+ Hugo Bellen, PhD | Funding period: February 1, 2017 - January 31, 2019
Suppressing the iron/sphingolipid/PDK/Mef2 pathway implicated in FA for therapeutic evaluation
+ David Corey, PhD | Funding period: July 1, 2016 - December 31, 2017
Development of Oligonucleotide Activators of FXN Expression
PI/Investigator: David Corey, PhD
- UT Southwestern, Dallas, Texas Award type:
General Research Grant Grant Title:
Development of oligonucleotide activators of FXN expression Lay summary:
Friedreich's ataxia (FRDA) is an incurable genetic disorder caused by reduced expression of the mitochondrial protein frataxin (FXN). Agents that increase expression of FXN would correct the disease-causing defect and are a promising approach to therapy. FRDA patients have an expanded GAA repeat region within intron one of FXN and this expanded repeat causes transcriptional silencing by a mechanism that has yet to be definitively described. We designed duplex RNAs and single stranded locked nucleic acid (LNA) oligonucleotides (short man-made strands of DNA) to recognize the repeat region and interfere with contacts that contribute to decreased transcription. We found that both duplex RNAs and LNA oligonucleotides caused increased expression of FXN protein in cells derived from FRDA patients. The increase in FXN expression was similar to the level found in normal cells. Both duplex RNAs and LNA oligonucleotides belong to classes of molecule that are being developed clinical. The combination of our promising initial results with experience using similar models in clinical trials suggests a path towards clinical translation towards treatment of FRDA. We now report extending this research to testing more compounds in more cells. Our findings extend the generality of our approach and provide a broader database for evaluating how to move forward with drug discovery.Publications
- Liu J, Hu J, Ludlow AT, Pham JT, Shay JW, Rothstein JD, Corey DR. (2017) "c9orf72 Disease-Related Foci Are Each Composed of One Mutant Expanded Repeat RNA." Cell Chem Biol. 24: 141-148.
- Matsui M and Corey DR. (2016) "Noncoding RNAs as drug targets." Nature Rev. Drug. Discov. 16: 167-179.
+ Richard Wade-Martins, PhD | Funding period: November 15, 2016 - March 31, 2019
Identification of FXN-increasing drug targets by CRISPR-mediated mutagenesis
PI/Investigator: Richard Wade-Martins, PhD
- University of Oxford, UK Award type:
General Research Grant Grant Title:
Identification of FXN-increasing drug targets by CRISPR-mediated mutagenesis Lay summary:
Currently, there is no disease-modifying treatment for Friedreich's ataxia (FRDA), a progressive neurodegenerative disease caused by low cellular levels of frataxin protein (FXN). Although a few therapeutic strategies are currently being explored, to design effective therapies for FRDA it is important to identify genes involved in the regulation of FXN levels (FXN level modifiers) since this could lead to the development of effective FXN up-regulating strategies. Identification of novel FXN level modifiers can be efficiently achieved by performing high-throughput functional screens using genome-wide mutagenesis. Here, we propose an unbiased screen to identify FXN level modifiers using the CRISPR-Cas9 technology, a recently established genome-editing tool which has gained considerable interest in the scientific community for its ability to provide specific and bi-allelic genomic disruptions at high efficiency. We will use the CRISPR-Cas9 technology to induce genome-wide mutagenesis and we will assess the effect of specific genome disruptions on FXN levels. This will be achieved by transducing two FRDA cell models with a lentiviral library of single-guide RNAs and by analysing changes in FXN protein levels, using flow cytometry. Cells with increased FXN protein expression will be isolated by Fluorescence-Activated Cell Sorting (FACS) and sequenced to identify the mutated genes responsible for the increase in FXN expression. Once we have identified novel targets, we will assess their ability to rescue the molecular hallmarks characteristic of FRDA on a series of in vitro FRDA models and assays. To the best of our knowledge this approach is novel in FRDA. Other groups are developing target identification screens for FRDA, however these are focused on the use of the RNAi technology, which presents important limitations such as off-target effects and partial target knockdown, which can ultimately decrease the efficiency of a screen. The CRISPR- Cas9 method has already shown to overcome the limitations associated with the use of RNAi. We believe that the experiments proposed in this application have the potential to identify novel therapeutic targets for FRDA as well as provide a better understanding of the regulation of FXN protein levels.Co-sponsor: Pfizer and Ataxia UKPublications
- Lufino MMP, Silva AM, Németh AH, Alegre-Abarrategui J, Russell AJ, Wade-Martins R. (2013) "A GAA repeat expansion reporter model of Friedreich's ataxia recapitulates the genomic context and allows rapid screening of therapeutic compounds." Human Molecular Genetics. 22: 5173-5187.
- Silva AM, Brown JM, Buckle VJ, Wade-Martins R and Lufino MMP. (2015) "Expanded GAA repeats impair FXN gene expression and reposition the FXN locus to the nuclear lamina in single cells." Human Molecular Genetics; 24:3457-71.
+ Michael Green, PhD | Funding period: November 1, 2017 - October 31, 2019
Systematic Identification of Pharmacological Activators of the Repressed FXN Gene to Treat Friedreich Ataxia
PI/Investigator: Michael Green, MD, PhD
- University of Massachusetts Medical School, USA Award type:
General Research Grant Grant Title:
Systematic identification of pharmacological activators of the repressed FXN gene to treat Friedreich AtaxiaLay summary:
Friedreich ataxia (FA) is an autosomal recessive disease characterized by progressive damage to the nervous system and severe cardiac abnormalities. The disease is caused by a GAA•TTC triplet repeat expansion in the first intron of the FXN gene (hereafter called the triplet repeat expansion (TRE)-FXN gene), which represses FXN transcription. The FXN gene encodes a protein called frataxin, a ubiquitous, nuclear-encoded mitochondrial protein that plays a key role in iron metabolism. FXN repression results in reduced levels of frataxin, leading to mitochondrial dysfunction, which is the underlying basis of the disease. Currently there are no effective treatments for FA. Reactivation of the repressed TRE-FXN gene is a potential therapeutic approach for FA that would correct the root cause of the disease rather than a secondary, downstream consequence of the frataxin deficiency. We hypothesize that there are a number of epigenetic repressors whose pharmacological inhibition will lead to upregulated transcription of the TRE-FXN gene, resulting in increased frataxin levels and decreased disease symptomatology. As proof-of-concept, we performed a small-scale candidate-based RNA interference (RNAi) screen, which identified 10 epigenetic regulators of the TRE-FXN gene; for convenience we refer to these epigenetic regulators as FXN Repressing Factors (FXN-RFs). We then showed that small molecule inhibitors of these FXN-RFs can also upregulate transcription of the TRE-FXN gene. Our preliminary results strongly support the feasibility of our approach for identifying small molecule FXN-RF inhibitors that upregulate TRE-FXN transcription. Experiments in this application are focused on the discovery of new small molecule FXN-RF inhibitors, and determination of whether transcriptional upregulation of TRE-FXN can correct well-characterized mitochondrial abnormalities in FA neurons and cardiomyocytes, which are the cell types most relevant to FA. The results of the proposed cell-based experiments will identify the most efficacious, least cytotoxic compounds that in future studies can be analyzed in FA mouse models. In the long-term, the results of our study are likely to have a major impact on the field of FA therapeutics and have the potential to lead to development of a new class of drugs that can ameliorate this devastating disease.Co-sponsor: FARA IrelandPublications
- Gazin C, Wajapeyee N, Gobeil S, Virbasius CM, and Green MR. (2007) "An elaborate pathway required for Ras-mediated epigenetic silencing." Nature. 449:1073-7.
- Wajapeyee N, Malonia SK, Palakurthy RK, and Green MR. (2013) "Oncogenic RAS directs silencing of tumor suppressor genes through ordered recruitment of transcriptional repressors." Genes Dev. 27:2221-6.
- Serra RW, Fang M, Park SM, Hutchinson L, and Green MR. (2014) "A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype." Elife. 3:e02313.
- Fang M, Ou J, Hutchinson L, and Green MR. (2014) "The BRAF oncoprotein functions through the transcriptional repressor MAFG to mediate the CpG island methylator phenotype." Mol Cell. 55:904-15.
- Bhatnagar S, Zhu X, Ou J, Lin L, Chamberlain L, Zhu LJ, Wajapeyee N, and Green MR. (2014) "Genetic and pharmacological reactivation of the mammalian inactive X chromosome." Proc Natl Acad Sci U S A. 111:12591-8.
+ Sathiji Nageshwaran, PhD | Funding period: January 1, 2018 - June 30, 2019
Chromatin structure, function and hierarchy of frataxin in FA
PI/Investigator: Sathiji Nageshwaran, PhD
- Harvard University, MA, USAAward type: General Research Grant - Postdoctoral FellowshipGrant Title:
Chromatin structure, function and hierarchy of frataxin in FA Lay summary:
This postdoctoral fellowship is an extension of my graduate studies work supported by FARA, which focused on epigenetic editing of the frataxin gene as a novel therapeutic approach for FA. Frataxin (FXN) gene silencing is the result of an abnormal trinucleotide (i.e., GAA) expansion within intron 1 of the FXN gene. This has been shown to result in an abnormal epigenomic environment within the frataxin gene locus. Although several components have been implicated in the establishment of this environment—from aberrant histone and DNA methylation, heterochromatin and R-loop formation and antisense transcription—the direct causality of each of these factors and its relevance to FXN gene silencing remains elusive. Specifically, it remains to be determined the relative importance of each of these factors with respect to different frataxin gene locus elements, such as the promoter region and the regions upstream and/or downstream of the GAA repeats. With the advent of RNA guided endonuclease technology (CRISPR), I intend to interrogate these factors directly at the frataxin locus. These include studies to interrogate the lysine methyltransferases, which I studied in during my graduate tenure. I showed that one such enzyme specifically silenced the FXN gene, supporting the notion that attenuation of its activity may provide a potential therapeutic avenue to de-repress FXN silencing. Additional FXN regulatory targets I will interrogate include the histone modifications H3K9me3 and H3K27me3, which have been found to be associated with the pathologically silenced FXN in FA. I have developed histone-mutant peptides that may provide the basis for small-molecule method of alleviating both silencing histone modifications. Furthermore, I am using the CRISPR/Cas9 technology to manipulate the expression of these histone modifications to establish a hierarchy of histone post-translational modifications involved in pathologically silencing the FXN locus. These studies will not only provide insight into the mechanism of aberrant FXN silencing but might also lead to a novel and radical therapeutic approach for FRDA and potentially other epigenetically regulated disorders.Publications
- Nageshwaran S, Festenstein R. (2015) "Epigenetics and Triplet-Repeat Neurological Diseases." Front Neurol. 6:262.
- Natisvili T, Yandim C, Silva R, Emanuelli G, Krueger F, Nageshwaran S, Festenstein R. (2016) "Transcriptional Activation of Pericentromeric Satellite Repeats and Disruption of Centromeric Clustering upon Proteasome Inhibition." PLoS One. 11:e0165873.
+ Diane Ward, PhD | Funding period: January 1, 2018 - December 31, 2019
Identification of the mechanism of oxidant-mediated Yfh1/frataxin turnover and screen for compounds that suppress the effects of reduced Yfh1/frataxin levels
PI/Investigator: Diane Ward, PhD
- University of Utah, USA Award type:
General Research GrantGrant Title:
Identification of the mechanism of oxidant-mediated Yfh1/frataxin turnover and screen for compounds that suppress the effects of reduced Yfh1/frataxin levels Lay summary:
Friedreich ataxia (FRDA) is a lethal human disorder resulting from mutations in the FXN gene, which give rise to decreased levels of the frataxin protein. The frataxin protein is targeted to the mitochondrial matrix where it plays a critical role in the synthesis of mitochondrial iron-sulfur (Fe-S) clusters. These clusters are essential prosthetic groups involved in oxygen binding and enzymatic activity. Frataxin, like many of the proteins involved in mitochondrial Fe-S cluster synthesis is highly conserved among eukaryotes. The yeast gene YFH1, encodes for the homologue of mammalian frataxin. Loss of YFH1 in yeast results in increased mitochondrial oxidants, increased mitochondrial iron accumulation and decreased Fe-S cluster synthesis. Importantly, overexpression of the human frataxin in yeast complements the loss of Yfh1 and corrects these phenotypes. As such, we utilize yeast as a model system to study FRDA and iron metabolism because of the conserved cellular function and relative ease of genetic manipulation. Using yeast, we discovered that mutation of a yeast gene ERG29 results in defective sterol synthesis, leading to increased levels of sterol intermediates. We found that these intermediates interact with iron generating mitochondrial oxidants, and that the increased mitochondrial oxidants resulted in the loss of Yfh1. Mutant ERG29-induced loss of Yfh1 led to decreased Fe-S cluster synthesis, and ultimately cell death due to decreased mitochondrial respiratory activity. The finding that mitochondrial oxidants affect Fe-S cluster activity through decreased frataxin is not restricted to yeast or to sterol intermediates (1). Together, these results support the hypothesis that conditions that increase mitochondrial oxidants will result in frataxin/Yfh1 degradation. Based on these findings, we propose to determine the mechanism of oxidant-induced loss of frataxin/Yfh1. We will also take advantage of the strong phenotype of Yfh1 and Erg29 knockdown in yeast to screen for compounds that can suppress the effects of loss of Yfh1. Determining the mechanism of how increased mitochondrial oxidants and iron affect frataxin/Yfh1 stability and identifying compounds that suppress the effects of reduced frataxin/Yfh1 levels will guide in identifying efficacious treatments for FRDA and may possibly provide novel treatments for human diseases in which mitochondrial function is compromised.Lay abstract references
- Mouli, S., Nanayakkara, G., AlAlasmari, A., Eldoumani, H., Fu, X., Berlin, A., Lohani, M., Nie, B., Arnold, R. D., Kavazis, A., Smith, F., Beyers, R., Denney, T., Dhanasekaran, M., Zhong, J., Quindry, J. and Amin, R. (2015) "The role of frataxin in doxorubicin-mediated cardiac hypertrophy." Am J Physiol Heart Circ Physiol. 309:H844-859.
- Li, L., Kaplan, J. and Ward, D. M. (2017) "The glucose sensor Snf1 and the transcription factors Msn2 and Msn4 regulate transcription of the vacuolar iron importer gene CCC1 and iron resistance in yeast." J Biol Chem. 292:15577-15586.
- Seguin, A, Takahashi-Makise, N, Yien, YY, Huston, NC, Musso, G, Wallace, JA, Bradley, T, Bergonia, H, Matsumoto, M, Igarashi, K, Phillips, JD, Paw, BH, Kaplan, J and Ward, DM. (2017)
- "Reductions in Abcb10 affect the heme biosynthesis transcriptional profile". J. Biol. Chem. 292:16284-16299.
- Yaish, HM, Farrell, CP, Christensen, RD, MacQueen, BC, Jackson, LK, Trochez-Enciso, J, Kaplan, J, Ward, DM, Salah WK and Phillips, JD. (2017) "Two novel mutations in TMPRSS6 associated with iron-refractory iron deficiency anemia in a mother and child." Blood Cells Mol. Dis. 65:38-40. PMID: 28460265.
+ Amylyx & Hong Lin, PhD | Funding period: November 1, 2017 – October 31, 2019
Evaluation of Amylyx Pharmaceutical's AM0035, a combination of tauroursodeoxycholic acid and sodium phenylbutyrate, in FRDA models
PI/Investigator: Kent Leslie
- Chief Scientific Officer of Amylyx PharmaceuticalsPI/Investigator: Hong Lin, PhD
- in the laboratory of David Lynch, MD, PhD at Penn and CHOPAward type:
General Research Grant Grant Title:
Evaluation of Amylyx Pharmaceutical's AM0035, a combination of tauroursodeoxycholic acid and sodium phenylbutyrate, in FRDA modelsLay summary:
Amylyx Pharmaceuticals has developed a novel therapeutic, AMX0035, a combination of two compounds, Sodium Phenylbutyrate (PB) and Tauroursodeoxycholic Acid (TUDCA), for the treatment of neurodegenerative disease. Amylyx discovered a synergy between these two compounds across multiple preclinical models when administered together in a specific range of doses. AMX0035 has demonstrated synergistic efficacy in models of neurodegeneration, oxidative insult, classical activation of neuro-inflammation, and bioenergetics deficits, through the combined targeting of pathways of mitochondrial dysfunction (ROS production, caspase-dependent apoptosis, mitochondrial channel formation) and ER stress. We hypothesize that reducing mitochondrial stress pathways whilst simultaneously increasing chaperone protein production could synergistically increase cellular viability in response to oxidative insults and provide an effective therapy for Friedreich's ataxia (FRDA) patients. Amylyx received FDA clearance for AMX0035's Investigational New Drug (IND) application in April 2017. Furthermore, Amylyx initiated a Phase II trial evaluating the safety and efficacy of AMX0035 in ALS patients in mid-2017 and expects to initiate a Phase II trial evaluating AMX0035 in Alzheimer's in early 2018. Taken together, Amylyx is excited and primed to explore therapeutic potential of AMX0035 in FRDA. In collaboration with Dr. Marek Napierala at the University of Alabama, Birmingham, we tested AMX0035 in FRDA patient-derived fibroblasts. We found that AMX0035 promotes cell viability, including protecting frataxin-deficient cells from metabolic stresses, potentially through a mitochondrial-targeted mechanism of action. With this FARA grant, we will continue studies to characterize the mitochondrial effects of AMX0035 in FRDA patient-derived fibroblasts. Working alongside Drs. David Lynch and Hong Lin at the Children's Hospital of Philadelphia, we will evaluate the target effects of AMX0035 in the KIKO FRDA mouse model. Here we plan to assess the early signals of mitochondrial biogenesis target engagement in vivo, as well as confirm the presence and blood-brain barrier permeability of AMX0035. Furthermore, we will evaluate the neurosensory, biochemical, and functional effects of AMX0035 in the KIKO FRDA mouse model. These industry-academia collaborations enable us to streamline the process of evaluating AMX0035 in translatable models of FA, with the potential to guide clinical drug development. These continued studies will build upon the preliminary efficacy data of AMX0035 in models of FRDA and will advance our understanding of the role of mitochondrial dysfunction in disease pathology.
+ Aseem Ansari, PhD | Funding period: December 15, 2017 – December 14, 2019
Elucidating the mechanism by which synthetic molecules stimulate Frataxin in Friedreich's ataxia
PI/Investigator: Aseem Ansari, PhD
- University of Wisconsin-Madison, USA Award type:
General Research GrantGrant Title:
Elucidating the mechanism by which synthetic molecules stimulate frataxin in Friedreich's ataxiaLay summary:
Over the years, several therapeutic routes have been explored to treat Friedreich's ataxia (FA/FRDA). An ideal therapeutic strategy is to target the mechanistic root of the disease — the massive increase in number of GAA trinucleotide repeats in the frataxin gene (FXN) of FA patients. The expanded repeats block the ability of the cellular machinery to synthesize (transcribe) frataxin RNA which in turn leads to a deficiency in levels of frataxin – a protein that is essential for the optimal functioning of mitochondria, the cell's powerhouse. The extent of this trinucleotide repeat "expansion" correlates with the severity and onset of the disease. While new advances in CRISPR-based genome engineering methods have enormous potential to target the GAA repeat expansion, it remains unclear if this approach will emerge as safe treatment option in the near future.
Our lab has created novel bifunctional molecules that selectively bind GAA repeat expansions and actively enable the cellular machinery to transcribe across the repressive GAA repeats within FXN. When scrutinized with genomic approaches, our synthetic transcription elongation factors (Syn-TEFs) display the ability to specifically overcome the repressive roadblocks to transcription at FXN without perturbing the function of other cellular genes. Based on this specificity of action, we propose to utilize Syn-TEFs to examine underlying molecular mechanisms that contribute to FXN silencing in patient cells. Deeper mechanistic understanding will guide the design of next generation Syn-TEFs that are precision-tailored for individuals with the disease. We further propose to test the efficacy of Syn-TEFs in patient-derived neurons and heart cells – cell types in which frataxin deficiency results in ataxia and morbidity. Taken together, the proposed experiments will aid in the development of this class of molecules as potential individual-tailored precision therapeutics.Publications
- Erwin GS, Grieshop MP, Ali A, Qi J, Lawlor M, Kumar D, Ahmad I, McNally A, Teider N, Worringer K, Sivasankaran R, Syed DN, Eguchi A, Ashraf M, Jeffery J, Xu M, Park PMC, Mukhtar H, Srivastava AK, Faruq M, Bradner JE, and Ansari AZ. (2017) "Synthetic transcription elongation factors license transcription across repressive chromatin." Science. 358:1617-1622
+ Jon Watts, PhD | Funding period: February 1, 2018 - January 31, 2020
Activating frataxin expression in animals using chemically modified oligonucleotides
PI/Investigator: Jon Watts, PhD
- RNA Therapeutics Institute, UMass Medical School, USA Award type:
General Research GrantGrant Title:
Activating frataxin expression in animals using chemically modified oligonucleotidesLay summary:
Friedrich's Ataxia (FA) is an incurable genetic disease caused by insufficient expression of the mitochondrial protein frataxin. This reduced expression is primarily driven by a GAA triplet repeat expansion within the first intron of the frataxin gene, and this mutant expansion leads to formation of an "R-loop" or heterochromatin formation and transcriptional gene silencing . A number of strategies are being developed for treating FA, including approaches to improve mitochondrial function, release epigenetic silencing, or modulate pathways downstream of frataxin . Nevertheless, specific activation of frataxin expression is the ideal therapeutic strategy, addressing the disease at its root cause. Pioneering work by David Corey's laboratory at the University of Texas Southwestern Medical Center showed that frataxin expression could be activated by treating FA patient-derived fibroblasts with oligonucleotides complementary to the expanded repeat . My group recently worked with Dr. Corey's group and others to further explore the chemistry of these repeat-targeted oligonucleotides . The next key step in the development of oligonucleotide therapeutics for FA is their testing in animal models. Gene expression and regulation can differ in one tissue relative to another, and in cultured cells relative to organisms. While animal models of FA are imperfect, it is essential to test whether the frataxin activation observed in cultured cells is robust and reproducible in the more complex context of in vivo use. My laboratory has made progress on optimizing the medicinal chemistry of oligonucleotides for the central nervous system, and we are actively working on methods to improve the potency of oligonucleotides in peripheral tissues including heart. Together with our colleagues at the RNA Therapeutics Institute, we have been working on strategies including conjugation of small molecules and hydrophobic groups, use of advanced sugar-modified nucleotides, and development of multimeric oligonucleotide structures. We aim to deepen the preclinical data on lead oligonucleotide compounds, as well as lay the groundwork for clinical development of these compounds.Grant made possible with support from the Crisp Family FundLay abstract references
- Groh M, Lufino MMP, Wade-Martins R and Gromak N. (2014) "R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome." PLOS Genetics. 10: e1004318.
- Strawser CJ, Schadt KA and Lynch DR. (2014) "Therapeutic approaches for the treatment of Friedreich's ataxia." Expert Rev. Neurotherapeutics. 14: 949-957.
- Li L, Matsui M and Corey DR. (2016). "Activating frataxin expression by repeat-targeted nucleic acids." Nat Commun. 7: 10606.
- Li L, Shen X, Liu Z, Norrbom M, Prakash TP, O'Reilly D, Sharma VK, Damha MJ, Watts JK, Rigo F and Corey DR. (2018) "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat." Nucleic Acid Ther., in press.
- Pendergraff HM, Krishnamurthy PM, Debacker AJ, Moazami MP, Sharma VK, Niitsoo L, Yu Y, Tan YN, Haitchi HM, and Watts JK. (2017) "Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA." Mol Ther Nucleic Acids. 8158-168.
- Khvorova A., Watts J.K., (2017) "The chemical evolution of oligonucleotide therapies of clinical utility." Nat. Biotechnol. 35: 238-248.
+ Joseph Baur, PhD & Shana McCormack, MD | Funding period: July 1, 2018 – June 30, 2020
NAD+ Precursor Supplementation in Friedreich’s Ataxia
PI/Investigator: Joseph Baur, PhD
- Perelman School of Medicine, University of PennsylvaniaPI/Investigator: Shana McCormack, MD
- Children’s Hospital of Philadelphia Award type:
Keith Michael Andrus Cardiac Research AwardGrant Title:
NAD+ Precursor Supplementation in Friedreich’s AtaxiaLay summary:
Nicotinamide adenine dinucleotide (NAD+) is important for bioenergetic and metabolic processes.
NAD+ deficiency has been implicated in heart failure and there is increasing interest restoring its concentration as a therapeutic strategy.
Intriguingly, hyperacetylation of mitochondrial proteins is emerging as a consistent feature of failing hearts,
and is dramatic in a severe model of FA cardiomyopathy (the cardiac-specific frataxin knockout). The sirtuin SIRT3 is
responsible for removing acetylation from many mitochondrial proteins. Thus, Martin et al. (JCI Insight, 2017) hypothesized that
supplementing cardiac NAD+ (via the precursor nicotinamide mononucleotide, NMN) might enhance SIRT3 activity, reduce acetylation,
and delay heart failure. NMN did indeed improve cardiac function in the heart specific frataxin KO in a SIRT3-dependent manner,
but paradoxically did not have a major effect on acetylation. These observations support NAD+ metabolism as a therapeutic target in FA,
but raise many important questions about the underlying mechanisms. We propose to look at the effects of NAD+ precursor supplementation
in two additional mouse models of FA.
We will also test short-term NMN for tolerability and effects on cardiac bioenergetics in adults with FA without overt heart failure. We will assess the feasibility and safety of short-term (1 week) NAD+ precursor supplementation (NMN, dosing based on studies including NCT03151239) in individuals with FA (n=6) via an open-label study. Although we will primarily focus on feasibility and safety, we will also look for normalization of bioenergetics. Together, these experiments will provide insight and, if promising, guide the design of a longer phase 2/3 interventional study of NAD+ supplementation in FA.
- Martin, A. S. et al. (2017) Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich's ataxia cardiomyopathy model. JCI Insight 2(14). doi: 10.1172/jci.insight.93885. PubMed PMID: 28724806; PMCID: PMC5518566
+ CRISPR Therapeutics & Marek Napierala, PhD | Funding period: Funding period: October 1, 2017 – September 30, 2019
CRISPR / Cas9 mediated deletion of the human FXN intronic trinucleotide repeat as a therapeutic approach for Friedreich's Ataxia
PI/Investigator: Tony Ho, MD
of Crispr Therapeutics PI/Investigator: Marek Napierala, PhD
- University of Alabama at Birmingham Award type: Kyle Bryant Translational Research Award Grant Title: CRISPR / Cas9
mediated deletion of the human FXN intronic trinucleotide repeat as a therapeutic approach for Friedreich's ataxiaLay summary:
Friedreich's ataxia (FA) is an incurable neurodegenerative disease caused most commonly by a pathologic expansion of a naturally occurring short DNA sequence repeat in both copies of the FXN gene. The FXN gene codes for the protein frataxin, which is needed for the proper functioning of mitochondria, the powerhouse of the cell. Having two copies of the FXN gene with expanded repeats significantly reduces the amount of frataxin protein produced and is associated with a number of serious cellular abnormalities. While frataxin protein is produced by most cells in the body, some cells are more sensitive to the degree of frataxin deficiency than others. In particular, neurons in the brain, eye, and spinal cord as well as muscle cells in the heart appear disproportionally vulnerable to damage and degeneration in the setting of reduced FXN protein. Dysfunction in these cells leads to progressive imbalance and discoordination, loss of visual acuity, and life-threatening cardiomyopathy that develops in patients diagnosed with FA. It has been shown that removing the expanded repeat sequences from FXN gene, using different DNA editing platforms (i.e. molecular scissors that can specifically excise the repeat sequences), can consistently restore the production of frataxin protein and prevent degeneration of affected cells (1-2). The CRISPR / Cas9 system, a recently discovered DNA editing strategy, is technically simpler compared to other forms of molecular scissors. In this grant, we propose to use the CRISPR / Cas9 system to remove the expanded repeat sequence from the FXN gene in FA patient-derived stem cells and in a FA mouse model. We will screen for and identify CRISPR reagents that efficiently remove the expanded repeat sequences and will optimize the delivery of these reagents to patient-derived cells and to the FA-model mouse. We will examine the restoration of frataxin protein function after repeat expansion removal. This proof of concept study will enable the generation of CRISPR-based therapeutic reagents and strategies to deliver these reagents to patient cells thereby potentially supporting the development of an effective therapy for FA. Co-sponsor: The Cure FA FoundationLay abstract references
- Li Y, Polak U, Bhalla AD, Rozwadowska N, Butler JS, Lynch DR, Dent SYR, Napierala M. (2015) "Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich's Ataxia." Mol Ther. 23:1055-1065.
- Oullet DL, Cherif K, Rousseau J, and Temblay JP. (2017) "Deletion of the GAA repeats from the human frataxin gene using CRISPR-Cas9 system in YG8R-derived cells and mouse models of Friedreich ataxia." Gene Therapy. 24: 265-274.
+ Stephanie Cherqui, PhD | Funding period: February 1, 2018 - January 31, 2019
Stem Cell Gene Therapy for Friedreich’s Ataxia
PI/Investigator: Stephanie Cherqui, PhD
- University of California, San Diego, USAAward type:
General Research GrantGrant Title:
Stem cell gene therapy for Friedreich’s ataxiaLay summary:
Friedreich’s ataxia (FRDA) is a multi-systemic autosomal recessive genetic disorder caused by a mutation in the frataxin gene, which reduces the amount of frataxin made by the cell. Frataxin is a mitochondrial protein involved in iron metabolism and is critical for proper mitochondrial function and health. Consistent with the cellular role of frataxin, FRDA is characterized by ataxia, neurodegeneration, muscle weakness, and cardiomyopathy – there is no treatment for this lethal disease. Our lab is interested in the role of inflammation in disease progression, and in the ability to modulate the immune system for therapeutic gain (1-3). Specifically, we study the capacity of hematopoietic stem and progenitor cells (HSPCs) to differentiate into immune cells that can migrate to sites of inflammation in the body and halt the progression of disease. Recently, we tested this therapeutic strategy in the Y8GR mouse model of FRDA (4). This mouse model expresses exclusively the mutated human FXN transgene, thus mimicking the transcriptional deficiency and clinical phenotype seen in FRDA patients. We transplanted normal (i.e., no frataxin mutation), control HSPCs from wild-type mice into 2-month old FRDA YG8R mice and found that this therapy worked quite beyond our expectation. Control HSPCs transplanted in the YG8R mouse differentiated into macrophages that migrated to all sites of inflammation injury, such as the brain, spinal cord, dorsal root ganglia, skeletal muscle and heart of the YG8R mouse. The neurologic and muscular complications in the YG8R mouse were completely prevented as observed 7-months post-transplantation (latest time point tested) after only a single infusion of control HSPCs. As well, it appears that part of the therapeutic gain from this treatment occurs from the transfer of functional frataxin protein from the control HPSC-derived macrophages to the affected tissues in the YG8R mouse. Taken together, we hypothesize that this strategy could also treat FRDA patients. Given the high risk of morbidity and mortality associated with HSPC transplantation from a donor (i.e., allogeneic transplantation), our objective is to develop "an autologous" HSPC gene therapy approach for FRDA, whereby patient cells are extracted, corrected and returned to the patient. To accomplish our objective, we will first develop a gene-correction therapy strategy for FRDA HSPCs and determine their ability of halting disease progression in YG8R mice. Furthermore, we will treat YG8R mice in late-stage disease and determine the ability of HSPCs to reverse preexisting complications. This work represents the first autologous gene-corrected HSPC transplantation treatment strategy for FRDA and builds the foundation for a clinical application of this strategy.Grant made possible with support from the Crisp Family FundLay abstract references
- Harrison F, Yeagy BA, Rocca CJ, Kohn DB, Salomon DR, Cherqui S. (2013) "Hematopoietic stem cell gene therapy in the mouse model of cystinosis." Mol Ther. 21:433-444.
- Naphade S, Sharma J, Gaide Chevronnay HP, Shook MA, Yeagy BA, Rocca CJ, Ur SN, Lau AJ, Courtoy PJ, Cherqui S. (2015) "Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes." Stem Cells. 33:301-309.
- Gaide Chevronnay HP, Jansen V, Van Der Smissen P, Rocca CJ, Liao XH, Refetoff S, Pierreux CE, Cherqui S*, and Courtoy P*. (2016) "Hematopoietic stem cell transplantation can normalize thyroid function in a cystinosis mouse model." Endocrinology. 57:1363-1371 *co-senior author
- Rocca CJ, Goodman SM, Dulin JN, Haquang JH, Gertsman I, Blondelle J, Smith JLM, Heyser CJ, Cherqui S. (2017) "Hematopoietic stem cell transplantation prevents development of Friedreich’s Ataxia in a humanized mouse model." Sci Transl Med. 9:eeaj2347.
+ Javier Santos, PhD | Funding period: March 15, 2019 - March 14, 2021
Structural Dynamics and the Consolidation of Protein Function in Protein Complexes Involved in the Biosynthesis of Iron-Sulfur Clusters: Quaternary Addition of Small Trojan Tutor Proteins
Javier Santos, PhD
- Departamento de Fisiología y Biología Molecular y Celular. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Consejo Nacional de Investigaciones Científicas y Técnicas, ArgentinaAward type:
General Research GrantGrant Title:
Structural Dynamics and the Consolidation of Protein Function in Protein Complexes Involved in the Biosynthesis of Iron-Sulfur Clusters: Quaternary Addition of Small Trojan Tutor ProteinsLay summary:
Iron-sulfur (Fe-S) clusters are essential cofactors present in all known forms of life. To exert their functions, hundreds of proteins require such cofactors. In eukaryotic organisms, the biogenesis of most Fe-S clusters takes place in the mitochondria. The process involves the interactions and activities of several key proteins, which form a supercomplex. Namely these proteins are: Frataxin, NFS1, ISCU, and ACP and ISD11. Mutations in these building blocks lead to severe human diseases. For instance, mutations in Frataxin, or a deficiency in its expression, results in Friedreich’s Ataxia (FRDA). In particular, Frataxin upregulates the activity of the supercomplex. Our laboratory studies the stability, internal motions, and functionality of Frataxin mutants to better understand the root cause of loss of function in the pathogenic variants. We propose delivering a small “Trojan” tutor protein with high affinity for Frataxin to the mitochondrial matrix, inside the cell. Modulation of Frataxin stability and activity may exert control on the supercomplex function. Furthermore, we will search for “Trojan” tutors against other proteins of the supercomplex. In this way, we will increase our chances of successfully improving Fe-S cluster biosynthesis.Publications:
Rescuing the Rescuer: On the Protein Complex between Human Mitochondrial Acyl Carrier Protein and ISD11. Herrera MG, Pignataro MF, Noguera ME, Cruz KM and Santos J. ACS Chemical Biology ACS Chemical Biology 2018 Jun 15;13(6):1455-1462.
Biophysical Characterization of the Recombinant Human Frataxin Precursor. Castro IH, Ferrari A, Herrera MG, Noguera ME, Maso L, Benini M, Rufini A, Testi R, Costantini P and Santos J*. 2018. FEBS Open Bio. doi:10.1002/2211-5463.12376.
3- Insights on the conformational dynamics of human frataxin through modifications of loop-1. Noguera ME, Aran M, Smal C, Vazquez DS, Herrera MG, Roman EA, Alaimo N, Gallo M, Santos J. Arch Biochem Biophys. 2017 Dec 15;636:123-137.
Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region. Faraj SE, González-Lebrero RM, Roman EA, Javier Santos*. Scientific Reports, Nature. 2016 Feb 9;6:20782
Structural characterization of metal binding to a cold-adapted frataxin. Noguera ME, Roman EA, Rigal RB, Cousido-Siah A, Mitschler A, Podjarny A, and Santos J*. J Biol Inorg Chem. 2015 Jun;20(4):653-64.
A helix-coil transition induced by the metal ion interaction with a grafted iron-binding site of the CyaY protein family. Vazquez DS, Agudelo WA, Yone A, Vizioli N, Arán M, González Flecha FL, González Lebrero MC, Santos J*. Dalton Trans. 2015 Feb 7;44(5):2370-9
The Alteration of the C-terminal Region of Human Frataxin Distorts its Structural Dynamics and Function. 7- Faraj SE, Roman EA, Aran M, Gallo M, Santos J*. FEBS J. 2014 Aug;281(15):3397-3419.
The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin. Faraj SE, Venturutti L, Roman EA, Marino-Buslje CB, Mignone A, Tosatto SCE, Delfino JM*, and Santos J*. FEBS OPEN BIO, 2013 Jul 24;3:310-20.
Frataxin from Psychromonas ingrahamii as a model to study stability modulation within the CyaY protein family. Roman EA, Faraj SE, Cousido-Siah A, Mitschler A, Podjarny A, Santos J*. Biochim Biophys Acta. 2013. Jun;1834(6):1168-80.
Protein Stability and Dynamics Modulation: The Case of Human Frataxin. Roman EA, Faraj SE, Gallo M, Salvay AG, Ferreiro DU, Santos J*. PLoS ONE 2012. 7(9): e45743.
+ Jill Napierala, PhD | Funding period: March 1, 2019 - Feb 28, 2021
Defining the pathogenic mechanism of the Frataxin G130V mutation
Jill Napierala, PhD
- University of Alabama at BirminghamAward type:
General Research GrantGrant Title:
Defining the pathogenic mechanism of the Frataxin G130V mutationLay summary:
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease caused by reduced expression of the mitochondrial protein Frataxin (FXN). Most individuals with FRDA have large expansions of repetitive DNA sequences in both copies of the FXN gene, while some have an expansion in one copy and a missense or nonsense mutation in the other. Both genotypes (homozygous and compound heterozygous) result in reduced levels of mature FXN protein when compared with healthy controls. The most prevalent missense mutation changes one amino acid at position 130 (G130V) in the FXN protein. Individuals with the FXN G130V mutation exhibit clinical features distinct from FRDA individuals with repeat expansions in both copies of FXN, including retained reflexes, preserved sensory responses, spared speech, and slower disease progression. Unexpectedly, a lower amount of FXN protein is detected in samples from FRDA G130V patients than in samples from FRDA patients with two FXN repeat expansions, who often endure a more severe and faster progressing disease. Therefore, the clinical presentation of FRDA G130V patients does not appear to agree with the extremely low levels of mature FXN protein detected in patient samples. This clinical distinction suggests a unique G130V-associated pathogenesis that has not yet been investigated. The FXN protein is shortened from a precursor form (FXN-P) to an intermediate (FXN-I) form, and finally a mature (FXN-M) form. We discovered that the ratio of FXN-G130V-I to FXN-G130V-M is higher than that observed for the FXN-WT protein, which is almost all completely shortened to the mature form (FXN-WT-M). We hypothesize that the G130V mutation impairs this processing of FXN and/or destabilizes the mature form. The unprocessed FXN-G130V-I form is functional and compensates for the decreased amount of FXN-M, thus slowing disease progression in FRDA G130V patients. Little is known regarding the levels, processing, and function of the FXN-G130V protein in cells due to lack of reagents and models that can distinguish the mutant G130V protein from the non-mutated protein produced from the FXN copy with the repeat expansion. We have designed and generated unique patient-derived cell line and mouse models to define the levels and function of FXN-G130V protein in living systems. The data collected from these studies will address whether the G130V mutation provides a protective effect to FRDA G130V patient cells by increasing the ratio of a functional FXN-I form that compensates for loss of the FXN-M form. This discovery could be developed into a therapeutic strategy to benefit all individuals living with FRDA.
+ Arnulf Koeppen, MD | Funding period: February 1, 2017 - January 31, 2019
The pathogenesis of Friedreich ataxia
PI/Investigator: Arnulf Koeppen, MD
- VA Medical Center and Albany Medical College, NY, USA Award type:
General Research Grant Grant Title:
The pathogenesis of Friedreich ataxia Lay summary:
Investigators of FA have established several firm observations: (1) In the vast majority of cases, FA is caused by a homozygous guanine-adenine-adenine (GAA) trinucleotide repeat expansion in intron 1 of the frataxin gene; (2) the mutation results in decreased biosynthesis of frataxin, a small mitochondrial protein; (3) frataxin is essential for the proper delivery of iron-sulfur clusters to complexes I, II, and III of the mitochondrial electron transport chain, the citric acid cycle enzyme aconitase, and to ferrochelatase; and (4) frataxin deficiency renders cultured cells sensitive to oxidative stress. It has been more difficult, however, to establish oxidative stress or defective anti-oxidant defenses in live transgenic animals or humans with FA [1-4]. It is still very uncertain how the mutation leads to the complex pathological and clinical phenotypes of FA. The key question is: how does deficiency of frataxin cause severe lesions in so many diverse locations—such as the dentate nucleus (DN) of the cerebellum, dorsal root ganglia (DRG), the Betz cells of the motor cortex, sensory peripheral nerves, heart, and the insulin-secreting ß-cells of the pancreas—while leaving other organs entirely unaffected? Most offered explanations have been speculative, and no unifying concept has emerged. We postulate that each one of the vulnerable tissues contains a unique set of proteins that undergo changes in the level of expression in response to frataxin deficiency. Therefore, the goal of this research is to identify those proteins-of-interest in FA that are up-regulated or down-regulated relative to levels in non-FA subjects, and to determine the functional consequences of these differences. Discovery and identification of FA-relevant proteins will use a combination of tissue-based proteomics with multiple antibodies (immunohistochemistry and immunofluorescence [IHC, IF]), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting (WB), and reverse-phase protein array or reverse-phase protein lysate microarray (RPA). While FA is still incurable, the investigation seeks to establish a firm understanding of the pathogenesis of the disease by a combination of techniques. The results are expected to fine-tune how we understand and classify the presentation of disease in FA. For example, these findings may replace words like "atrophy" and "degeneration" typically used to describe pathologic changes to the DRG, with the words "hypoplasia" and "hyperplasia". This distinction is important, because therapy for hypoplasia and hyperplasia in any "degenerative" condition is generally unsuccessful. Additionally, our research strategy includes an effort to "discover" proteins that are relevant to FA. Lastly, the described methods will also contribute to an understanding of the emerging maladaptive mitochondriogenesis and possible toxic effects of frataxin replacement therapy.Lay abstract references
- Hayashi G and Cortopassi G. (2015) "Oxidative stress in inherited mitochondrial diseases." Free Radic Biol Med. 88:10-7.
- Hayashi G, Shen Y, Pedersen TL, Newman JW, Pook M, and Cortopassi G. (2014) "Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia." Hum Mol Genet. 23: 6838-47.
- Schulz JB, Dehmer T, Schols L, Mende H, Hardt C, Vorgerd M, Bürk K, Matson W, Dichgans J, Beal MF, and Bogdanov MB. (2000) "Oxidative stress in patients with Friedreich ataxia." Neurology 55:1719-21.
- Shan Y, Schoenfeld RA, Hayashi G, Napoli E, Akiyama T, Iodi Carstens M, Carstens EE, Pook MA, and Cortopassi GA. (2013) "Frataxin deficiency leads to defects in expression of antioxidants and Nrf2 expression in dorsal root ganglia of the Friedreich's ataxia YG8R mouse model." Antiox Redox Signal. 19:1481-93.
- Koeppen AH, Becker AB, Qian J, Gelman BB, Mazurkiewicz and JE. (2017) "Friedreich Ataxia: developmental failure of the dorsal root ganglion." J Neuropathol Exp Neurol. 76: 969-977.
- B. Koeppen AH, Becker AB, Qian J, and Feustel PJ. (2017) "Friedreich ataxia: hypoplasia of spinal cord and dorsal root ganglia." J Neuropathol Exp Neuorol. 76:101-108.
- C. Koeppen AH, Becker AB, Feustel PJ, Gelman BB, and Mazurkiewicz JE. (2017) "The significance of intercalated discs in the pathogenesis of Friedreich cardiomyopathy." J Neurol Sci. 367:171-176.
+ Zhen Yan, PhD | Funding period: March 1, 2017 - February 28, 2019
Endurance and resistance mitigate Friedreich's ataxia
PI/Investigator: Zhen Yan, PhD
- University of Virginia, VA, USA Award type:
General Research Grant Grant Title:
Endurance and resistance mitigate Friedreich's ataxia Lay summary:
The most common clinical symptoms of Friedreich's ataxia (FRDA) are ataxia, muscle weakness, diabetes and heart failure; the latter three conditions are directly related to loss of mitochondrial function and progressive oxidative damage in skeletal muscle and heart. Exercise is a potent means to promote mitochondrial function in various tissue types. This includes endurance (aerobic) exercise of the heart and skeletal muscle, which involves lower-body strength training such as running, jogging, or swimming. The role of endurance exercise in FRDA, however, has not been studied. Over the last decade, my lab has focused on the impact of endurance exercise on mitochondrial remodeling. We have recently obtained exciting new data to show that a single bout of treadmill running induces mitophagy [i.e., self-eating (autophagy) of mitochondria] in the muscles of mice, supporting a model of exercise-mediated improvement of mitochondrial quality. This improvement is achieved by simultaneous processes of the addition of new mitochondria through biogenesis, and the removal of damaged mitochondria through mitophagy. We propose that every bout of exercise acts as a "stress test" for all mitochondria, promoting the removal of suboptimal mitochondria and signaling for the making of new mitochondria. This dynamic process underscores the improvement of mitochondrial function with endurance exercise training. More recently, we have also developed a novel mouse model of resistance (strength) training involving voluntary weightlifting. Our resistance exercise paradigm shows evidence of enhanced contractile and metabolic adaptations in skeletal muscle following training. Altogether, the objective of this proposal is to elucidate how exercise affects mitochondria in skeletal muscle and heart in a mouse model of FRDA. I propose the following two specific aims to thoroughly examine the impacts of two major types of exercise on the FRDA mice: 1) to determine if resistance training and endurance exercise are equally effective in preventing symptomatic FRDA in these mice; and 2) to elucidate the mechanism by which endurance exercise improves exercise capacity and metabolic functions in the FRDA mouse. The findings will likely lead to paradigm shifting practice for FRDA prevention and treatment and pave the way for the development of effective therapeutics for this detrimental disease.Publications
- Yan Z, Lira VA, and Greene NP. (2012) "Exercise training-induced regulation of mitochondrial quality." Exerc Sport Sci Rev. 40:159-64.
- Lira VA, Okutsu M, Zhang M, Greene NP, Laker RC, Breen DS, Hoehn KL, and Yan Z. (2013) "Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance." FASEB J. 27:4184-93.
- Drake JC, Wilson RJ, and Yan Z. (2016) "Molecular mechanisms for mitochondrial adaptation to exercise training in skeletal muscle." FASEB J. 30:13-22.
- Laker RC, Drake JC, Wilson RJ, Lira VA, Lewellen BM, Ryall KA, Zhang M, Saucerman JJ, Goodyear LJ, Kundu M, and Yan Z. (2017) "AMPK phosphorylation of Ulk1 is required for lysosome targeting of mitochondria in mitophagy induced by exercise." Nat Commun. 8:548.
+ Jordi Magrane, PhD | Funding period: September 1, 2017 - July 31, 2019
Functional analysis of primary sensory neurons and (proprio) sensory pathology in Friedreich's ataxia
PI/Investigator: Jordi Magrane, PhD
- Cornell University, NY, USA Award type:
General Research Grant Grant Title:
Functional analysis of primary sensory neurons and (proprio) sensory pathology in Friedreich's ataxia Lay summary:
Friedreich's ataxia (FA) is a physiologically complex disorder that affects several tissues over the course of the disease. Early clinical symptoms in FA patients include areflexia, sensory loss, progressive limb and gait ataxia, and weakness. Impairments in patient's sensory systems consist of a loss of large dorsal root ganglion (DRG) neurons, atrophy of sensory neuron (SN) axons in the dorsal columns, atrophy in the dorsal nucleus of Clarke. Additionally, the patient sensory system exhibits degeneration of the dorsal spinocerebellar tract, corticospinal tract, primary sensory nuclei (cochlear nucleus, lateral geniculate nucleus), and dentate nucleus of the cerebellum. Functionally, defects in conduction velocity along sensory fibers of FA patients have also described. These observations raise the question whether the neuronal pathology in FA results from abnormalities in sensory circuitry. We initially focused on several aspects of mitochondria morphology and dynamics in in vitro and in vivo models of FA, and studied the peripheral nervous system and spinal cords of adult FA mouse models. This proposal aims to study whether reduced frataxin (Fxn) levels have an early impact on DRG SNs, with ambitions to shed light into the progression of sensory loss in FA. Our objectives are: 1) To explore the mechanisms of mitochondrial and neuronal dysfunction in embryonic SNs derived from a FA mouse model; and 2) to investigate early abnormalities in the sensory circuitry by using in vivo and ex vivo imaging.Publications
- Bolea I, Gan WB, Manfredi G, Magrane J (2014) "Imaging of mitochondrial dynamics in motor and sensory axons of living mice." Methods Enzymol, 547:97-110.
- Lin H, Magrane J, Rattelle A, Stepanova A, Galkin A, Clark EM, Dong Y, Halawani SM, and Lynch DR. (2017) "Early cerebellar deficits in mitochondrial biogenesis and respiratory chain complexes in the KIKO mouse model of Friedreich ataxia." Disease Models & Mechanisms. 10:1343-1352.
+ Katia Aquilano, PhD | Funding period: March 15, 2017 - March 14, 2019
Studying the role of brown fat in Friedreich's ataxia
PI/Investigator: Katia Aquilano, PhD
- University of Rome Tor Vergata, Italy Award type:
General Research Grant Grant Title:
Studying the role of brown fat in Friedreich's ataxia Lay summary:
Friedreich's ataxia (FRDA) is characterized by a variable phenotype, which includes pervasive neurological and cardiomyopathy symptoms (1), as well as diabetes mellitus (2). To date, the major cause of diabetes mellitus occurrence in FRDA patients seems to be related to impairment of mitochondria in pancreatic ß-cells (3)— that are fundamental in generating signals that trigger and amplify insulin secretion and regulate body adiposity (i.e., body fat). Defective pancreatic ß-cells cause insulin resistance and increased body adiposity in FRDA patients (2). The increased fat storage is due to the accumulation of triglycerides (TGs), the main constituent of body fat in humans. Typically, the expansion of TG fat depots is commonly found in overweight and obese individuals and occurs in what is called "white adipose tissue" (WAT) and "brown adipose tissue" [BAT; (4)]. BAT is a metabolically active, specialized tissue that can break down TGs (i.e., lipolysis) through a process called thermogenesis. For many years, the conventional belief was that BAT existed only in infants but not in adults, thus little research was performed on this tissue in adult humans. Recently, it has been discovered that metabolically active BAT is present also in adult humans and its activity contributes in maintaining glycaemia (5). Given the accumulation of intracellular TGs found in many cells and tissues of FRDA patients, we predict that the activity of BAT in these patients is defective. Therefore, we want to characterize BAT function in a FRDA mouse model of FXN deficiency. We aim to address whether BAT is dysfunctional in this model and if dysfunction contributes to the progression towards diabetes. We intend to unravel whether thermogenic function is impaired in mouse FRDA BAT in terms of defective mitochondrial, TG lipolytic activity, lipogenesis (i.e., fat production) and adipogenesis (i.e. adipose cell generation). After evaluation of body metabolic parameters by metabolic chambers and blood biochemical tests (e.g. glycaemia, lipid profile, OGTT), we will determine BAT morphology and activity (e.g. by assaying uncoupling respiration, thermogenic and lipolytic proteins). Adipogenesis will be also studied in mouse-derived brown adipocytes precursors. Increased thermogenesis via BAT has beneficial effects on overall body metabolism. Thus, we will induce lipolysis and thermogenesis in adipocytes via treatment of the FRDA mouse with butyrate (6). Butyrate is a metabolite with significant anti-diabetic effects including enhancing the thermogenic cascade in WAT and BAT (7). The goal of this butyrate supplementation strategy is to test if butyrate might restore BAT function and halt disease progression in the FRDA mouse model. The results obtained will provide insights into whether BAT is a prospective therapeutic target to combat metabolic disturbances observed in the FRDA mouse. Lastly, our work will explore butyrate as a valuable and safe molecule to be employed for future clinical research.Lay abstract references
- Koeppen AH, Ramirez RL, Becker AB, Bjork ST, Levi S, Santambrogio P, Parsons PJ, Kruger PC, Yang KX, Feustel PJ, and Mazurkiewicz JE. (2015) "The pathogenesis of cardiomyopathy in Friedreich ataxia." PLoS One. 10:e0116396.
- Cnop M, Mulder H and Igoillo-Esteve M. (2013) "Diabetes in Friedreich ataxia." J Neurochem. 126:94-102.
- Cnop M, Igoillo-Esteve M, Rai M, Begu A, Serroukh Y, Depondt C, Musuaya AE, Marhfour I, Ladriere L, Moles Lopez X, Lefkaditis D, Moore F, Brion JP, Cooper JM, Schapira AH, Clark A, Koeppen AH, Marchetti P, Pandolfo M, Eizirik DL, and Fery F. (2012) "Central role and mechanisms of beta-cell dysfunction and death in friedreich ataxia-associated diabetes." Ann Neurol. 72:971-982.
- Tseng YH, Cypess AM, and Kahn CR. (2010) "Cellular bioenergetics as a target for obesity therapy." Nat Rev Drug Discov. 9:465-482.
+ Javier Santos, PhD | Funding period: January 15, 2017 - January 14, 2019
Structural Dynamics and the Consolidation of Protein Function in Protein Complexes Involved in the Biosynthesis of Iron-Sulfur Clusters: Quaternary Addition of Small Trojan Tutor Proteins
PI/Investigator: Javier Santos, PhD
- University of Buenos Aires, Argentina Award type:
General Research Grant Grant Title:
Structural dynamics and the consolidation of protein function in protein complexes involved in the biosynthesis of iron-sulfur clusters: quaternary addition of small trojan tutor proteins Lay summary:
Iron-sulfur (Fe-S) clusters are essential cofactors present in all known forms of life, and hundreds of proteins require such cofactors to work. In eukaryotes, the biogenesis of most Fe-S clusters takes place in the mitochondria. The process involves the interactions and activities of several key proteins, namely frataxin (FXN), NFS1, ISCU, and ISD11. Mutations in these proteins lead to very serious human diseases, among them mutations in FXN results in Friedreich's Ataxia (FRDA). The Fe-S biosynthesis process can be summarized in three main steps: The first step is critical and involves the activity of cysteine desulfurase NFS1/ISD11, ISCU and FXN as a protein complex. Iron, sulfur, and electrons are transferred to a protein scaffold (ISCU); the second step is the assembly of Fe-S cluster itself; and the third step is the transfer of the Fe-S cluster from the scaffold to a receiving Fe-S protein. NFS1 is stabilized by its interactions with the ISD11 protein. FXN upregulates the transfer process of the sulfur group from NSF1 to the nascent Fe-S cluster and, given that FXN maintains iron soluble, it is thought that this protein assists in iron transfer. Our lab studies the stability, internal motions, and functionality of FXN variants. We study human FXN mutants to better understand the root cause of FXN instability in pathogenic variants. Inspired by the stabilizing interactions of NFS1 and ISD11, we propose delivering a small anchoring protein—or "Trojan" tutor protein—to the cell to modulate and stabilize FXN. In stabilizing FXN, we aim to improve FXN structural dynamics and its activity in the Fe-S cluster biosynthesis protein complex. We have selected the protein Sac7d to prepare Trojan tutor proteins with high affinity for FXN. Moreover, we will prepare TAT-signal peptide-Sac7d variants, which will enable the Sac7d protein to enter the mitochondrial matrix, where Fe-S biosynthesis occurs. To guide our study toward a feasible protein therapy, we will test the immunogenicity of the Sac7d variants. Furthermore, we will search for other Trojan tutor or scaffolding proteins to aid FXN. In this way, we will increase our chances of successfully stabilizing FXN and improving Fe-S cluster biosynthesis.Lay abstract references
- Faraj SE, Gonzalez-Lebrero RM, Roman EA, and Santos J. (2016) "Human frataxin folds via an intermediate state. Role of the C-terminal region." Nature Scientific Reports. 6: 20782.
- Faraj SE, Roman EA, Aran M, Gallo M, Santos J. (2014) "The Alteration of the C-terminal Region of Human Frataxin Distorts its Structural Dynamics and Function." FEBS J. 281: 3397-3419.
+ Sara Anjomani Virmouni, PhD |
Funding period: April 1, 2018 – March 31, 2020
Elucidation of the metabolic signature of Friedreich's ataxia
PI/Investigator: Sara Anjomani Virmouni, PhD
- Brunel University London & Insitute of Cancer research, UK. Award type:
General Research Grant Grant Title:
Elucidation of the metabolic signature of Friedreich's ataxia Lay summary:
In recent years, there has been a growing interest in the use of metabolomics in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, insufficient resources have been dedicated to studying alterations in the levels of small molecules, metabolites, and lipids in FRDA. Therefore, we aim to identify unique metabolic signatures of FRDA human and mouse model samples using mass spectrometry–based metabolomics approaches. To our knowledge, the current, novel YG8LR mouse model represents the largest GAA repeat expansion-containing model of all available FRDA mouse models. Due to the presence of the very large hyperexpansions in these mice, similar to those detected in FRDA patients, and also due to the inverse correlation between the GAA repeat size and the disease severity, we propose to employ the YG8LR mice for this project. Our approach to studying the role of metabolic dysfunction in FA disease pathogenesis is threefold: 1) We aim to evaluate the intracellular oxidative stress and mitochondrial health and function in the novel YG8LR transgenic mouse model, and to assess the bioenergetics profiles of FRDA human and mouse cell lines using tools to analyze oxidative metabolism; 2) We aim to investigate the effect of frataxin reduction on metabolic pathways involved in mitochondrial function and energy metabolism in FRDA human and mouse model samples using several highly-sensitive mass spectrometric-based methods; and lastly 3) We aim to delineate the impact of targeting relevant metabolic enzymes to rescue the deficits of central metabolism in FRDA both in vitro and in vivo. We expect that the findings of this proposal will provide a unique opportunity to devise novel therapeutic strategies for FRDA patient diagnosis and treatment through targeting their unique metabolism. Efforts to use metabolic phenotyping as a novel therapeutic approach may not only be limited to FRDA therapeutics, but could be also useful for pharmaceutical companies which are active in developing drugs for different metabolic diseases and neurological disorders.
- Santoro A, Anjomani Virmouni S, Paradies E, Villalobos V, Al-Mahdawi S, Khoo M, Porcelli V, Vozza A, Perrone M, Denora N, Taroni F, Merla G, Palmieri L, Pook MA, Marobbio CMT: Diazoxide as a novel frataxin-increasing therapy for Friedreich Ataxia. Human Molecular Genetics 2018, ddy016, https://doi.org/10.1093/hmg/ddy016.
- Gupta A, Anjomani-Virmouni S, Koundouros N, Dimitriadi M, Choo-Wing R, Valle A, Zheng Y, Chiu YH, Agnihotri S, Zadeh G, Asara JM, Anastasiou D, Arends MJ, Cantley LC, Poulogiannis G. (2017) "PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation." Mol Cell. 65:999-1013.e7.
- Mardakheh FK, Sailem HZ, Kümper S, Tape CJ, McCully RR, Paul A, Anjomani-Virmouni S, Jørgensen C, Poulogiannis G, Marshall CJ, Bakal C. (2016) "Proteomics profiling of interactome dynamics by colocalisation analysis (COLA)." Mol Biosyst. 13:92-105.
- Anjomani-Virmouni S, Al-Mahdawi S, Sandi C, Yasaei H, Giunti P, Slijepcevic P, Pook MA: Identification of telomere dysfunction in Friedreich ataxia. Molecular Neurodegeneration 2015, 10:22.
- Anjomani Virmouni S, Ezzatizadeh V, Sandi C, Sandi M, Al-Mahdawi S, Chutake Y, Pook MA: A novel GAA-repeat-expansion-based mouse model of Friedreich's ataxia. Disease models & mechanisms 2015, 8:225-235.
- Anjomani Virmouni S, Sandi C, Al-Mahdawi S, Pook MA: Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich ataxia. PLoS One 2014, 9:e107416.
+ Massimo Pandolfo, MD & Hélène Puccio, PhD |
Funding period: October 1, 2018 – September 30, 2020
Inflammation and metabolic changes in the nervous system in Friedreich ataxia:
relevance for pathogenesis and identification of biomarkers
PI/Investigator: Massimo Pandolfo, MD
- Hôpital Erasme, BelgiumPI/Investigator: Hélène Puccio, PhD
- IGBMC, FranceAward type:
General Research GrantGrant Title:
Inflammation and metabolic changes in the nervous system in Friedreich ataxia:
relevance for pathogenesis and identification of biomarkersLay summary:
The overall objective of this project is to investigate the pathogenic role of metabolic changes and neuroinflammation in Friedreich ataxia (FRDA) neuropathology, and to identify and validate related biomarkers to be used as candidate surrogate outcomes in clinical trials. The neuropathology of FRDA is characterized by marked differences in the vulnerability of neuronal systems. The reason(s) for such specific vulnerabilities are still unknown. Exploring changes in RNA and protein levels, metabolites, and inflammatory markers in different nervous system structures and biofluids from FRDA models may provide clues about pathogenesis and specific vulnerability. Furthermore, data from models can guide the search and allow cross-validation of biomarkers of disease status and/or progression in human patients. We plan to perform unbiased proteomic analysis and focused analysis of metabolic and inflammation markers in plasma and CSF of FRDA patients. Analysis of both plasma and CSF will be performed to dissect contribution from central nervous system (CNS) and peripheral tissues, both affected in FRDA but with different time courses. Data will be cross-validated with findings in two mouse models, , as well as in human induced pluripotent cell (hiPSC)-derived neurons, including proprioceptive neurons.
+ Vijayendran Chandran, PhD | Funding period: January 1, 2018 - December 31, 2019
Effect of early and late intervention of frataxin restoration in frataxin knockdown mouse model
PI/Investigator: Vijayendran Chandran, PhD
- University of Florida, USAAward type:
General Research Grant Grant Title:
Effect of early and late intervention of frataxin restoration in frataxin knockdown mouse modelLay summary:
Friedreich's ataxia (FRDA) the most commonly inherited ataxia is caused by severely reduced levels of frataxin (Fxn). The generation of mouse models for FRDA is vital for understanding and designing better therapeutic strategies. Existing fxn knockout FRDA animal models are either mildly symptomatic or restricted in their ability to recapitulate and evaluate the spatial and temporal aspects of FRDA pathology, as they are engineered to be tissue-specific conditional knockouts. We have developed an inducible mouse model for FRDA that permits reversible frataxin knockdown and detailed studies of the temporal progression or recovery following restoration of frataxin expression. We targeted a single copy shRNA against the Fxn transgene (doxycycline- inducible) under the control of H1 promoter gene into the rosa26 genomic locus. Fxn knockdown was achieved to control the onset and progression of the disease depending on the dose of doxycycline (dox). We observed behavioral deficits including ataxia, early mortality, defect in muscle strength, as well as degeneration of dorsal root ganglia, cardiomyopathy, and iron deposition, parallel to what is observed in FRDA patients. It is important to note that this is the first FRDA animal model that exhibits several symptoms parallel to FRDA, which can be rescued. Rescue experiments were carried out by withdrawal of dox to reverse the acceleration of disease progression even after significant motor dysfunction was observed. In this proposed project, we plan to characterize and identify molecular mechanism due to the effect of early and late frataxin restoration after Fxn knockdown in our mouse model. We plan to ask three critical questions: (1) What are the early molecular changes due to Fxn knockdown and restoration? (2) Which behavioral, physiological and pathological deficits can be rescued when Fxn levels are restored at the end stage of the disease? (3) What are the structural, molecular and functional architectural changes in intact biological tissues due to Fxn knockdown and rescue? These questions and this mouse model will enable us to better understand the Fxn function and will be essential for establishing various therapeutic strategies for FRDA.Publications
- Chandran V, Gao K, Swarup V, Versano R, Dong H, Jordan MC, and Geschwind DH. (2017) "Inducible and reversible phenotypes in a novel mouse model of Friedreich's Ataxia." Elife. 6:e30054.
+ Joriene De Nooij, PhD |
Funding period: June 1, 2018 – May 31, 2020
Modeling Friedreich Ataxia in human iPSC-derived sensory neuron subtypes.
Joriene De Nooij, PhD
- Columbia University, USA Award type:
General Research Grant Grant Title:
Modeling Friedreich ataxia in human iPSC-derived sensory neuron subtypes.Lay summary:
Friedreich ataxia (FA) patients present a complex set of clinical features, including ataxia, cardiomyopathy, diabetes mellitus, dysarthria, hearing loss, scoliosis, and visual loss (1-3). Distinctive characteristics of the FA disease phenotype are the progressive limb and gait ataxia and the absence of tendon reflexes (areflexia), symptoms that are consistently observed at early stages of the disease (4,5). The gait ataxia and areflexia correlates with a progressive loss of sensory neurons (SNs) in dorsal root ganglia (DRG) (6-7). Interestingly, while the frataxin gene (Fxn) appears expressed in most DRG neurons (8-10), individual SN subclasses (e.g., mechanoreceptors, proprioceptors or nociceptors) appear differently affected by a loss of Fxn. For example, most if not all FA patients experience a loss in the sense of touch or limb position – senses that are mediated by skin mechanoreceptors and proprioceptors, respectively (8). In contrast, few patients exhibit a reduced sensitivity to pain or temperature - senses associated with nociceptors (8, but see 12). However, exactly which or how each of the specific SNs in DRG are affected by a Fxn deficiency remains poorly understood. This proposal aims to define the molecular pathways that underlie the vulnerability of DRG SNs subtypes to the loss of Fxn. We seek to do this by modeling FA using patient-derived induced pluripotent stem cells (iPSCs) that we differentiate into these distinct SN subtypes. We will use CRISPR/Cas9 gene-editing strategies to generate selective fluorescent labels for these SN subtypes, allowing us to create reporter lines to correctly identify and characterize each subtype in both FA and control SNs. The underlying therapeutic goal of this work is to identify the sensory neuronal differences in FA, and to exploit that knowledge in the development of better treatment strategies.Lay abstract references:
- Pandolfo M. (2009) "Friedreich ataxia: the clinical picture." J Neurol. 256:Suppl 1:3-8.
- Parkinson MH, Boesch S, Nachbauer W, Mariotti C, Giunti P. (2013) "Clinical features of Friedreich's ataxia: classical and atypical phenotypes." J Neurochem. 126:Suppl 1:103-17.
- Abrahao A, Pedroso JL, Braga-Neto P, Bor-Seng-Shu E, de Carvalho Aguiar P, Barsottini OG. (2015) "Milestones in Friedreich ataxia: more than a century and still learning." Neurogenetics. 16:151-60.
- Harding AE. (1981) "Friedreich's ataxia: a clinical and genetic study of 90 families with an analysis of early diagnostic criteria and intrafamilial clustering of clinical features." Brain. 104:589-620.
- Stephenson J, Zesiewicz T, Gooch C, Wecker L, Sullivan K, Jahan I, Kim SH. (2015) "Gait and balance in adults with Friedreich's ataxia." Gait Posture. 41:603-7.
- Caruso G, Santoro L, Perretti A, Massini R, Pelosi L, Crisci C, Ragno M, Campanella G, Filla A. (1987) "Friedreich's ataxia: electrophysiologic and histologic findings in patients and relatives." Muscle Nerve. 10:503-15.
- Koeppen AH, Morral JA, Davis AN, Qian J, Petrocine SV, Knutson MD, Gibson WM, Cusack MJ, Li D. (2009) "The dorsal root ganglion in Friedreich's ataxia." Acta Neuropathol. 118:763-76
- Jiralerspong S, Liu Y, Montermini L, Stifani S, Pandolfo M. (1997) "Frataxin shows developmentally regulated tissue-specific expression in the mouse embryo." Neurobiol Dis. 4:103-13.
- Koutnikova H, Campuzano V, Foury F, Dollé P, Cazzalini O, Koenig M. (1997) "Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin." Nat Genet. 16:345-51.
- Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, Chen L, Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG, Duncan BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R, Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart NN, Stumpf KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB, Young RC, Youngstrom BL, Yuan XF, Zhang B, Zwingman TA, Jones AR. 2007) "Genome-wide atlas of gene expression in the adult mouse brain." Nature 445:168- 176.
- Saunders, P.W. (1913) "Sensory changes in Friedreich's disease." Brain. 36:166.
- Nolano M, Provitera V, Crisci C, Saltalamacchia AM, Wendelschafer-Crabb G, Kennedy WR, Filla A, Santoro L, Caruso G. (2001) "Small fibers involvement in Friedreich's ataxia." Ann Neurol. 50:17-25.
- de Nooij JC, Simon CM, Simon A, Doobar S, Steel KP, Banks RW, Mentis GZ, Bewick GS, Jessell TM. (2015) "The PDZ-domain protein Whirlin facilitates mechanosensory signaling in mammalian proprioceptors." J Neurosci. 35:3073-84.
- de Nooij JC, Doobar S, Jessell TM. (2013) "Etv1 inactivation reveals proprioceptor subclasses that reflect the level of NT3 expression in muscle targets." Neuron 77:1055-68.
- Kramer I, Sigrist M, de Nooij JC, Taniuchi I, Jessell TM, Arber S. (2006) "A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification." Neuron. 49:379-93.
+ Joel Gottesfeld, PhD & Bettina Lehman, PhD |
Funding period: July 1, 2018 – June 30,, 2020
Development of a novel iPSC-derived neuronal cell model for Friedreich’s ataxia and stem cell therapies
Bettina Lehman, PhD - Scripps Research Institute, CAPI/Investigator: Joel Gottesfeld, PhD
- Scripps Research Institute, CA
Postdoctoral FellowshipGrant Title:
Development of a novel iPSC-derived neuronal cell model for Friedreich’s ataxia
and stem cell therapiesLay summary:
This grant provides postdoctoral fellowship support for Dr. Bettina Lehman’s efforts in the laboratory of Dr. Joel Gottesfeld, at The Scripps Research Institute, La Jolla, California, for training in Friedreich’s ataxia research. Studies in our lab and other groups have failed to uncover a clear pathological phenotype in neuronal cells derived from Friedreich’s ataxia (FRDA) patient induced pluripotent stem cells (iPSCs). While Pandolfo and colleagues have recently reported that the abundance of various iron-sulfur cluster proteins is reduced in such cells , mitochondrial dysfunction has not been observed despite several years of effort. We hypothesize that this failure could be due to the immature nature of the neuronal cells, and the fact that most FRDA patients present with symptoms between the ages of 8 to 15 years, with no symptoms at birth. To better reveal the molecular mechanisms and disease-related phenotypes of FRDA, it is vital to take into account the age of the neuronal cells derived from patient iPSCs. Since iPSC-derived neuronal cells are generally quite immature, it is not that surprising that these cells fail to recapitulate the degenerative hallmarks of FRDA, such as mitochondrial dysfunction and oxidative stress . To circumvent these limitations of iPSC-derived neurons, we have generated stable doxycycline-dependent inducible expression of progerin in FRDA patient iPSC-derived cells. Progerin is a truncated form of the nuclear protein lamin A and is responsible for the early onset aging disease Huntchinson Gilford Progeria. Based on previous studies in the literature, forced expression of progerin in neuronal cells has been demonstrated to successfully model late-onset diseases, taking into account age progression properties . We hypothesize that similar expression of progerin in FRDA neurons will allow us to establish reproducible phenotypes of FRDA, such as mitochondrial dysfunction, oxidative stress, etc. We have demonstrated aging phenotypes and mitochondrial dysfunction in progerin-expressing FRDA iPSC-derived neurons. Additionally, previous studies have been performed with unspecified neuronal cells, rather than peripheral sensory neurons, which are the cell types primarily affected in FRDA. We propose to derive sensory neurons, which will be beneficial to both study the underlying molecular mechanisms in FRDA and to screen for therapeutic compounds that can reverse hallmarks of disease, including FXN expression, epigenetic modifications and mitochondrial function. We will also embark on two collaborative efforts with other laboratories in the FRDA field, providing neurons to those labs and helping test potential therapeutics.Publications cited in summary
- Codazzi, F., Hu, A., Rai, M., Donatello, S., Salerno Scarzella, F., Mangiameli, E., Pelizzoni, I., Grohovaz, F., and Pandolfo, M. (2016).
Friedreich ataxia-induced pluripotent stem cell-derived neurons show a cellular phenotype that is corrected by a benzamide HDAC inhibitor. Hum Mol Genet 25, 4847-4855.
- Martelli, A., and Puccio, H. (2014). Dysregulation of cellular iron metabolism in Friedreich ataxia: from primary iron-sulfur cluster deficit to mitochondrial iron accumulation. Front Pharmacol 5, 130.
- Miller, J.D., Ganat, Y.M., Kishinevsky, S., Bowman, R.L., Liu, B., Tu, E.Y., Mandal, P.K., Vera, E., Shim, J.W., Kriks, S., Taldone, T., Fusaki, N., Tomishima, M.J., Krainc, D., Milner, T.A., Rossi, D.J., and Studer, L. (2013). Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13, 691-705.
+ Manuela Corti, PhD | Funding period: September 1, 2016 – March 1, 2019
Clinical outcome measures of efficacy in the treatment of Friedreich's ataxia
PI/Investigator: Manuela Corti, P.T., PhD
- University of Florida, FL, USA Award type:
General Research GrantGrant Title:
Clinical outcome measures of efficacy in the treatment of Friedreich's ataxia Lay summary:
The purpose of this multi-year longitudinal study is to identify the natural progression of Friedreich's Ataxia (FA), including disease phenotypes and their correlation with disease severity, current standard of care and adverse events. A key goal of this study is to develop and validate outcome measures that can be used in the development of future clinical trials for definitive treatment of FA. The development of a successful treatment is dependent not only on the understanding of the pathophysiological mechanisms of the disease and the attributes of the proposed therapeutic product, but also on the identification of sensitive and non-invasive, or minimally invasive outcome measures to demonstrate the potential effect of treatment. The identification of sensitive outcome measures evaluating both the cardiac, metabolic, and neuromuscular function in FA will be crucial to: 1) Evaluate the naturally-occurring changes in neural function as the result of growth, maturation, disease progression, or secondary consequences of the disease; and 2) Detect the effectiveness of potential therapeutic strategies. In this study, we will characterize measures of cardiac performance, neuromuscular physiology, speech and swallowing, as well as frataxin protein quantification in children and adults with FA. To achieve these objectives, we will use cutting edge techniques, including echocardiography and magnetic resonance imaging (MRI), metabolic exercise testing, neurophysiological and mass spectrometry measures. We are planning a 5-year, bi-annual, longitudinal study of 50 individuals with a confirmed diagnosis of Friedreich's Ataxia. Completion of this project will be an important milestone to advance the study of the natural history, discovery and validation of clinical outcome measures and biomarkers in FA. In addition, results of this study might be implemented in potential clinical trials to evaluate the effect of treatment. Publications
- Smith BK, Renno MS, Green MM, Sexton TM, Lawson LA, Martin AD, Corti M, and Byrne BJ. (2016) "Respiratory motor function in individuals with centronuclear myopathies." Muscle Nerve. 53:214-21.
- Doerfler P, Nayak S, Corti M, Morel L, Herzog R, and Byrne BJ. (2016) "Targeted Approaches to Induce Immune Tolerance for Pompe Disease Therapy." Molecular therapy. Methods & clinical development. 3:15053.
- Corti M, Cleaver B, Clément N, Conlon TJ, Faris KJ, Wang G, Benson J, Tarantal AF, Fuller D, Herzog RW, and Byrne BJ. (2015) "Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning. Hum Gene Ther Clin Dev. 26:185-93.
- Corti M, Elder M, Falk D, Lawson L, Smith B, Nayak S, Conlon T, Clement N, Erger K, Lavassani E, Green M, Doerfler P, Herzog R, and Byrne B. (2014) "B-Cell Depletion is Protective Against Anti-AAV Capsid Immune Response: A Human Subject Case Study." Mol Ther Methods Clin Dev. 1. pii: 14033.
+ Chad Heatwole, MD | Funding period: October 1, 2018 – September 30, 2020
Developing a Clinically Relevant Disease Specific Patient Reported Outcome Measures for use in Friedreich’s Ataxia Therapeutic Trials and FDA Drug Labeling Claims
Chad Heatwole, MD
- University of Rochester Medical Center, NY Award type:
General Research GrantGrant Title:
Developing a Clinically Relevant Disease Specific Patient Reported Outcome Measures for use in Friedreich’s Ataxia Therapeutic Trials and FDA Drug Labeling ClaimsLay summary:
This research will utilize existing methods and infrastructure to develop and validate disease-specific, patient-reported outcome measures for clinical trials of patients with Friedreich's ataxia. This proposal will shift and refine current research paradigms by producing instruments that will efficiently identify relevant changes in several areas of Friedreich's ataxia patient health. All instruments will be developed and validated in accordance with FDA guidelines for use in drug labeling claims. In addition, input will be obtained from the Friedreich's ataxia research community to optimize the acceptance and use of each of these instruments. These measures will provide researchers with valuable tools to use in clinical trials of pediatric and adult Friedreich's ataxia patients. Although the validation techniques proposed in this study are considered industry standard by many, they have never been implemented on this scale for pediatric and adult Friedreich's ataxia. At the completion of our work, the Friedreich's ataxia research community will have valid and highly responsive outcome measures to aid in therapeutic assessment and therapeutic development in Friedreich's ataxia.Publications
- Chad Heatwole, Rita Bode, Nicholas Johnson, Jeanne Dekdebrun, Nuran Dilek, Katy Eichinger, James E. Hilbert, Eric Logigian, Elizabeth Luebbe, William Martens, Michael P. McDermott, Shree Pandya, Araya Puwanant, Nan Rothrock, Charles Thornton, Barbara G. Vickrey, David Victorson, Richard T. Moxley, III (2017) The Myotonic Dystrophy Health Index: Correlations with Clinical Tests and Patient Function. Muscle Nerve. Author manuscript; available in PMC 2017 Feb 1
- Heatwole C, Bode R, Johnson N, et al. Patient-reported impact of symptoms in myotonic dystrophy type 1 (PRISM-1) (2012) . Neurology. 79(4):348-357
- Heatwole C, Bode R, Johnson N, et al. (2013) The myotonic dystrophy health index: Initial evaluation of a new outcome measure. Muscle Nerve.
+ David Herrmann, MD & Peter Creigh, MD | Funding period: September 1, 2018 – August 31, 2020
In-Vivo Confocal Imaging of Meissner’s Corpuscles as a Biomarker in Friedreich’s Ataxia (FA)
David Herrmann, MBBCh – University of Rochester Medical Center, NYPI/Investigator:
Peter Creigh, MD – University of Rochester Medical Center, NYAward type:
General Research GrantGrant Title:
In-Vivo Confocal Imaging of Meissner’s Corpuscles as a Biomarker in Friedreich’s Ataxia (FA) Lay summary:
This is an extension of an observational study designed to determine the potential role of Meissner Corpuscle (MC) Imaging as a biomarker in FA. In the first phase, 16 FA patients and 16 healthy controls were recruited and studied over 12 months with a series of potential biomarkers. Several of these biomarkers looked promising, and this study is being extended to include an additional 11 participants with FA, and the study will continue for up to 24 months. This is a two-part study. An initial cross-sectional phase assessed the utility of MC imaging, quantitative sensory testing (QST) (touch-pressure, vibration (timed and quantitative) and cold detection thresholds), as biomarkers in FA. These candidate biomarkers will now be assessed longitudinally in a larger cohort of patients and for a longer period in this extension phase. The study will include a screening and baseline visit on the same day and 3 subsequent visits, conducted 6, 12 and 24 months after the initial study visit. The original study included only screening, baseline 6 and 12 month visits, and in this extension a 24 month measurement will be added.Co-Sponsor: Voyager Therapeutics
+ Ian Harding, PhD | Funding period: October 1, 2018 – September 30, 2020
Neuroinflammation in Friedreich Ataxia: Mechanism, Biomarker, and Therapeutic Target
PI/Investigator: Ian Harding, PhD
- Monash University, AustraliaAward type:
General Research GrantGrant Title:
Neuroinflammation in Friedreich Ataxia: Mechanism, Biomarker, and Therapeutic TargetLay summary:
This study aims to establish if in vivo biomarkers of inflammation exist in the brain and spinal cord of individuals with Friedreich ataxia (FRDA). Although clinical presentation and progression are variable in individuals with FRDA, a universal feature is ataxia and loss of motor control secondary to the significant neuropathology that typifies FRDA. Sustained activation of immune-responsive cells in the brain – termed neuroinflammation – may represent one mechanism contributing to this progressive neuropathology. Recent preclinical and post-mortem studies in FRDA report increased inflammatory metabolites and gliosis in the nervous system. Importantly, cell line and animal studies indicate that blocking the inflammatory response in FRDA may ameliorate cell death. The link between chronic neuroinflammation and progressive neurodegeneration has also become increasingly well-establish in other degenerative disorders, including Alzheimer’s and Parkinson’s diseases. This project will be the first to evaluate in vivo neuroinflammation and its link with measures of neurodegeneration in individuals with FRDA using a novel combination of magnetic resonance imaging (MRI) and positron emission tomography (PET) brain imaging approaches. Given there are currently no treatments that are proven to alter the devastating natural history of FRDA, identifying markers of neuroinflammation and uncovering its role in driving or exacerbating neuropathology in FRDA will be key to improving the understanding of disease mechanisms, tracking disease progression, and identifying and monitoring novel treatment approaches.Co-sponsor: fara Australia and FARA Ireland
- Selvadurai LP, Harding IH, Corben LA, Georgiou-Karistianis N. (2018). Cerebral abnormalities in Friedreich Ataxia: A review. Neuroscience & Biobehavioral Reviews 84: 394-406.
- Harding IH, Corben LA, Delatycki MB, Stagnitti MR, Storey E, Egan GF, Georgiou-Karistianis N. (2017). Cerebral compensation during motor function in Friedreich ataxia: The IMAGE-FRDA study. Movement Disorders 32(8): 1221-1229.
- Selvadurai LP*, Harding IH*, Corben LA, Stagnitti MR, Storey E, Egan GF, Delatycki MB, Georgiou- Karistianis N. (2016). Cerebral and cerebellar grey matter atrophy in Friedreich ataxia: The IMAGE-FRDA study. J Neurology 263(11): 2215-2223. *Equal Contribution
- Harding IH, Raniga P, Delatycki MB, Stagnitti MR, Corben LA, Storey E, Georgiou-Karistianis N, Egan GF. (2016). Tissue atrophy and elevated iron concentration in the extrapyramidal motor system in Friedreich ataxia: The IMAGE-FRDA study. J Neurology Neurosurgery and Psychiatry 87: 1261-1263.
- Harding IH, Corben LA, Storey E, Egan GF, Stagnitti MR, Poudel GR, Delatycki MB, Georgiou- Karistianis N. (2016). Fronto-cerebellar dysfunction and dysconnectivity underlying cognition in Friedreich ataxia: The IMAGE-FRDA study. Human Brain Mapping 37: 338-350.
+ Louise Corben, PhD | Funding period: July 1, 2018 - June 30, 2019
Developing a clinically meaningful instrumented measure of upper limb function in Friedreich ataxia
Louise Corben, PhD - Murdoch Children’s Research Institute, Melbourne. AustraliaAward type: AFAF-FARA Ride Ataxia Europe Research AwardGrant Title:
Developing a clinically meaningful instrumented measure of upper limb function in Friedreich ataxiaLay summary:
This prospective longitudinal study aims to develop and evaluate the capacity of an upper limb measure to capture change in function over a 4.5 and 9 month period in 40 individuals with Friedreich ataxia (FRDA). We have developed an instrumented measure of upper limb function which has the potential to be a sensitive and clinically relevant biomarker for use in future clinical trials. This measure has arisen from a conceptual framework that has evaluated the strengths and limitations of current tools, identified specific motor components that contribute to upper limb impairment in FRDA as well as the upper limb activities that are meaningful and relevant to individuals with FRDA. Importantly the conceptual framework underlying this new measure has ensured the specific functional capacity as measured by this tool would enhance quality of life if it was maintained or improved by therapeutic intervention. Moreover, not only does this tool measure functional capacity it correlates with and detects upper limb ataxia as measured by clinical testing thus ensuring measurement of ataxia while completing a functional task, as well as overcoming assessor bias and skill. Our compelling preliminary data using this measure, a BioKin-WMS wireless motion capture device in the pre-oral phase of eating in individuals with FRDA, has demonstrated the face and discriminate validity of this tool, but indicates that a prospective longitudinal study with greater power is now required to evaluate both the reliability and capacity of the tool to measure change over time. This study has the potential to validate this novel clinically meaningful instrumented upper limb measure for FRDA as a biomarker for use in clinical trials. Importantly, if validity and reliability are established, this upper limb measure will provide a functionally meaningful outcome measure that will reflect the clinical trajectory of FRDA and enable inclusion in future clinical trials of those individuals who are currently unable to complete the traditional measures, in particular those that involve ambulation.Co-sponsor: Association Française de l'Ataxie de FriedreichPublications
- Milne SC, Corben LA, Roberts M, Murphy A, Tai G, Georgiou-Karistianis N, Yiu EM, Delatycki MB. (2018) Can rehabilitation improve the health and wellbeing in Friedreich's ataxia: a randomised controlled trial. Clinical Rehabilitation 32(5):630-643
- Milne SC, Murphy A, Georgiou-Karistianis N, Yiu EM, Delatycki MB, Corben LA (2018) Psychometric properties of outcome measures evaluating decline in gait in cerebellar ataxia: a systematic review. Gait and Posture, 61: 149-162
- Tai G, Yiu EM, Delatycki MB, Corben LA. (2017) How does performance of the Friedreich Ataxia Functional Composite compare to rating scales? Journal of Neurology, 264(8): 1768-1776.
- Corben LA, Klopper F, Stagnitti MM, Georgiou-Karistianis N, Bradshaw JL, Rance G, Delatycki MB. (2017) Measuring Inhibition and Cognitive Flexibility in Friedreich Ataxia. Cerebellum, 16(4):757-763.
For more informational about grants awarded for Outcome Measures
please visit our page on the
Center of Excellence in FA
+ Hélène Puccio, PhD | Funding period: July 1, 2016 - June 31, 2018
Investigation of cardiac pathophysiological mechanism and relevant biomarker in the context of FXN deficiency and FXN overexpression induced toxicity
PI/Investigator: Hélène Puccio, PhD
- INSERM and IGBMC, France Award type: Keith Michael Andrus Cardiac Research Award Grant Title:
Investigation of cardiac pathophysiological mechanism and relevant biomarker in the context of FXN deficiency and FXN overexpression induced toxicity Lay summary:
Many therapeutic strategies for FA aim at increasing the cellular level of FXN or alleviating the secondary pathological events which are both impairing mitochondria function and bioenergetics. However, most of the clinical trials have relied on peripheral cells (blood or buccal cells) to validate the short-term bio-availability and the biological effect of the investigational drug. In the present proposal, we aim at developing a comprehensive framework for facilitating the preclinical development and validation of therapeutic strategies for FA cardiomyopathy, with a particular focus on the cardiac gene therapy protocol that we have developed. We have previously shown the proof of concept for the prevention and the correction of FA cardiomyopathy by in vivo gene therapy . More recently, we have conducted a series of dose response studies, which have defined the therapeutic thresholds (histological and molecular) conditioning the stabilization or the correction of the cardiac function in the MCK mouse model (funded by FARA and AAVLife/Annapurna Therapeutics). On the basis of these results, AAVlife/Annapurna is optimizing the administration paradigm in large animal model. The vector that has currently been used in proof of concept studies in the MCK model is under the control of a ubiquitous promoter that expresses levels of frataxin that are 10 to 50 fold higher than the endogenous level. Although no toxicity has been seen with this level of frataxin in the MCK mouse model over 1 year, it is difficult to predict the potential of toxicity of such high amounts of frataxin over several years, as it would be the case in FA patients. Therefore, to finalize the preclinical development of this gene therapy protocol, we have started to investigate the effect of high level of FXN overexpression in order to identify the upper threshold limit. In parallel, we are trying to identify potential biomarkers reflecting the physiological state of the cardiomyocytes in the context of FXN deficiency or FXN overexpression. In preliminary experiments, we have shown in the MCK mouse model that cardiac overexpression of FXN at a level over 100 fold the endogenous level is associated with severe mitochondria and cardiomyocytes dysfunction, which is eventually deleterious for cardiac function. The first objective of this project is to study the pathological mechanisms associated with these toxic events and to identify clinical relevant biomarkers reflecting the mitochondria function and metabolic state of cardiomyocytes in order to provide biosafety guidelines in FA clinical trials (gene or pharmacological therapies). The second objective is to generate with a minimum time and resources, a specific cardiac conditional KO mouse model recapitulating the FA cardiac phenotype similarly to the MCK mouse model, but without the artefactual peripheral myopathy observed at 15 weeks of age in the MCK mice model . This new mouse model will be available to the FA community and should facilitate greatly the therapeutic evaluation of new drug and the investigation of specific plasmatic biomarker. The third objective is to identify potential biomarkers relevant for FXN deficiency and FA cardiomyopathy, either plasmatic or by in vivo imaging, which could be used as secondary endpoints in clinical trial, the clinical management of FA patients, as a diagnostic and if possible a predictive tool of the cardiac phenotype.Lay abstract references
- Perdomini M, Belbellaa B, Monassier L, Reutenauer L, Messaddeq N, Cartier N, Crystal RG, Aubourg P, Puccio H. (2014) "Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia." Nat Med. 20:542-7.
+ Arnulf H. Koeppen, MD | Funding period: Jan 15, 2019-Jan 14, 2020
Cytoskeletal, heat shock, and blood vessel proteins in Friedreich cardiomyopathy
Arnulf H. Koeppen, MD
- VA Medical Center, AlbanyAward type: Keith Michael Andrus Cardiac Research Award Grant Title:
Cytoskeletal, heat shock, and blood vessel proteins in Friedreich cardiomyopathyLay summary:
Heart failure with or without arrhythmia is the most common cause of death in patients with
Friedreich ataxia (FA). However, in patients with modest expansions of guanine-adenine-adenine (GAA) trinucleotide repeats in the shorter allele, heart disease may be entirely absent (Koeppen, et al., 2016). Several different mechanisms may contribute to heart failure in FA, directly and indirectly associated with reduced frataxin. Inflammation may be the principal mechanism underlying fiber necrosis and myocardial scarring (Koeppen, et al., 2015), but it cannot account for cardiomyocyte hypertrophy or the remarkable disorganization of intercalated discs (ICD) (Koeppen, et al., 2016). In the human heart, ICD are fully developed at the age of 6 years (Peters, et al., 1994), and it is unclear how ICD become so chaotic in most cases of FA. Hearts in FA may also be subject to ischemia, but the literature contains little attention to this mechanism. In most cases of FA, cardiomyocytes contain small perinuclear iron-positive granules (Lamarche, et al., 1980).
This study set out to look at the mechanisms of progressive cardiomyopathy in FA patients. Based on previous data collected by the investigator, he hypothesized (a) that frataxin deficiency in mitochondria disables the function of the desmin interactome in the maintenance of normal heart fiber cytoskeleton, including ICD; (b) that frataxin deficiency leads to the addition of extra fragmentary ICD along the course of heart fibers; and (c) that αB-crystallin and desmin aggregation impair iron export from cardiomyocytes. He also proposed that capillary hyperplasia in FA hearts causes ischemia due to "luxury perfusion" and that FA hearts do not respond to oxidative stress in FA as reflected by the unexpected downregulation of HSP72 and HSP27. The objectives of this study are therefore (1) to establish the destruction of the desmin interactome and its effect on ICD by immunohistochemistry and single- and double-label immunofluorescence of multiple heart proteins, including those of the ICD; (2) to provide semiquantitative analysis of these proteins by Western blots with validated antibodies; and (3) to explore the microvascularity of FA hearts by a quantitative morphological method.
- The significance of intercalated discs in the pathogenesis of Friedreich cardiomyopathy.
Koeppen AH, Becker AB, Feustel PJ, Gelman BB, Mazurkiewicz JE.
J Neurol Sci. 2016 Aug 15;367:171-6. doi: 10.1016/j.jns.2016.06.006. Epub 2016 Jun 4. PMID: 27423584
- Pathology of Intercalated Discs in Friedreich Cardiomyopathy.
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