CURE RTD NEWS

RTD type 2 is caused by mutations in the SLC52A2 gene, which encode for the riboflavin transporter protein RFVT2. Although RFVT2 have been shown to be responsible for transport of riboflavin across cell membranes it remains unclear why lack of this protein specifically damages sensory and motor neurons.  It is Prof. Barile’s hypothesis that the absorption of riboflavin in neuronal cells mediated by RFVT2 is the limiting step of biosynthesis of FAD (Flavin Adenine Dinucleotide), which is an essential cofactor of more than 100 enzymes collectively known as flavoproteins. Flavoproteins are crucial for energy metabolism in neurons and are mostly located in the mitochondria. It is expected that defects in RFVT2 will be the cause of alterations in the whole content of flavin cofactors and mitochondrial bioenergetics, which can result in cellular damage.

Despite the importance of the issue, very little is understood about RFVT2’s role, and the functional consequence of various SLC52 mutations, on mechanisms of transport and metabolism of riboflavin in neurons, as well as the biogenesis of adequate amounts of intracellular flavoproteins. This proposal plans to help address these important questions using several unique model systems, including an animal model and cell samples from individuals with RTD. These findings can provide the fundamental steps necessary toward the improvement of therapies or design of novel drugs for RTD.

Meet Maria Barile

Maria Barile has a broad background in enzymology and riboflavin metabolism.  She is an Associate Professor of Biochemistry and Researcher at the University of Bari Aldo Moro in Italy.  She has been involved in numerous national and international projects over the past 20 years, which have focused on the characterization of human enzymes involved in riboflavin metabolism, as well as in the pharmacological and clinical aspects of riboflavin therapy. She has published 63 peer-reviewed publication and her research group was responsible for cloning the human gene responsible for FAD synthase.

Meet Cesare Indiveri

Cesare Indiveri is an expert in the field of membrane transporters biology with over 120 peer-reviewed publications in top journals including Nature and Cell. He is currently the Director of the Department of DiBEST (Biologia Ecologie Scienze della Terra) and Full Professor in Biochemistry at University of Calabria in Italy. Cesare Indiveri’s research group has been focused mainly on transporters of nutrients such as amino acids, carnitine and riboflavin with specialization in the measurement of transport activity in intact cells, in isolated organelles and in artificial membrane systems called proteoliposomes.

How will this project work?

These research teams will use several unique model systems to address the questions noted above including:

1)  Motor neurons developed using induced pluripotent stem cells (iPSCs) from individuals with RTD type 2 and type 3.

2)  A novel C. Elegans (Roundworm) model in which specific genes related to flavin homeostasis are silenced. 

3)  A model in which the riboflavin transporter RFVT2 is reconstituted in proteoliposomes, which are systems used to mimic lipid membranes.

What is the significance of this study?

These tissues and model systems have the potential to significantly impact the understanding that RFVT2 play in RTD disease processes and may identify novel pathways for therapeutic intervention that could complement RTD-targeting therapies.

Cure RTD Research Funding

This grant to Prof. Maria Barile is part of Cure RTD’s Basic Research and Drug Discovery programs that we’re currently announcing for our 2019-2020 grant cycle.

Basic research is the first step in our comprehensive research model. We fund basic research to investigate the biology and cause of RTD, in order to identify the most effective treatment strategies. We also use this funding to develop tools that facilitate RTD research.

Over the past few years it has become clear that riboflavin treatment merely slows the disease progression of riboflavin transporter deficiency (RTD) rather than completely halting disease progression in a some patients.  In addition, the long-term outcomes for all RTD patients on riboflavin treatment alone remain unknown.  Hence, there is a great need to identify complementary, novel therapeutic strategies that can act synergistically with riboflavin treatment to halt RTD disease progression.

While it is known that the riboflavin transporters RFVT2 and RFVT3 are responsible for the transport of riboflavin in the body, the exact mechanism by which riboflavin deficiency causes neuronal damage in RTD is not understood. The development of an animal model with RTD will enable the investigation of these mechanisms, in an effort to subsequently develop novel therapeutic strategies for RTD.

In this proposal, Dr. Manoj Menezes and his team will develop an animal model of RTD using Caenorhabditis elegans (Roundworms) to clarify the underlying mechanism of neuronal injury in RTD.  Areas of investigation in this model will include neuronal and muscle morphology, mitochondrial function, synaptic transmission, life span as well as motor and sensory behaviors.  This will be the first RTD animal model to carry the genetic mutations which are the human equivalent to SLC52A2 gene mutations causing RTD type 2.

Meet Dr. Manoj Menezes

Dr Manoj Menezes is considered an international expert in the pathophysiology and therapy of RTD, having published several research papers describing its phenotype, pathophysiology and use of disease-modifying therapy.  Dr. Manoj Menezes completed his MD in Paediatrics from MS University, Baroda, India and completed his PhD in inherited peripheral neuropathies including RTD from the University of Sydney.  He is a Staff Specialist in Neurology and Neurogenetics at CHW and a Clinical Senior Lecturer at the CHW Clinical School at The University of Sydney.  He is also Director of the Peripheral Neuropathy Management Clinic, CHW and co-team leader of the Neuropathy Group at the Kids Neuroscience Centre, Kids Research.

How will this project work?

Caenorhabditis elegans (C. elegans) is one of the most widely used animal model systems to address questions in neurobiology.  C. elegans possess a simple, but extremely well-defined nervous system that functions similarly to mammalian systems.  In this project, C. elegans  will be generated carrying mutations in the worm riboflavin transporter gene (rtf-1),  which is the human equivalent for the SLC52A2 gene causing RTD type 2.  This model will enable the study of neurons and other areas affected by RTD at different developmental stages.

What is the significance of this study?

Findings from this study have the potential to significantly impact our understanding of how mutations in the SLC52A2 genes cause neuronal damage in individuals with RTD.  This animal model may help identify novel pathways for therapeutic intervention, leading to the development of complementary novel therapeutic strategies that can act synergistically with riboflavin treatment to halt RTD disease progression.  In addition, once validated this animal model can be used for future studies to test novel compounds to treat RTD.

Cure RTD Research Funding

This grant to Dr. Manoj Menezes is part of Cure RTD’s Basic Research and Drug Discovery programs that we are currently announcing for our 2019-2020 grant cycle.

Basic research is the first step in our comprehensive research model. We fund basic research to investigate the biology and cause of RTD in order to identify the most effective treatment strategies.

One of Cure RTD’s primary goals is to support research that identifies disease-modifying therapies that can act synergistically with riboflavin to stop disease progression.  Clinical trials of disease-modifying therapies for RTD require a clear understanding of natural disease progression of RTD and ability to measure the benefit of any intervention.  However,  reproducible, valid and responsive outcome measures of disease progression in RTD currently do not exist.

In this project, Dr. Manoj Menezes and his team will develop and validate a consistent method to evaluate children and young adults with RTD.  Once developed, this tool will be made available free-of-charge, worldwide, to be used by clinicians for tracking RTD patient progress and also for human clinical trials of novel therapeutic strategies, to understand if they truly modify disease and improve patient outcomes.

Meet Dr. Manoj Menezes

Dr Manoj Menezes is considered an international expert in the pathophysiology and therapy of RTD, having published several research papers describing its phenotype, pathophysiology and use of disease-modifying therapy.  Dr. Manoj Menezes completed his MD in Paediatrics from MS University, Baroda, India and completed his PhD in inherited peripheral neuropathies including RTD from the University of Sydney.  He is a Staff Specialist in Neurology and Neurogenetics at CHW and a Clinical Senior Lecturer at the CHW Clinical School at The University of Sydney.  He is also Director of the Peripheral Neuropathy Management Clinic, CHW and co-team leader of the Neuropathy Group at the Kids Neuroscience Centre, Kids Research.

How will this project work?

CMTPedS is a standardized clinical outcome measure for children and young adults with inherited neuropathy.  Over the last six years, Dr Manoj Menezes and his team have performed 26 assessments in children and young adults with RTD using CMTPedS both at baseline and while on riboflavin supplementation.  This data will be used to develop and validate an RTD specific scale, using retrospectively collected data to measure disease progression and prospectively evaluate the convergent validity of this scale.

What is the significance of this study?

Carefully developed and validated outcome measures have the potential to influence the result of clinical trials and ultimately improve patient care. A reproducible, valid and responsive outcome measure is required for human clinical trials of novel compounds identified in the laboratory and tested on animal models, in order to understand if they truly modify disease and improve patient outcomes.

Cure RTD Research Funding

This grant to Dr. Manoj Menezes is part of Cure RTD’s Clinical Trials programs that we’re currently announcing for our 2019-2020 grant cycle.

Riboflavin Transporter Deficiency (RTD) is characterized by a loss of specific cells in the spinal cord and brainstem called motor and sensory neurons.  As part of this study, skin cells (fibroblasts) obtained from individuals with RTD Type 2 and 3 are reprogrammed back into an embryonic-like pluripotent state, called induced pluripotent stem cells (iPSCs). These iPSCs can then be used for the development of an unlimited source of living human cells, such as motor neurons, for investigating the pathogenetic mechanisms in RTD and also the response to various treatments, such as riboflavin and antioxidants.

Dr. Compagnucci has shown that motor neurons developed using iPSCs from individuals with RTD have several abnormalities compared to motor neurons derived from healthy individuals. These abnormalities include reduced axon elongation and perturbation in the neurofilament composition.  When these RTD motor neurons are treated with high-dose riboflavin, they are only partially rescued, suggesting the need to develop complementary novel therapeutic strategies.

In this proposal, Dr. Compagnucci will build upon these preliminary results to better understand the pathogenetic mechanisms in RTD and investigate complementary novel therapeutic strategies, which can lead to new and improved treatment approaches for individuals with RTD.

Meet Dr. Compagnucci

Who are you?

I am a Biologist at Bambino Gesu` Children’s Hospital (OPBG) working in the Unit of Neuromuscular and Neurodegenerative Disorders led by Dr. Enrico Bertini. I obtained my PhD in Craniofacial Development at King’s College London (UK). At OPBG my main research interest is to unveil the biological mechanisms regulating neuronal development and function in health and disease.

How did you first become involved with RTD research?

I first became interested in the cellular and molecular biology that characterizes RTD when I moved to OPBG in 2012. Our lab director, Dr. Enrico Bertini, was an author of one of the first research reports about RTD Type 2, which described a young boy with genetically confirmed RTD.

What is your current role in RTD research?

I am currently involved in characterizing RTD pathogenetic mechanisms and testing various protocols for treatment of RTD. Over the past 6 years I have worked to characterize the modelling of iPSCs in order to deepen our knowledge of the system and be able to use iPSCs to model RTD. In our research so far we have discovered emerging pathogenetic mechanisms in RTD neurodegeneration, and investigated the biological rationale behind riboflavin supplementation. We have found that although riboflavin is important to ameliorate the RTD phenotype it is not sufficient to fully rescue the cells. Since riboflavin supplementation has some limits in rescuing all of the disease features in our models I am interested in developing complementary novel therapeutic strategies.

What do you hope to learn from this research project?

I hope to determine other biological mechanisms altered in the neurons derived from the patients’ iPSCs and to characterize the molecular and cellular features when cells are treated with combined riboflavin and antioxidants.

How will this project work?

Patients’ iPSCs will be differentiated into motor neurons. The main focus of our investigation will be on the cytoskeletal features of these motor neurons, since cytoskeletal dynamics are fundamental to build functional neuronal networks and particularly in motor neurons to extend and maintain neurites for long distances. In addition to this, we will study the redox features of RTD cells and look for possible amelioration of the RTD phenotype following treatment with riboflavin plus combinations of antioxidants.

What is the significance of your study?

Based on the available literature and our preliminary results, the combination of riboflavin and antioxidants supplementation could provide a sound foundation for future patient therapies, aimed at progressively ameliorating RTD manifestations. Furthermore, working on patients’ specific iPSCs may allow us to develop custom therapies, considering individual variability of pathogenic mechanisms and responses to drug treatment. Therefore, this study may lead to life improvement for individuals with RTD.

Cure RTD Research Funding

This grant to Dr. Compagnucci is part of Cure RTD’s Basic Research and Drug Discovery programs that we’re currently announcing for our 2018-2019 grant cycle.

Basic Research is the first step in our comprehensive research model. We fund basic research to investigate the biology and cause of RTD, in order to identify the most effective treatment strategies. We also use this funding to develop tools that facilitate RTD research.

Drug discovery converts what we have learned about the causes and biology of RTD through basic research into new drug candidates and treatment approaches that can be tested in clinical trials. This funding will be used strategically to help accelerate research, and to ensure we are developing treatments for all types, ages, and stages of RTD.