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Frataxin Deficiency Drives a Shift from Mitochondrial Metabolism to Glucose Catabolism, Triggering an Inflammatory Phenotype in Microglia (bulk RNA-Seq)

ABSTRACT: Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreich's ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.

ORGANISM(S): Mus musculus

PROVIDER: GSE261653 | GEO | 2024/04/16

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.

Project description:Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by a homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that a single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in the prevention of the neurologic and cardiac complications of FRDA in YG8R mice, and the rescue was mediated by FXN transfer from tissue engrafted HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation of this approach, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions in the cells. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. To this end, we generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. FRDA neurons generated from these iPSCs displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. These results were validated by molecular and functional studies, and gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe structural damage to the ER in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage and function in FRDA. In addition, these results validate the efficacy profile of our FXN gene editing approach, in a disease relevant model improving mitochondrial and cellular, and supporting our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia.

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Frataxin Deficiency Drives a Shift from Mitochondrial Metabolism to Glucose Catabolism, Triggering an Inflammatory Phenotype in Microglia (bulk RNA-Seq)

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