02 July 2014

Who is to blame for Alzheimer's disease – beta-amyloid or APOE4?

Alzheimer's disease: the gene I'm crazy about

Inna Burkova, "Biomolecule" based on Nature – Alzheimer's disease: The forgetting geneAt the beginning of the XX century, with the help of the German psychiatrist Alois Alzheimer, the world learned about the existence of a new neurodegenerative disease.

And although for a long time researchers did not perceive genetic predisposition as an important factor for the development of Alzheimer's disease, the situation soon changed. However, even now there are fierce disputes about the nature of this disease: who is to blame for everything – beta-amyloid or APOE4?

One day in 1991, neurologist Warren Strittmatter asked his supervisor, director of Duke University Allen Roses, to look at the results of his experiment. Strittmatter studied beta-amyloid, the main component of molecular clots found in the brains of people with Alzheimer's type dementia. He was looking for proteins binding to amyloid in the cerebrospinal fluid, and as a result came across apolipoprotein E (ApoE), which, it seems, had no direct connection with the development of the disease.

Beta-amyloid, which forms insoluble plaques in nerve cells in Alzheimer's disease, has traditionally been considered the main cause of neurotoxicity in this disease, but in reality the situation is not so simple. In particular, not only fibrillar, but also intermediate spherical aggregates of beta-amyloid can be toxic, lack of sleep positively affects the likelihood of developing AD, but sweetness – in reality, amyloid may not be a neurotoxin at all, but a component of innate immunity in the human nervous system.

Professor Roses immediately realized that his colleague had found something important. Two years earlier, they had discovered that the expression of some genes from chromosome 19 contributes to the development of dementia, and since Roses knew that the gene encoding ApoE is also on this chromosome, he was instantly struck by the idea of ApoE's involvement in the development of Alzheimer's disease (AD).

There are three variants of the APOE gene encoding the isoforms of the protein E2, E3 and E4 in the human body, and Roses decided to find out their effect on the development of Alzheimer's disease. To determine individual alleles, it was necessary to conduct a polymerase chain reaction (PCR). Since the scientist had very modest experience with PCR, he wanted to attract neurophysiologists to his team, but was refused: although they were busy hunting for the genes that underlie Alzheimer's disease, AROE seemed to them an unsuitable candidate. Roses recalls how later there were conversations in the laboratory: "... the boss completely lost his head from his crazy ideas."

But Roses didn't give up. He asked for help from his wife, geneticist Ann Saunders, who used PCR in her research. She had just given birth to a daughter and was on maternity leave, so they made an agreement. "She did all the experiments while I was looking after the child," says the professor. Within three weeks, the couple collected data, which later formed the basis of a series of landmark publications. They found out that the APOE4 allele contributes to the development of AD.

Today, twenty years later, APOE4 remains the leading risk factor for the most common form of dementia. Inheritance of one copy of APOE4 quadruples the risk of the disease, two copies – 12 times (Fig. 1).

Figure 1. Carriers of the APOE4 allele are more susceptible to the development of Alzheimer's disease
compared to those who inherited two copies of the APOE3 allele (according to Raber J et al., 2004)

However, Roses' data was mostly not taken seriously or criticized. Subsequently, even when the opinion about ApoE changed, most scientists still continued to work with beta-amyloid, as if "fixated" on the classics. But some laboratories still investigated ApoE, despite the indifference of funding institutions and the scientific community and the lack of resources needed to conduct large-scale experiments.

For a long time it was unknown what functions the ApoE protein performs in the brain, and gradually this puzzle began to interest many neurophysiologists. Interest in lipoproteins continued to grow, partly because clinical trials of drugs targeting beta–amyloid often ended in failure. Many researchers began to scrupulously study the ApoE4 protein and, as a result, attracted the attention of pharmaceutical companies. "Amyloid approaches" gradually ceased to be used, but they began to develop drugs aimed at apolipoprotein.

"Despite the lack of solid evidence, the amyloid hypothesis has become a strong scientific postulate in its time," says Zaven Khachaturian, president of the non–profit company Prevent Alzheimer's Disease 2020 and former coordinator of activities related to the study of AD at the US National Institutes of Health. Until recently, according to him, "no one tried to ask the fundamental question – have we correctly identified the basic premise of the disease?".

Tough competitionThere are various arguments as to why the discovery of Roses was ignored.

Many agree that the geneticist chose the wrong time to publish his results. In 1991, John Hardy and David Allsop proposed the "amyloid cascade hypothesis". They claimed that Alzheimer's disease is the result of an abnormal accumulation of beta-amyloid plaques in brain tissues. The scientific community supported the proposed idea, which was soon actively funded.

But Roses did not subscribe to this theory: "... amyloids are one of the many substances that form plaques; they eventually destroy cells and cause brain atrophy. I had no idea that this was the cause of dementia." By saying so, he may have wanted to hide a possible ApoE/Ab connection, and accidentally created a competition between the two hypotheses for funding. Unfortunately, Roses never received grants to work with ApoE.

There were also technical obstacles to learning ApoE. The protein is a part of various plasma lipoproteins and is a rather complex pharmacological target when working with the brain. ApoE has a lipophilic part and therefore, during biochemical analysis, it can aggregate with other molecules. Working with such proteins requires a deep understanding of the biochemistry of lipoproteins and methods of working with them.

Amyloid, on the contrary, was an easy target. After two decades of careful observation, a number of drugs have been created that alter the metabolism of amyloid, but they still have not met expectations. Of the six drugs that were clinically tested on patients with stage II or III of the disease in 2012, half immediately disappeared for safety reasons or lack of efficacy. And this situation is happening against the background of an aging population, a shaky health system and a shortage of medicines for Alzheimer's disease. "The number of unsuccessful trials aimed at treating Alzheimer's dementia has increased dramatically," says Lennart Mucke, director of the Gladstone Institute of Neurological Diseases (University of California, San Francisco). "It really rocked the pharmaceutical industry."

The three remaining drugs that target beta-amyloid are currently being tested on patients, as well as on people at high risk of AD who have not yet developed symptoms. Positron emission tomography has shown that the brains of subjects at high risk of developing AD differ from healthy brains over decades (!) before beta-amyloid begins to accumulate or neurons are destroyed. As a result of research that will be conducted over the next six years, scientists will understand whether these drugs are able to delay the onset of the disease or not. There is a feeling among researchers and representatives of the pharmaceutical industry that this is the last chance for the amyloid hypothesis. Against the background of these doubts, ApoE once again found itself in the spotlight.

According to Mack, if the trials fail, scientists will report to investors, providing all data from preclinical and early clinical trials. He hopes that AroE researchers will soon gain a great advantage. Despite the obstacles in this area, scientists continue to have a growing suspicion that ApoE4 is a prerequisite for the onset of the disease. This fact is confirmed by the Mack and Holtzman groups in experiments on transgenic mice that carry human ApoE isoforms.

Most likely, ApoE is involved in the development of AD in two different ways, one of which is amyloid-dependent. In both animals and humans, ApoE4 promotes the deposition of Beta-amyloid in the medulla, while ApoE3 is considered a "neutral" isoform, and ApoE2 is a "protective" form that reduces plaque accumulation. "This is quite convincing data," says Holtzman.

Another mechanism does not provide for a relationship with amyloid. When neurons are under stress, they express ApoE for their recovery. The "bad" form, ApoE4, is usually broken down into toxic fragments that damage the mitochondria and modify the cytoskeleton.

Figure 2. Two divergent hypotheses about how AroE contributes to the disease (Fulmer, 2012)

It is extremely difficult to assess the contribution of these two mechanisms to the risk of developing Alzheimer's disease, Holtzman says, but he and his colleagues believe that the transformation of the harmful ApoE isoform into a "neutral" one may become a promising approach for the treatment of AD. At Gladstone, researchers have begun to study this issue, and small regulatory molecules have already been found that transform ApoE4 into an ApoE3-shaped protein and thereby reduce the abnormal fragmentation of the first. In cell culture, even low concentrations of these molecules can reduce mitochondrial destruction and neuron dysfunction. Currently, these molecules are being tested on animals, and if they ultimately prove to be safe and effective, doctors will prescribe them to patients predisposed to AD, as well as statins to patients with high cholesterol and an increased risk of cardiovascular diseases.

More than enoughSuch drugs can also be effective for the treatment of other diseases.

"The mitochondrial hypothesis quite logically and concisely explains what the expression of ApoE4 leads to," says Mack, "not only in the context of Alzheimer's disease, but possibly also in other diseases." There is evidence that the appearance of this isoform is a possible risk factor also in Parkinson's disease and epilepsy. This protein is also associated with the development of destructive processes after traumatic brain injury and accelerated development of HIV infection. Fifteen biotech companies are already collaborating with Gladstone to develop drugs that operate on a similar principle.

Despite the lack of ApoE research grants, Roses never gave up. But a few years later, when his group discovered a link between ApoE and Alzheimer's disease, he got tired of the constant monetary confrontation and left science. After working in the pharmaceutical industry for ten years, during which he did not stop researching ApoE, in 2008 he returned to Duke University again.

In 2009, his group described a section of non-coding DNA with the TOMM40 gene, which is located next to APOE on the nineteenth chromosome. This section of DNA (abbreviated 523) varies in length and, depending on this, can determine the level of expression of the TOMM40 and AOE genes.

According to Roses, this was an important discovery, since the protein encoded by the TOMM40 gene – Tom40 – is essential for "healthy" mitochondria. Tom40 forms a channel in the outer mitochondrial membrane through which proteins necessary for the normal division of this organelle are imported. "We have known about the existence of such a mechanism for 10 years," says the scientist, "but we did not suspect that it leads to Alzheimer's disease."

Roses went on to claim that site 523 could be used to develop therapies and more accurately predict disease. The vast majority of people are at risk of encountering their Alzheimer's if they only live long enough, and only 25% of the population are carriers of the APOE4 allele. This means that the test for the carrier of this allele will never be an accurate predictor to the end. But genotyping for both genes – AROE and TOMM40 – can significantly increase accuracy, says Roses. In his laboratory, it was discovered that APOE3, the most common isoform, is usually inherited together with either a short or very long section 523. And in carriers of two APOE3 alleles, the age of onset of the disease will depend on the specific variant of site 523 inherited together with APOE.

Some laboratories managed to find evidence confirming the hypothesis of Roses, but others could not repeat the studies on TOMM40, and doubts arose about the reality of the effect of this gene on the risk of developing AD. However, Roses does not doubt the correctness of his hypotheses and believes that genomic studies that did not confirm his results had insufficient power to detect the coupling of TOMM40 and Alzheimer's disease.

Roses hopes that soon he will be able to back up his results with clinical studies that will be conducted at the company he founded Zinfandel Pharmaceuticals. Together with the Japanese pharmaceutical company Takeda, Zinfandel is currently funding phase III clinical trials (called TOMMORROW), designed to test Roses' ideas in practice. TOMMORROW should assess the risks of developing AD depending on the age of the patient and the variants of APOE and TOMM40. About 6,000 healthy elderly people will be selected to launch the program, and the research will continue for about 5 years.

The program will also investigate the possibility that pioglitazone, a drug for the treatment of patients with type 2 diabetes mellitus, in small doses will delay the development of AD in individuals classified as at high risk of developing Alzheimer's disease. The verification of this idea is caused by the fact that the results of experiments on animals and even on humans have been published, which indicate the ability of pioglitazone to prevent or reduce the pathology and symptoms associated with Alzheimer's disease. Roses thinks a possible mechanism for this is stimulation of mitochondrial division.

Even if it is not possible to get a powerful drug against Alzheimer's disease, there will still be a sense in these trials: by learning to delay the development of AD for at least two years, you can reduce the number of patients in the United States by 2 million people in 50 years, which is very, very good. In addition, the results of these trials will force researchers around the world to take a fresh look at dementia. Such a complex disorder as Alzheimer's disease cannot be studied only from one side, even if it includes ApoE4 or something else. Apparently, neurophysiologists are close to recognizing the limitations of their previous views and partially revising them, directing research along new paths, at the end of which there will be a solution to the problem that deprives us of our mind.

Portal "Eternal youth" http://vechnayamolodost.ru02.07.2014

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