Proteomic Analysis of Induced Pluripotent Stem Cells Derived from an HD Patient.
In our recent articles on induced pluripotent stem cells (iPSCs), we reported on the effective use of iPSCS derived from an HD patient with a 72 CAG count to treat a neurotoxin rat model of Huntington’s disease. We noted that these cells were also being used to gain insight into early pathology in Huntington’s disease. By learning which harmful changes occur early in the disease process, researchers can better prioritize targets for treatment. The goal is to address those early pathologies rather the many downstream ones that follow.
A new report analyzes changes in gene expression in this same line of cells. A team of researchers, again led by Dr. Jihwan Song, Associate Professor and Director of Laboratory of Developmental & Stem Cell Biology at CHA Stem Cell Institute, CHA University, Seoul, South Korea, compared the HD iPSCs, normal iPSCs , and normal embryonic stem cells before these stem cells differentiated into specific cell types (such as neurons). They found, strikingly, that there were already significant changes in the expression of genes even in this earliest stage of development. The expression of ten genes was regulated upward and seven were regulated downward as compared to normal iPSCs. The expression of ten genes were regulated upwards and ten were down-regulated as compared to normal embryonic stem cells. In other words, some proteins were produced at greater levels and others at lower levels than normal. This is the first proteomic analysis (study of proteins) to be conducted with HD iPSCs.
Most of these genes fell into categories. Proteins associated with oxidative stress were affected, suggesting that this known pathology in HD is an early and important one. Several proteins associated with protecting the cell from oxidative damage were down-regulated in the HD iPSCs, suggesting the cells with the HD protein are especially vulnerable to oxidative stress. These proteins were SOD1 (superoxide dismutase 1), GST (glutathione-S-transferase, and Gprx1 (glutathione peroxidase 1).
On the other hand, some proteins associated with oxidative damage were up-regulated. Proteins in the peroxiredoxin family (PRXs) were more strongly expressed in the HD cell lines than in the normal lines. These proteins defend against oxidative damage. Normally proteins found in the cytoplasm, one of these, PRX1, can be found in the nucleus of the cell under conditions of oxidative stress. This occurred in the HD cell lines, suggesting that the up-regulation of these proteins is a defense mechanism. However, the researchers think that this attempt to handle ROS (the reactive oxidative species molecules that cause oxidative damage) in the nucleus of the cell could also lead to the cell’s eventual degeneration.
Cells from all lines were differentiated into neurons and the same pattern was found. SOD1, GST, and Gprx1 were down-regulated while the PRXs were up-regulated.
Oxidative stress can lead to apoptosis, or programmed cell death, and the researchers examined whether there were more apoptotic cells in the HD lines. They found that there indeed were more apoptotic cells in the HD induced pluripotent stem cells than in the normal embryonic stem cells and the normal iPSCs. In addition there were also more apoptotic cells in the HD cells compared to the normal cell lines after differentiation into neurons.
The researchers were interested in learning whether DNA breakage and fragmentation resulted from the oxidative stress and caused the apoptotic cell death. If so, they expected to see the up-regulation of two proteins, BTF3 and ATM. BTF3 is a transcription factor which targets ATM, a gene which produces a protein which sends apoptotic signals when oxidation damages DNA. They found that both proteins were up-regulated in the HD cell lines.
The researchers also expected to see the phosphorylation of ATM on serine 1981 because this normally occurs in reaction to oxidative stress and is necessary for the sustained occupany of ATM on DNA double-stranded break sites. As expected, the phosphorylated form of ATM was increased in the HD cell lines.
The phosphorylation of ATM leads to modification through phosphorylation of additional regulators of apoptosis. The researchers looked at two of these, P53 and H2A.X, and found increased levels of phosphorylation at the expected sites on both proteins. In addition, they examined the expression of central regulators and effectors involved in apoptosis. There were high levels of the active forms of Bid (a pro-apoptotic protein upregulated by P53), of caspases 9, 3, and 7 (proteases that play an essential role in apoptosis), and of PARP (a DNA repair-involved protein which is a marker for cells undergoing apoptosis).
Putting these findings together, the researchers conclude that, “These results strongly suggest that cellular oxidative stress in HD-iPSCs can cause DNA damage, followed by activation of consecutive ATM-mediated signaling (i.e., phosphorylation of p53 and H2A.X) via its kinase activity. Activation of substrates of ATM in HD-iPSCs can lead to DNA damage-induced apoptotic cell death through mitochondrial pathway.”
The researchers then investigated whether the increased apoptotic cell death in the HD-iPSCs which occurred in response to oxidative stress affected the expression of cytoskeleton-associated proteins. The cytoskeleton is a network of fibers which helps to maintain the cell’s shape and plays an important role in trafficking within the cell and cell division.
The cytoskeleton of a cell: actin fibers are shown in red, microtubules are in green. The nucleus of the cell is in blue.
They found that the expressions of proteins associated with the cytoskeleton were in fact sharply reduced. These proteins are Cofilin-1 (Cfl-1), Stathmin (Stmn-1), Fascin-1 (Facn-1) and Septin-2 (Sept-2). Although it is currently unknown whether these proteins play a direct role in cell differentiation, the researchers have some evidence that there might be a connection and they speculated that cell differentiation might be negatively affected. The researchers looked at how efficiently the stem cells differentiated into neurons. They found that neuronal differentiation and neurite outgrowth were significantly reduced in the HD iPSCs.
The researchers will repeat the study with different lines of iPSCs with differing CAG counts, but this first example of a proteomic analysis shows that the results are likely to provide important insights into the beginning of the disease process.
Jung-Il Chae, Dong-Wook Kim, Nayeon Lee, Young-Joo Jeon, Iksoo Jeon, Jihye Kwon, Jumi KIM, Yunjo Soh, Dong-Seok Lee, Kang Seok Seo, Nag-Jin Choi, Byoung Chul Park, Sung Hyun Kang, Joohyun Ryu, Seung-Hun Oh, Dong Ah Shin, Dong Ryul Le, Jeong Tae Do, In-Hyun Park, George Q. Daley and Jihwan Song. “Quantitative proteomic analysis of induced pluripotent stem cells derived from a human Huntington’s disease patient.” Journal of Biochemistry 2012 Jun 13. [Epub ahead of print].
– Marsha L. Miller, Ph.D., August 17, 2012