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Comprehensive Analysis of Gene Expression Patterns in Friedreich’s Ataxia Fibroblasts by RNA Sequencing Reveals Altered Levels of Protein Synthesis Factors and Solute Carriers

ABSTRACT: Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients have low FXN mRNA and frataxin protein levels when compared with heterozygous carriers or healthy controls. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells, and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Transcriptome profiling of FRDA skin fibroblasts revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in the FRDA cells. Finally, comparison of genes differentially expressed in FRDA fibroblasts to 3 previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE104288 | GEO | 2017/09/27

SECONDARY ACCESSION(S): PRJNA412241

REPOSITORIES: GEO

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Project description:Background Friedreich’s ataxia (FRDA) is a rare genetic disorder characterized by motor discoordination and predominant cerebellar involvement. It is caused by mutations leading to decreased transcription of frataxin (FXN), a gene encoding for a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron handling. Many advances have been achieved in understanding the pathophysiology of FRDA and the mechanisms whereby FXN deficiency causes cerebellar degeneration. However, the issue concerning the contribution of central nervous system-resident non-neuronal cells to the pathogenesis of FRDA is still poorly explored. In this work, we set out to verify if specific vulnerabilities of cerebellar microglia could be involved in the pathogenesis of FRDA. Methods Primary cerebellar microglia were obtained from the KIKO mouse model of FRDA. We analyzed their phenotype and functions by a multilayer study, encompassing morphological analysis, migration and phagocytosis assays, gene expression by RNA-seq, RT-PCR, western blot, and immunofluorescence. Mitochondria functionality was evaluated by flow cytometry and real-time metabolic analysis. We assessed the effect of FRDA microglia on cell viability and neurite length in primary neurons. We analyzed in vivo microglia phenotype in the cerebellum of KIKO miceduring an early developmental stage by means of morphology analysis, RT-PCR and immunofluorescence. Results We demonstrated that FRDA microglia exhibit traceable morphological, transcriptional and functional changes, particularly regarding inflammatory pathways and mitochondria functionality. In addition, we found that FRDA microglia impair neuron survival and neurite length in vitro. Finally, we showed that cerebellar microglia in vivo exhibit dysregulations at early postnatal age of FRDA mice. Conclusions These results indicate that FRDA microglia display aberrant features and may participate in neuron dysfunction, evidencing a non-cell-autonomous mechanism in FRDA. Therefore, microglia targeting could be a valuable strategy in ameliorating cerebellar degeneration in the disease.

Project description:Friedreich's Ataxia (FRDA), a rare, inherited, progressive degenerative disease, is caused by a defective expression of mitochondrial protein frataxin (FXN), due to homozygous hyper-expansion of GAA triplets in the gene which severely reduce its transcription. FXN is crucial for cell survival and its deficiency in humans critically affects viability of neurons, cardiomyocytes and pancreatic beta cells. Around two thirds of individuals with FRDA show typical manifestation of hypertrophic cardiomyopathy, that can progress in heart failure and death. Except for FXN, very few genes have been correlated with ataxia and cardiac disease in FRDA. To identify other genes involved in pathogenesis of FRDA, a microarray analysis on FRDA patient's lymphoblastoid cells stably reconstituted with FXN was performed. HS-1 associated protein X-1 (HAX-1), a family of proteins playing important roles in the protection of cardiomyocytes from apoptosis, is the highest up-regulated transcript (FC=+2, p<0.0006). HAX-1 is highly expressed in heart and HAX-1 heterozygous-deficient hearts exhibit increases in infarct size. The microarray result was further assessed with qRT-PCR and western blot analysis performed on HEK293 stably transfected with empty vector or FXN-vector and lymphoblasts from FRDA patients compared to clinically unaffected heterozygous parents, confirming a strong co-expression of HAX-1 and FXN both at mRNA and protein level. Moreover, analysis of FXN and HAX-1 in peripheral blood mononuclear cells (PBMCs) of FRDA patients (n=13), heterozygous parents (n=6) and non-correlated healthy controls (n=13) revealed that their expressions are correlated. The positive correlation is more significant in FRDA patients showing a cardiac disease (10/13). The positive correlation between frataxin and HAX-1 expression level suggests a potential link of HAX-1 expression to pro-survival or protective cellular pathways related to frataxin. Thus, HAX-1 may be considered a specific biomarker of cardiomyopathy in FRDA and may represent a prognostic implement in these patients. Moreover, the discovery of novel genetic "risk" or "protective" factors in cardiomyopathy is crucial to improve risk stratification strategies and to identify new therapeutic targets.

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Comprehensive Analysis of Gene Expression Patterns in Friedreich’s Ataxia Fibroblasts by RNA Sequencing Reveals Altered Levels of Protein Synthesis Factors and Solute Carriers

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