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Learning more about physiological endoplasmic reticulum stress using the ERAI system

Visualization of stress in the cell’s protein factory, the endoplasmic reticulum, may lead to new treatments for many debilitating diseases
Takao Iwawaki
Initiative Research Scientist
Iwawaki Initiative Research Unit
RIKEN Advanced Science Institute
The endoplasmic reticulum is a cell organelle that acts as a processing factory for secreted proteins and membrane proteins. The accumulation of abnormal proteins in endoplasmic reticulum causes a functional disorder called ‘endoplasmic reticulum stress’, which if not arrested leads to cell death. When and where does endoplasmic reticulum stress occur? How do cells avoid this stress? Trying to solve these mysteries, Initiative Research Scientist Takao Iwawaki has developed the world’s first endoplasmic reticulum stress visualization system, and is using the system to advance his research. Endoplasmic reticulum stress has recently been found to play a role in the development of various diseases including Alzheimer's disease, diabetes and cancer. Research into endoplasmic reticulum stress is expected to lead to the development of therapeutic agents for these diseases.

The endoplasmic reticulum — a protein processing factory

The endoplasmic reticulum stress visualization system developed by Iwawaki has been dubbed ‘ERAI’, short for the endoplasmic reticulum stress activated indicator. Erai also means ‘great’ in Japanese. “Please call it ‘e-ra-i’,” says Iwawaki. “I think names are very important, because people are attracted by names that have a strong impact.”

What kind of a system is ERAI? Is it really as great as it seems?

At the microscopic level, a cell in a living organism contains many multi-layered sack-like cell organelles called endoplasmic reticulum. The functions of the endoplasmic reticulum can be divided into three groups: (1) synthesis, modification, transport and quality control of secreted proteins and membrane proteins; (2) synthesis of lipids and maintenance of homeostatic properties; and (3) storage of calcium ions. Iwawaki focuses on the first category of functions.

The formation of proteins in a cell starts with the DNA in the cell nucleus. The DNA contains genes, which encode information on when, where and what kinds of proteins are to be produced. In the process of making a protein, the four base sequences adenine, thymine, guanine and cytosine in part of the gene are transcribed into RNA. Non-coding regions, called introns, are then removed to form messenger RNA (mRNA), which is carried away from the nucleus. The mRNA base sequences are, in turn, translated into amino acids in cell organelles called ribosomes, leading finally to the formation of proteins. The newly produced ‘nascent’ proteins, however, are merely long strings of amino acids, and still do not fulfill their intended function. Secreted and membrane proteins, such as digestive enzymes, hormones and antibodies, are carried to endoplasmic reticulum as they are, and there the nascent proteins are folded and combined with sugar molecules. Properly folded and functional proteins then emerge from the endoplasmic reticulum to be carried to the areas where they are needed.

“The endoplasmic reticulum serves as a protein processing factory in which proteins, at this stage merely long strings of amino acids or ‘raw materials’, are ‘manufactured’ into properly folded proteins,” says Iwawaki. “As you know, no factory can avoid producing defective products. The accumulation of improperly folded proteins causes the endoplasmic reticulum to lose its functions. This situation is called ‘endoplasmic reticulum stress’. I want to elucidate the mechanisms of occurrence and avoidance of endoplasmic reticulum stress.”

Persistent endoplasmic reticulum stress leads to the death of cells. Thus, cells are equipped with a stress response mechanism for avoiding endoplasmic reticulum stress. “In a factory, when the rate of defective products reaches a certain level, the production line will be stopped. This is also the case with the endoplasmic reticulum. A signal is issued to repress the translation of mRNAs to prevent further new materials from being carried to it. This is the first stress response. Additionally, the number of helpers that serve to fold proteins is increased as an effective measure to process the accumulated proteins. This is the second stress response, called the unfolding protein response (UPR), and it is this action that is the focus of my research. We can also observe a stress response in which defective products are removed from endoplasmic reticulum.”

The world’s first visualization of endoplasmic reticulum stress

Iwawaki had a question when he started to study the endoplasmic reticulum in graduate school. The mammalian UPR has three pathways, involving either PERK, ATF6 or IRE1 molecules, which are present in the membrane of the endoplasmic reticulum. These molecules can sense the accumulation of altered proteins, and serve to induce the transcription of molecular chaperone genes that help fold proteins. Yeast cells, however, have only the IRE1 pathway, and it can develop under ordinary conditions even in the absence of IRE1 molecules. Iwawaki’s question: “Is an endoplasmic reticulum stress response essential for living organisms to survive?”

This question was answered in 2000, when it was found that mice have two types of IRE1, IRE1á and IRE1â, and that fetal mice die before birth if IRE1á is absent. This showed that an endoplasmic reticulum stress response is essential for the survival of mammals. At about the same time, a paper was published reporting a relationship between endoplasmic reticulum stress and Alzheimer’s disease. It has since been found that endoplasmic reticulum stress may play a role in diseases such as Parkinson’s disease, bipolar disorder, diabetes, arteriosclerosis, rheumatism, viral infections and cancer. “These diseases do not appear in yeast or cultured cells, which made me think that I should use animals to study endoplasmic reticulum stress at the level of the whole organism.”

“When and where does endoplasmic reticulum stress occur? This was the first question I wanted to answer,” says Iwawaki. The common method at the time to investigate the occurrence of endoplasmic reticulum stress was to measure molecular chaperone expression. However, such measurements are performed after grinding the cells and, therefore, it is not possible to tell where the stress occurred. Iwawaki then began the development of a system for visualizing endoplasmic reticulum stress. Fortuitously, as a result of an academic meeting, he happened to become aware that when IRE1 senses stress, the introns of the XBP1 gene are cut off, and the rest of the gene then acts as a factor to induce molecular chaperone transcription. “I immediately thought I could use this mechanism. First, we bring XBP1 genes and genes for green fluorescent protein, or GFP, together to create ERAI genes. Then, when endoplasmic reticulum stress occurs in animal cells bearing the introduced ERAI gene, IRE1 acts to cut off the introns of the XBP1 gene, which results in the formation of proteins in which XBP1 proteins and GFP are connected, causing light to be emitted. In the absence of endoplasmic reticulum stress, IRE1 remains inactive, and XBP1 also remains inactive without emitting light because IRE1 is not active. This is the basis of the ERAI system.”

The ERAI process is shown in Figure 2, and the results of an investigation of endoplasmic reticulum stress in the kidney of an ERAI gene-bearing mouse are shown in Figure 3. “The kidney did not emit light when a normal saline solution was administered, but emitted green light when tunicamycin, a medical agent, was administered. Thus we succeeded in visualizing endoplasmic reticulum stress for the first time in the world.”

Iwawaki also made another great discovery: the pancreas of the ERAI gene-bearing mouse glowed even when the mouse was given a normal saline solution. “The pancreas produces many secreted proteins, such as insulin. Thus, it was thought that the pancreas is under endoplasmic reticulum stress because it is constantly in an overloaded state. ERAI clearly demonstrated that the pancreas is constantly under endoplasmic reticulum stress. The light from the pancreas became intense from about 18 days after birth. An average baby mouse starts weaning at that time, and starts eating solids. This may change the way insulin or digestive enzymes are secreted, or how stress occurs in the pancreas.”

When these findings were published in Nature Medicine in December 2003, Iwawaki received many requests for ERAI genes and ERAI mice. For researchers who want to study the relationship between endoplasmic reticulum stress and diseases, ERAI is an effective research tool. However, Iwawaki felt the necessity to tackle new challenges in ERAI development. “To me, the development of ERAI at that time was not a complete success.”

Endoplasmic reticulum stress observed even in fetuses

Fetuses cannot survive in the absence of IRE1á. This means that some organs in fetuses are constantly under endoplasmic reticulum stress. IRE1á acts even during the fetal development of a normal mouse, thus providing a means of relieving this stress. Iwawaki was hoping that he would be able to use the ERAI system to visualizing endoplasmic reticulum stress in fetuses. The fetus, however, failed to emit light. “I thought that this was due to a lack of sensitivity. So I developed the ERAI-LUC system in which luciferase, a luminescent enzyme used in place of GFP genes, and XBP1 genes were brought together.”

GFP, used in the original ERAI scheme, fluoresces when exposed to light. Thus, light is required to investigate internal organs using GFP genes. Luciferase, on the other hand, emits light by oxidizing photosubstrates called luciferins, thus eliminating the need to use light. Using a dedicated system and administering luciferins at the last minute allows observation of the entire organ without causing damage.

Figure 4 (click link to article to see figures 4 and 5) shows the results obtained from investigating endoplasmic reticulum stress in the fetus of an ERAI-LUC mouse. “A widely accepted theory says that endoplasmic reticulum stress occurs in the liver of the fetus, but I found this to be incorrect. My investigation proved that the stress occurs in the placenta. This result was a surprise, but also showed that an endoplasmic reticulum stress response occurs through a new and different pathway.”

It is thought that endoplasmic reticulum stress occurs, in a strict sense, in a portion of the placenta called the placental labyrinth, a network of maternal and fetal blood vessels where nutrients, waste, oxygen and carbon dioxide are exchanged. IRE1á-deficient mice are known to have fewer blood vessels in this area, and to have difficulty in exchanging these necessities. Endoplasmic reticulum stress in the placenta, Iwawaki believes, is due to a shortage of sugar molecules as a result of the interruption of placental exchange. Proteins folded in the endoplasmic reticulum are only completed when combined with sugar molecules. “When sugar molecules are deficient, endoplasmic reticulum stress cannot be relieved, no matter how active the expression of molecular chaperones. New blood vessels need to be created and sugar molecules supplied. The conventionally known endoplasmic reticulum stress response completes within a single cell. However, we found that there is a pathway through which the endoplasmic reticulum stress can be relieved by exerting an effect on the extracellular environment beyond the bounds of the cells. This result was obtained by investigating the organism as a whole.”

As his next target, Iwawaki aims to develop effective treatments for diseases in which endoplasmic reticulum stress may play a role. “Mating an ERAI-LUC mouse and a disease-model mouse will allow us to discover when and where endoplasmic reticulum stress occurs as part of the disease. This knowledge will provide an important clue on how to treat the disease. If the mechanism of the endoplasmic reticulum stress response can be clarified, we will be able to adjust the level of the stress response.”

The ERAI-LUC system has been further improved, and now allows for the expression of ERAI genes only at specific sites. “The ERAI-LUC system has now been perfected,” Iwawaki says with confidence. However, the system cannot be used on humans because gene recombination is required. “We need to develop a better technique for visualizing human endoplasmic reticulum stress. This will also serve to help develop therapeutic agents for disease.”

Inspired by Tonegawa’s Nobel Prize in Physiology or Medicine

“I was an arts student in high school,” says Iwawaki. He belonged to an athletics club, and devoted all his time to training because he wanted to become a physical education teacher. However, he experienced a turning point in his life when he was in his third year in 1987. “Dr Susumu Tonegawa was awarded the Nobel Prize in Physiology or Medicine in 1987, and our high school biology teacher told us passionately about Dr Tonegawa’s research results.” Tonegawa is now director of the RIKEN Brain Science Institute. “Physical and chemical laws can serve as tools to elucidate ambiguous processes like life phenomena. I was deeply affected by this fresh and interesting idea, and it made me think that I would like to be involved in those kinds of research activities in the future.” However, at the time of the announcement, in November, it had become too late to shift his direction from humanities to science before the university entrance exams. He took the entrance examination for his originally intended university, and was admitted into the department of economics. Dissatisfied, he abandoned that course in his second year and reenrolled in biology. He now works as an initiative research scientist at RIKEN.

This year is the fifth and final year of the Iwawaki Initiative Research Unit. “We have achieved some of our initial targets, but at the same time, new questions have been raised. Research activities do not have clear goals.”

Takao Iwawaki

Takao Iwawaki was born in Osaka, Japan, in 1969. He graduated from the Faculty of Education, Nara University of Education, in 1995, and obtained his PhD in 2001 from the Nara Institute of Science and Technology (NAIST). He then worked as a special postdoctoral researcher at the RIKEN Brain Science Institute for two years, and as a JST PRESTO Researcher at NAIST for another two years. In 2005, he returned to RIKEN as an initiative research scientist leading his own research group. His research presently focuses on functional analysis of endoplasmic reticulum stress responsive molecules using transgenic and gene targeting mice and on development of imaging methods for monitoring physiological and pathological endoplasmic reticulum stress in vivo.

Saeko Okada | Research asia research news
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