Alexander disease: A lifetime’s work in the hope of saving lives

By Emily Leclerc, Waisman Science Writer

Albee Messing
Albee Messing, VMD, PhD

It’s hard to predict how a small decision may make a major impact on a person’s life. For Albee Messing, VMD, PhD, Waisman investigator, the decision to pursue what others deemed a ridiculous idea has led to a potential treatment for a devastating neurological disease.

Messing, professor emeritus of comparative biosciences at the University of Wisconsin Madison and Michael Brenner, PhD, professor emeritus in the University of Alabama at Birmingham’s Department of Neurobiology had been working together for several years. Together, they had been studying how the gene GFAP was regulated in mouse models. After publishing several research papers together, Messing asked Brenner if he would help him with a new study. Messing wanted to study if the overexpression of GFAP resulted in a certain reactive response in the brain. But Brenner initially refused.

The GFAP gene codes for a protein called GFAP, which stands for glial fibrillary acidic protein. This protein is predominantly made in the central nervous system (CNS), primarily in astrocyte cells. Astrocytes are found in both the spinal cord and the brain wherein they perform a range of tasks including acting as a support and immune cell for the CNS. Change in GFAP expression is also a prominent feature in activated astrocytes. “If there is any kind of injury to the CNS – be it a traumatic injury, disease, or genetic defect – GFAP tends to be upregulated in astrocytes,” Brenner says. That upregulation is an important characteristic of astrocytes’ reactive response to injury, which includes an increase in size, forming a scar to protect the injured region, and releasing molecules that aid in recovery.

Messing was curious if the overexpression of GFAP would result in a similar reactive response and he needed Brenner’s help in designing the study. “I told him it was a bad idea and I’m not going to do that,” Brenner says.

This didn’t dissuade Messing. Eventually, after more than six months of constantly asking, Brenner caved and decided that it would be far less effort to design the construct than to keep saying no to Messing.

Together, they designed mice to overexpress the GFAP gene by adding extra copies to the mouse genome. To their surprise, the mice died soon after birth. When they examined the brains of these mice, they were shocked to find clumps of proteins strewn throughout, localized in the astrocytes. “We had, really without intending to, created a mouse that produced these classic lesions of Alexander disease,” Messing says.

Finding the genetic cause of AxD

Alexander disease (AxD) is an extremely rare neurological disorder that can cause the destruction of white matter in the brain. It is progressive and often fatal. The classic marker of AxD is the presence of abnormal protein clumps in the brain called Rosenthal fibers of which a main component is GFAP. At the time of Messing and Brenner’s experiments – the late-1990s – it was suspected that AxD had a genetic cause but no one knew which gene was responsible. Then Messing and Brenner’s mice with extra copies of GFAP appeared with Rosenthal fibers.

Rosenthal Fibers
Rosenthal fibers, a hallmark of Alexander disease, pictured here (red) in the rat model designed by Messing and Hagemann.

“Only one other candidate gene had been examined in the past and it was negative,” Messing says. “So, the question was just hanging out there waiting for somebody to come along and try something.” The fact that the only change they had made in the mice to cause them to have Rosenthal fibers was to add additional copies of the GFAP gene, led Messing and Brenner to suggest that some malfunction of the GFAP gene was what caused Alexander disease.

Messing quickly applied for funding to study GFAP’s relationship to AxD. Messing collected DNA samples from 13 patients who had died from AxD and sent them off to Brenner for sequencing. 12 out of the 13 samples had mutations in the GFAP gene which was causing the GFAP protein to misfold. “The sequencing was just unbelievably definitive,” Messing says. “We had very strong statistical evidence that these were pathogenic mutations and that GFAP mutations accounted for nearly all cases of Alexander disease.” They published their results in 2001. Further research has shown that an estimated 90% of AxD cases are caused by a mutation in the GFAP gene. The cause of the remaining 10% is still unknown.

A shift in research focus

With Messing and Brenner’s discovery of the causative gene behind AxD, several things happened on both the clinical and research fronts. Clinical neurologists now had an easy way to diagnose the disease through a blood test to identify the mutation. Messing decided to dedicate his lab exclusively to researching AxD and research into GFAP and the GFAP protein exploded.

Tracy L. Hagemann, PhD
Tracy L. Hagemann, PhD

Right around this time, Tracy Hagemann, PhD, associate research professor at Waisman, joined Messing’s lab. Hagemann had studied the genetics of rare diseases at Rush University Medical Center in Chicago and was looking to work with animal models to better understand disease mechanisms when she stumbled across Messing’s lab. “I sent him an email and asked if he was hiring. It was too much of a coincidence that he had just discovered the cause of a rare disease with mouse models,” Hagemann says.

She traveled to Waisman, interviewed with Messing, and was promptly hired. She joined his lab as Messing was analyzing the data from the 13 samples Brenner had sequenced. Hagemann has been with Messing’s lab ever since. Messing credits her with being an integral part of his research. Without her, he doesn’t know if his work would have progressed the way it has.

Armed with the knowledge of AxD’s causative gene and Hagemann at his side, Messing jumped on the opportunity to develop a hugely collaborative project involving contributors across the globe to study AxD. Brenner would continue to help with genetics and experts were added in chemistry and GFAP’s molecular pathways. But with this new scientific and analytical capability came the need for a better mouse model.

“We realized the mice that started this all just added a couple copies of GFAP, which wasn’t what was going on. People don’t have duplications, they have mutations,” Messing says. “So, our project was to make a mouse containing mutations that were identical to the human mutations.”

Hagemann developed the DNA constructs for this new model, and with the help of senior technicians Denice Springman and Heide Peickert, Messing and Hagemann were able to develop a more accurate mouse model of AxD. “When we first saw that the mice had the same protein aggregation that people did, it was so exciting. It was actually Albee’s birthday, so I brought him to the microscope to show him the pathology and said, ‘Happy Birthday’,” Hagemann says.

First steps towards a treatment

Various astrocytes with early signs of GFAP pathology (green). One astrocyte is labeled with a red dye to show its processes. Nuclei are blue.

The new mouse model allowed for an important step forward in the research. With a more accurate representation of the disease, Messing and Hagemann could begin the search for effective therapies. While AxD is not caused by an overexpression of GFAP, the levels of the protein still play an important role in disease severity. Higher levels of the abnormal GFAP protein tend to result in more severe cases. A drug that is able to reduce the amount of GFAP may work as an effective treatment. Messing and Hagemann started by testing candidates based on other research reports and scanning through the Food and Drug Administration’s library of approved drugs, looking for any that may have activity against the GFAP protein. Because, then as it is now, there is no approved treatment for AxD.

Messing, Hagemann, and the graduate students and postdocs in Messing’s lab, did not have much luck finding a drug. They identified a few that had an effect on GFAP, but it was not a strong effect and the drugs had a very small safety window. A decade or so of this work yielded very little in the way of results. It did, however, showcase the mouse model’s shortcomings.

Eventually, Messing and Hagemann once again began toying with the idea of developing a more accurate model, this time in rats. This happened to align perfectly with Messing convincing the pharmaceutical company Ionis to develop antisense oligonucleotides to suppress GFAP. “It just so happened about the time we began a collaboration with Ionis, we had started generating what we hoped would be a better model in rats. In the end, those two things came together perfectly at the same time,” Hagemann says.

Antisense oligonucleotides or ASOs are small pieces of DNA that can be used to stop the production of a targeted protein. Messing and Hagemann worked with Ionis to test ASOs designed to halt the production of the GFAP protein. Their hope was that eliminating the protein would treat AxD at its source. “I want to take something that is now progressive and fatal and turn it into something you can manage, like a chronic disease,” Messing says. After initially testing the ASO treatment in mouse models of AxD, Messing and Hagemann provided the first evidence that direct suppression of GFAP could reduce symptoms of the disease.

Then with their newly developed rat model that more accurately portrayed the white matter damage and physical manifestations seen in humans, Hagemann and Messing set about testing to see if their ASOs were effective at treating clinically relevant symptoms. To their surprise and delight, they were extremely effective. Rats treated early with the ASOs showed impressive disease prevention, staying virtually indistinguishable from their healthy littermates. Rats that were treated after they were severely impaired, showed a drastic improvement in symptoms and even some reversal of white matter damage. These remarkable results in two different animal models persuaded Ionis to generate ASOs to target human GFAP for a potential treatment in people with AxD.

Moving into clinical trials

Ionis’ clinical trial is currently conducting a combined phase 1-3 and actively recruiting patients at sites across the country. They started treatment with their first patient in the summer of 2021. Messing is hopeful that the ASO treatment will perform well and be able to help people with AxD.

Lab photo with logo
Albee Messing (left) and Tracy Hagemann (next to Messing) stand in Messing’s lab with other lab members in 2001

Part of that hope comes from Messing’s close connections with the AxD community. Ever since his work on the disease started in the 90s, Messing has kept in close contact with the community. “When we wanted to do the genetics in the late 90s, we needed their permission,” Messing says. “I started developing these contacts and it was important to them that somebody was finally working on Alexander disease. And it was important to us that they are helping us do it. So, I formed a commitment to them early on and kept it.”

Those early relationships have flourished into a dedicated community that Messing works hard to keep informed of all of the latest research in AxD. He used to hold regular webinars and organized family conferences. He wrote a book on AxD and established a Facebook group that now has close to 1000 followers. He sends out monthly newsletters, does a monthly podcast, and posts a weekly graphic on Instagram from the research literature. Messing does all this to keep this small and information-starved community abreast of the most recent developments.

Over the years, the families of patients have contributed a great deal to Messing’s research through their time, money, and dedication. For both Messing and Hagemann the decades of research that has led to this clinical trial may finally provide a way to give back to the community that has supported them for so long.

“In 2018, Messing organized a family conference at the Waisman Center. After the presentations covering our progress using GFAP-targeted ASOs in rodent models, one of the parents raised the point that she was watching her child deteriorate every day. Knowing that the treatment may not be approved in time to help her child, she asked what could be done to move things faster and how could she help,” Hagemann says. “You can never do enough to meet that kind of need. As much as Messing has contributed to the patient community, they have also been a driving force and have contributed significant energy and funds toward our research. If we come up with something that finally helps them, then I will be proud… or let’s just say happy.”

There are still many questions left to be answered. The research is far from over with many molecular mysteries waiting to be unraveled. Messing is now semi-retired and passing the reins of his lab to Hagemann. She is excited at the prospect of continuing Messing’s work which she has been a part of for more than 20 years.  And with all of the information left to uncover, there is no shortage of questions to ask.

“I am very gratified that it worked out the way it has. I think early on, a lot of people like me do things just out of curiosity,” Messing says. “But it’s an added benefit when it turns out to be of real value. It’s nice that people appreciate it and that it has made a difference. “

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