Retrograde response to mitochondrial dysfunctions associated to LOF variations in FLAD1 exon 2: unraveling the importance of RFVT2

Abstract

Flavin adenine dinucleotide (FAD) synthase (EC 2.7.7.2), encoded by human flavin adenine dinucleotide synthetase 1 (FLAD1), catalyzes the last step of the pathway converting riboflavin (Rf) into FAD. FLAD1 variations were identified as a cause of LSMFLAD (lipid storage myopathy due to FAD synthase deficiency, OMIM #255100), resembling Multiple Acyl-CoA Dehydrogenase Deficiency, sometimes treatable with high doses of Rf; no alternative therapeutic strategies are available. We describe here cell morphological and mitochondrial alterations in dermal fibroblasts derived from a LSMFLAD patient carrying a homozygous truncating FLAD1 variant (c.745C > T) in exon 2. Despite a severe decrease in FAD synthesis rate, the patient had decreased cellular levels of Rf and flavin mononucleotide and responded to Rf treatment. We hypothesized that disturbed flavin homeostasis and Rf-responsiveness could be due to a secondary impairment in the expression of the Rf transporter 2 (RFVT2), encoded by SLC52A2, in the frame of an adaptive retrograde signaling to mitochondrial dysfunction. Interestingly, an antioxidant response element (ARE) is found in the region upstream of the transcriptional start site of SLC52A2. Accordingly, we found that abnormal mitochondrial morphology and impairments in bioenergetics were accompanied by increased cellular reactive oxygen species content and mtDNA oxidative damage. Concomitantly, an active response to mitochondrial stress is suggested by increased levels of PPARγ-co-activator-1α and Peroxiredoxin III. In this scenario, the treatment with high doses of Rf might compensate for the secondary RFVT2 molecular defect, providing a molecular rationale for the Rf responsiveness in patients with loss of function variants in FLAD1 exon 2.

HIGHLIGHTS

• FAD synthase deficiency alters mitochondrial morphology and bioenergetics;

• FAD synthase deficiency triggers a mitochondrial retrograde response;

• FAD synthase deficiency evokes nuclear signals that adapt the expression of RFVT2.

Keywords:

• FLAD1
• LSMFLAD
• MADD
• RFVT2
• mitochondrial myopathy

Acknowledgments

The helpful collaboration of Graziana Dipace and Morena Cardinale, who participated as students in the early stages of this work, and biomedical laboratory technician Helle Highland Nygaard, who performed SLC52A2 sequence analysis, is gratefully acknowledged.

Author contributions

Maria Tolomeo: Writing—Original Draft, Visualization and Conceptualization. Guglielmina Chimienti: Investigation: immunoblotting analysis. Martina Lanza: Investigation: cell culturing and management. Roberto Barbaro: Writing Original Draft, Visualization, and Investigation: cell imaging and immunofluorescence analyses. Alessia Nisco: Investigation: transcript quantifications. Tiziana Latronico: Investigation: cell microscopy and ROS assays. Piero Leone: Investigation and Data Curation. Giuseppe Petrosillo: Investigation: bioenergetics. Grazia M. Liuzzi: Supervision and resources. Bryony Ryder: Investigation clinical care of patient. Michal Inbar-Feigenberg: Supervising clinical care of patient and cell collections. Matilde Colella: Supervision and Investigation. Rikke K.J. Olsen: Supervision, Writing—Review & Editing. Angela M. S. Lezza: Investigation: mtDNA content, Writing—Review & Editing. Maria Barile: Writing—Review & Editing, Supervision, Project administration, Funding acquisition.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by University of Bari “A. Moro” to (M.B.) under Grant “Progetti Competitivi”—Effetto di mutazioni di FLAD1 e di alterazioni dell’omeostasi delle flavine sullo stato redox e sulla biogenesi mitocondriale: uno studio integrato su fibroblasti umani -. Piero Leone was recipient of a research fellowship financed by “Cure RTD Foundation” (Year 2019) http://curertd.org/news/new/.