MU scientists develop model to study complex pregnancy disease
By Danielle Pycior | Bond LSC
Researchers have been exploring the complicated and difficult world of pluripotent stem cells for 15 long years on the second floor of the University of Missouri’s Bond Life Sciences Center.
A type of stem cell that can be turned into any cell in the human body and self-replicate, scientist Michael Roberts and his lab have used these cells to study the early stages of pregnancy in the hopes of understanding preeclampsia — the number one cause of maternal mortality worldwide.
Their work has focused on the complex interaction between a mother’s body and the placenta. This temporary organ connects a fetus via an umbilical cord to the uterine wall of the mother to provide nutrients, gas exchange and waste elimination for the developing child.
“The very fact that you can remove the placenta to cure the patient suggests that this maternal condition has its origin in the placenta” said Roberts.
Preeclampsia comes in two main forms, early-onset starting around 20 weeks of pregnancy, and late-onset occurring after about 35 weeks. Symptoms can range from high blood pressure to headaches, fatigue and other forms of stress in the mom. Severe forms of preeclampsia are life-threatening for mother and child, and the only treatment is removing the placenta, which forces the mother to undergo cesarean section, invariably prematurely.
Researchers have struggled to understand this disease for multiple reasons. While the underlying defect is generally believed to be shallow formation of the placenta and failure of certain cells called trophoblasts to invade sufficiently deeply into the wall of the womb, early detection in the first trimester is almost impossible right now, and, by the time doctors can detect it, the placenta has faced so much damage that it is of little use to researchers.
Working with University of Missouri Health Care physician Danny Schust and basic scientists Laura Schulz and Bond LSC’s Toshihiko Ezashi, Roberts’ lab acquired real-world samples of cells related to the disease. Mothers who came into MU Women’s and Children’s Hospital in Columbia diagnosed with early onset preeclampsia agreed to give part of the tissue from their umbilical cord after childbirth, which was then used to grow primary cultures of connective tissue cells. These were reprogrammed into stem cells that are essentially identical to embryonic stem cells. These induced pluripotent stem cells were then converted to trophoblast cells — the cells in the fetus that make up the outer layer of the placenta — and examined for abnormalities associated with the disease.
“If you do this successfully, you essentially recapitulate the early stages of the pregnancy that led to preeclampsia.” Roberts said.
After this, the researchers tested the cells under various conditions to try and figure out the early stages of the disorder, something that had been relatively impossible previously. Since preeclampsia is difficult to detect in early stages and early placentas from such patients are unavailable for ethical reasons, this approach has allowed researchers a unique look at what happens during a preeclamptic pregnancy.
They compared normal pregnancy cells with cells from preeclampsia pregnancies. By putting the cells in incubators, they observed them under different conditions and levels of stress. Under 5% oxygen, the cells acted similarly to the normal pregnancy, but under 20% oxygen, the cells were highly stressed, resulting in their inability to function properly.
“So, we essentially showed that at least one feature of these preeclampsia cell lines was that they weren’t able to cope with high oxygen and so invaded poorly,” Roberts said. “What we assume is that in the disease there is some sort of stress occurring that causes these cells to not behave properly.”
Roberts’ lab members Toshihiko Ezashi, a research professor at Bond LSC, and Megan Sheridan, a former MU graduate student in the Roberts’ lab who is now at University of Cambridge in England, discovered that there isn’t one gene that leads to preeclampsia, but a few genes within a large cohort of genes involved in controlling a trophoblast cell’s invasive properties.
While this discovery narrows the search for answers, it still leaves a lot of questions being asked.
“We are still nowhere near creating a test for preeclampsia and we are certainly far from a cure because how can you prevent a disease before you know a woman has it?” Roberts said.
This arduous, complicated and incredibly intriguing work looks ahead to dissecting the genetic abnormalities of the disorder in the hopes of creating a successful test and treatment, which could make all the difference for women worldwide.
“This is a disease that affects a large number of women,” Roberts said. “In this country, women can usually find proper care, but in countries outside the U.S. without hospitalization, the woman is going to die and so is the baby.”
Ten years ago, Lucas Woods stepped into Gary Weisman’s lab with a fresh perspective on P2 cell receptors. Now, as an experienced lab manager, Woods dives deeper into the role of these receptors in a myriad of diseases.
Woods came to the Bond Life Sciences Center after graduating from Missouri State University in Springfield with a degree in cell and molecular biology. He studied P2 receptors as a part of his undergraduate research with Dr. Richard Garrad, who completed his post-doctoral research in Weisman’s lab. Garrad recognized Woods’s interest in the area of study and recommended he continue his research with Weisman at MU.
“The initial plan was to work in Dr. Weisman’s lab for a year or two then enroll in graduate school, but after I started here, one thing led to another and here I am, 10 years later, still doing the same research I have been doing for the past 12 years,” Woods said.
Although Weisman’s lab researches the function of P2 receptors in diseases such as Alzheimer’s disease, autoimmune diseases and oral cancers, they are currently investigating the manipulation of P2 receptors in Sjögren’s syndrome.
Sjögren’s syndrome is an autoimmune disease primarily affecting women at later stages in life where the immune system attacks and damages the salivary and lacrimal glands, reducing the ability to produce saliva and tears. This can lead to dental caries, periodontitis, yeast and bacterial infections, digestive disorders, loss of taste and difficulty swallowing, all of which reduce the quality of life for patients. The progression of Sjögren’s syndrome can also lead to the development of lymphoma.
“Usually people don’t show up to the doctor until they notice they have dry mouth and dry eye,” Woods said. “There’s no real solution for them. There are current treatments that try to alleviate the symptoms, such as artificial saliva and tears, rather than treat the cause of the disease. The idea of this research is to make better treatments.”
Currently, Weisman’s lab is focusing on two drugs that target P2 receptors to systemically reduce the inflammation in the salivary gland and improve saliva flow. The lab uses mouse models of human Sjögren’s syndrome to test the effectiveness of these two drugs in blocking the response of P2 receptors to extracellular ATP, which can exacerbate inflammation if left unchecked.
“We were joking the other day that when people ask us what we do for a living and we say we collect mouse saliva, they don’t believe us,” Woods said.
Rather than trying to generate a specific drug for clinical use, the lab aims to establish the idea that if a safe drug is found that can block these receptors and improve the Sjögren’s-like symptoms in mice, then it may translate as a therapeutic strategy in humans.
After working with Weisman for 10 years, it’s safe to say Woods has learned a lot. But the main lesson that he has taken from his experience is that effective communication of science is key.
“Success in science is as much dependent on your ability to communicate science as your ability to do science,” Woods said. “That communication is often in the form of writing papers for publication or writing grants, but the ability to effectively communicate the work that you did and the work you are going to do is absolutely essential.”
In conjunction with his research, Woods is also taking classes toward completing a graduate degree.
“I’m in a unique position right now where I am both a research staff member in the biochemistry department and I’m a student in the department as well,” Woods said.
Woods’s interest in science sparked when he realized that it could lead to limitless discovery. The idea that he could be the first to discover something that the entire world doesn’t know about yet drives him in his research.
When not in the lab, Woods enjoys running and playing with his two kids, Audrey and Edison.
Eventually, Woods hopes to run his own lab. When asked whether he would continue his research with P2 receptors, Woods said it would be in his best interest because of his long history in the field of study.
“P2 receptors are so ubiquitous in the body, there’s no shortage of questions to ask that need answering,” Woods said.
For David Mendoza, a scientist at Bond Life Sciences Center, it’s not an inconsequential question. He works to decipher the answer in an effort to better fortify the food we eat.
“We need to understand how plants accumulate iron,” Mendoza said. “Iron is really important for plants and for us because iron is a critical part of proteins that help produce energy, especially for plants in photosynthesis. Without iron, plants are sick. Without iron, we get sick.”
His most recent published work further pinpoints where and how a plant senses the amount of iron, focusing on the leaves. Initially, previous researchers believed the iron sensor was in the roots because plants absorb water and nutrients from the ground. Scientists now describe a circuit of sensors in plants where the leaves sense the iron status of the entire plant and tell the roots to continue or stop uptake.
“It’s counterintuitive that plants sense iron in the leaves, but when you think about it, it does make sense,” Mendoza said. “Although the roots are the first tissue in contact with the soil, their sensor is local and can’t control the entire plant. Leaf signals are systemic and can integrate signals from the entire plant to tell the roots the plant status.”
After studying and comparing iron availability in the model plant Arabidopsis using different growth conditions, Mendoza and his team found that the abundance of different proteins, called transporters, were vital. Transporters can move water, nutrients and other molecules within the plant. In the leaves, they found that a lack of one transporter — Oligopeptide Transporter 3 — causes an over-accumulation of iron throughout the plant. Additionally, they found that this transporter mediates a ‘shoot-to-root’ signaling that prevents excess, potentially fatal, iron uptake at the root level.
By moving plants from iron-available conditions to iron-absent conditions, the team tracked the abundance of OPT3 and other genes over time. They found that the responsive genes in the veins of the plants respond quickly to varying levels of iron. Because iron is an extremely reactive metal, and technically a micronutrient, too much can be as bad as too little.
“There has to be a right balance of having the right concentration inside the cells, and how exactly plants achieve this homeostasis is not known,” Mendoza said.
Mendoza predicts he and his team will find several more iron sensors in the roots, leaves and vascular tissues of their plants.
Standing on the shoulders of scientists before him
Mendoza credits the work of researchers before him for providing the toolbox necessary for his research.
“The reason we are where we are right now is because other researchers have found responsive markers for iron deficiency and, over the years, we have found that there are specific markers for roots and for leaves,” Mendoza said. “In the leaves, there are also different markers for different cell types.”
Knowing that there are iron sensing markers in different cell types, Mendoza and his team were able to ask a simple question — which cell responds faster to iron deficiency?
“The toolbox was already there, we just used it to answer this question and we discovered not only the leaves, but the veins are really sensitive to changes in iron deficiency and sufficiency.”
For Mendoza and his team, the challenge lies in the fact that plants don’t like to accumulate iron. They are working to better understand how to convince the plant to safely store more iron in a way that is available for humans.
“The trick in biofortification is to convince the plant to accumulate more iron in available tissues in a safe and bio-available way,” Mendoza said.
However, increasing the amount of iron in plants can have harmful effects if not careful. Mendoza proposes a double strategy to increase iron uptake in plants and express an iron storage mechanism to prevent iron toxicity.
We are what we eat
According to the World Health Organization, 80% of people globally do not have enough iron in their bodies and 30% of people are anemic. Iron is necessary for the body to generate hemoglobin so that red blood cells can carry oxygen to the rest of the body. This is why some people with low iron levels may experience dizziness, weakness and fatigue.
The main contributor to iron deficiency is our diets. Staples such as corn and rice supply a large portion of our calorie intake, however, they don’t have nearly as many nutrients as greens.
In the future, Mendoza also hopes to look into making plants more selective for nutrients even if they are grown in the presence of other toxic elements, such as cadmium or arsenic.
“Plants are one of the very few organisms that can do absolutely everything with raw elements,” Mendoza said. “They can synthesize everything. I want to understand how they are capable of doing that to fortify the food we eat.”
This research was published in the Journal “Plant, Cell & Environment” in March 2018 and was funded by the US National Science Foundation, CAREER, NSF EPSCoR, Department of Energy and the Vietnam Education Foundation Training Program.
Researchers are one step closer to understanding HIV
By Danielle Pycior | Bond LSC
Usually, the human immune system is good at recognizing infected cells and then killing them, but in the case of the human immunodeficiency virus (HIV), the virus has ways to hide. One of the ways is by using a viral protein called Vpu.
Vpu helps HIV survive by hiding the fact that it is infected from its host cells. For the past few years, researchers at the University of Missouri have helped uncover how this works.
“If you delete Vpu, those virus-infected cells are killed more efficiently,” said Marc Johnson, a Bond LSC scientist and professor in the Department of Molecular Microbiology and Immunology at the MU School of Medicine.
Understanding the Basics
Johnson and his lab study the connection between viruses and their hosts, trying to understand how viruses convince the host to allow them to keep replicating. In the case of HIV, Johnson and his colleagues discovered that Vpu only functions with the help of B TrCP, another protein.
“The virus doesn’t do anything by itself,” Johnson said. “It only has nine genes, while we have 30,000. It works by tricking its host genes to doing its bidding for it.”
Yul Eum Song, a graduate researcher in Johnson’s lab, explained that CRISPR technology allowed them to alter the genome to see how the proteins operate. CRISPR is a gene editing technique adapted from the DNA of bacteria that allows scientists to add, remove or edit specific locations in a genome.
“So, we use this to target and knock out genes to see if they’re necessary for the mechanism of HIV,” Song said.
They used CRISPR to remove the two types of B TrCP strands from the genome to see if Vpu would still hide HIV without its help.
“CRISPR has lots of applications, but the simplest, and the one I use is basically just molecular scissors that you can put into cells. It will make cuts in the genome wherever you want,” Johnson said.
By removing a certain gene, researchers can see how cells will react with and without certain proteins. In this case, Johnson and his team discovered that without either type of B TrCP strand expressed in the genome, Vpu couldn’t function, so it couldn’t avoid being killed by the immune system.
The magnitude of the HIV problem
“Thirty-five million people are infected with HIV. There are more people living with HIV today than ever in history, and the number keeps going up every year because once they’re infected, they’re infected for life,” Johnson said.
Johnson said that in terms of public health, HIV is a huge concern. Though treatments have gotten substantially better, they can have nasty side effects and don’t always work for everyone, sometimes resulting in death.
“By understanding the mechanisms of HIV, researchers can help combat the virus and create treatments,” Song said.
Both researchers pointed out how discoveries in the area of HIV can translate into other fields. The knowledge that virologists researching HIV find can help other scientists figure out what questions to ask and what functions to look for.
“We’ve learned about the cell and how our own cells work by studying the virus, but the virus is constantly utilizing and counteracting in ways that we’re still figuring out,” Johnson said.
Looking forward, Johnson and his lab are trying to find compounds that will block Vpu, and consequently allow the immune system to kill the HIV infected cells. Though the virus can never be completely removed from someone’s system, virologists search for treatments that can target and kill those cells that are expressing HIV.
“Part of the process is nailing down how VPU works, and a big part of what my lab is doing now is actually screening and testing compounds that do block VPU activity,” Johnson said. “That means if you have an infected virus, you treat them with this compound, and then they behave just like VPU was not there.”
By understanding how various proteins function in cells, researchers can get closer to understanding ways to combat viruses, and closer to understanding the micro-complexities that exist inside the human body.
This research was published in the Journal“Viruses” in Oct. 2018 and was funded by the National Institute of Allergy and Infectious Disease of the National Institutes of Health.
Joint press release by University of Missouri and Hokkaido University
What causes rats without a Y chromosome to become male?
A look at the brains of an endangered spiny rat off the coast of Japan by University of Missouri (MU) Bond Life Sciences Center scientist Cheryl Rosenfeld could illuminate the subtle genetic influences that stimulate a mammal’s cells to develop as male versus female in the absence of a Y chromosome.
The root of the answer is in the chromosomes of this particular mammal. Males of the Amami spiny rat (Tokudaia osimensis) are not like most therian mammals — a name used to group animals that give live birth including placental mammals and marsupials. Unlike in most mammals, these males have no Y chromosome, which has been shed over eons of evolution. And they only have one X chromosome.
“I’d been interested in these rats for many years now, and it’s unclear how sexual differentiation of the gonads and brain occur in this species since both males and females have a single X chromosome,” said Cheryl Rosenfeld, lead author on the study and an MU researcher.
Since most mammals inherit chromosomes from both parents — an X from our mother and either an X or Y chromosome from our father — the only way to develop as male is to inherit a Y chromosome. That male chromosome contains a sex-determining region Y (SRY) gene that stimulates male sexual differentiation in many mammalian species, including humans. SRY triggers the fetus to become male by producing a protein that binds DNA, which leads to development of testes and subsequent production of testosterone. This steroid hormone then stimulates development of the rest of the male reproductive tract. This surge in testosterone from the testes also programs masculinization of the brain.
Naturally, this got Rosenfeld curious about how the absence of a Y chromosome and SRY might affect gene expression differences in male and female Amami spiny rats. In collaboration with Asato Kuroiwa from Hokkaido University Takamichi Jogahara of the Frontier Science Research Center in Japan, and Scott Givan, associate director of the MU Informatics Research Core Facility, her laboratory received brain samples from both males and females of this species.
They took those brains, isolated the RNA from them and sequenced the samples of the male and females to compare transcripts between the two sexes. Transcripts are the step between a gene and a protein, essentially a piece of RNA that encodes the information to make a particular protein. Subtle variations in the resulting RNA can change the final protein, giving rise to alternative forms that can exhibit increased or decreased potency. Investigators found major differences between males and females.
“Several different transcripts or isoforms encode the same gene, and while there might be, for example, 10 transcripts for a particular gene, some transcripts are more potent than others and have more effect in individual cells,” Rosenfeld said. “When we compared males to females, there were several hundred more transcripts upregulated in males than females. Our thought is that since both have the same sex chromosomes, the resulting differences could be originating from the fact that the males might have more of one of the more potent transcripts.”
Expression differences in such transcripts might also arise due to epigenetic changes, which are alterations that affect the turning on/off of certain DNA regions but without affecting the DNA itself. Potentially, in females, these more effective transcripts are not expressed because of such epigenetic changes.
When the team – including MU student Madison Ortega who is in the MU Initiative for Maximizing Student Diversity (IMSD) program — looked closer, they realized many of the transcripts expressed in males encode for various zinc finger protein genes. In males, these genes can be turned on by SRY and are thought to significantly influence sex development.
“What we think might be happening is the males might be turning on all these other zinc finger transcripts that may compensate for the absence of SRY, so they influence undifferentiated gonad to become a testes and help program the brain to be male. Without these zinc finger protein transcripts, female sexual differentiation of the gonad and brain might result,” Rosenfeld said. “It’s possibly it’s more of a potential gradient within these rodents, where you have to turn on all these other zinc finger protein transcripts in males to stimulate male sexual differentiation and compensate for the absence of SRY.”
With the potential extinction of Amami spiny rats on the horizon, furthering this research has a degree of urgency.
“By elucidating more of the mechanisms in this endangered species, I think it might help us save the species or facilitate them being bred in captivity,” Rosenfeld said.
She hopes this research will continue with her team fully sequencing the genome of the Amami spiny rats to look more closely at what’s going on and how that might lead to a better understanding of the nuances of how animals without a Y chromosome undergoes male sexual differentiation.
“If you move outside the mammals, there are all sorts of sex chromosomes and exceptions like the duckbill platypus who has five sets of X and Y, and they can’t find differences between males and females,” Rosenfeld said. “People are focused on sex chromosomes and the SRY gene that they might be forgetting other contributing factors. By understanding these anomalous species, it opens up the idea that the mechanisms regulating gonadal and brain sexual differentiation are quite complex and not fully understood.”
For scientists, studying a disease presents a puzzle looking for an answer, but there are real people behind the research that may one day cure the illnesses that turned their lives upside down. Chris Lorson and Monir Shababi work on one of these puzzles in Bond LSC.
Find out more about their work and the faces behind SMARD, a rare, often fatal, genetic motor neuron disease in the following story courtesy of the College of Veterinary Medicine.
Monir Shababi, an assistant research professor in veterinary pathobiology, and Christian Lorson, Bond LSC principal investigator, College of Veterinary Medicine professor and associate dean for research and graduate studies, have invested countless hours during the past five years to solving a cruel medical mystery. A family who has endured the agonizing ordeal of having two children born with the same disease has invested funding for the research being conducted at MU’s Bond Life Sciences Center.
The disease is called spinal muscle atrophy with respiratory distress, or SMARD. SMARD is a progressive motor neuron disease that has no treatment or cure. At least, not yet.
Shababi, PhD, and Lorson, PhD, and the Sims family — mother Jill, father Eric, grandparents Grant and Patricia — have teamed up in an effort to change that.
The disease is so rare that it is largely unknown, even to most medical professionals. When you are the parent of a child with SMARD, you are in a daily, nonstop, life-and-death struggle.
It is exhausting. It is frustrating. It is a battle that requires an endless reserve of endurance and willpower. And, it requires cutting-edge, scientific discoveries that are just coming to light at MU’s Bond Life Sciences Center.
Catherine Sims lives on a ventilator and needs around-the-clock care. Yet, now age 5, her life is a victory.
“Our first daughter was born healthy, so we had no idea that we carried such a terrible disease,” Jill Sims says. “Then, our second child, Bobby, — who is named after my dad — was born very small, which was unusual given our family history, and he was very quiet as an infant. Those were the only things I noticed. He was three weeks old and we were driving back from Thanksgiving at my parents’ house. I fed him and put him in his car seat. I checked on him 30 minutes later and he had died. He had aspirated. The disease causes the diaphragm not to work, so he couldn’t breathe and eat at the same time.”
Bobby Sims, born Oct. 31, 2012, died on Nov. 30, 2012. His death was attributed to unknown respiratory failure, and he was considered a victim of Sudden Infant Death Syndrome (SIDS). Catherine Sims was born in August 2013; her diagnosis came four months later.
“I went on to have Catherine next, and then we knew something was up,” Sims says. “Catherine was very similar to Bobby, very small and very quiet. That, of course, led us to figure out something was going on.
“In the period of time when Catherine was having problems and was hospitalized but undiagnosed, Catherine had a test done that put her group of symptoms into a specific category of neuromuscular diseases,” Sims says. “A good friend of mine Googled that category and the search produced a WordPress blog that Lisa Porter Werner had contributed to.”
The blog contained personal stories of families who had children with a disease named SMARD. The goal of the blog was to put SMARD on the radar, for families who didn’t have a diagnosis and needed to find answers as well as find support. Porter Werner had posted her own family’s story.
“My friend forwarded me Lisa’s particular story regarding her two children with SMARD, and the story almost identically matched my own,” Sims recalls.
Porter had read extensively and combed the internet for information and cases similar to those of her children. Porter eventually found a modicum of information about something called SMARD, which had been diagnosed in approximately 60 children.
“Lisa Porter’s blog contained the personal stories of families who had children with SMARD,” Sims recalls. “My friend forwarded me Lisa’s particular story regarding her two children with SMARD, and the story almost identically matched my own.
“The Werner’s first daughter died at six weeks of age. It was called a SIDS case; she just died in her sleep,” Sims says. “They had Silas, their son who is living with SMARD, shortly thereafter and she put him in a sleep study when he was three weeks old. She said, ‘No, my daughter didn’t just die. There was a reason.’ It turned out that Silas was having major breathing problems during sleep.
“I was convinced after reading about Lisa’s family that my two children had SMARD as well, and I asked Catherine’s doctors to test her for it,” Sims says. “Catherine’s test came back positive four weeks later. A year or so later, I connected with Lisa through a Facebook group for families with children with SMARD. We began talking more, once my in-laws funded SMARD research at the Jackson Lab, and continued to talk once we found out about Dr. Shababi’s paper that came out in 2016.”
In order to know what SMARD is, it is important to know what it is not. Despite the obvious similarities in name, spinal muscular atrophy (SMA) and spinal muscular atrophy with respiratory distress have sharp differences.
Both conditions affect the lower motor neuron cells of the spinal cord that control voluntary muscle activities like walking, talking, breathing and swallowing. Both are sometimes characterized as “like ALS in babies.”
SMA, which can range from type 1-4, is caused by mutations in or the absence of the SMN1 gene. SMA typically causes weakness in the core first and the baby or child may present as hypotonic, or having low muscle tone — sometimes called floppy baby syndrome. Babies or children with SMA may eventually develop respiratory compromise over time.
SMA is the leading genetic killer of infants; one in 40 people are carriers of SMA.
SMARD, in contrast, is extremely rare. The exact number of cases is unknown, but it has clearly occurred in more than the approximately 100 children worldwide who now carry that tragic diagnosis. SMARD is branded an “orphan” disease, a term commonly applied to any debilitating medical condition that affects fewer than 200,000 Americans. There is little information and few resources available regarding SMARD.
SMARD is a genetic disease, caused by mutations or loss of the IGHMBP2 gene, Immunoglobulin MU-binding protein 2. The condition is inherited in a recessive pattern, meaning both parents must be carriers of the gene mutation and each parent must pass along a copy of the mutation in order for the child to be affected. In essence, every time two carriers have a baby, there is a one in four chance their child will be affected.
Onset of the disease usually occurs suddenly, in what seems to be an otherwise healthy baby, typically between 6 weeks and 6 months of age. Once the diaphragm is paralyzed, the infant must depend on their accessory muscles to breathe. These muscles also weaken as the disease progresses, until the child needs mechanical ventilation.
Many children die in the first year of life, often in their sleep or from a respiratory illness. Past the age of 1 year, almost all children living with SMARD require a tracheostomy, a ventilator and a wheelchair.
Simply put, SMA usually presents as a hypotonic or “floppy” baby who gradually develops respiratory distress. SMARD presents as a baby in respiratory distress who gradually becomes hypotonic.
SMA and SMARD share a similarity in that both are monogenic disorders, conditions caused by mutations or loss of a single gene. Shababi and Lorson have an established history of working with SMA. Now, their focus is SMARD.
“In 2009 and 2010, a lab at the Ohio State University used a viral vector to introduce the SMN gene in SMA mice,” Shababi, the CVM researcher, says. “The viral vector does not contain the necessary genes required for the virus to cause infectious disease. You can replace viral genes with the specific gene you want and keep only the part of the virus that is required to enter the body, find its receptor and produce the desired protein from the gene it carries.
“They (researchers at Ohio State) put a human SMN gene into a viral vector — adeno-associated virus 9 (AAV9) — that has the potential to pass the blood brain barrier in humans. This virus has the capability to enter into the brain, the spinal cord, muscles and peripheral organs,” Shababi continues. “The AAV9 virus carrying the SMN gene was injected into SMA mice. They were able to rescue the affected mice. That was a huge step toward treating SMA. That vector is currently in Phase 2 clinical trials with AveXis/Novartis.
“With SMARD, there is also a single gene involved in the disease — the IGHMBP2 gene,” Shababi continues. “So, we took a human IGHMBP2 gene, in the form of cDNA, and placed it into the same AAV9 vector and injected it into the brain of SMARD pups that were 2 days of age. Our virus did the job and the SMARD mice were cured.”
“Dr. Shababi posted a paper, I believe in March 2016, that reported the results of her work on SMARD,” Sims says. “Lisa found the paper and contacted Dr. Shababi and had a wonderful reception. They had several very long conversations about what Monir was doing, what she had already been doing, and they immediately had a strong connection.
“Dr. Shababi was very personable over the phone, and was very passionate and very approachable about her work,” Sims relates. “Sometimes, it’s hard to get ahold of people, but Monir answers her own phone, and she was very clear with Lisa about what had already been done, which was pretty cool for us because we didn’t know — we didn’t realize how much work Dr. Shababi and Dr. Lorson had already done on SMARD. We were impressed by how much of a handle they already had on the disease. They were ahead of the game. That was great news for us on the family side; at the time, we were aware of only one other lab in the country — the Jackson Lab in Maine — doing work in this area. We couldn’t believe that, wow, there’s a second lab and they are already in gear, they already have a lot of good things going.
“Then, Lisa got me in the loop with Monir, and I talked to her a few times,” Sims continues. “They were having a funding issue, which is not surprising because of how rare the disease is. When we first learned about the work being done at the Jackson Lab, my in-laws agreed to fund SMARD research at Jackson. After learning what Dr. Shababi and Dr. Lorson were doing, I talked to my in-laws again and asked if they would be interested in funding Monir’s research. My father-in-law and I had a few conversations with Monir and Chris Lorson, and then my in-laws decided to do another fund, this time at Missouri, that started this past December.”
“If you look back a number of years, there has been a gene therapy on the translational side that has had exceptionally powerful results in SMA,” says Lorson. “AveXis now has a Phase 2 clinical trial going for their gene therapy product, which has the potential to be very impactful. It has demonstrated efficacy in SMA, but also provides an important proof of principle for gene therapy as a whole. So, it was really exciting to know that there’s only one gene responsible for each of these horribly devastating diseases, SMA and SMARD. It allows you to consider following a similar path. Knowing that, Monir started developing a project that was gene therapy, gene replacement for SMARD.
“Whenever I talk about this, I give about 110 percent of the credit to Monir,” Lorson explains. “Monir has really been the driver of this entire project. Originally, I said, ‘Monir, I’d really like you to develop this gene therapy for SMARD, I think it’s a really exciting area of research. I’ll check back in about six months.’ When I did, we had the mice, we had the vector and she was doing the experiments. That’s exactly the kind of gumption that you hope to find. She did all of that. My role was to say, ‘Good job, Monir!’
“She was the first author on an important paper in Molecular Therapy published in 2016,” Lorson continues. “Based upon that, and the level of excitement, people found her. Through Facebook and Facebook friends, they started to communicate back and forth. Monir is driving it. Monir is doing it.
“AAV9 is in clinic for a number of other diseases, but every time you put a new gene in, you have to go through the Food and Drug Administration,” Lorson says. “That’s why the process isn’t as simple as it might appear to be. Every single time you change that vector — that gene delivery vehicle — you have to get it approved.”
“My in-laws have been very generous, but you need a lot of capital to do this research,” Jill Sims says. “SMARD is so rare that progress will probably come only from academic research. You really need a lot of support and you need a lot of funding from various sources. Right now, our life continues the same. It’s great that everybody is doing this great research, but you need so much more for a cure. That’s what everybody wants; we want our kids to be normal.
“A day in the life of someone with SMARD is very difficult,” Sims says. “There’s a lot that has to be done to have a normal life, and there are a lot of obstacles to that, so you’re constantly trying to overcome those.
“This disease is devastating,” Sims continues. “It can take away every basic human function: the ability to sit, crawl, stand, walk, talk, swallow, feed oneself, clean oneself, use writing utensils and so on. The disease also makes the person more likely to have respiratory problems since they can’t breathe or even cough on their own. It is hard as a parent. Every day we live with the potential fatality of this disease. If their trach tubes come out, they cannot breathe. These trachs sit in their windpipes, held in by ties, like a tight necklace. It is not secure.
“You may go months without anything happening then, all of a sudden, it’s coming out. When that happens, she may only have 60 or so seconds to live,” Sims says. ”You have to have someone always watching them, either a specially trained nurse or a parent, who is a trained caregiver.
“That’s the hard part that we always live with,” says Sims. “Yes, she looks good, and she goes to school, and she’s in activities, to some degree. We adapt everything so she can do as much as possible. But, she is living with a fatal disease that is non-treatable. We basically just manage her symptoms. We know very well that we could lose a second child. That’s what is hardest on us. Even though there are these great advances, she is alive because of amazing machines. Every day presents the chance that she could die.
“When we take Catherine places, there are always at least 10 machines that go with her,” Sims says. “Everything just takes longer. We have a special van with a lift, because she’s in a wheelchair. You are in the thick of trying to make what is not normal to be normal.
“You can’t just pick up your child and go, you can’t feed them a different way, or put a different outfit on them,” Sims continues. “Those are the silly things I took for granted having had a healthy child before. I just did her hair, brushed her teeth, and put her in whatever, and fed her whatever I wanted. Catherine cannot do that. It’s the small things that you take for granted, and there are so many ‘small’ things. We are fortunate to have excellent in-home nursing care, but this also means that my husband and I have had to sacrifice a lot of our privacy. And, I’ve had to give up a lot of my mothering, because I have someone else that always needs to know what I’m doing. That’s hard.
“So, we want a cure,” Sims states. “We are all in. We are always fighting the disease. Our goal would be to have a cure as fast as possible, because the older the kids get, the less chance you have of curing them. This is a neurologic disease; it is hard to get those nerves back. We realize that our kids may be too old. Catherine will be 5; Lisa’s Silas is 8 or 9. They’re kind of old. The ideal time would be right at birth or shortly thereafter. So, that’s what we want. We want the big places — the big funding sources — to realize how important this is, even though it affects only a small number of people.”
“Our gene therapy vector is a very powerful tool,” Lorson says. “It is early days, in terms of trying to push it to the clinic, but we’re trying to do all the important pre-clinical questions.
“There are a number of questions you have to ask,” Lorson continues. “When do you deliver that kind of vector? Does it work only if you do it right at birth, before disease develops? Can you correct the disease, in other words, once the research animals have the disease, can you bring them back to more of a normal state? Or, once that happens, is it just too late for something like gene therapy? We want to deliver what they want to see, in terms of working hard and getting results out. That is what we are trying to do.
“I want to say, ‘Thank you,’ in the biggest way possible to the Sims family,” Lorson says. “Their generosity is really amazing. We consider this an exceptional honor. We want to be the best stewards they could possibly find, of their trust and of their funds. People go out and raise these funds — in some cases, through car washes and bake sales — so you have to put a particularly high value on those dollars. My fondest hope is that we do that every day.”
If you would like to help in the battle against diseases that could someday be relieved through gene therapy, please visit this page.
It takes a lot to move a discovery from lab bench to an application that can provide therapeutic benefits to those suffering from disease.
Bond LSC’s Chris Lorson is making moves to bridge that gap with the start of Shift Pharmaceuticals. With its formation in March 2017, Lorson adds co-founder and Chief Science Officer of the company to his list of titles that include Bond LSC investigator, professor of veterinary pathobiology and associate dean for research and graduate studies.
Shift Pharmaceuticals builds off of years of progress the Lorson Lab has made in understanding spinal muscular atrophy (SMA), which is the leading genetic cause of infant deaths. The disease causes neurons to die, leading to muscle failure, including those that affect walking, arm movement, and respiratory function. While SMA is technically a rare disease, it is remarkably common, affecting nearly 1/10,000 births.
“It’s a devastating disease for patients and families; while the primary defect in is nerves, this leads to problems in muscles, bones, and other vital systems,” Lorson said. “Historically, the majority of kids who develop SMA do not survive beyond 3-5 years.”
Lorson knew his research had potential for drug development, and MU’s Office of Technology Management & Industry Relations (OTMIR) pursued patent protection for the technology. This process helps safeguard the innovations resulting from the research and allows MU to better attract commercial interest to develop and market medical treatments originating from the technology. But it took a partnership with co-founder Steve O’Connor to get the ball rolling. O’Connor is MU’s Entrepreneur in Residence and has significant experience in starting drug development businesses and now serves as CEO of Shift.
“I never knew how to start a company,” Lorson said. “I would argue most academics don’t – this isn’t part of our traditional training. I sat around not knowing what to do, realizing I had this thing that could do a lot, but the practical steps of setting up a biotech start-up were beyond me.”
“Steve thought this technology sounded really cool, so in late March he submitted paperwork and by the end of March we were a company. Shift’s first grant was submitted 3 days later.”
The goal of Shift Pharmaceuticals is to move their lead compound into the clinic for SMA.
“When you look at the disease, it’s not just one cell type and not just one clinical type of patient,” Lorson said. “It really is a disease that is complex and the idea is to bring more options to the fight.”
With the start of any new business, money is always a necessity. Funding from the advocacy group CURE SMA provided the initial funding for the discovery of this compound, while several other foundations including FightSMA, the Gwendolyn Strong Foundation, and Muscular Dystrophy Association have further contributed to the pre-clinical development. MU’s OTMIR negotiated an option agreement with Shift, giving them the exclusive rights to the technology, which helped them obtain a recent $2.73 million grant from the Department of Defense Congressionally Directed Medical Research Program (CDMRP). This will move the company toward the first phase of investigating a new drug.
The root of SMA
Lorson has spent most of his scientific career chasing the underlying causes of SMA.
That search focused in on a few key genes in those suffering from the disease. Two genes — named survival motor neuron-1 and -2 (SMN1 and SMN2, respectively) — are central to SMA development. In patients, SMN1 is mutated and doesn’t process enough of a key protein (SMN) that helps neurons function. While SMN2 acts as a backup gene for this function, a miniscule change in the SMN2 gene causes it to make less SMN protein than required by the body.
In 2016, the Lorson Lab at Bond LSC produced a compound that increases the lifespan of SMA mice . They targeted the back-up gene, SMN2, to produce more functional protein and discovered an increase of protein causing a significant lifespan extension in treated mice. This discovery showed promise for creating a cure for those with SMA.
Shift Pharmaceuticals will be working on developing a drug that builds off Lorson’s work and targets all forms of SMA.
Shift’s first two employees, Mizzou alumnus Paul Morcos and UMKC alumnae Diane Beatty, will help move toward that goal. With Morcos in research and development and Beatty negotiating regulatory affairs, the company hopes to move the drug toward FDA approval.
Thanks to the recent Department of Defense grant, the next step looks to testing in larger animals and at things Lorson did not consider before.
“The experiments we will be doing are not academic in nature, rather, they are focused on the singular goal of preparing our lead compound for an FDA submission. From a traditional academic lab perspective, these might sound rather boring,” Lorson said. “All of these things are not things you do in an academic setting, but that is exactly the point. This is drug development, not the quest for another paper.”
Within the Business Incubator, MU will provide space for the company as well for their research. This sort of partnership is just another part that may one day help translate vital basic research into future treatment.
“Almost everybody has the possibility of doing something that is translational, it is just envisioning it in a different way,” Lorson said.
By Roger Meissen | Bond LSC After a decade of work, Cheryl Rosenfeld is no stranger to bisphenol A (BPA), and her most recent study challenges the dangers posed by developmental exposure the chemical.
Her results continue to raise concerns about how BPA can potentially turn on or off genes in animals and subsequent effects on that early exposure can have on the development and brains of rats. Their research was published in the journal Epigenetics in July.
Rosenfeld and the University of Missouri joined experts from University of Cincinnati and FDA researchers as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA Consortium project. This collaboration is one of several across the United States meant to judge the chemical’s effect using standardized protocols established by the FDA to determine whether BPA exposure, especially during perinatal life, leads harmful effects.
“This is the first study published since a February 2018 BPA statement that challenges the FDA assertion that there is no concern for BPA,” Rosenfeld said. “We’ve shown using the FDA models and studies done right there at their facility that, indeed, early life exposure to BPA can result in gene expression and epigenetic changes that persist into adulthood.”
The study looked at gene expression changes in two brain regions — the hippocampus and the hypothalamus. The hippocampus is associated with long-term learning and memory and the hypothalamus plays a large role in hormone production that influence both the endocrine and nervous systems and affects diverse behaviors, including socialization, sexual behaviors, and appetite control.
Partners at the FDA/National Center for Toxicological Research fed Sprague-Dawley groups of rats — a standardized animal model in this research — diets of BPA, the synthetic estrogen present in birth control pills, ethinyl estradiol, or a chemical-free diet during a developmental period.
The brains from these animals were sent to Rosenfeld’s laboratory, who took biopsies from specific regions of the brain. They used these samples to evaluate whether a group of 10 genes, shown to be affected by BPA exposure in other studies, was affected by this exposure. They also examined the DNA methylation patterns for the promoters of three of these genes to determine whether prior BPA exposure led to persistent epigenetic changes. Epigenetic modifications do not affect the DNA sequence itself but gene and/or eventual protein expression.
Investigators determined that for several of the genes examined BPA exposure altered the expression pattern relative to animals not exposed to either chemical. Sex differences in gene expression in these two brain regions exists in normal animals, and such differences might thus contribute to masculinization or feminization of the brain manifesting as differences in various behavioral patterns, such as male or female sexual behavior. However, previous exposure to BPA abolished many of these gene expression differences between males and females, suggesting that it could disrupt male- and female-typical behaviors. For a gene, brain derived neural factor (BDNF), involved in learning and memory, BPA exposure led to increased methylation of its promoter, which could affect the expression of this key gene. Hippocampal expression of several genes was associated with prior performance in a test designed to measure learning and memory.
“It has become increasingly apparent that BPA can act as a weak estrogen, but what we’re seeing in these results is that it can elicit other effects in addition to those mirroring estrogen and likely independent of estrogen receptor pathways,” Rosenfeld said.
Initiatives like this and other CLARITY-BPA studies aim to answer questions that may later inform government regulators on how to limit or balance the health effects of manufactured chemicals that end up in the environment and may affect human and animal growth in previously unknown ways. With more than 15 billion pounds of BPA were estimated to be produced in 2013, its ubiquitous use in making plastics, lining cans and other manufacturing is of concern. Rosenfeld hopes a closer look at its epigenetic effects may lead to better regulation of the chemical.
“When people are thinking about the effects of BPA, they need to be thinking about it on a molecular scale,” she said. “These results might be subtle, but they can lead to dramatic consequences with long-standing, irreversible changes. Once BPA exposure resculpts an animal’s brain through DNA methylation and other epigenetic changes, it may be permanent.”
Research at the undergraduate level offers more than meets the eye. With students from every year of their undergraduate careers working in Bond LSC, it’s a great opportunity to acquire skills and experience.
Linda Blockus, head of the Undergraduate Research office in 150 Bond LSC, advises students to get started early and be proactive.
“I encourage students who are interested in research to talk to people and network,” Blockus said. “Talk to your professors, advisors and other students to find out what is available. Then, pursue those opportunities.”
It isn’t all as intimidating as it might appear. Students have a number of resources available to find out more about research on campus.
“There’s no one way to get involved,” Blockus said. “Students can go directly through our website, undergradresearch.missouri.edu, come to our office or go to their professors.”
That’s exactly what students involved in the Freshman Research in Plant Sciences (FRIPS) program have done. Sarah Unruh, a Ph.D. student who serves as a Graduate Student Coordinator for the program, boasts of the program’s ability to guide research-minded students along their path at Bond LSC.
“They do 10 hours of research in lab,” Unruh said. “We try to give them skills that are helpful moving forward, so things like finding papers and keeping up with a lab notebook.”
Each of the students selected for the program works in a lab they find the most interesting, but the program assists with those relationships to help students adjust to the process.
“Students lead the way in which lab they go to,” Unruh said. “They interview with different faculty, but we facilitate the match-making.”
Those interactions and networking opportunities open doors down the line.
“I think what they get the most out of FRIPS is that they’re actually doing science, so they get an idea of what it looks like,” Unruh said. “They’re making connections on a different level than just the classroom with teaching assistants and professors.”
Jenna Bohler — one of the students involved in FRIPS this year — has benefited from its connection-facilitating.
“Paula McSteen, Norman Best and Jenn Brown have taught me so much this year in particular,” Bohler said. “They’ve been great resources whenever I’ve had questions.”
Bohler is about to finish her FRIPS experience and can attest to the program’s influence on her first year at Mizzou.
“I knew coming into college I wanted to be involved in research, and FRIPS allowed me to get involved really early so I have four years instead of two or three,” Bohler said.
And it’s not only helpful in the lab.
“What I’ve learned from FRIPS has helped with my classes,” Bohler said. “I learn things before I’m taught them in class, which makes them easier to understand.”
Some FRIPS students have even extended their research opportunities beyond their freshman year.
“Students have used their time wisely in the lab and then gone on to do summer research programs,” Unruh said.
For those who aren’t freshman but find themselves interested in research, there are a number of programs available.
The Society of Undergraduate Researchers in Life Sciences (SURLS) is a group of undergraduate researchers who meet twice a month to explore the options they have within their field. It helps participants to network, meet people with similar interests and better understand a number of components of research.
Alec Wilken, a junior bioscience major who works in the Holliday lab in the medical school, served as the vice president and will be the president for his senior year. He’s been part of SURLS since he was a freshman and has seen first-hand how it’s shaped his path in the field of research.
“SURLS helped me find what I was interested in,” Wilken said. “We have professors come in, and we visit labs, which helps undergraduates grasp how interesting research on campus really is.”
SURLS provides students with the opportunity to grow throughout their undergraduate careers.
“I stayed in SURLS after joining my lab because it became a vehicle that helped me be better in my lab,” Wilken said.
The organization’s impact has allowed Wilken to uncover the path he wants his career to take, as he now plans to earn a Ph.D.
“I found a home in research, and SURLS helped me do that,” Wilken said.
For those with plans to pursue a Ph.D. in their future, MU’s Initiative for Maximizing Student Diversity (IMSD) is the perfect fit.
The grant is funded by the National Institutes of Health (NIH), but at Mizzou there’s the addition of Express to the program’s title. It stands for Exposure to Research for Science Students, which emphasizes the scientific aspect of the program.
Brian Booton, is the undergraduate director for IMSD-Express at Mizzou, acknowledges the prestige that goes along with being an IMSD scholar.
“It’s a highly selective grant,” Booton said. “There are only 49 programs in the country.”
With stiff competition for the program at universities across the nation, it’s important to focus on the students’ experiences.
“The ways in which IMSD-Express helps students is more than just research,” Booton said. “We try to expose students to the different pathways where further education can take them.”
Part of that is through the way the weekly meetings breakdown.
“I break programing down into three areas: personal, academic and professional development,” Booton said.
Doing so helps guide students in the right direction because it is set up to further their education by developing skills for success.
But it’s not all lectures and typical meetings. IMSD-Express offers a peer mentorship program for underclassmen apprentices to be paired with upperclassmen fellows.
“Even if you have a professor you really admire, there’s some distance there,” Booton said. “Someone that’s only two years older than you is more relatable; it’s spending time with your future self.”
The various research opportunities at Mizzou make it possible for students to supplement their classroom learning in a way unlike any other.
“It’s part of your education,” Blockus said. “Taking advantage of research is a great way to set yourself up for the future.”
For more information and to apply for these opportunities, visit:
Endocrine disruptors alter baby mice calls generations later
By Roger Meissen | Bond LSC
The sounds can seem like a mix between a bird tweet and a high-pitched scream to us, but these vocalizations that baby California mice make are essential to how they communicate with their parents and siblings.
Exposure of grandparent mice to bisphenol A (BPA) and related endocrine disrupting chemicals (EDCs) may alter that communication in their grandoffspring, potentially affecting the communication between pups and their parents and the resulting parental care provided to them.
According to a new study, MU Bond Life Science Center’s Cheryl Rosenfeld and an interdisciplinary team of researchers from the US and Germany looked at how this communication alters from normal patterns across multiple generations of California mice.
“We specifically wanted to see if grandparents were exposed, would that affect the communication of the grandoffspring?” Rosenfeld said. “What we saw was that in some cases, some aspects of their vocalizations became even more pronounced. It might be a response to multigenerational exposure to EDCs or they might be calling more because they aren’t receiving sufficient parental care in an effort to say, ‘hey, you’re neglecting me; please pay attention and provide warmth and nutritional support to me.’”
Studies from Rosenfeld previously found that BPA caused lax parenting and neglect in first-generation mice when their parents were developmentally exposed to the chemical. This chemical acts as an endocrine disruptor and mimics the effect of hormones like estrogen in animals, altering their development. BPA is prevalent in the environment because it’s heavily used in manufacturing and leaches out of our plastics, linings of food cans and dozens of other sources.
The study showed female babies tended to make shorter calls out to parents early on after being born, but as they aged they called out more, and male babies made longer calls in early postnatal periods and spoke more as they aged. These patterns were different from controls not exposed to the chemicals.
“Exposure of the their grandparents to EDC’s is altering these grandoffspring behaviors and that could have important ramifications to human babies and how EDCs might affect their initial form of communication, crying,” Rosenfeld said. “This follow up work is clearly important because children with autism have communication deficits, as evidenced even in their early crying patterns, and altered social skills. We’re always trying to find animal models like this that might explain whether exposure to environmental chemicals is increasing the incidence of autism or autistic-like signs in animal models.”
California mice are an especially useful model for studying behavior changes, because these mice are monogamous and both mom and dad are essential in rearing their pups, similar to most human societies. This allows scientists to potentially extrapolate their behavior changes to humans.
In this study, both female and male grandparents were fed one of three diets — A BPA diet that contained an environmentally relevant concentration of this chemical, an ethinyl estradiol diet or diet free of any EDCs. Ethinyl estradiol is another disruptor found in birth control that mimics the effect of estrogen in the body. All offspring were fed the chemical-free food after being weaned off the parents. They had babies, and these grandchildren were the generation scientists looked at to study their communication.
The grandchildren were recorded with special microphones that could pick up the calls of the babies in isolation booths. These sounds range from communications humans can’t even hear as they are high in the ultrasonic range- greater than 20,000 hertz- to communications that begin in the range of human hearing and then project into ultrasonic range. When researchers lowered the frequency of these high-pitched calls to a range we can hear they sound like a mix between owl screeches and bird tweets (how the vocalizations appear and sound are included below for the reader to decide for themselves) . They then compared them to normal mice, looking at the length of each call and the pattern of the calls, what they refer to as “syllables.” Each syllable is akin to an individual sentence or phrase in humans.
These calls from BPA exposed mice were compared to the ethinyl estradiol and the mice not exposed to any chemicals.
“We’re seeing clear traits emerge in this F2 generation with the vocalizations and I think it lends credence to the idea that these things could tamper with vocalization patterns, which are incredibly important in how pups communicate with each other and their parents, whether it’s because they are trying to get more attention from exposed parents or what we call multigenerational effects in that the exposure of their grandparents directly affected their later grandoffspring traits.”
The study, “Multigenerational effects of Bisphenol-A or Ethinyl Estradiol Exposure on F2 California Mice (Peromyscus californicus) pup vocalizations,” was funded by the National Institute of Environmental Health Sciences Grant (5R21ES023150) and was published in the journal PLOS One June 18, 2018.