PAF Grant Maclean

PAF Awards $50,000 New Research Grant

Ken Maclean, PhD, University of Colorado Denver 

“Chemical Chaperone Treatment to Restore Enzyme Activity in Folding Mutations of Propionyl-Co-A Carboxylase: Towards a Personalized Therapeutic Strategy in Propionic Acidemia (PA)” – In Summer 2020, PAF awarded a $50,000 grant.”  

Propionic acidemia (PA) is a severe life-threatening disease for which there is currently no truly effective treatment. The disease is caused by mutation in one of the two genes that code for the enzyme propionyl-CoA carboxylase (PCC). This enzyme is made up of two different proteins that fold around each other into a complex structure with six of each of these two molecules. This is a very unusual and complex structure for a metabolic enzyme and recent work in our laboratory has found that a number of specific mutations that cause PA cause problems by interfering with the protein folding and/or assembly process leading to a non-functional enzyme and thus the disease. In cells, proteins with complicated folding patterns are often assisted in their folding by other proteins called chaperones. We have observed that a number of mutant forms of PCC can be restored to normal activity if they are helped to fold correctly using these chaperone proteins. In our study, we will examine a number of chemicals that can also function as chaperones and assist with protein folding with a view towards restoring full activity in mutant forms of PCC. This work will initially occur in a bacterial PCC expression system to identify promising compounds and then depending upon progress, move into treating human PCC patient derived cells. These studies have the potential to serve as an initial first step in the rational design of a personalized medicine strategy for patients with specific mutations causing PA.

PAF research summary Elango

PAF Awards $44,253 New Research Grant

Rajavel Elango, PhD, University of British Columbia

“Optimizing amino acids in medical foods to manage propionic acidemia”  

Propionic Acidemia (PA) is primarily caused by an enzymatic defect, propionyl-CoA carboxylase (PCC), in the catabolic pathway of valine, isoleucine and other propiogenic precursors. The dietary management of PA mainly depends on protein restriction from food to reduce supply of propiogenic amino acids, and the use of special medical foods. These medical foods contain all essential amino acids and nutrients, but no propiogenic compounds. Recently, concerns have been raised about their use, due to the imbalanced content of the Branched Chain Amino Acids (BCAA) – high leucine, to minimal or no valine and isoleucine. The imbalanced mixture of BCAA negatively impacts plasma concentrations of valine and isoleucine, and has been proposed to affect growth in pediatric PA patients. 

In an ongoing retrospective natural history study (n=4), patients with PA treated at our center from birth (or diagnosis) to age 18y, we observed that higher intake of medical food (compared to intact protein) results in lower ht-for-age Z scores. Based on these pilot data, we propose that there is an immediate need to determine the optimal amounts of leucine to be present in the medical foods.

Therefore, the specific objectives of the current study are to:

  1. Stable isotope studies
    1. Determine the ideal ratio among BCAA in children using the stable isotope-based indicator amino acid method to optimize protein synthesis in a Proof-of-Principle approach.
    2. Test the ratio among BCAA using the same stable isotope-based method in our cohort of PA patients to determine impact on protein synthesis, and plasma metabolite responses.
  2. Determine the impact of the use of natural (intact) vs formula (medical food) protein on anthropometric, biochemical and clinical outcomes via a retrospective natural history study of PA patients treated at BC Children’s Hospital.

Recent dietary guidelines for PA are discouraging the reliance on medical foods as a sole dietary source. However most individuals with PA are at risk for malnutrition and depend on these medical foods as an easy tolerable source of energy and protein. Thus, determining the optimal ratio of BCAA in PA medical foods is necessary to optimize protein synthesis, promote anabolism, growth and prevent the accumulation of toxic metabolites. 

Our laboratory, equipped with use of novel stable isotope tracers to examine protein and amino acid metabolism, is ideally suited to address the question of the ideal BCAA ratio to be used for dietary management of PA and potentially impact health outcomes.

 

Propionic Acidemia Foundation Research Grant – Richard

PAF Awards $33.082.12  Research Grant in 2019

PAF Awards $30,591  Continuation Grant in 2020

Eva Richard, PhD, Universidad Autonoma de Madrid, Spain

“Cardiomyocytes derived from induced pluripotent stem cells as a new model for therapy development in propionic acidemia”

Understanding the cellular and molecular mechanisms that occur in genetic diseases is essential for the investigation of new strategies for their prevention and treatment. In this context, induced pluripotent stem cells (iPSC) offer unprecedented opportunities for modeling human disease. One of the fundamental powers of iPSC technology lies in the competency of these cells to be directed to become any cell type in the body, thus allowing researchers to examine disease mechanisms and identify and test novel therapeutics in relevant cell types.

The main objective of this project is focused on the generation of human iPSC-derived cardiomyocytes (hiPSC-CMs) from propionic acidemia (PA) patients as a new human cellular model for the disease.In PA, cardiac symptoms, namely cardiac dysfunction and arrhythmias, have been recognized as progressive late-onset complications resulting in one of the major causes of disease mortality. Using hiPSC-CMs we will study cellular processes, such as mitochondrial function and oxidative stress which have been recognized as main contributors for PA pathophysiology. In addition, our aim is to unravel novel altered pathways using high-throughput techniques such as RNAseq and miRNA analysis. We will also examine the potential beneficial effects of an antioxidant and a mitochondrial biogenesis activator in PA cardiomyocytes. The results that derive from this project will be relevant for the disease providing insight into the affected biological processes, and thus providing tools and models for the identification of novel adjuvant treatments for PA.

Update September 2019 – Eva Richard PhD

There is an unmet clinical need to develop effective therapies for propionic acidemia (PA). Advances in supportive treatment based on dietary restriction and carnitine supplementation have allowed patients to live beyond the neonatal period. However, the overall outcome remains poor in most patients, who suffer from numerous complications related to disease progression, among them cardiac alterations, a major cause of PA morbidity and mortality. In our research, we developed a new cellular model of PA based on induced pluripotent stem cells (iPSC) with the goal of defining new molecular pathways involved in the pathophysiology of PA which would be potential treatment targeting.

Traditionally, disease pathophysiology has been studied in immortalized or human cell lines and in animal models. Unfortunately, immortalizedcells often do not respond as primary cells and animal models do not exactly recapitulate patients‘ symptoms. So far, patients-derived fibroblasts have been mainly usedas cellular models in PAdue to theiravailability and robustness, but they have important limitations.

The ability to reprogram somatic cells to iPSCs has revolutionized the way of modeling human disease. To study rare diseases, stem cell models carrying patient-specific mutations have become highly important as all cell types can be differentiated from iPSCs. We have generated and characterized two iPSC lines from patients-derived fibroblasts with defects in PCCA and PCCB genes. These iPSC lines can be differentiated into cardiomyocytes that mimic the tissue-specific hallmarks of the disease. The presence of PA cardiomyocytes has been easily established by visual observation of spontaneously contracting regions, and the expression of several cardiac markers. We have observed that PCCA-deficient cardiomyocytes present an increase in degradation products and in lipid droplets, and exhibit mitochondrial dysfunction compared to control cells. We further discovered the down-regulation of several miRNAs in PCCA cardiomyocytes compared to control ones, and several miRNAs targets are currently being analyzed in order to investigate underlying cellular pathological mechanisms. Interestingly, we have performed several experiments to analyze the effect of the mitochondrial biogenesis activator, MIN-102 compound (PPAR agonist, derivative of pioglitazone) in cardiomyocytes.

Preliminary results showed an increase in the oxygen consumption rateof PCCA and control cells. In our next steps, we plan to complete the analysis in the PCCA cardiomyocyte line, characterize PCCB cardiomyocytes and to study in depth the therapeutic potential of MitoQ and MIN-102 compounds.

We would like to sincerely thank the Propionic Acidemia Foundation for supporting our research.

Update March 2020

 “Cardiomyocytes derived from induced pluripotent stem cells as a new model for therapy development in propionic acidemia.”

Eva Richard, Associate Professor

There is an unmet clinical need to develop effective therapies for propionic acidemia (PA). Advances in supportive treatment based on dietary restriction and carnitine supplementation have allowed patients to live beyond the neonatal period. However, the overall outcome remains poor in most patients, who suffer from numerous complications related to disease progression, among them cardiac alterations, a major cause of PA morbidity and mortality. In our research, we developed a new cellular model of PA based on induced pluripotent stem cells (iPSC) with the goal of defining new molecular pathways involved in the pathophysiology of PA which could be potential therapeutical targets.

Traditionally, disease pathophysiology has been studied in immortalized or human cell lines and in animal models. Unfortunately, immortalized cells often do not respond as primary cells and animal models do not exactly recapitulate patients‘ symptoms. So far, patients-derived fibroblasts have been mainly used as cellular models in PA due to their availability and robustness, but they have important limitations.

The ability to reprogram somatic cells to iPSCs has revolutionized the way of modeling human disease. To study rare diseases, stem cell models carrying patient-specific mutations have become highly important as all cell types can be differentiated from iPSCs. We have generated and characterized two iPSC lines from patients-derived fibroblasts with defects in the PCCA and PCCB genes. These iPSC lines can be differentiated into cardiomyocytes that mimic the tissue-specific hallmarks of the disease. The presence of cardiomyocytes has been easily established by visual observation of spontaneously contracting regions, and the expression of several cardiac markers. PCCA iPSC-derived cardiomyocytes exhibited an alteration of autophagy process with an accumulation of residual bodies and mitochondrial dysfunction characterized by reduced oxygen consumption and alteration of mitochondrial biogenesis due to a deregulation of PPARGC1A. We also evaluated the expression of heart-enriched miRNAs previously associated with cardiac dysfunction and several miRNAs were found deregulated. Furthermore, we found increased protein levels of Herp, Grp78, Grp75, sigma-1R and Mfn2 suggesting ER stress and calcium perturbations in these cells.

We are planning to analyze PCCB cardiomyocytes to compare the results with PCCA and control data. We are working to obtain mature cardiomyocytes in order to perform electrophysiology studies (K+ currents) using a whole-cell patch clamp method. We are interested in the study of the tissue-specific bioenergetic signature comparing cardiomyocytes derived from control and PA patients´ iPSCs by reverse phase protein microarrays (RPPMA). Future work also includes testing the effect of the mitochondrial biogenesis activator, MIN-102 compound (PPAR agonist, derivative of pioglitazone) and of the mitochondrial targeting antioxidant MitoQ in PA cardiomyocytes.

We would like to sincerely thank the Propionic Acidemia Foundation for supporting our research.

 

 

 

Propionic Acidemia Foundation Research Grant Guofang Zhang

PAF Awards $48,500 Research Grant

Guofang Zhang, PhD, Duke University

“Propionyl-CoA and propionylcarnitine mediate cardiac complications in patients with propionic acidemia”

Energy production is the central cardiac metabolism for continuous mechanical work. An average human adult heart consumes ~ 6 kg ATP/day. ATP storage in the heart is only sufficient to sustain the heart beat for a few seconds. A tightly coupled cardiac energy metabolism from various substrates is critical for sufficient ATP production required by normal heart function.

One molecule of palmitic acid (fatty acid) generates much more ATP than one molecule of glucose does after their complete metabolism.Fatty acids contribute ~70-90% cardiac energy production in normal condition. However, heart still maintains high flexibility of fuel switch in response to various available substrates. Acetyl-CoA is the first convergent metabolite derived from the diverse fuel substrates via different pathways and enters tricarboxylic acid cycle (TCAC) for energy production. Therefore, the level of acetyl-CoA or the ratio of acetyl-CoA/CoA tightly controls the metabolic fluxes from two major fuels, i.e.,glucose and fatty acid, in the heart. Acetyl-CoA or CoA level is also finely tuned by carnitine acetyltransferase (CrAT) that catalyzes the reversible interconversion between short-chain acyl-CoAs and acylcarnitines.Acetylcarnitine level is ~10-100 fold greater than that of acetyl-CoA in heart and is seen as the buffer of acetyl-CoA. CrAT is highly expressed in high energy demanding organs including heart and mediates fatty acid and glucose metabolism possibly by dynamically interconverting acetyl-CoA and acetylcarnitine into each other.The deficiency of CrAT has been shown to change cardiac fuel selection.

Propionic acidemia (PA) is often associated with cardiac complications. However, the pathological mechanism remains unknown. We have demonstrated that high exogenous propionate led to the propionyl-CoA accumulation and cardiac fuel switch from fatty acid to glucose in the perfused normal rat hearts (Am. J Physiol. Endocrinol. Metab.,2018,315:E622-E633). The deficiency of propionyl-CoA carboxylase in PA also induces the accumulation of propionyl-CoA. Next, we will attempt to understand whether and how the elevated propionyl-CoA in the Pcca-/- heart (collaboration with Dr. Michael Barry)could interrupt cardiac energy metabolism by investigating the fuel switch flexibility, CrAT mediated metabolism, and buffer capacity of acetylcarnitine using stable isotope-based metabolic flux analysis (J. Biol. Chem., 2015,290:8121-32). We hope that the outcome of this project will provide meaningful therapeutic recommendation for patients with PA, especially with the cardiac complication.

PAF Awards grant for Dr. Oleg Shchelochkov and Dr. Charles P. Venditti for $32,912

PAF awarded a  $32,912 research grant to Oleg Shchelochkov, M.D. and Charles P. Venditti MD, PhD at National Human Genome Research Institute, National Institutes of Health  – 2018

“Diversion of Isoleucine and Valine Oxidative Pathway to Reduce the Propionogenic Load in Propionic Acidemia.”

Patients with propionic acidemia require lifelong protein restriction. In addition to taking a protein restricted diet, many propionic acidemia patients are also prescribed medical formulas. This dietary approach aims to decrease the intake of four amino acids that can become propionic acid. These four amino acids – isoleucine, valine, threonine, and methionine – are called essential, because they cannot be made in the human body and need to be supplied from foods. Too much protein intake creates a situation where excess can lead to a buildup of propionic acid in the body. On the other hand, limiting these four amino acids too much can lead to poor growth. Therefore, patients’ diets are optimized to minimize propionic acid production while encouraging good growth. We wonder whether it is possible to increase dietary protein intake while minimizing the risk of propionic acid buildup.

To answer this question, we are planning to do a series of experiments in zebrafish. Why use zebrafish? Zebrafish share significant similarity to humans in how they process propionic acid. In addition, zebrafish reproduce and mature quickly, which are very important qualities to help search for new drugs that could be used to treat propionic acidemia. Our zebrafish are kept in a special building where the animals are being cared for by a dedicated team that includes scientists, veterinarians, engineers, aquatic specialists, and many others. They check on fish and feed them several times a day, maintain fish tanks, and keep their water very clean.

This type of facility is unique and had enabled our studies of metabolic diseases in zebrafish. Our ongoing studies have shown that zebrafish affected by metabolic diseases have symptoms that are very similar to patients. Even with treatment, affected fish have difficulty growing, get tired easily, have poor appetites and sometimes perish before adulthood. Using special genomic tools, we are planning to change in how the fish processes protein to direct it away from becoming propionic acid. As we make these changes to the biochemical pathways of propionic acidemia zebrafish, we will be carefully watching how these treatments improve their growth, development, appetite and survival. These experiments will help us understand how we can potentially reduce propionic acid toxicity while helping patients achieve a less restrictive diet.

Interview with Joel Pardo  – Summer  2020

Can you tell me about yourself and how you became interested in science?

I was always interested in the sciences. I think ultimately what propelled me towards a career in science was my research experience at the University of California, San Diego. The mentorship I received from Dr. Joshua Bloomekatz helped me develop the ability to reason scientifically and appreciate the opportunities to grow professionally. I learned from him how to design experiments to answer important scientific questions. We often had lengthy discussions about the direction of my project. He helped me make sense of the collection of observations coming from different sources and nurtured my own independent thinking.In gaining an appreciation for his analytical method of thinking, I began to see myself as someday contributing to scientific thinking as a physician-scientist.

During your training at NIH, you worked on a project to find new treatments using zebrafish. What did you find exciting and challenging about studying zebrafish?

Most people are familiar with mice, which are often used in science to find and test new drugs. Working with mice requires a lot of work to have enough animals needed for an experiment. Zebrafish, on the other hand, can produce hundreds of offspring after one breeding cycle. Zebrafish lay eggs directly into water, which also makes it easier to study them soon after they hatch. Somewhat surprisingly,the zebrafish enzymes that handle propionic acid are very similar to the enzymes in humans. These two properties of zebrafish make them an exciting model to study a disease like propionic acidemia.

One of the most challenging parts of my research in zebrafish was their size. Zebrafish offspring are very small, measuring less than a quarter of an inch. I had to spend a lot of time looking at zebrafish under the microscope and learn how to move them around without hurting them. This can be difficult as these small animals are fragile at this young age.

Can you tell us about your PA project?

Earlier in my work, we were able to get zebrafish, which had mutations in the genes linked to propionic acidemia. I needed to understand what propionic acidemia does to zebrafish. We were able to show that propionic acidemia in zebrafish looks a lot like the disease we see in patients. Fish with propionic acidemia had poor appetite, did not grow well, and had difficulty moving. Using special genetic tools, we then attempted to change how zebrafish processed propionic acid and helped them survive longer. Our preliminary results are proving promising, but more work is still needed.

What are your plans after you complete your training at NIH?

The NIH postbac program is a full-time research award for students that have recently completed a bachelor’s degree and are considering a career in science or medicine. I was fortunate enough to join Dr. Charles Venditti’s lab 2 years ago to work on the zebrafish project under Dr. Oleg Shchelochkov. I thoroughly enjoyed my post-bac experience. Looking back on the past 2 years, I feel the lab, and in particular the mentorship of Dr. Shchelochkov, has facilitated and nurtured my growth as a future physician-scientist with roots in propionic acidemia research. In 2019 I applied to MD/PhD programs at several US universities. After having traveled to over half a dozen states and interviewing at many fantastic universities, I ultimately decided upon the physician-scientist training program at University of Minnesota. As I plan my transition to the program, I am currently looking for winter coats.