Stem cells are the key to understanding Alzheimer’s disease

Lawrence Goldstein of UCSD transforms skin cells into neurons with a pluripotent induction technique to investigate what happens in the brains of people with the disease.

By Karina Toledo

Agência FAPESP – Stem cells will be the key to finding the cure for Alzheimer’s disease. Lawrence Goldstein, professor at the University of California San Diego and one of the leading researchers on the subject, is sure of it.

However, Goldstein sees little future in research aimed at developing therapies for the substitution of defective neurons. To him, stem cells are, in reality, tools that will allow for an understanding of what goes wrong in the brain and leads to development of the disease.

With the help of technology that allows scientists to create induced pluripotent stem cells (IPS), Goldstein developed a method of transforming the cells of Alzheimer’s patients into neurons. The objective, now, is to study the neurons of patients with a hereditary form of the disease to discover the altered biochemical processes that can be manipulated through drugs or genetic methods to reverse the problem.

Visiting Campinas to participate in the workshop “Advanced Topics in Genomics and Cell Biology” – held this past May at the Central Laboratory of High Performance Technologies (LaCTAD) and the Center for Molecular Biology and Genetic Engineering, with FAPESP funding – Goldstein revealed the details of his ongoing research.

Agência FAPESP – You are considered one of the top advocates of stem cell research in the United States. Do you believe that they will allow for the development of a therapeutic treatment for Alzheimer’s disease?
Lawrence Goldstein – This is not my laboratory’s approach. There are groups that attempt to develop therapies for Alzheimer’s using stem cells, but I am skeptical in relation to this. Once the disease sets in and spreads to the brain, I do not believe that it is possible to substitute defective neurons – at least not with the technology that is currently available.

Agência FAPESP – How can stem cells be useful when it comes to Alzheimer’s disease?
Goldstein – They are important tools to understand what goes wrong in the brain and leads to development of the disease. It has always been very difficult to test the hypothesis because the only methods available were animal models, the brains of human cadavers and human non-neuronal cells in which they attempted to mimic the conditions of the disease. Removing brain cells from a live patient with Alzheimer’s to study them is complicated, so we developed a method to create neurons in vitro based on skin cells from the patient – which are easy to obtain through a biopsy. We did this with technology that allows for the creation of induced pluripotent stem cells developed by Shinya Yamanaka (a researcher at the University of Kyoto who won the 2012 Nobel Prize in Medicine for the method). Afterwards, we induced differentiation in neuronal stem cells and, later, in neurons that we can treat with drugs and manipulate genetically to see what happens with the cerebral biochemistry.

Agência FAPESP – Why did you opt for IPS and not embryonic stem cells? For facility and ethical questions?
Goldstein – Embryonic stem cells were used to develop parts of the method. However, the type of genetic experiment that we are doing – to attempt to discover how mutations cause certain effects in neurons – requires the IPS technology. We are attempting to capture the unique genetic architecture of each individual. Instead of doing a brain biopsy on these people, we create neurons using a skin cell. We began experiments soon after Yamanaka published the method in 2007. However, it took time to create the lines of cells, characterize them and test them. We published our method in Nature in 2012.

Agência FAPESP – What experiments have you done so far?
Goldstein – We obtained skin cells from six volunteers – two healthy, two with sporadic forms of the disease, which correspond to 99% of the cases, and two with a hereditary form of Alzheimer’s. Patients with this hereditary mutation have an extra copy of a gene that codifies the amyloid precursor protein (APP). Instead of having two APP genes, they have three, or rather, they produce 50% more of this protein, and this gives them a practically 100% chance of developing the disease around 40 years of age. From the samples of each of the six volunteers, we cultivated three or more lines of IPS cells, totaling 18 lines. We induced differentiation in each of the 18 lines and cultivated a type of cell called neuronal progenitor or neuronal stem cells and then purified it. Afterwards, we induced differentiation in neurons. The preliminary results showed that the biochemical behavior is abnormal in the neurons of patients with the hereditary form of disease. In the neurons of healthy volunteers, we still have not found consistent biochemical abnormalities. However, of the neurons of healthy volunteers with sporadic Alzheimer’s, half were normal, and the other half were altered. We believe that this biochemical abnormality is an important part of the initial phase of the disease.

Agência FAPESP – Is this APP protein related to the formation of brain plaque that is believed to be the cause of neuron degeneration?
Goldstein – The majority of research on Alzheimer’s has been focused on the so-called “amyloid cascade hypothesis.” According to this theory, the amyloid beta peptide (A-beta) – a fragment of the APP protein – tends to aggregate and form plaques that have a toxic effect on neurons, impeding the synapses and leading to cellular death. However, the attempt to prove this hypothesis has failed in all clinical trials with humans and experiments with animals. Pharmaceutical research has attempted to create inhibitors of the gamma secretase enzyme for cleaving the APP. However, this enzyme acts on more than a hundred proteins, which means that the drug alters several biological processes. Despite many side effects, the cognitive function of patients did not improve in the patients that were tested. Another strategy has been to stimulate the immune system and create a response against A-beta peptide in an attempt to clear the brain and avoid plaque formation. This technique also failed to show results.

Agência FAPESP – What is the role of APP in the brain?
Goldstein – There is also a lot of discussion about this. Studies have shown that if you completely remove this protein from the brain of a mouse through genetic manipulation, the animal will not die, but its development will be compromised. There are three different versions of this gene in the genome of all mammals. When you silence the APP gene, the other two take over some of the functions. So, it is difficult to really know what happens. We argue that when you remove the APP protein, there are alterations in the transportation of certain materials to synapses. However, it is not a catastrophic alteration. These are quantitative changes and it is not easy to perceive them in an experiment.

Agência FAPESP – What would be the relationship of physical trauma with the disease?
Goldstein – Most likely, what happens in these situations is interruptions in these paths of long distance communication and APP processing. It could be that APP cleavage is induced, and the resulting products accumulate. It is a very important research path to follow, but nothing has been performed yet because the entire field is struggling with low funding. In the United States, by conservative estimates, people spend US$ 200 billion per year on the treatment of Alzheimer’s patients, and we spend roughly US$ 500 million solely on the type of research that we do to attempt to discover the primary causes of the disease. The ratio is 400 to 1. It is ridiculous. We are throwing money away.

Agência FAPESP – Are the other biochemical alterations related to Alzheimer’s disease?
Goldstein – One of the main changes observed in the brains of people who die of the disease, in addition to amyloid plaques, is the formation of neurofibrillary tangles. Those who defend the A-beta peptide toxicity theory believe that they somehow activate enzymes, which modify a protein called TAU. These enzymes add a phosphate group to the TAU, and this makes this protein aggregate with axon microtubules [through which the essential materials for synapses are transported], forming these tangles. I support the hypothesis that in response to this alteration in transportation of materials, the neuron attempts to remove these tangles that act as small speed bumps. By removing the aberrant TAU protein from the microtubule, however, the behavior of several of the loads is altered, and this could be the beginning of a degenerative process. However, it is still not clear what happens to the normal biology to induce the disease. What we have attempted to discover is how a small change leads to the long, slow decline, and in some people – not all – no hypothesis to date adequately describes the events that lead to this aberrant behavior of the TAU protein. However, the tools are limited. Now, we believe that with real human neurons submitted to the biochemical alterations of the disease in vitro, we can solve the puzzle.

Agência FAPESP – How do you intend to do this?
Goldstein – In the neurons of patients with the hereditary form of the disease, we want to see what the effect of the increased concentration of APP – caused by the extra copy of the gene – on the TAU protein is. Afterwards, we can test drugs to attempt to modify this result. These will certainly be very useful drugs. But, before this drug screening, we need to better standardize the method so that the neurons behave the same way every day in all tests. Our plan is to test 50,000 drugs, which is a small number. When large laboratories do their initial screenings, they evaluate approximately 1 million. This number is what we have managed to do in the academic world.