One mutation, many challenges: Unraveling the mystery behind fragile X syndrome

By Charlene N. Rivera-Bonet, Waisman Science Writer

Xinyu Zhao Lab
Waisman Center July 12, 2022. (Photo © Andy Manis)

One mutation in a single gene, as straightforward as it may sound, can cause a broad range of symptoms and severity among those who carry it. This is the case for fragile X syndrome (FXS), the most common hereditary form of intellectual disability. The exact function of the gene that causes fragile X syndrome remains a mystery, but it is one of Waisman Center investigator Xinyu Zhao’s goals to unravel it.

Fragile X syndrome is caused by a single genetic mutation in the FMR1 gene on the X chromosome.  FMR1 makes a protein called FMRP that is important for brain development, but individuals with FXS fail to produce this protein. Under a microscope, the tip of their X chromosome can appear “fragile,” which gives the condition its name. It is more common in males with approximately 1 in 4,000 versus 1 in 7,000 for females. The diversity of characteristics presented by individuals with FXS makes finding and developing treatments a little trickier.

Xinyu Zhao, PhD, professor of neuroscience and Jenni & Kyle Professor in Novel Neurodevelopmental Diseases, is taking multiple novel approaches to understand the role of FMR1 in human brain development, and seeking ways to tailor treatments based on the characteristics of the patients for optimal outcomes.

Individuals with FXS can present developmental delays, learning disabilities, and social and behavioral issues. About half of the individuals with FXS are also co-diagnosed with autism spectrum disorder (ASD). “Because [FXS] is a single gene disease, it provides a window of opportunity for us to understand autism, which is even more diverse in its phenotype and genetics,” Zhao says. An individual’s phenotype is the set of observable characteristics that result from the interaction between their genes and their environment.

Zhao’s lab focuses on understanding the function of FMR1 gene in brain development by studying mutant mice that do not have FMRP and by investigating the characteristics of neurons derived from induced pluripotent stem cells (iPSC) of individuals with FXS, with the hopes of developing better treatments.

Zhao’s work in FXS started somewhat serendipitously. She had an interest in the formation of neurons from stem cells – or neurogenesis – in the post-natal brain, and how that contributes to the process of learning and memory. During a symposium, she met scientists who worked on FXS and learned that individuals with the syndrome had lifelong learning and memory deficits. “So, I decided to investigate whether in fragile X the learning and memory deficits may have something to do with neurogenesis in post-natal adult brains,” Zhao says.

Zhao’s work in the field started with her showing that FMRP is important for neurogenesis and the deletion of the gene led to impaired learning and memory in mice. “These mouse models give us a platform to try to understand what happens when this gene is not expressed in human neurons,” Zhao says. Her work has come a long way since, furthered by the multiple fruitful collaborations at Waisman.

Neurons derived from stem cells of individuals with fragile X syndrome
Neurons derived from stem cells of individuals with fragile X syndrome. The body of the neuron is shown in green, and nucleus are shown in blue. Credit: Yu Gao, PhD.

Repurposing a cancer drug to treat FXS

As Zhao’s research evolved, her lab discovered that the lack of FMR1 led to a cascade of events that affected the pathway that controls adult neurogenesis. They found that FMRP is important for repressing a protein called MDM2, which is involved in the differentiation of neural stem cells. In the absence of FMRP, MDM2 levels are too high.

MDM2 then became a new target. In an attempt to regulate the levels of MDM2, Zhao’s lab used a drug called Nutlin-3, an MDM2 inhibitor. Nutlin-3 and its derivatives are FDA-approved compounds for clinical trials for cancer treatments, since MDM2 also supports tumor formation.

In the mouse models of FXS, Nutlin-3, at a dosage much lower than those tested for targeting cancer, was able to rescue neurogenesis in the brain and reverse the cognitive and behavioral deficits in both young adult and older mice. Yue Li, PhD, and Zhao are co-inventors on a patent for this discovery filed by the Wisconsin Alumni Research Foundation (WARF). This reversal was long-lasting in the young adult mice. The drug, they discovered, alters both the intrinsic properties of adult neural stem cells and the microenvironment surrounding the neural stem cells leading to increased generation of new neurons.

However, there has been some resistance on repurposing cancer drugs such as compounds like Nutlin-3 as an FXS treatment. “Cancer and autism share many common molecular pathways,” Zhao says. She believes that repurposing some of the cancer drugs might lead to faster advancements in developing a treatment for FXS, as there is currently no treatment for the condition.

Different brain waves call for different treatments

Human fragile X neurons (bright cells)
Human fragile X neurons (bright cells) plated onto multi-electrode array (MEA), in which extracellular electrodes (black) are embedded in cell culture plates to measure electrical activities of human neurons. Credit: Carissa Sirois

In parallel to mouse models, Zhao works with human models using iPSCs derived from individuals with FXS. “Using the human model allows us to identify the function of this gene in humans, which will be really important for effective clinical trial development,” Zhao says.

There is a high diversity of phenotypes, drug response, and symptom severity among individuals with fragile X. For example, an autism diagnosis is present in less than half of the patients, and anxiety and social behavior deficits are more severe in certain cases. “So, one question is, how can changes in a single gene yield such differences among different human patients?” Zhao asks.

Craig Erickson, MD, a clinician at Cincinnati Children’s Medical Center, has demonstrated that individuals with FXS show different resting brain electrical activity as measured through noninvasive electroencephalogram (EEG). The electrical activity was classified as low gamma and high gamma. Low gamma versus high gamma FXS patients also present different symptom severity and responded differently to treatment. “And [Erickson] thinks that resting EEG may be a way to stratify patients before clinical trials,” Zhao says. Since clinical trials are expensive, it will be more efficient and cost effective to test the drugs in cultured neurons first. “If we can establish a cellular model for this differential EEG phenotypes of FXS, maybe we can also identify the molecular mechanism underlying these differential phenotypes, then design smartly the kind of drug, combination, or dosage to treat the two different populations of FXS,” Zhao explains.

Erickson, Zhao, and Anita Bhattacharyya, PhD, assistant professor of cell and regenerative biology, started a collaboration to study these differences by characterizing iPSCs derived from the patients. Initially funded by The John Merck Fund, they generated iPSC lines from high gamma and low gamma FXS patients and controls. Carissa Sirois, PhD, the postdoctoral fellow leading this project, has received fellowships from Fragile X Research Foundation (FRAXA), Autism Science Foundation, and UW-Center for Stem Cell and Regenerative Medicine to carry out the initial phase of this study.  The study has now received $3 million in funds from the Department of Defense.

Their preliminary results show that once the stem cells from high and low gamma individuals are differentiated into neurons, the neurons also show different electrical characteristics.

The group will also seek to understand what is happening at the molecular scale, first by doing gene expression analysis, and second by creating three-dimensional organoids. Grown from stem cells, these organoids are given the appropriate nourishing environment to develop into neurons and glia in three dimensions representing the frontal cortex of the brain. They have limited organ functionality, but can be used to more accurately represent the natural environment of the cells in a living organism. This allows the scientist to measure the development of fragile X neurons over long term, since organoids can be kept in culture dishes for months or even over a year allowing for long term differentiation needed for human brain development. Soraya Sandoval, a neuroscience graduate student, has received an NIH Diversity Supplement to Zhao’s NIH grant to work on FXS organoids. Natasha Mendez-Albelo, a molecular cellular pharmacology graduate student, together with Yu Guo, PhD a postdoctoral fellow, is investigating the contributions of other neuronal cell types as well as other genes to the altered electrical activities of FXS neurons.

Neurons derived from stem cells of individuals with fragile X syndrome
Neurons derived from stem cells of individuals with fragile X syndrome. Neuron are seen in green. Red fluorescent protein was introduced as a label in an experiment where the gene expression of these neurons was altered. Credit: Natasha Mendez

This project will integrate the expertise of many scientists such as Daifeng Wang, PhD, assistant professor of computer sciences, and biostatistics and medical informatics, Ari Rosenberg, PhD, associate professor of computational neuroscience, André Sousa, PhD, assistant professor of neuroscience, and Qiang Chang, PhD, professor of medical genetics and neurology, and director of the Waisman Center.

“I think this new project is really exciting. It will provide the first comprehensive analysis of differential phenotypes among fragile X patients and then provide, not only a stellar model that can be used for preclinical testing of drugs, but also reveal the mechanisms that may underlie the differences among this population,” Zhao says.

It also may shed light on how single gene disorders are treated, and provide a template on how to study neurological diseases without even knowing the gene that is causing it by using iPSCs derived from patients.

Extended reach and future directions

Zhao’s work in FXS also extends beyond the walls of the Waisman Center. In addition to all the research advances and important findings Zhao’s lab has accomplished for the field, they have also created cell lines and molecular reagents to study FXS that have been widely distributed into both industry and academic labs. “We are generating important data, but also important resources for the field,” Zhao says.

The Zhao group’s work with Nutlin-3 and patient stratification based on brain waves are two of the many efforts they are running to better understand FXS and find treatments for it. They are also using human FXS neurons developed from stem cells and organoids to test other drug targets they previously identified in mouse models, such as the mitochondrial pathway, a project led by postdoctoral fellow Minjie Shen, PhD, who also received a FRAXA fellowship for the project.

One big question for the field, Zhao says, is ‘can the FMR1 gene be reactivated?’. In addition, gene delivery of FMR1 is also under consideration. However, one big question is, “how early and how much FMR1 should be reactivated or introduced to achieve therapeutic effect? “That’s the other question I would like to ask for the next few years,” Zhao says.

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