related to mitochondrial function were downregulated in DS-iPSCs, suggesting that mitochondria were dysfunctional in these cells (Qiu et al., 2017). To further demonstrate the impairment of mitochondrial function in DS-iPSCs, specific changes in mitochondrial MMP, ROS and ATP content were investigated to provide further evidence of impaired mitochondrial function. As shown in Figs. 1A and 1B, a, a high green fluorescence intensity was noted in the DS-iPSCs. The quantification of green fluorescence intensity was analyzed by ImageJ (Alafnan et al., 2021), and the results suggested that the contents of ROS were increased 2.24-fold in DS-iPSCs (0.076 ± 0.004 vs 0.034 ± 0.003;
Previous studies have shown that RUNX1 is involved in the pathogenesis of DS (Bourquin et al., 2006; Edwards et al., 2009; Patel et al., 2011). Furthermore, overexpression of RUNX1 was detected in DS-iPSCs and peripheral blood of children with DS (Supplementary Fig. S1), leading to the speculation that RUNX1 may be a key gene regulating mitochondrial function in DS. To determine whether RUNX1 affects mitochondrial function, RUNX1 was overexpressed in N-iPSCs (Supplementary Fig. S2). The results indicated that mitochondrial dysfunction was present in N-iPSCs following RUNX1 overexpression, which was similar to that in DS-iPSCs. JC-1 staining indicated that the green fluorescence intensity was considerably enhanced in RUNX1-overexpressing N-iPSCs compared with that observed in N-iPSCs expressing normal levels of RUNX1 (0.090 ± 0.017 vs 0.037 ± 0.0039;
Accumulation of ROS in mitochondria causes abnormalities in mitochondrial structure and function, including ATP synthesis obstruction, reduced mitochondrial MMP, and impaired respiratory chain function. Mitochondrial respiratory chain deficiencies block OXPHOS and inhibit ROS production, which aggravates mitochondrial dysfunction and creates a vicious cycle of mitochondrial damage (Bhatti et al., 2017; Hu and Liu, 2011). Thus, mitochondrial function was evaluated in iPSCs overexpressing RUNX1. The results indicated that overexpression of RUNX1 led to a 3.17-fold decrease in ATP content compared with that of control cells (4.75 ± 0.36 vs 2.11 ± 0.26;
High levels of mitochondrial ROS can induce oxidative damage in mitochondria, which impairs the function of the mitochondrial respiratory chain, leading to the accumulation of intracellular ROS. The membrane ROS levels of iPSCs were determined using BODIPYTM C11, and the data confirmed that RUNX1 overexpression increased mitochondrial and cell membrane ROS levels compared with those of control cells (0.10 ± 0.0034 vs 0.077 ± 0.0015;
To further determine whether RUNX1 overexpression could affect mitochondrial function, DS-iPSCs were transfected with RUNX1 shRNA vector, and the mitochondrial function was measured. The results indicated that the green fluorescence intensity was markedly decreased when the expression levels of RUNX1 were inhibited in DS-iPSCs (0.068 ± 0.015 vs 0.029 ± 0.0020;
RNA-seq analysis indicated that 51 differentially expressed genes were identified between the experimental and control groups using the edgeR R package (Fig. 4A). This included 39 upregulated and 12 downregulated genes (Fig. 4B). KEGG analysis revealed a set of 39 upregulated genes, and the results indicated that PI3K-Akt was identified as the most significant signaling pathway, in which five genes (
A previous study has shown that the activation of mTOR signaling via the PI3K/Akt signaling pathway is involved in the regulation of mitochondrial ROS production in PC-12 cells, which can ultimately affect cell apoptosis (Li et al., 2020). In the present study, PI3K/Akt inhibitor LY294002 was used to suppress the expression of Akt in DS-iPSCs. The results showed that the expression level of Akt in DS-iPSCs was significantly decreased, accompanied by a significant increase in ATP content and a decrease in ROS level (Supplementary Fig. S3).
Akt is a major downstream effector molecule of the PI3K/Akt signaling pathway, and increased phosphorylation implies activation of the PI3K signaling pathway (Hart and Vogt, 2011; McCubrey et al., 2012). The present results revealed a significant increase in the phosphorylation of Akt, S6 and 4EBP1 in N-iPSCs + RUNX1 cells (Fig. 5A). These results indicated that upregulation of RUNX1 in iPSCs activated the PI3K-Akt signaling pathway by increasing the phosphorylation of Akt, S6 and 4EBP1.
The effects of RUNX1 overexpression on the induction of apoptosis of iPSCs were investigated by flow cytometry. A significant increase in apoptosis was observed in RUNX1-overexpressing N-iPSCs (19.00 ± 1.79 vs 32.46 ± 1.54;
The most apparent clinical features of DS are neurological complications, including early onset of Alzheimer’s disease (AD) and mental retardation. The central nervous system requires high quantities of energy. Redox homeostasis and adequate ATP levels are essential for normal neurodevelopment (Arrázola et al., 2019; Kann and Kovács, 2007). Mitochondria supply virtually all eukaryotic cells with energy through ATP production by OXPHOS. Accordingly, maintenance of the mitochondrial function is fundamentally important to sustain cellular health. Previous studies reported that mitochondrial abnormalities, including aberrant mitochondrial morphology and function, were detected in both patients with DS and in DS cells (Aburawi and Souid, 2012; Shah et al., 2019). In the present study, mitochondrial abnormalities were confirmed in DS-iPSCs. Therefore, it is reasonable to speculate that the aberrant development of the nervous system in DS is closely associated with mitochondrial dysfunction, as iPSCs have the pluripotency to differentiate into various nerve cell types. However, the mechanism leading to mitochondrial dysfunction in DS is still not clear, and the key genes responsible for this process have not been identified thus far.
RUNX1 is an important transcriptional regulator located in DSCR, which is closely associated with the development and regeneration of the nervous system. A recent study indicated that large-scale hypermethylation of RUNX1 (~28 kb) was the strongest signal within the DS differentially methylated regions in peripheral blood (Laufer et al., 2021), which was also confirmed by the current study. In the present study, overexpression of RUNX1 was observed in both DS-iPSCs and peripheral blood samples of children with DS. It was therefore speculated that RUNX1 may be a crucial regulatory gene involved in the pathogenesis of DS. In a recent study,
The present study investigated whether RUNX1 affects the neurological development of DS by regulating mitochondrial function. The results revealed that overexpression of RUNX1 induced impaired mitochondrial functions, characterized by excessive generation of ROS, decreased MMP and reduced overall energy metabolism. It was also observed that RUNX1 could reduce the number of active lysosomes and cause lysosomal dysfunction. Furthermore, the data confirmed that impaired mitochondrial functions, including excessive generation of ROS, and decreased MMP and ATP content, could be reversed when the expression levels of RUNX1 were inhibited in DS-iPSCs. All these results strongly suggested that the abnormal expression levels of RUNX1 may be a critical factor (although not the sole factor) in the occurrence of mitochondrial dysfunction in DS-iPSCs, and indicated that overexpression of RUNX1 led to neurological abnormalities by affecting mitochondrial function in DS.
It is well known that the neuropathology of AD is an important clinical feature of DS, which is almost always present in patients with DS >40 years of age. Perluigi et al. (2014) demonstrated that the PI3K/Akt signaling pathway displayed increased activity in patients with DS and those with DS/AD. Notably, the PI3K/Akt signaling pathway plays a critical role in the regulation of cell proliferation, death, and metastasis (Lim et al., 2015), RUNX1 overexpression increased the phosphorylation levels of PI3K, Akt, and mTOR proteins (Liu et al., 2020).
Akt is a well-known pro-survival kinase and activated by phosphorylation at Ser 473 via the PI3K signaling pathway. Activated Akt plays a critical role in inhibiting neuronal death through further activation or inhibition of its downstream target proteins (Ye et al., 2015). In the present study, it was further found that overexpression of RUNX1 may activate the PI3K/Akt signaling pathway via increasing the phosphorylation of Akt, S6 and 4EBP1 in iPSCs. Certain upregulated genes in the PI3K-Akt signaling pathway, including
Previous reports showed that Akt plays a central role in apoptosis inhibition through its regulatory effects on various downstream targets such as anti-apoptotic Bcl-2 (Yu et al., 2016). Normal mitochondrial function depends on the integrity of the mitochondrial membrane, which is strictly regulated by the Bcl-2 family of proteins (D'Orsi et al., 2017). The balance of cell death and survival signals in the Bcl-2 protein family determines the cell fate. As a pro-apoptotic member of the Bcl-2 family, Bim induces the release of cytochrome
In conclusion, the present study demonstrated overexpression of RUNX1 in DS-iPSCs, and found that RUNX1 could regulate mitochondrial function and mitochondria-mediated apoptosis via the modulation of the PI3K-Akt signaling pathway in iPSCs. However, further investigation is required to assess whether RUNX1 promotes the neural differentiation of DS-iPSCs by regulating mitochondrial function.
This research was supported by the National Key Research and Development Program of China (2019YFA0801402) and the National Natural Science Foundation of China (81971421, 81471485), Shanghai key clinical specialty project (shslczdzk05705), Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212200).
Y.L., J.Y., and F.Z. conceived and performed experiments, wrote the manuscript, and secured funding. Y.L. and Y.Z. performed experiments. J.Y., Z.R., and F.Z. provided expertise and feedback. All authors have read and agreed to the published version of the manuscript.
The authors have no potential conflicts of interest to disclose.
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