Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich’s ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system (Zhang et al. 2012). The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that, in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53 and EXO1 belonging to this pathway rescued the increased GAA expansions. Further, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA Polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.

Very late-onset Friedreich’s ataxia with rapid course mimicking as possible multiple system atrophy cerebellar type

This 55-year-old man was admitted to the hospital with an insidious onset, progressive backward fall (due to severe truncal ataxia), dysarthria, stiffness in extremities, distal dominant muscle wasting along with behavioural changes and urinary incontinence. Clinical assessment indicated mild cognitive decline (Mini-Mental State Examination 22/27) with cerebellar, pyramidal and peripheral nerves involvement. On investigations, nerve conduction studies revealed symmetrical, sensorimotor peripheral neuropathy affecting both lower limbs. Brain and whole spine MRI revealed widespread cerebral and mild cerebellar atrophy, pons and medulla volume loss, and a normal spinal cord. Transthoracic echocardiography revealed concentric left ventricular hypertrophy. His gene analysis revealed eight GAA repeats on allele 1, and 37 GAA repeats on allele 2 in the first intron of the frataxin gene. Considering his clinical profile and genetic analysis, he was diagnosed as a case of very late-onset Friedreich’s ataxia with likely compound heterozygous genotype.

Charcot - Marie - Toots disease: description of 2 clinical cases of the disease in members of the same family (father and daughter)

Charcot — Marie — Tooth disease belongs to a genetically heterogeneous group of monogenic diseases with a predominant lesion of the peripheral nervous system associated with predominant degeneration of the myelin sheath and/or axon of the motor and sensory nerves and spinal roots, accompanied by motor disorders and specific pain syndrome. The article presents a description of 2 clinical cases of patients (father and daughter), whose disease was manifested by chronically progressive weakness and atrophy of the distal limb muscles, reflexes reduction, foot and hand deformation, gait changes and sensory disorders. Genetic tests were performed to detect duplication/deletion of the PMP22 gene and the expansion of GAA repeats in the FXN gene, which yielded negative results. Based on complaints, neurological examination, family medical history and data of instrumental research (EMNG), we made a clinical diagnosis of the CMT with an autosomal dominant type of inheritance and a high degree of penetrance. At the department, patients received the infusion therapy (neurometabolic therapy, vitamin therapy), physical therapy courses according to an individual plan, physiotherapy and reflexotherapy with a moderate positive effect in the form of a subjective increase in muscle strength in the lower extremities, increasing walking stability and confidence. The article also highlights the peculiarities of the pathogenesis of different genetic variants of CMT and prospect for pathogenetic treatment of this disease.

Molecular and Cellular Substrates for the Friedreich Ataxia. Significance of Contactin Expression and of Antioxidant Administration

In this study, the neural phenotype is explored in rodent models of the spinocerebellar disorder known as the Friedreich Ataxia (FA), which results from mutations within the gene encoding the Frataxin mitochondrial protein. For this, the M12 line, bearing a targeted mutation, which disrupts the Frataxin gene exon 4 was used, together with the M02 line, which, in addition, is hemizygous for the human Frataxin gene mutation (Pook transgene), implying the occurrence of 82–190 GAA repeats within its first intron. The mutant mice phenotype was compared to the one of wild type littermates in regions undergoing differential profiles of neurogenesis, including the cerebellar cortex and the spinal cord by using neuronal (β-tubulin) and glial (Glial Fibrillary Acidic Protein) markers as well as the Contactin 1 axonal glycoprotein, involved in neurite growth control. Morphological/morphometric analyses revealed that while in Frataxin mutant mice the neuronal phenotype was significantly counteracted, a glial upregulation occurred at the same time. Furthermore, Contactin 1 downregulation suggested that changes in the underlying gene contributed to the disorder pathogenesis. Therefore, the FA phenotype implies an alteration of the developmental profile of neuronal and glial precursors. Finally, epigallocatechin gallate polyphenol administration counteracted the disorder, indicating protective effects of antioxidant administration.

Induced pluripotent stem cell-derived primary proprioceptive neurons as Friedreich ataxia cell model

Human induced pluripotent stem cells (iPSCs) are used to generate models of human diseases that recapitulate the pathogenic process as it occurs in affected cells. Many differentiated cell types can currently be obtained from iPSCs, but no validated protocol is yet available to specifically generate primary proprioceptive neurons. Proprioceptors are affected in a number of genetic and acquired diseases, including Friedreich ataxia (FRDA).FRDA is a recessive neurodegenerative and systemic disease due to epigenetic suppression of frataxin (FXN) expression caused by the presence of expanded GAA repeats at the FXN locus. The most characteristic early neuropathologic finding in FRDA is the loss of large primary proprioceptive neurons in the dorsal root ganglia (DRGs), with associated loss of large myelinated fibers in the dorsal roots and in the posterior columns of the spinal cord. Both a developmental deficit and progressive neurodegeneration are thought to underlie the loss of proprioceptors in FRDA, though the relative contribution of these two components is unclear. The basis of the high specific vulnerability of proprioceptors in FRDA is also unknown. In order to address these open questions about FRDA pathogenesis and at the same time develop a cell model that can be applied to other conditions primarily affecting proprioceptors, we set up a protocol to differentiate iPSCs into primary proprioceptive neurons. We modified the dual-SMAD inhibition/WNT activation protocol, previously used to generate nociceptor-enriched cultures of primary sensory neurons from iPSCs, to favor instead the generation of proprioceptors. We succeeded in substantially enriching iPSC-derived primary sensory neuron cultures in proprioceptors, largely exceeding the proportion normally represented by these cells in dorsal root ganglia. We also showed that almost pure populations of proprioceptors can be purified from these cultures by fluorescence-activated cell sorting. Finally, we demonstrated that iPSCs from a FRDA patient can generate normal appearing proprioceptors but have subtle differentiation deficits and more limited survival.

SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.

Epigenetics of Friedreich’s Disease: Methylation of the (GAA)n-Repeats Region in FXN Gene

Background: Friedreich’s disease (FD) is the most common hereditary ataxia. It is associated, most frequently, with homozygous GAA repeats expansion in intron 1 of the FXN gene. Methylation of the FXN gene can play an important role in the pathogenesis of FD. Aims: to study methylation pattern in CpG sites flanking GAA-expansion in intron 1 of the FXN gene in patients with FD and their heterozygous relatives as well as its relationship with clinical features. Materials and methods: We studied DNA samples from patients with FD (n=18), their relatives carrying heterozygous GAA expansion (n=12), and control group (n=15). Pattern of methylation was studied by direct sequencing of DNA regions after bisulphide processing. Results: We analyzed 18 CpG sites in the UP-GAA region of the gene (before GAA-repeats) and 12 CpG sites in the DOWN-GAA region (after GAA-repeats). In the UP-region, the mean methylation level of CpG sites in FD patients was higher compared to controls (n=15) (р0.05), while in the DOWN-region there was a decrease of mean methylation level in FD compared to controls (р0.05). Analysis of methylation level in different CpG sites in the UP-GAA region revealed hypermethylation for 15 of 18 CpG-sites as compared to controls (р0.05). The most significant differences in methylation level in the UP-GAA region were seen for CpG sites 50−54, 57 and 58. In contrast, in the DOWN-GAA region almost all CpG sites were fully methylated in the control group, while in FD patients methylation was significantly lower (р0.05). We revealed positive correlation of mean methylation level and more expanded allele length for the UP-GAA region in FD (r=0.63; p=0.03), and no correlations for the DOWN-GAA region. In heterozygous carriers we observed an analogous positive correlations in the UP-GAA region for CpG site 50 (r=0.77; p=0.04), while in the DOWN-GAA region there was inverse correlation of methylation with GAA repeat number in the expanded allele (r=-0.83, p=0.02). Negative correlation was found between the hypermethylation of some CpG-sites in the UP-GAA region and age of the disease onset (p0.05). Conclusion: We revealed hypermethylation in the UP-GAA region and hypomethylation in the DOWN-GAA region in patients with FD compared to controls and correlations of methylation level with the GAA expansion length and age of disease onset.

Oxidative stress and loss of Fe-S proteins in Friedreich ataxia induced pluripotent stem cell-derived PSNs can be reversed by restoring FXN expression with a benzamide HDAC inhibitor

Epigenetic suppression of frataxin (FXN) expression caused by the presence of expanded GAA repeats at theFXNlocus is the key pathogenic event in Friedreich ataxia (FRDA), a recessive neurodegenerative and systemic disease. FXN is involved in iron-sulfur (Fe-S) cluster biogenesis in mitochondria, its deficiency causes multiple Fe-S protein deficiencies, mitochondrial dysfunction and oxidative stress. Primary sensory neurons (PSNs) in the dorsal root ganglia (DRGs) are the most vulnerable cells in FRDA, whose abnormal development and degeneration leads to the onset and early progression of ataxia. We generated PSNs from induced pluripotent stem cells (iPSCs) from FRDA patients and showed that they recapitulate the key pathogenic events in FRDA, including low FXN levels, loss of Fe-S proteins and impaired antioxidant responses. We also showed that FXN deficiency in these cells may be partially corrected by a pimelic benzamide histone deacetylase inhibitor, a class of potential therapeutics for FRDA. We generated and validated a cellular model of the most vulnerable neurons in FRDA, which can be used for further studies on pathogenesis and treatment approaches.