LiveLattice: Real-time visualization of tilted light-sheet microscopy data using a memory-efficient transformation algorithm.

Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D; x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), rely on a tilting of the sample plane with respect to the light sheet of 30-45 degrees to ease sample preparation. Data from such tilted-sample-plane LSFMs require subsequent deskewing and rotation for proper visualization and analysis. Such transformations currently demand substantial memory allocation. This poses computational challenges, especially with large datasets. The consequence is long processing times compared to data acquisition times, which currently limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopy datasets without significant hardware demand, we have developed WH-Transform, a novel GPU-accelerated memory-efficient algorithm that integrates deskewing and rotation into a single transformation, significantly reducing memory requirements and reducing the preprocessing run time by at least 10-fold for large image stacks. Benchmarked against conventional methods and existing software, our approach demonstrates linear scalability. Processing large 3D stacks of up to 15 GB is now possible within one minute using a single GPU with 24 GB of memory. Applied to 4D LLSM datasets of human hepatocytes, human lung organoid tissue, and human brain organoid tissue, our method outperforms alternatives, providing rapid, accurate preprocessing within seconds. Importantly, such processing speeds now allow visualization of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on-the-fly data processing, visualization, and analysis on standard workstations, thereby revolutionizing biological imaging applications for LLSM, SPIM and similar light microscopes.

Mitochondrial impairment, decreased sirtuin activity and protein acetylation in dorsal root ganglia in Friedreich Ataxia models.

Friedreich ataxia (FA) is a rare, recessive neuro-cardiodegenerative disease caused by deficiency of the mitochondrial protein frataxin. Mitochondrial dysfunction, a reduction in the activity of iron-sulfur enzymes, iron accumulation, and increased oxidative stress have been described. Dorsal root ganglion (DRG) sensory neurons are among the cellular types most affected in the early stages of this disease. However, its effect on mitochondrial function remains to be elucidated. In the present study, we found that in primary cultures of DRG neurons as well as in DRGs from the FXNI151F mouse model, frataxin deficiency resulted in lower activity and levels of the electron transport complexes, mainly complexes I and II. In addition, altered mitochondrial morphology, indicative of degeneration was observed in DRGs from FXNI151F mice. Moreover, the NAD+/NADH ratio was reduced and sirtuin activity was impaired. We identified alpha tubulin as the major acetylated protein from DRG homogenates whose levels were increased in FXNI151F mice compared to WT mice. In the mitochondria, superoxide dismutase (SOD2), a SirT3 substrate, displayed increased acetylation in frataxin-deficient DRG neurons. Since SOD2 acetylation inactivates the enzyme, and higher levels of mitochondrial superoxide anion were detected, oxidative stress markers were analyzed. Elevated levels of hydroxynonenal bound to proteins and mitochondrial Fe2+ accumulation was detected when frataxin decreased. Honokiol, a SirT3 activator, restores mitochondrial respiration, decreases SOD2 acetylation and reduces mitochondrial superoxide levels. Altogether, these results provide data at the molecular level of the consequences of electron transport chain dysfunction, which starts negative feedback, contributing to neuron lethality. This is especially important in sensory neurons which have greater susceptibility to frataxin deficiency compared to other tissues.

Evolution of an Iron-Detoxifying Protein: Eukaryotic and Rickettsia Frataxins Contain a Conserved Site Which Is Not Present in Their Bacterial Homologues.

Friedreich's ataxia is a neurodegenerative disease caused by mutations in the frataxin gene. Frataxin homologues, including bacterial CyaY proteins, can be found in most species and play a fundamental role in mitochondrial iron homeostasis, either promoting iron assembly into metaloproteins or contributing to iron detoxification. While several lines of evidence suggest that eukaryotic frataxins are more effective than bacterial ones in iron detoxification, the residues involved in this gain of function are unknown. In this work, we analyze conservation of amino acid sequence and protein structure among frataxins and CyaY proteins to identify four highly conserved residue clusters and group them into potential functional clusters. Clusters 1, 2, and 4 are present in eukaryotic frataxins and bacterial CyaY proteins. Cluster 3, containing two serines, a tyrosine, and a glutamate, is only present in eukaryotic frataxins and on CyaY proteins from the Rickettsia genus. Residues from cluster 3 are blocking a small cavity of about 40 Å present in E. coli's CyaY. The function of this cluster is unknown, but we hypothesize that its tyrosine may contribute to prevent formation of reactive oxygen species during iron detoxification. This cluster provides an example of gain of function during evolution in a protein involved in iron homeostasis, as our results suggests that Cluster 3 was present in the endosymbiont ancestor of mitochondria and was conserved in eukaryotic frataxins.

Mice harboring the FXN I151F pathological point mutation present decreased frataxin levels, a Friedreich ataxia-like phenotype, and mitochondrial alterations.

Friedreich Ataxia (FA) is a rare neuro-cardiodegenerative disease caused by mutations in the frataxin (FXN) gene. The most prevalent mutation is a GAA expansion in the first intron of the gene causing decreased frataxin expression. Some patients present the GAA expansion in one allele and a missense mutation in the other allele. One of these mutations, FXNI154F, was reported to result in decreased content of mature frataxin and increased presence of an insoluble intermediate proteoform in cellular models. By introducing this mutation into the murine Fxn gene (I151F, equivalent to human I154F) we have now analyzed the consequences of this pathological point mutation in vivo. We have observed that FXNI151F homozygous mice present low frataxin levels in all tissues, with no evidence of insoluble proteoforms. Moreover, they display neurological deficits resembling those observed in FA patients. Biochemical analysis of heart, cerebrum and cerebellum have revealed decreased content of components from OXPHOS complexes I and II, decreased aconitase activity, and alterations in antioxidant defenses. These mitochondrial alterations are more marked in the nervous system than in heart, precede the appearance of neurological symptoms, and are similar to those observed in other FA models. We conclude that the primary pathological mechanism underlying the I151F mutation is frataxin deficiency, like in patients carrying GAA expansions. Therefore, patients carrying the I154F mutation would benefit from frataxin replacement therapies. Furthermore, our results also show that the FXNI151F mouse is an excellent tool for analyzing tissue-specific consequences of frataxin deficiency and for testing new therapies.

Mitochondrial iron and calcium homeostasis in Friedreich ataxia.

Friedreich Ataxia is a neuro-cardiodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. Many evidences indicate that frataxin deficiency causes an unbalance of iron homeostasis. Nevertheless, in the last decade many results also highlighted the importance of calcium unbalance in the deleterious downstream effects caused by frataxin deficiency. In this review, the role of these two metals has been gathered to give a whole view of how iron and calcium dyshomeostasys impacts on cellular functions and, as a result, which strategies can be followed to find an effective therapy for the disease.

Calcitriol increases frataxin levels and restores mitochondrial function in cell models of Friedreich Ataxia.

Friedreich ataxia (FA) is a neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. In primary cultures of dorsal root ganglia neurons, we showed that frataxin depletion resulted in decreased levels of the mitochondrial calcium exchanger NCLX, neurite degeneration and apoptotic cell death. Here, we describe that frataxin-deficient dorsal root ganglia neurons display low levels of ferredoxin 1 (FDX1), a mitochondrial Fe/S cluster-containing protein that interacts with frataxin and, interestingly, is essential for the synthesis of calcitriol, the active form of vitamin D. We provide data that calcitriol supplementation, used at nanomolar concentrations, is able to reverse the molecular and cellular markers altered in DRG neurons. Calcitriol is able to recover both FDX1 and NCLX levels and restores mitochondrial membrane potential indicating an overall mitochondrial function improvement. Accordingly, reduction in apoptotic markers and neurite degeneration was observed and, as a result, cell survival was also recovered. All these beneficial effects would be explained by the finding that calcitriol is able to increase the mature frataxin levels in both, frataxin-deficient DRG neurons and cardiomyocytes; remarkably, this increase also occurs in lymphoblastoid cell lines derived from FA patients. In conclusion, these results provide molecular bases to consider calcitriol for an easy and affordable therapeutic approach for FA patients.

PPAR gamma agonist leriglitazone improves frataxin-loss impairments in cellular and animal models of Friedreich Ataxia.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence in diabetes. The underlying pathophysiological mechanism of FRDA, driven by a significantly decreased expression of frataxin (FXN), involves increased oxidative stress, reduced activity of enzymes containing iron­sulfur clusters (ISC), defective energy production, calcium dyshomeostasis, and impaired mitochondrial biogenesis, leading to mitochondrial dysfunction. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcriptional factor playing a key role in mitochondrial function and biogenesis, fatty acid storage, energy metabolism, and antioxidant defence. It has been previously shown that the PPARγ/PPARγ coactivator 1 alpha (PGC-1α) pathway is dysregulated when there is frataxin deficiency, thus contributing to FRDA pathogenesis and supporting the PPARγ pathway as a potential therapeutic target. Here we assess whether MIN-102 (INN: leriglitazone), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and animal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+ exchanger (NCLX), resulting in an improvement of mitochondrial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase frataxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid ß-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. The results reinforced the different tissue requirement in FRDA and the pleiotropic effects of leriglitazone that could be a promising therapy for FRDA.

Frataxin-deficient cardiomyocytes present an altered thiol-redox state which targets actin and pyruvate dehydrogenase.

Friedreich ataxia (FA) is a cardioneurodegenerative disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Previously, we set up a cardiac cellular model of FA based on neonatal rat cardiac myocytes (NRVM) and lentivirus-mediated frataxin RNA interference. These frataxin-deficient NRVMs presented lipid droplet accumulation, mitochondrial swelling and signs of oxidative stress. Therefore, we decided to explore the presence of protein thiol modifications in this model. With this purpose, reduced glutathione (GSH) levels were measured and the presence of glutathionylated proteins was analyzed. We observed decreased GSH content and increased presence of glutahionylated actin in frataxin-deficient NRVMs. Moreover, the presence of oxidized cysteine residues was investigated using the thiol-reactive fluorescent probe iodoacetamide-Bodipy and 2D-gel electrophoresis. With this approach, we identified two proteins with altered redox status in frataxin-deficient NRVMs: electron transfer flavoprotein-ubiquinone oxidoreductase and dihydrolipoyl dehydrogenase (DLDH). As DLDH is involved in protein-bound lipoic acid redox cycling, we analyzed the redox state of this cofactor and we observed that lipoic acid from pyruvate dehydrogenase was more oxidized in frataxin-deficient cells. Also, by targeted proteomics, we observed a decreased content on the PDH A1 subunit from pyruvate dehydrogenase. Finally, we analyzed the consequences of supplementing frataxin-deficient NRVMs with the PDH cofactors thiamine and lipoic acid, the PDH activator dichloroacetate and the antioxidants N-acetyl cysteine and Tiron. Both dichloroacetate and Tiron were able to partially prevent lipid droplet accumulation in these cells. Overall, these results indicate that frataxin-deficient NRVMs present an altered thiol-redox state which could contribute to the cardiac pathology.