Frataxin-deficient cardiomyocytes present an altered thiol-redox state which targets actin and pyruvate dehydrogenase

Rosa Purroy, Marta Medina-Carbonero, Joaquim Ros, Jordi Tamarit; Redox Biology, Volume 32, 2020, 101520, doi:10.1016/j.redox.2020.101520.


We identified two proteins with altered redox status in frataxin-deficient NRVMs: electron transfer flavoprotein-ubiquinone oxidoreductase and dihydrolipoyl dehydrogenase (DLDH). As DLDH is involved in protein-bound lipoic acid redox cycling, we analyzed the redox state of this cofactor and we observed that lipoic acid from pyruvate dehydrogenase was more oxidized in frataxin-deficient cells. Also, by targeted proteomics, we observed a decreased content on the PDH A1 subunit from pyruvate dehydrogenase. Finally, we analyzed the consequences of supplementing frataxin-deficient NRVMs with the PDH cofactors thiamine and lipoic acid, the PDH activator dichloroacetate and the antioxidants N-acetyl cysteine and Tiron. Both dichloroacetate and Tiron were able to partially prevent lipid droplet accumulation in these cells. Overall, these results indicate that frataxin-deficient NRVMs present an altered thiol-redox state which could contribute to the cardiac pathology.

Calpain-Inhibitors Protect Frataxin-Deficient Dorsal Root Ganglia Neurons from Loss of Mitochondrial Na+/Ca2+ Exchanger, NCLX, and Apoptosis

Elena Britti, Fabien Delaspre, Jordi Tamarit & Joaquim Ros; Res (2020). doi:10.1007/s11064-020-03020-3

Calpains are calcium-dependent proteases activated in apoptotic cell death and neurodegeneration. Friedreich Ataxia is a neurodegenerative rare disease caused by frataxin deficiency, a mitochondrial protein. Dorsal root ganglion (DRG) sensory neurons are among the cellular types most affected in this disease. We have previously demonstrated that frataxin-deficient DRGs show calpain activation, alteration in calcium levels and decreased content of the Na+/Ca2+ exchanger (NCLX). This transporter is involved in mitochondrial calcium efflux. In this study, we have performed a time-course analysis of several parameters altered in a frataxin-deficient DRGs. These include decline of NCLX levels, calcium accumulation, mitochondrial depolarization, α-fodrin fragmentation and apoptotic cell death. Furthermore, we have analysed the effect of the calpain inhibitors MDL28170 and Calpeptin on these parameters. We have observed that these inhibitors increase NCLX levels, protect sensory neurons from neurite degeneration and calcium accumulation, and restore mitochondrial membrane potential. In addition, calpain 1 reduction alleviated neurodegeneration in frataxin-deficient DRG neurons. These results strengthen the hypothesis of a central role for calcium homeostasis and calpains in frataxin-deficient dorsal root ganglia neurons.

Calcitriol increases frataxin levels and restores altered markers in cell models of Friedreich Ataxia

Elena Britti, Fabien Delaspre, Marta Medina-Carbonero, Arabela Sanz, Marta Llovera, Rosa Purroy, Stefka Mincheva-Tasheva, Jordi Tamarit, Joaquim ROS; bioRxiv 2020.04.09.034017; doi: https://doi.org/10.1101/2020.04.09.034017

Friedreich Ataxia (FA) is a neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. In primary cultures of dorsal root ganglia neurons, we showed that frataxin depletion resulted in decreased levels of the mitochondrial calcium exchanger NCLX, neurite degeneration and apoptotic cell death. Here we describe that frataxin-deficient dorsal root ganglia neurons display low levels of ferredoxin 1, a mitochondrial Fe/S cluster-containing protein that interacts with frataxin and, interestingly, is essential for the synthesis of calcitriol, the active form of vitamin D. We provide data that calcitriol supplementation, used at nanomolar concentrations, is able to reverse the molecular and cellular markers altered in DRG neurons. Calcitriol is able to recover both ferredoxin 1 and NCLX levels and restores mitochondrial membrane potential. Accordingly, apoptotic markers and neurite degeneration are reduced resulting in cell survival recovery with calcitriol supplementation. All these beneficial effects would be explained by the finding that calcitriol is able to increase the mature frataxin levels in both, frataxin-deficient DRG neurons and cardiomyocytes; remarkably, this increase also occurs in lymphoblastoid cell lines derived from FA patients. In conclusion, these results provide molecular bases to consider calcitriol for an easy and affordable therapeutic approach for FA patients.

Particle-mediated delivery of frataxin plasmid to a human sensory neuronal model of Friedreich's ataxia

Ewa Czuba-Wojnilowicz, Serena Viventi, Sara E. Howden, Simon Maksour, Amy E. Hulme, Christina Cortez-Jugo, Mirella Dottori and Frank Caruso; Biomater. Sci., 2020, Advance Article doi:10.1039/C9BM01757G

Increasing frataxin protein levels through gene therapy is envisaged to improve therapeutic outcomes for patients with Friedreich's ataxia (FRDA). A non-viral strategy that uses submicrometer-sized multilayered particles to deliver frataxin-encoding plasmid DNA affords up to 27 000-fold increase in frataxin gene expression within 2 days in vitro in a stem cell-derived neuronal model of FRDA.

Long-term voluntary running prevents the onset of symptomatic Friedreich’s ataxia in mice

Henan Zhao, Beven M. Lewellen, Rebecca J. Wilson, Di Cui, Joshua C. Drake, Mei Zhang & Zhen Yan; Sci Rep 10, 6095 (2020). doi:10.1038/s41598-020-62952-6

Importantly, long-term (4 months) voluntary running in KIKO mice starting at a young age (2 months) completely prevented the functional abnormalities along with restored Irp1 expression, improved mitochondrial function and reduced oxidative stress in skeletal muscle without restoring Fxn expression. We conclude that endurance exercise training prevents symptomatic onset of FRDA in mice associated with improved mitochondrial function and reduced oxidative stress. These preclinical findings may pave the way for clinical studies of the impact of endurance exercise in FRDA patients.

Progress towards drug discovery for Friedreich’s Ataxia: Identifying synthetic oligonucleotides that more potently activate expression of human frataxin protein

Xiulong Shen, Johnathan Wong, Thahza P. Prakash, Frank Rigo, Yanjie Li, Marek Napierala, David R. Corey; Bioorganic & Medicinal Chemistry,
2020, 115472, doi:10.1016/j.bmc.2020.115472.

We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of “gapmer” oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo.

High levels of frataxin overexpression leads to mitochondrial and cardiac toxicity in mouse models

Brahim Belbellaa, Laurence Reutenauer, Nadia Messaddeq, Laurent Monassier, Helene Puccio; bioRxiv 2020.03.31.015255; doi:10.1101/2020.03.31.015255

AAV-mediated gene therapy constitutes a promising approach for FA, as demonstrated in cardiac and neurological mouse models. While the minimal therapeutic level of FXN protein to be restored and biodistribution have recently been defined for the heart, it is unclear if FXN overexpression could be harmful. Indeed, depending on the vector delivery route and dose administrated, the resulting FXN protein level could reach very high levels in the heart, cerebellum, or in off-target organs such as the liver. The present study demonstrates safety of FXN cardiac overexpression up to 9-fold the normal endogenous level, but significant toxicity to the mitochondria and heart above 20-fold. We show gradual severity with increasing FXN overexpression, ranging from subclinical cardiotoxicity to left ventricle dysfunction. This appears to be driven by impairment of mitochondria respiratory chain, ultrastructure and homeostasis, which lead to myofilaments alteration, cell death and fibrosis. Overall, this study underlines the need, during the development of gene therapy approaches, to consider appropriately vector potency, long term safety and biomarkers to monitor such events.

Disarrangement of Endoplasmic reticulum-mitochondria communication impairs Ca2+ homeostasis in FRDA

Laura R Rodríguez, Pablo Calap-Quintana, Tamara Lapeña-Luzón, Federico V Pallardó, Stephan Schneuwly, Juan Antonio Navarro Langa, Pilar Gonzalez-Cabo; bioRxiv 2020.03.27.011528; doi:10.1101/2020.03.27.011528

Friedreich ataxia (FRDA) is a neurodegenerative disorder characterized by neuromuscular and neurological manifestations. It is caused by mutations in gene FXN, which results in loss of the mitochondrial protein frataxin. Endoplasmic Reticulum-mitochondria associated membranes (MAMs) are inter-organelle structures involved in the regulation of essential cellular processes, including lipid metabolism and calcium signaling. In the present study, we have analyzed in both, unicellular and multicellular models of FRDA, an analysis of calcium management and of integrity of MAMs. We observed that function of MAMs is compromised in our cellular model of FRDA, which was improved upon treatment with antioxidants. In agreement, promoting mitochondrial calcium uptake was sufficient to restore several defects caused by frataxin deficiency in Drosophila Melanogaster. Remarkably, our findings describe for the first time frataxin as a member of the protein network of MAMs, where interacts with two of the main proteins implicated in endoplasmic reticulum-mitochondria communication. These results suggest a new role of frataxin, indicate that FRDA goes beyond mitochondrial defects and highlight MAMs as novel therapeutic candidates to improve patient's conditions.

Cofilin dysregulation alters actin turnover in frataxin-deficient neurons

Diana C. Muñoz-Lasso, Belén Mollá, Pablo Calap-Quintana, José Luis García-Giménez, Federico V. Pallardo, Francesc Palau & Pilar Gonzalez-Cabo; Sci Rep 10, 5207 (2020). https://doi.org/10.1038/s41598-020-62050-7.

In this work, we demonstrate the dysregulation of the actin cytoskeleton in frataxin-deficient neurites of DRG neurons from the YG8R mice as a result of hyperactivation of cofilin-1 and the complex ARP2/3, which could affect the dynamics of growth cones and neurite growth. Recent work has determined that cofilin hyperactivation is age-dependent57, so it would be interesting to analyze these results at the embryonic stage. Assessing if there is a dysregulation of cofilin that could contribute to a failure in the neurite growth of embryonic neurons would help to understand the hypoplastic phenomena previously described in FRDA patients1. As a whole, our results show for the first time an imbalance in the activity of cofilin that could explain, at least partially, the neuropathy of FRDA. Future research will determine if cofilin is a potential molecular target for a therapeutic approach in FRDA.

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells

Gabriela Vilema-Enríquez, Robert Quinlan, Peter Kilfeather, Roberta Mazzone, Saba Saqlain, Irene del Molino del Barrio, Annalidia Donato, Gabriele Corda, Fengling Li, Masoud Vedadi, Andrea H Németh, Paul E Brennan, Richard Wade-Martins; bioRxiv 2020.03.26.010439; doi:10.1101/2020.03.26.010439

Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogues were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression, and highlight SUV4-20 H1 as a potential novel therapeutic target for FRDA.