Stem cells help explain the brain development of primates

Brazilian Maria Carolina Nasser Marchetto is part of a group of scientists at the Salk Institute for Biological Studies who are trying to determine the factors responsible for the superior cognitive ability of the human brain.

By Karina Toledo, in Campinas

Agência FAPESP – Understanding what happened during the evolutionary process to give the human brain superior cognitive ability over that of other primates is the goal of a group of researchers at the Salk Institute for Biological Studies in the United States.

Using technology that enables the creation of induced pluripotent stem (IPS) cells from skin cells, followed by their transformation into neurons, the group’s scientists, led by Fred Gage, are comparing the gene expression patterns of human brain genes with that of their closest living evolutionary relatives: chimpanzees and bonobos. They are also investigating how these patterns control the development of brain cells.

Preliminary findings were presented by Brazilian Maria Carolina Nasser Marchetto, former FAPESP scholarship recipient and member of the Salk Institute team since 2005, during the FAPESP-sponsored event “Advanced Topics in Genomics and Cell Biology,” which was held August 4-6, 2014 at the University of Campinas (Unicamp).

In an interview with Agência FAPESP, Marchetto said that the knowledge generated by this type of study can help identify genes that are important for cognition and that could serve as targets for therapies to treat diseases such as autism, schizophrenia and Alzheimer’s.

Agência FAPESP – What research will you be presenting at this event?
Maria Carolina Nasser Marchetto – The project uses IPS cell technology to understand the differences between humans and our closest evolutionary relatives: chimpanzees and bonobos. Instead of using this technology to answer questions about a particular disease, we’ll try to answer questions about evolution.

Agência FAPESP – What kind of questions?
Marchetto – From the perspective of genetic conservation, we are very similar. There is nearly 98% similarity between the human genome and the genomes of non-human primates. The question we try to answer is: how are we different? As neuroscientists, we think that much of this difference is found in the brain because the physiology of other organs is highly conserved in mammals. Studies that compare human brains to those of chimpanzees point to differences in the number of neurons, the types of neurons found, and the degree of dendritic arborization. Certain structures present in the human brain, mainly in the cortex, are not found in the brain of chimpanzees. On example is Broca’s area, which is connected to language. So our plan is to compare the morphology and functioning of human neurons with those of chimpanzee and bonobo neurons, using neurons obtained via IPS cell technology in both cases. It’s an exploratory project whose initial idea is to investigate the differences between these two systems.

Agência FAPESP – And what have your findings been up to now?
Marchetto – During the pluripotency phase, even before inducing differentiation in the parent cells, we’ve already seen a difference in the expression of two proteins: APOBEC-3B and PIWIL2. These two factors act as brakes, limiting the ability of mobile elements, also called jumping genes, to skip within the genome, and they are found at increased levels in human IPS cells. In other words, humans have greater control over the mobile elements that generate genetic variability. However, in chimpanzees and bonobos, these proteins are expressed at lower levels, producing a genome that is genetically more varied. As a result, if we compare two chimpanzees from neighboring regions in Africa, they will have more genetic differences between each other than I, in America, would have with someone from Africa. We’ve published an article in the journal Nature with the hypothesis that the price for human cultural evolution was giving up excess genetic variability. In other words, during the process of evolution, the human genotype gained variability until it reached an ideal point. Mechanisms then appeared to contain genetic evolution, and, simultaneously, cultural evolution began to occur.

Agência FAPESP – Have you reached the point of inducing differentiation in neurons and then comparing human cells with non-human primate cells?
Marchetto – Yes. We started from the assumption that if there is 98% similarity among humans, chimpanzees and bonobos, then there would have to be at least one distinct protein encoding gene, but there would only be a few. We assumed that the big difference would be in the dynamics of gene expression. Studies conducted using post-mortem brain tissue have shown that genes expressed in the pre-frontal cortex of humans and chimpanzees are very similar during the initial years of development. All signs indicate that several genes are expressed earlier in chimpanzees and diminish over time, whereas in humans, expression begins more slowly but continues to develop, allowing the neurons to acquire characteristics of maturity for a longer period of time. We are using in vitro and in vivo experiments to investigate this assumption.

Agência FAPESP – How?
Marchetto – We transplanted the parent cells of human and chimpanzee neurons into the brains of mice and are evaluating the developmental stage of the neurons after two, four, six, eight, 19 and 26 weeks. We’re comparing the time course of morphological changes associated with the degree of maturity, such as the cell body size, dendrite length and degree of arborization. We are also looking at the density of the spines, which are where the neurotransmitters that allow communication between neurons are released. More mature neurons have higher numbers of spines. Analyses conducted during the second week revealed that the human neuron is less developed that that of the chimpanzee. Between the sixth and eighth week, this relationship begins to reverse. The neurons of chimpanzees remain at a static stage of development, or sometimes even regress, whereas the human cells continue to develop until the 26th week.

Agência FAPESP – And what have been the results in vitro?
Marchetto – We have just received test results and still need to analyze them. However, we are looking at messenger RNA expression, which is the signal that gives rise to proteins, to identify any differences. In vitro results are more difficult to analyze over a prolonged period because neurons do not have enough support to survive for 26 weeks. However, we have been able to reach eight weeks. It’s also more difficult to evaluate morphology in vitro because we’re dealing with a veritable cellular tossed salad. During in vivo experiments, we’re exclusively comparing pyramidal neurons of the cerebral cortex.

Agência FAPESP – What’s the next step?
Marchetto – These preliminary findings have opened the door to several paths for further investigation. With regard to pluripotent cells, for example, we plan to determine why those two proteins are expressed at lower levels in chimpanzees and bonobos. What regulates this process? We’re also studying cells from a group of Brazilian patients who were recently discovered to harbor a mutation that prevents expression of the APOBEC-3B protein. We’re going to create IPS cells from these patients to study what happens to these mobile elements in the genome without this protein.

Agência FAPESP – What types of benefits can this knowledge of the differences between human and primate brains offer?
Marchetto – If we’re able to understand how evolution occurred and how humans and chimpanzees differ, the genes that are important for cognition will become clear. This knowledge can be used to identify therapy targets for diseases that affect cognition. It is basic scientific knowledge that facilitates the process of developing new drugs.

Agência FAPESP – Are you also using IPS cell technology to study the diseases that affect the brain?
Marchetto – The biggest problem we’ve faced in studying neurodegenerative and neuropsychiatric diseases or diseases of development, such as autism, is that we don’t fully understand which mutations are involved. It is therefore hard to create animal models to study mechanisms or test new drugs. However, IPS cell technology allows us to use cells from the patient herself as a study model. Thus, the mutation will certainly be present, even if we do not know its location. We can take the skin cell of a patient, induce pluripotency and then differentiation into a neuron parent cell, then a neuron. This process allows us to compare neurons from a group of volunteers with a severe form of autism, patients who don’t speak and whose brain has grown as they develop, with neurons from a control group of neurotypical volunteers.

Agência FAPESP – What features are you comparing?
Marchetto – The number of connections that each neuron forms, the number of cell divisions and the neuronal communication pattern are some examples.