Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

Daniel Fil, Balu K. Chacko, Robbie Conley, Xiaosen Ouyang, Jianhua Zhang, Victor M. Darley-Usmar, Aamir R. Zuberi, Cathleen M. Lutz, Marek Napierala, Jill S. Napierala; Disease Models & Mechanisms 2020 : dmm.045229 doi: 10.1242/dmm.045229 Published 25 June 2020

We report generation of the first mouse model harboring a Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure to thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA (mtDNA) lesions and fragmentation are accompanied by marked amplification of mtDNA in FxnG127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of FxnG127V cells upon alteration of fatty acid availability. Importantly, substitution of FxnWT with FxnG127V is compatible with life and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose FxnG127V cells as a simple and robust model for testing therapeutic approaches for FRDA.

DNA Hypermethylation and Unstable Repeat Diseases: A Paradigm of Transcriptional Silencing to Decipher the Basis of Pathogenic Mechanisms

Poeta, Loredana; Drongitis, Denise; Verrillo, Lucia; Miano , Maria Giuseppina; Genes 11, no. 6: 684. doi:10.3390/genes11060684

We report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders.

NRF2 Regulation Processes as a Source of Potential Drug Targets against Neurodegenerative Diseases

Cores, Á.; Piquero, M.; Villacampa, M.; León, R.; Menéndez, J.C; Biomolecules 2020, 10, 904. doi:10.3390/biom10060904

NRF2 acts by controlling gene expression, being the master regulator of the Phase II antioxidant response, and also being key to the control of neuroinflammation. NRF2 activity is regulated at several levels, including protein degradation by the proteasome, transcription, and post-transcription. The purpose of this review is to offer a concise and critical overview of the main mechanisms of NRF2 regulation and their actual or potential use as targets for the treatment of neurodegenerative diseases.

[Epigenetic Regulation of Clinical Manifestations of Friedreich's Disease]

E P Nuzhny, N Yu Abramycheva, N S Nikolaeva, M V Ershova, S A Klyushnikov, S N Illarioshkin, E Yu Fedotova. Épigeneticheskaia reguliatsiia klinicheskikh proiavleniĭ bolezni Fridreĭkha [Epigenetic regulation of clinical manifestations of Friedreich's disease]. Zh Nevrol Psikhiatr Im S S Korsakova. 2020;120(1):20‐26. doi:10.17116/jnevro202012001120

Correlations between the methylation level of CpG-sites in UP-GAA and DOWN-GAA and the number of GAA repeats in both expanded FXN alleles in patients with FD were found. An analysis revealed an earlier onset and a more severe course of FD in cases with hypermethylation of several CpG-sites in the UP-GAA region. The correlation between the methylation pattern and the presence of extraneural manifestations of FD was also revealed. In FD patients with cardiomyopathy, a hypomethylated CpG-site in the promoter region was found. In FD patients with carbohydrate metabolism disorders, two hypomethylated CpG-sites in the DOWN-GAA region were observed.

EXERCISE MAY OFFER ‘PROFOUND’ BENEFITS FOR FRIEDREICH'S ATAXIA, RESEARCH SUGGESTS

UVA Today. University of Virginia, June 15, 2020 Josh Barney.

Yan, of UVA’s Departments of Medicine, Pharmacology and Molecular Physiology and Biological Physics, and his colleagues have published their findings in Scientific Reports. Friedreich’s ataxia typically limits patients’ ability to exercise. But Zhen Yan’s findings suggest that well-timed exercise programs early in life may slow the progression of the disease, which robs patients of their ability to walk.
We conclude that endurance exercise training prevents symptomatic onset of FRDA [Friedreich’s ataxia] in mice associated with improved mitochondrial function and reduced oxidative stress,” the researchers report in a scientific paper on the findings. “These preclinical findings may pave the way for clinical studies of the impact of endurance exercise in FRDA patients.”

Substrate-dependent suppression of oxidative phosphorylation in the Frataxin-depleted heart

Cesar Vasquez, Monika Patel, Aishwarya Sivaramakrishnan, Carmen Bekeova, Lauren Anderson-Pullinger, Nadan Wang, Erin L Seifert
bioRxiv 2020.06.12.148361; doi:10.1101/2020.06.12.148361

Friedreich's ataxia is an inherited disorder caused by depletion of frataxin (Fxn), a mitochondrial protein involved in iron-sulfur cluster biogenesis. Cardiac dysfunction is the main cause of death; pathogenesis remains poorly understood but is expected to be linked to an energy deficit. In mice with adult-onset Fxn loss, bioenergetics analysis of heart mitochondria revealed a time- and substrate-dependent decrease in oxidative phosphorylation (oxphos). Oxphos was lower with substrates that depend on Complex I and II, but preserved for lipid substrates, especially through electron entry into Complex III via the electron transfer flavoprotein dehydrogenase. This differential substrate vulnerability is consistent with the half-lives for mitochondrial proteins. Cardiac contractility was preserved, likely due to sustained β-oxidation. Yet, a stress response was stimulated, characterized by activated mTORC1 and the p-eIF2α/ATF4 axis. This study exposes an unrecognized mechanism that maintains oxphos in the Fxn-depleted heart. The stress response that nonetheless occurs suggests energy deficit-independent pathogenesis.

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Dr. Jixue Li, Dr. Yanjie Li, Dr. Jun Wang, Dr. Trevor J. Gonzalez, Dr. Aravind Asokan, Dr. Jill Napierala, and Dr. Marek Napierala; Hum Gene Ther. 2020;10.1089/hum.2020.053. doi:10.1089/hum.2020.053

Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site-specifically integrated into the genome of HEK293T and human induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

Treatment of Dilated Cardiomyopathy in a Mouse Model of Friedreich’s Ataxia using N-acetylcysteine and Identification of Alterations in MicroRNA Expression that Could be Involved in its Pathogenesis

S. Chiang, M.L.H. Huang, D.R. Richardson, Pharmacological Research, 2020, 104994, doi:10.1016/j.phrs.2020.104994.

Deficient expression of the mitochondrial protein, frataxin, leads to a deadly cardiomyopathy. Our laboratory reported the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation after frataxin deletion in the heart. This was due, in part, to a pronounced increase in Keap1. To assess if this can be therapeutically targeted, cells were incubated with N-acetylcysteine (NAC), or buthionine sulfoximine (BSO), which increases or decreases glutathione (GSH), respectively, or the NRF2-inducer, sulforaphane (SFN). While SFN significantly (p < 0.05) induced NRF2, KEAP1 and BACH1, NAC attenuated SFN-induced NRF2, KEAP1 and BACH1. The down-regulation of KEAP1 by NAC was of interest, as Keap1 is markedly increased in the MCK conditional frataxin knockout (MCK KO) mouse model and this could lead to the decreased Nrf2 levels. Considering this, MCK KO mice were treated with i.p. NAC (500- or 1500-mg/kg, 5 days/week for 5-weeks) and demonstrated slightly less (p > 0.05) body weight loss versus the vehicle-treated KO. However, NAC did not rescue the cardiomyopathy. To additionally examine the dys-regulation of Nrf2 upon frataxin deletion, studies assessed the role of microRNA (miRNA) in this process. In MCK KO mice, miR-144 was up-regulated, which down-regulates Nrf2. Furthermore, miRNA screening in MCK KO mice demonstrated 23 miRNAs from 756 screened were significantly (p < 0.05) altered in KOs versus WT littermates. Of these, miR-21*, miR-34c*, and miR-200c, demonstrated marked alterations, with functional clustering analysis showing they regulate genes linked to cardiac hypertrophy, cardiomyopathy, and oxidative stress, respectively.

Vestibular Impact of Friedreich Ataxia in Early Onset Patients

A Maudoux, N Teissier, M Francois, Th Van Den Abbeele, C Alberti, I Husson, S R Wiener-Vacher; Cerebellum Ataxias. 2020;7:6. Published 2020 May 28. doi:10.1186/s40673-020-00115-z

We show that Friedreich ataxia, even at onset, frequently associate saccadic intrusions, abnormal ABRs and decreased vestibulo-ocular and vestibulospinal responses progressing over time. These sensory impairments combined with ataxia further impair patient's autonomy. These vestibular, auditory and visual impairments could be used as markers of the severity and progression of the disease. Adding vestibular and auditory testing to Friedreich patient's evaluation may help physicians improve patient's management.

A Novel Solution-Gated Graphene Transistor Biosensor for Ultrasensitive Detection of Trinucleotide Repeats

Zhiqi Ge, Mingyu Ma, Zang Chang, Meijun Chen, Hanping He, Xiuhua Zhang and Shengfu Wang, 2020, doi:10.1039/D0AN00205D

A new way to detect GAA trinucleotide repeats (TNR) based on solution-gated graphene transistor (SGGT) with high performance was developed. Friedreich's ataxia (FRDA) is a neurodegenerative disease where the first intron of the frataxin (FXN) gene exhibits an extended GAA repeat region. Herein, a SGGT biosensor was constructed based on G-quadruplex DNAzymes and graphene channels. The DNAzymes quantify the captured target DNA by producing a strong catalytic current signal depending on the peroxidase-like activity. The higher the target DNA quantity captured on the gate electrode is, the higher is the concentration of DNAzymes on the surface of the gate electrode, which generates a high catalytic current. Due to the excellent self-amplifying performance of the transistor, the current signal of the SGGT is several hundreds of times larger than in conventional electrochemistry under identical detection conditions. Moreover, a large current signal can be obtained in the case of a low concentration of H2O2 when compared to the case of an enzyme-catalyzed transistor. The SGGT biosensor also exhibits ultra-low detection limit (32.25 fM), a wide linear range (100 fM–100 nM), and excellent selectivity. The results show that the SGGT biosensor shows a great potential in the early diagnosis of neurodegenerative diseases.

Cerebellar cognitive disorder parallels cerebellar motor symptoms in Friedreich ataxia

Naeije, G., Rai, M., Allaerts, N., Sjogard, M., De Tiège, X. and Pandolfo, M.; Ann Clin Transl Neurol. doi:10.1002/acn3.51079

Dentate nuclei (DN) are involved in cerebellar modulation of motor and cognitive functions, whose impairment causes ataxia and cerebellar cognitive affective syndrome (CCAS). Friedreich ataxia (FRDA) disease progression relates to degeneration of the dentate nucleus and dentato‐thalamic pathways, causing cerebellar ataxia. Volumetric MRI also shows mild loss in the cerebellar cortex, brainstem, and motor cortex. Cognitive deficits occur in FRDA, but their relationship with ataxia progression is not fully characterized. We found a significant positive correlation between severity of patients’ ataxia and more marked CCAS as assessed with the CCAS‐Scale. This relation could be related to progressive DN impairment.

HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich’s Ataxia

Sherzai, Mursal; Valle, Adamo; Perry, Nicholas; Kalef-Ezra, Ester; Al-Mahdawi, Sahar; Pook, Mark; Sara Anjomani Virmouni. Frontiers. Collection. doi:10.3389/fgene.2020.00584

Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.

Functional coupling of presequence processing and degradation in human mitochondria

Kücükköse, C., Taskin, A.A., Marada, A., Brummer, T., Dennerlein, S. and Vögtle, F.‐N. (2020), FEBS J. doi:10.1111/febs.15358

The mitochondrial proteome is built and maintained mainly by import of nuclear‐encoded precursor proteins. Most of these precursors use N‐terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce.