The energy-making organelles called mitochondria (shown in green) that work inside cells to make energy aren’t working as they should in the neurons (shown in red) of people with fragile X syndrome. UW–Madison researchers have identified a protein and gene involved in this mitochondrial dysfunction, as well as a potential treatment. Image by: Minjie Shen

Fragile X syndrome, the most common form of inherited intellectual disability, may be unfolding in brain cells even before birth, despite typically going undiagnosed until age 3 or later.

A new study published today in the journal Neuron by researchers at the University of Wisconsin–Madison showed that FMRP, a protein deficient in individuals with fragile X syndrome, has a role in the function of mitochondria, part of a cell that produces energy, during prenatal development. Their results fundamentally change how scientists understand the developmental origins of fragile X syndrome and suggest a potential treatment for brain cells damaged by the dysfunction.

Xinyu Zhao is a neuroscience professor and neurodevelopmental diseases researcher at UW–Madison’s Waisman Center. Four postdoctoral fellows in her lab led the study.

The study, led by four postdoctoral fellows — Minjie Shen, Carissa Sirois, Yu (Kristy) Guo and Meng Li — working in the lab of the lab of Xinyu Zhao, neuroscience professor and neurodevelopmental diseases researcher at UW–Madison’s Waisman Center, found FMRP regulating a gene called RACK1 to promote mitochondrial function. Using a drug to enhance mitochondrial function, they were able to rescue brain cells damaged by lack of FMRP.

Individuals with FXS may present developmental delays — not sitting, walking or talking at expected ages — as well as mild to severe intellectual disability, learning disabilities and social and behavioral problems. About half are also diagnosed with autism spectrum disorder.

In previous research, Zhao found that mitochondria in mice with an FMRP deficiency that imitates FXS were smaller and unhealthy. Diving deeper, they also discovered that FMRP regulates genes involved in mitochondria fission-fusion, a process in which mitochondria fuse into a bigger shape in order to produce more energy for the cell.

For the study, researchers grew brain cells called neurons grown from induced pluripotent stem cells. Because the stem cells came from people with FXS, the researchers could study the development of the disorder at a cellular level, determining whether mitochondria in human cells experienced issues similar to those in mice.

“And indeed, we found that human neurons also have fragmented (smaller) mitochondria,” Zhao says. They also found fewer mitochondria in neurons derived from FXS patients, which they did not see in the neurons of the mice modeling FXS.

“In human neurons, it’s a deficit in twofold. Not just fission-fusion, but also likely in the production of mitochondria,” Zhao says.

Although it has long been known that FMRP is deeply involved in FXS, the new discovery pinpoints a role for the protein in early development of the condition.

Symptoms of FXS present long after the baby is born. Many babies appear to be developing typically before showing slower development, autistic features or developmental deficits. Children with FXS are typically diagnosed at three years of age or older.

“Which means many scientists have been thinking that FMRP is more important for the postnatal maturation state,” Zhao says.

FMRP is protein that regulates the use of messenger RNA, sort of a working copy of DNA used to produce the proteins that make things happen in cells. The researchers found that many of the mRNA strands that interact with FMRP are implicated in autism, providing a molecular link between FXS and autism spectrum disorder. Unexpectedly, many FMRP-bound mRNAs are expressed by genes classified as essential — genes that are very busy during prenatal development but less active after birth.

“This means that FMRP has a function in prenatal development that we have not really thought about before,” Zhao says. “The fact that we found that FMRP also regulates prenatal development is really interesting and is actually indicating that what we see in fragile X syndrome, some of the effects already happened within the prenatal development.”

One of those essential genes is RACK1, identified for the first time as playing a role in FXS.

“When RACK1 is lower in fragile X neurons, the mitochondria are suffering and the neurons exhibit mitochondrial deficit and hyperexcitability, like immature neurons. But when we reintroduce RACK1, we can rescue this,” Zhao says.

Using cultured neurons derived from individuals with FXS to screen for drugs, the researchers found a drug called leflunomide that corrected mitochondrial deficits. The treatment improved mitochondrial function and reduced the neurons’ hyperexcitability.

Next, Zhao wants to do a detailed biochemical analysis of mitochondrial dysfunction and figure out which key proteins are less present in FXS-affected neurons. She is also working on better understanding how RACK1 and leflunomide work to rescue mitochondrial function.

Other collaborators on the study include Waisman Center investigators Qiang Chang, Anita Bhattacharyya, Andre Sousa, Daifeng Wang, Donna Werling and UW–Madison neuroscience professor Jon Levine.

This research was supported by grants from the National Institutes of Health (R01MH118827, R01NS105200, R01MH116582, R01MH118827, R01HD064743, R01NS064025, R01AG067025, U01MH116492, P51 OD011106, U54HD090256, P50HD105353, R24HD000836 and T32 GM141013) and the Department of Defense (W81XWH-22-1-0621), as well as funding from the Brain Research Foundation, Wisconsin Alumni Research Foundation, Brain and Behavior Research Foundation, Simons Foundation, FRAXA Research Foundation, Autism Science Foundation and UW–Madison awards including the Jenni and Kyle Professorship, Vilas Faculty Mid-Career Investigator Award, Kellett Mid-Career Award, SciMed scholarships, Stem Cell and Regenerative Medicine Center postdoctoral fellowship and Hilldale Undergraduate Research Fellowship.

Researchers analyzed genetic material from cells in the prefrontal cortex (the area shaded in each brain) from four closely-related primates to characterize subtle differences in cell type and genetics.

While the physical differences between humans and non-human primates are quite distinct, a new study reveals their brains may be remarkably similar. And yet, the smallest changes may make big differences in developmental and psychiatric disorders.

Understanding the molecular differences that make the human brain distinct can help researchers study disruptions in its development. A new study, published recently in the journal Science by a team including University of Wisconsin–Madison neuroscience professor Andre Sousa, investigates the differences and similarities of cells in the prefrontal cortex — the frontmost region of the brain, an area that plays a central role in higher cognitive functions — between humans and non-human primates such as chimpanzees, Rhesus macaques and marmosets.

The cellular differences between these species may illuminate steps in their evolution and how those differences can be implicated in disorders, such as autism and intellectual disabilities, seen in humans. Sousa, who studies the developmental biology of the brain at UW–Madison’s Waisman Center, decided to start by studying and categorizing the cells in the prefrontal cortex in partnership with the Yale University lab where he worked as a postdoctoral researcher.

“We are profiling the dorsolateral prefrontal cortex because it is particularly interesting. This cortical area only exists in primates. It doesn’t exist in other species,” Sousa says. “It has been associated with several relevant functions in terms of high cognition, like working memory. It has also been implicated in several neuropsychiatric disorders. So, we decided to do this study to understand what is unique about humans in this brain region.”

Andre Sousa Photo by Andy Manis

Sousa and his lab collected genetic information from more than 600,000 prefrontal cortex cells from tissue samples from humans, chimpanzees, macaques and marmosets. They analyzed that data to categorize the cells into types and determine the differences in similar cells across species. Unsurprisingly, the vast majority of the cells were fairly comparable.

“Most of the cells are actually very similar because these species are relatively close evolutionarily,” Sousa says.

Sousa and his collaborators found five cell types in the prefrontal cortex that were not present in all four of the species. They also found differences in the abundancies of certain cell types as well as diversity among similar cell populations across species. When comparing a chimpanzee to a human the differences seem huge — from their physical appearances down to the capabilities of their brains. But at the cellular and genetic level, at least in the prefrontal cortex, the similarities are many and the dissimilarities sparing.

“Our lab really wants to know what is unique about the human brain. Obviously from this study and our previous work, most of it is actually the same, at least among primates,” Sousa says.

The slight differences the researchers found may be the beginning of determining some of those unique factors, and that information could lead to revelations about development and developmental disorders at a molecular level.

“We want to know what happened after the evolutionary split between humans and other primates,” Sousa says. “The idea is you have a mutation in a gene or in several genes and those genes now have slightly different functions. But if these genes are relevant for brain development, for example, how many of a certain cell is produced, or how cells are connecting to other cells, how is it affecting the neuronal circuitry and their physiological properties? We want to understand how these differences lead to differences in the brain and then lead to differences we can observe in adults.”

The study’s observations were made in the brains of adults, after much of the development is complete. This means that the differences may be occurring during the brain’s development. So, the researchers’ next step is to study samples from developing brains and extend their area of investigation past the prefrontal cortex to potentially find where and when these differences originate. The hope is that this information will lead to a more robust foundation to lay developmental disorder research on top of.

“We are able to do extraordinary things, right? We are studying life itself, the universe, and so much more. And this is really unique when you look around,” says Sousa, whose team included graduate students Ryan Risgaards and Zachary Gomez-Sanchez, research intern Danielle Schmidt, and undergraduate students Ashwin Debnath and Cade Hottman. “If we have these unique abilities, it has to be something in the brain, right? There is something in the brain that allows us to do all of that and we are really interested in knowing what it is.”

It’s a significant step in efforts to help people with the disease, says UW–Madison Waisman Center senior scientist Tracy Hagemann, who led the study alongside Albee Messing, professor emeritus of comparative biosciences and founder of the Alexander Disease Lab. With University of Alabama at Birmingham colleague Michael Brenner, Messing discovered the gene responsible for Alexander disease more than 20 years ago.

People born with Alexander disease may develop an enlarged brain and head, experience seizures or delayed development, have stiffness in their arms and legs, and have intellectual disabilities. The disease, which involves destruction of the white matter of the brain, is often not diagnosed until symptoms are pronounced, says Hagemann.

Tracy Hagemann

Albee Messing

The new study, published Nov. 17 in Science Translational Medicine, provided preliminary data instrumental for a human clinical trial currently being led by Ionis Pharmaceuticals. Hagemann, Messing, and the Alexander Disease Lab are not directly involved.

However, working with Ionis Pharmaceuticals, the researchers developed a treatment that consists of small pieces of DNA called antisense oligonucleotides, which in their rat model was able to target mRNA in cells and tag the mRNA for destruction, effectively halting it from creating proteins.

One feature of Alexander disease is the formation of abnormal protein aggregates called Rosenthal fibers, caused by mutations in the gene that makes a protein called GFAP. The connection between this abnormal GFAP and the white matter destruction seen in Alexander disease is not yet clear, but changes in the protein are an intrinsic part of the disease in almost all cases.

Studies with a mouse model developed by Hagemann, Messing and their collaborators, and published three years ago, showed that antisense oligonucleotides were able to reduce GFAP and clear Rosenthal fibers. However, mice display only subtle symptoms of Alexander disease and researchers can’t measure important improvements in behavior or quality of life that may result from treatment.

The research team was able to develop a rat model that better represents the white matter damage and physical manifestations seen in humans. The model also provides better opportunities to assess symptom improvement in response to antisense oligonucleotide treatment.

A hallmark of Alexander disease is the buildup of abnormal proteins called Rosenthal fibers, pictured here (red) in brain tissue from a study of a rat model of the disease. The study identified a potential treatment and helped provide early data for a human clinical trial now underway. Tracy Hagemann et al.

“Alexander disease is considered a leukodystrophy, where white matter deficits develop, and we don’t see evidence of that or motor impairment in the mouse model,” says Hagemann. “So, for a preclinical model, the rats are much improved compared to the mice.”

The rats treated with antisense oligonucleotides before they developed major physical symptoms stayed virtually indistinguishable from their healthy littermates. When treatment began after the rats were severely impaired, their symptoms not only drastically improved; they also experienced a reversal in some of the damage to their white matter.

The antisense oligonucleotides, she explains, “clear out the GFAP aggregates (or Rosenthal fibers), and not only can we prevent the disease from happening by treating animals at an early stage before they’re really showing significant clinical signs, we can treat them when they’re at their worst and see reversal of some of the disease phenotypes.”

In people, Hagemann says, “we’ll be happy if we can stop the disease from progressing. But if you can actually see some reversal of symptoms that have already occurred, that would be wonderful.”

In addition to creating a foundation for clinical trials, the rat model has also paved the way to study aspects of the disease that are not yet understood, Hagemann notes, including the first opportunity to study the link between the GFAP mutations and white matter deficits in mammals.

These developments have been possible because of Messing’s extensive work on Alexander disease over the last 25 years, as well as the contributions of colleagues around the world, Hagemann adds. Messing’s “dedication and commitment to Alexander disease research has deepened our understanding of the disorder immensely.”

Study co-authors include Robert F. Berman of the University of California, Davis; Mel B. Feany at Brigham and Women’s Hospital, Harvard Medical School and Boston Children’s Hospital; and Ming-Der Perng of National Tsing Hua University. The study was supported by grants from the National Institutes of Health, including the National Institutes of Health’s Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD076892, HD03352, HD090256, and HD103526) and the National Institute of Neurological Disorders and Stroke. It was also supported by Children Living with Inherited Metabolic Disease and the Juanma Fund. 

In a new study addressing these hurdles, University of Wisconsin–Madison researchers demonstrated a proof-of-concept stem cell treatment in a mouse model of Parkinson’s disease. They found that neurons derived from stem cells can integrate well into the correct regions of the brain, connect with native neurons and restore motor functions.

The key is identity. By carefully tracking the fate of transplanted stem cells, the scientists found that the cells’ identity — dopamine-producing cells in the case of Parkinson’s — defined the connections they made and how they functioned.

Coupled with an increasing array of methods to produce dozens of unique neurons from stem cells, the scientists say this work suggests neural stem cell therapy is a realistic goal. However, much more research is needed to translate findings from mice to people.

Su-Chun Zhang talks with a postdoctoral student in his research lab at the Waismam Center. Photo: Jeff Miller

The team, led by UW–Madison neuroscientist Su-Chun Zhang, published its findings Sept. 22 in the journal Cell Stem Cell. The research was led by Zhang lab postdoctoral researchers Yuejun Chen, Man Xiong and Yezheng Tao, who now hold faculty positions in China and Singapore.

“Our brain is wired in such an accurate way by very specialized nerve cells in particular locations so we can engage in all our complex behaviors. This all depends on circuits that are wired by specific cell types,” says Zhang, a professor of neuroscience and neurology at UW–Madison’s Waisman Center. “Neurological injuries usually affect specific brain regions or specific cell types, disrupting circuits. In order to treat those diseases, we have to restore these circuits.”

To repair those circuits in the Parkinson’s disease mouse model, the researchers began by coaxing human embryonic stem cells to differentiate into dopamine-producing neurons, the kind of cells that die in Parkinson’s. They transplanted these new neurons into the midbrains of mice, the brain region most affected by Parkinson’s degeneration.

Several months later, after the new neurons had time to integrate into the brain, the mice showed improved motor skills. Looking closely, Zhang’s group was able to see that the transplanted neurons grew long distances to connect to motor-control regions of the brain. The nerve cells also established connections with regulatory regions of the brain that fed into the new neurons and prevented them from being overstimulated.

Both sets of connections — feeding in and out of the transplanted neurons — resembled the circuitry established by native neurons. This was only true for dopamine-producing cells. Similar experiments with cells producing the neurotransmitter glutamate, which is not involved in Parkinson’s disease, did not repair motor circuits, revealing the importance of neuron identity in repairing damage.

To finally confirm that the transplanted neurons had repaired the Parkinson’s-damaged circuits, the researchers inserted genetic on-and-off switches into the stem cells. These switches turn the cells’ activity up or down when they are exposed to specialized designer drugs in the diet or through an injection.

When the stem cells were shut down, the mice’s motor improvements vanished, suggesting the stem cells were essential for restoring Parkinson’s-damaged brains. It also showed that this genetic switch technology could be used to fine-tune the activity of transplanted cells to optimize treatment.

Zhang found that neurons derived from stem cells can connect with native neurons and restore motor functions. But more research is needed to translate the findings from mice to people.

The Zhang group and other researchers have spent years developing methods to turn stem cells into the many different types of neurons within the brain. Each neurological disease or injury would require its own specialized nerve cells to treat, but the treatment plans would likely be broadly similar. “We used Parkinson’s as a model, but the principle is the same for many different neurological disorders,” says Zhang.

The work has personal meaning to Zhang. As a physician and scientist, he often receives letters from families desperate for help treating neurological disorders or brain trauma. It’s also an experience he can relate to. Six years ago, Zhang was in a bike accident and broke his neck. When he awoke partially paralyzed in the hospital, his first thought was of how stem cells — which he had already researched for years — could help him recover.

Now, largely rehabilitated after years of physical therapy, Zhang still believes that the right stem cell treatments could, in the future, help people like him and the families he hears from.

To that end, Zhang’s group is currently testing similar treatments in primates, a step toward human trials.

“There is hope, but we need to take things one step at a time,” he says.

This work was supported in part by the National Institutes of Health (grants NS096282, NS076352, and NS086604, MH099587 and MH100031).

With the help of experts at Waisman Biomanufacturing, within the University of Wisconsin–Madison’s Waisman Center, UW–Madison School of Veterinary Medicine Professor Yoshihiro Kawaoka will lead a $3 million effort to produce as many as 1,000 doses of an experimental vaccine that has already been proven to work safely in monkeys.

“The goal is to produce a safe and effective vaccine against Ebola virus for people,” says Kawaoka, a world expert on Ebola and influenza. The vaccine is planned for use in a phase 1 clinical trial in Japan and is the only whole-virus Ebola vaccine candidate under development.

It will be produced at Waisman Biomanufacturing, a specialized facility whose mission is to help translate scientific discovery into early-stage clinical trials. The staff of the facility provides expert help with manufacturing processes, quality control and overall product development in addition to regulatory support.

“Waisman Biomanufacturing produces many different types of biopharmaceutical products, keeping our range of expertise broad in order to serve any University of Wisconsin investigator who has a biological that they wish to bring into the clinic,” says Carl Ross, the facility’s managing director. “We have made many prophylactic and therapeutic vaccines for use in human clinical trials.”

The technology behind the new Ebola vaccine was devised nearly a decade ago by Peter Halfmann, a research scientist in Kawaoka’s lab who is also an expert on the Ebola virus. It is known as “Delta VP30,” and is a form of Ebola virus that is noninfectious and safe to work with under routine laboratory conditions such as those at Waisman Biomanufacturing. The virus is missing a critical gene — one of only eight genes that make up the virus genome — that makes a protein the virus needs to reproduce in host cells.

Vaccines work by exposing the immune system to viruses or parts of viruses. The Delta VP30-based vaccine may offer better protection against Ebola virus than others in the pipeline, Kawaoka says, because it is a whole-virus vaccine. Other Ebola vaccine candidates use vector viruses to ferry a single Ebola protein, a surface antigen, to prime the immune system.

“Here, we have a whole-virus vaccine that presents all the viral proteins to the immune system, which may result in increased and broadened immune responses compared to vaccines that present only a single viral antigen to the immune system,” Kawaoka explains.

The need for an Ebola vaccine is acute. Periodic outbreaks of the disease in sub-Saharan Africa, including an epidemic between 2013 and 2016, caused major loss of life and serious economic disruption in the three countries where it occurred: Sierra Leone, Guinea and Liberia.

The technology devised in 2008 by Halfmann in Kawaoka’s lab provides a safe way to explore countermeasures for Ebola, a disease whose high mortality rate is amplified by a lack of clinically-tested vaccines and antiviral compounds. The Delta VP30 technology has been approved by the National Institutes of Health for use under Biosafety Level 2 conditions and has been utilized safely for a decade to study the basic biology of the virus, identify potential antiviral compound candidates, and make the whole-virus vaccine.

The need for an Ebola vaccine is acute. Periodic outbreaks of the disease in sub-Saharan Africa caused major loss of life and serious economic disruption.

“We have 10 years of experience with this system,” says Kawaoka of work performed in the UW–Madison School of Veterinary Medicine and the Influenza Research Institute (IRI) located in University Research Park. “That includes data that demonstrates that the vaccine does not replicate in and is not pathogenic in animals, including mice with deficient immune systems and nonhuman primates.”

Waisman Biomanufacturing, notes Ross, has a long history of producing experimental vaccines for clinical trials, including for HIV, influenza, hepatitis, herpes and human papillomavirus, among others. In addition to its emphasis on producing vaccines, the lab specializes in gene and cell therapies, including stem cell products.

The Ebola vaccine work at Waisman Biomanufacturing will begin in March, with the clinical vaccine doses for the Japanese trial produced by December of 2018.

The new vaccine project will be the subject of an informational meeting to be held Feb. 27 at 4:30 p.m. at the Friends of the Waisman Center Auditorium on the first floor of the West Annex. The Waisman Center is located at 1500 Highland Ave. Free parking is available after 4:30 p.m. in Lot 82, behind the Waisman Center and accessible from Highland Avenue.

With the help of experts at Waisman Biomanufacturing, within the University of Wisconsin–Madison’s Waisman Center, UW–Madison School of Veterinary Medicine Professor Yoshihiro Kawaoka will lead a $3 million effort to produce as many as 1,000 doses of an experimental vaccine that has already been proven to work safely in monkeys.

Ebola virus swarms the surface of a host cell in this electron micrograph. Like most viruses, Ebola requires the help of a host cell to survive and replicate. Photo: Takeshi Noda, University of Tokyo

“The goal is to produce a safe and effective vaccine against Ebola virus for people,” says Kawaoka, a world expert on Ebola and influenza. The vaccine is planned for use in a phase 1 clinical trial in Japan and is the only whole-virus Ebola vaccine candidate under development.

It will be produced at Waisman Biomanufacturing, a specialized facility whose mission is to help translate scientific discovery into early-stage clinical trials. The staff of the facility provides expert help with manufacturing processes, quality control and overall product development in addition to regulatory support.

“Waisman Biomanufacturing produces many different types of biopharmaceutical products, keeping our range of expertise broad in order to serve any University of Wisconsin investigator who has a biological that they wish to bring into the clinic,” says Carl Ross, the facility’s managing director. “We have made many prophylactic and therapeutic vaccines for use in human clinical trials.”

The technology behind the new Ebola vaccine was devised nearly a decade ago by Peter Halfmann, a research scientist in Kawaoka’s lab who is also an expert on the Ebola virus. It is known as “Delta VP30,” and is a form of Ebola virus that is noninfectious and safe to work with under routine laboratory conditions such as those at Waisman Biomanufacturing. The virus is missing a critical gene — one of only eight genes that make up the virus genome — that makes a protein the virus needs to reproduce in host cells.

Yoshihiro Kawaoka

Carl Ross

Vaccines work by exposing the immune system to viruses or parts of viruses. The Delta VP30-based vaccine may offer better protection against Ebola virus than others in the pipeline, Kawaoka says, because it is a whole-virus vaccine. Other Ebola vaccine candidates use vector viruses to ferry a single Ebola protein, a surface antigen, to prime the immune system.

“Here, we have a whole-virus vaccine that presents all the viral proteins to the immune system, which may result in increased and broadened immune responses compared to vaccines that present only a single viral antigen to the immune system,” Kawaoka explains.

The need for an Ebola vaccine is acute. Periodic outbreaks of the disease in sub-Saharan Africa, including an epidemic between 2013 and 2016, caused major loss of life and serious economic disruption in the three countries where it occurred: Sierra Leone, Guinea and Liberia.

The technology devised in 2008 by Halfmann in Kawaoka’s lab provides a safe way to explore countermeasures for Ebola, a disease whose high mortality rate is amplified by a lack of clinically-tested vaccines and antiviral compounds. The Delta VP30 technology has been approved by the National Institutes of Health for use under Biosafety Level 2 conditions and has been utilized safely for a decade to study the basic biology of the virus, identify potential antiviral compound candidates, and make the whole-virus vaccine.

The need for an Ebola vaccine is acute. Periodic outbreaks of the disease in sub-Saharan Africa caused major loss of life and serious economic disruption.

“We have 10 years of experience with this system,” says Kawaoka of work performed in the UW–Madison School of Veterinary Medicine and the Influenza Research Institute (IRI) located in University Research Park. “That includes data that demonstrates that the vaccine does not replicate in and is not pathogenic in animals, including mice with deficient immune systems and nonhuman primates.”

Waisman Biomanufacturing, notes Ross, has a long history of producing experimental vaccines for clinical trials, including for HIV, influenza, hepatitis, herpes and human papillomavirus, among others. In addition to its emphasis on producing vaccines, the lab specializes in gene and cell therapies, including stem cell products.

The Ebola vaccine work at Waisman Biomanufacturing will begin in March, with the clinical vaccine doses for the Japanese trial produced by December of 2018.

The new vaccine project will be the subject of an informational meeting to be held Feb. 27 at 4:30 p.m. at the Friends of the Waisman Center Auditorium on the first floor of the West Annex. The Waisman Center is located at 1500 Highland Ave. Free parking is available after 4:30 p.m. in Lot 82, behind the Waisman Center and accessible from Highland Avenue.

Using a mouse model for this disease, which in humans involves the destruction of white matter in the brain, a research team led by Albee Messing, director of the UW–Madison Waisman Center, found that a protein behind the symptoms of the disease, called GFAP, is broken down more rapidly in the body than researchers previously found in cell culture studies.

The results were published recently in a new study in the Journal of Biological Chemistry.

Albee Messing

Laura Moody

That’s a paradigm shift, because “the popular idea was that the GFAP protein would not be degraded as quickly,” says Laura Moody, a former postdoctoral researcher in Messing’s lab and first author of the new study. “But nobody had really tested this idea.”

Scientists already knew that GFAP accumulates to excess in some cells within the nervous system, called astrocytes, leading to the loss of motor and cognitive functions in people with the disease, and in some cases even death. However, they previously thought its accumulation was due to the fact that the cells created too much protein and did not break enough of it down.

The new finding could change the way researchers think about and try to solve Alexander disease.

“This study is an essential foundation for figuring out how to reduce or prevent GFAP accumulation in cells,” says Messing, a professor of neuropathology. “Previously, we thought that decreasing synthesis or increasing degradation of GFAP would be the way to go. But now it appears as if the cells have already tried to adapt to the higher levels of GFAP by increasing degradation, so we can now focus on finding ways to decrease GFAP synthesis.”

Moody and Messing worked with colleagues at the UW–Madison Biotechnology Center to calculate the rate at which GFAP protein was being made and degraded in mice with or without Alexander disease mutations.

The UW-Madison Waisman Center

They fed the mice food that contained known quantities of two different versions, or isotopes, of nitrogen, a component of all proteins. The two isotopes — one heavy and one light — are not radioactive and don’t harm the animals.

The heavy isotope of nitrogen occurs very rarely in nature, so usually it makes up only a minute fraction of proteins. But the mice were fed food containing higher-than-normal levels of the heavy isotope so that when the mice ate this food, their bodies absorbed the nitrogen isotopes and used it for several purposes, including to make GFAP molecules.

The researchers then used a technique called mass spectrometry to track the increase of heavy nitrogen in GFAP protein in the mice. Then, they used that information to calculate how quickly GFAP was being made and degraded, or turned over in cells.

“We found that in tissue culture there was no difference in how quickly GFAP was being turned over,” says Moody. “But surprisingly, in the animal models, GFAP was turning over more quickly in animals with Alexander disease mutations than in ones without those mutations.”

The new finding could change the way researchers think about and try to solve the rare, fatal disease.

While not a direct measure of protein degradation, the increased rate of GFAP turnover in mouse models of Alexander disease strongly indicates that degradation is, in fact, increasing as well, says Messing.

Moody says the finding will change the way therapeutics for Alexander disease are devised.

“Now that we know that GFAP is being synthesized and degraded more quickly in Alexander disease, it opens up new avenues for research,” she says. “It seems that if we can slow down the synthesis of GFAP, we should also be able to slow down its accumulation and develop therapies to treat Alexander disease.”

Other authors of the study include Gregory Barrett-Wilt and Michael Sussman, both at UW-Madison. The work was largely supported by donors to the Jelte Rijkaart Fund.

Since newly formed neurons were discovered in the hippocampus more than 20 years ago, scientists have identified the many roles they play in learning and memory. However, mystery continues to surround the genetic controls that regulate the formation of the delicate structures and the chemicals necessary for neural communication, Zhao says.

In a new study of mice, neuroscience professor Xinyu Zhao of University of Wisconsin-Madison identifies genetic activity in the adult hippocampus that causes stem cells to mature into fully formed neurons. Green: neural stem cells, some differentiating into neurons. Red: immature neurons. Image: Yu Gao, Waisman Center, University of Wisconsin-Madison

In the 1990s, Zhao worked as a postdoctoral fellow with Fred Gage at the Salk Institute for Biological Studies in California, site of groundbreaking discoveries on the formation of new neurons in the hippocampus. Until the discovery of this “adult neurogenesis,” scientists thought that the brain essentially had to make do with the supply of neurons it acquired at birth.

The limited supply of new neurons that grows in the hippocampus throughout the lifespan has a role in memory, and perhaps in recovery from degenerative disease.

Zhao, now a professor of neuroscience at UW-Madison, has been probing the adult neurogenesis puzzle for most of her scientific career. “While trying to understand the mechanism that regulates adult neurogenesis, I am also using adult neurogenesis as a model to study brain development and developmental diseases,” she says. “In lab animals, adult neurogenesis provides a source of cells that can show us how a neural stem cell develops into a fully-formed neuron.”

Xinyu Zhao

As might be expected for an organ as fantastically complex as the brain, “neurons come in many varieties,” Zhao says, “and their genetic activity changes as they mature. To really understand this process, we need to know which genes are active, and when.”

Under study, a group of neurons might average out, which would mask key distinctions at different stages. “When you look at individual cells you can see a lot more information than you can in groups,” Zhao says.

Imagine describing the monarch butterfly. Observing the larval stage, you would conclude it’s a caterpillar. Watching the insect stage, you would call it a butterfly. But what would you conclude from looking at a mix of caterpillars and butterflies?

The situation in developing neurons is even more confusing, Zhao says. “There are so many different types of neurons, and the stage of development is also critical for understanding the development process.”

The technology to leap this hurdle, which can isolate individual neurons so their genes can be extracted, was first used on neurons in 2014.

Taking young neurons from the hippocampus of adult mice, postdoctoral fellows Yu Gao and Feifei Wang (now a member of the faculty at Fudan University in Shanghai) isolated 84 single neurons that had differentiated from neural stem cells at least three days before — meaning they had started, but not completed, the transformation to a mature neuron.

Once the data on gene activation was assembled, Brian Eisinger, another postdoctoral fellow with expertise in bioinformatics, performed intensive analysis on the rising and falling activity of thousands of genes.

The V-shaped blue structure is part of the adult hippocampus, where new neurons are produced throughout life in most mammals, including humans. Red: new, immature neurons. Green: mature neurons. Image: Yu Gao, Waisman Center, University of Wisconsin-Madison

The gene activation profiles of individual cells revealed that the developing neurons go through four stages. But the results also revealed hints about the origin of common neurological conditions.

First, the most active genes during the stem cell phase overlapped significantly with genes implicated in Alzheimer’s and Parkinson’s diseases. “It surprised us to see that genes associated with the stem cell life stage are highly represented in these neurodegenerative diseases,” says Zhao. “We do not know the significance, but it’s possible that these conditions are more related to stem cell impairment than we thought.”

Second, the gene exhibiting increased activity during neuron maturation overlapped with genes associated with autism. That picture is complex, Zhao acknowledges, as thousands of genes are affected in some way in autism.

“This was an exploratory analysis that may open a new window on understanding complex disorders like autism,” Zhao says. “Imagine if you unexpectedly saw a mountain lion’s paw print in the forest. Now you would wonder if the lion explained a sudden shortfall of deer in the forest, but you would not know until you looked further.”

“This was an exploratory analysis that may open a new window on understanding complex disorders like autism.”

Xinyu Zhao

Junior biochemistry student Laurel Kelnhofer and graduate student Emily Jobe also contributed to the study, published today in the journal Cerebral Cortex.

Although papers using single-cell analysis of mammalian neurons have begun to appear in the last year, they focused on large populations of neurons, Zhao says. “Ours is the first single-cell analysis in adult-born new neurons. Our challenge was that these are scarce, so we had to work hard to optimize our cell isolation procedure.”

The study could only be performed at a world-class research institution like UW-Madison, Zhao added. “The Comprehensive Cancer Center provided the cell sorter we needed to get started. The Waisman Center, where I study mechanisms of brain development, had state-of-the-art confocal microscopes and an early model of the single-cell analyzer. The Biotechnology Center has the latest instruments for RNA quantification and ultra-fast RNA sequencing. To advance science, we needed all of these instruments — and the scientists who know how to run them.”

The research was funded by the National Institutes of Health, the Waisman Center, the Wisconsin Alumni Research Foundation and the Vilas Trust.