Objective Age-related macular degeneration (AMD) is the leading cause of visual impairment in older adults. Despite extensive studies on AMD, the mechanism of drusen formation remains unknown, limiting the development of therapeutic approaches. Therefore, our goal was to establish a reproducible primary porcine retinal pigment epithelium (RPE) model featuring dry AMD and characterize subsequent RPE morphological changes, the oxidative stress response, and the transcriptome profile at different stages of drusen formation. Methods Primary porcine RPE cells cultured on permeable Transwell and impermeable plastic surfaces were compared. Barrier functions were assessed via transepithelial electrical resistance measurement and immunostaining. Hydroxyapatite (HAP) deposition was assessed via Alizarin Red S staining and scanning electron microscopy with a focused ion beam. Extracellular glutathione was quantified via Ellman’s reagent. RT‒qPCR and RNA sequencing were used to analyze transcriptomic changes at different stages of drusen progression. Results In primary porcine RPE cells cultured on plastic dishes, initial ~ 2 µm HAP-containing spherules were detected after 2 weeks that progressed into large ~ 30 µm drusen by week 8. Robust deposit accumulation is accompanied by oxidative stress, RPE hyperpigmentation, and apoptosis. RNA-seq analysis revealed gradual upregulation of key epithelial‒mesenchymal transition (EMT) markers ( FN1, VIM , and ACTA2 ) and AMD-related genes ( C3, CFH, VEGFA, CLU , and TIMP3 ) over the 8-week culture period. Conclusion We established a highly reproducible primary porcine RPE model of dry AMD. Our RPE model enables the investigation of drusen formation and the RPE response across different AMD stages and provides a valuable platform for developing and testing novel therapeutic approaches.

The crucian carp (Carassius carassius) is one of the most anoxia-tolerant vertebrates. While physiological underpinnings of its ability to withstand O2 deprivation are well studied, the ability to tolerate the return to normoxia is still enigmatic. Such reoxygenation is associated with detrimental oxidation damage in other organisms, where mitochondria play a central role in the damaging effects. This leads to the question whether mitochondrial adaptations play a central role in the anoxia and reoxygenation tolerance of crucian carp. We here address whether mitochondria from crucian carp circumvent the negative effects of anoxia-reoxygenation exposure, namely the generation of reactive oxygen species (ROS) and subsequent oxidative stress. Crucian carp brain and heart mitochondria generated up to 4-fold less hydrogen peroxide (H2O2; a major ROS) compared with the closely related, anoxia-intolerant, common carp (Cyprinus carpio). The lower H2O2 emission was partly explained by higher (∼15-30%) total oxidant scavenging capacity. Complex II-mediated flux was ∼40% reduced after anoxia-reoxygenation in crucian carp heart mitochondria. Mitochondrial H2O2 generation measured in vivo was unaffected by anoxia-reoxygenation exposure in heart, brain and gill, but reduced by ∼25% in liver. There were also tissue-specific increases in protein carbonylation (∼1.8-fold in brain and gills) and mitochondrial DNA (mtDNA) damage (∼1.5-fold in liver and heart), indicating that biphasic oxidative stress responses affect tissues differently. Our data show that crucian carp avoid excessive mitochondrial ROS generation upon exposure to anoxia-reoxygenation. The tissue-specific distribution of protein and mtDNA oxidation indicate that crucian carp balance body redox signalling to secure resilience during fluctuating O2 availability.

Huntington's disease (HD) is a neurodegenerative autosomal dominant hereditary disease caused by a CAG triplet repeat expansion mutation in the gene encoding the huntingtin (HTT) protein. The main feature of HD is the loss of striatal neurons, accompanied by metabolic and transcriptional alterations in both neural and peripheral tissues. Induced pluripotent stem cells (iPSCs) derived from a transgenic HD (TgHD) minipig model expressing a mutant HTT construct were generated to investigate early metabolic, antioxidant and DNA integrity changes associated with HD development. Gene expression analysis showed increased expression of vascular endothelial growth factor (VEGF), pyruvate dehydrogenase kinase 1 (PDK1) and glutamine-oxaloacetic transaminase 1 (GOT1), implying early metabolic alteration in TgHD iPSCs. Moreover, upregulated FANCD2/FANCI-associated nuclease 1 (FAN1) expression indicated genotoxic stress linked to early HD development. These findings suggest metabolic shifts and putative genotoxic events in the pluripotent stem cell state of the TgHD model and point to early effect of the HD mutation. The model may be suitable for evaluating potential cell therapy and in vitro differentiation of iPSCs to neurons and other cells affected in HD.

Mitochondrial DNA (mtDNA) damage is strongly implicated in age-related macular degeneration (AMD), the most frequent cause of age-mediated visual impairment in developed countries. Here, we investigated and compared the fate of acutely induced oxidation damage in primary retinal pigment epithelial (RPE) cells. The individual RPE clones responded heterogeneously to hydrogen peroxide, both with respect to cell sensitivity and mtDNA damage formation. Peroxide-induced mtDNA damage in human RPE (hRPE) was fully repaired within 4 h. In parallel, hRPE cells secreted mtDNA from both apical and basolateral surfaces (AP-mtDNA and BL-mtDNA, respectively) independent of peroxide exposure and that could not be explained by detached cells. Most mtDNA was released apically, and the quality of AP-mtDNA was comparable to that of intracellular mtDNA (c-mtDNA). In contrast, BL-mtDNA constituted only 3% of AP-mtDNA, and exhibited a 10-fold higher damage burden. The difference in mtDNA quality suggests a non-random selection of mtDNA molecules to be targeted for export in apical versus basolateral direction. To investigate this, we analyzed epigenetic marks (m.545 methylation) and SNPs (heteroplasmies) in extracellular and cellular mtDNA. Extracellular mtDNA in general appeared to be more modified than c-mtDNA and that AP-mtDNA differs from BL-mtDNA, which is indicative of a targeted secretion. Porcine RPEs (pRPEs) from a minipig model of Huntington's Disease associated with impaired epithelial polarity (TgHD) displayed approximately 2-fold higher leakage of AP-mtDNA than control pRPEs, but lower than hRPEs. Together, our data imply that extracellular mtDNA originating from RPE shapes the outer retina and the implications for polar secretion are discussed.

Glutathione (GSH) and its oxidized form, glutathione disulfide (GSSG), are key regulators of cellular redox homeostasis, with their ratio serving as a critical biomarker for oxidative stress assessment in cell culture explants. This study presents a non-selective electrochemical sensor array combined with machine learning (ML) as a proof-of-concept platform for simultaneous GSH/GSSG quantification directly in cell culture medium, a complex and biologically relevant matrix widely used for explant and mammalian cell studies.The array comprises screen-printed sensors with a carbon-based working electrode without modification or modified with carbon nanotubes, quantum dots, and metal oxide/metal (nano)particles, selected for their partially overlapping responses and resilience to drift and to interferents inherent to undiluted cell culture. Among evaluated chemometric methods, Artificial Neural Networks excelled, achieving high-resolution discrimination and quantification of GSH/GSSG using a limited dataset with minimal control over experimental variables.These promising results highlight how/ML enables robust performance in challenging biological matrices by deconvoluting multivariate signals, compensating for interferents, and reducing data requirements, demonstrating feasibility for explant-based oxidative stress monitoring without extensive sample pretreatment or large training sets.

Adult neurogenesis in the hippocampus, involving the generation and integration of new neurons, is essential for behavioral pattern separation, which supports accurate memory recall and cognitive plasticity. Here, we explore the role of the DNA repair protein NEIL3 in adult hippocampal neurogenesis and behavioral pattern separation. NEIL3 is required for efficient proliferation and neuronal differentiation of neonatal NSPCs and adult-born NPCs in the hippocampus following a behavioral pattern separation task. NEIL3-depleted mice exhibited a reduced preference for the novel object location, indicating a deficit in pattern separation. NEIL3-deficient adult-born neurons exhibited a significant reduction in mature-like membrane properties, indicating impaired functional maturation. Interestingly, these impairments were not associated with the decreased genomic integrity but with the altered transcriptional regulation of the Wnt signaling pathway. Given the importance of adult neurogenesis in cognitive function, targeting NEIL3 could offer therapeutic potential for addressing age-related hippocampal dysfunction and cognitive decline.

Branched-chain fatty acids (BCFAs) are predominantly saturated fatty acids with one or more methyl branches on the carbon chain, typically found in dairy products and measured in micromolar concentrations in human plasma. The biological function of BCFAs in humans remains ill-defined, but a relationship between circulating BCFAs and cardiometabolic health has been suggested. The objective of this study was to evaluate the impact of BCFAs on energy metabolism in human myotubes. The results revealed distinct effects of BCFAs. 12-Methyltetradecanoic acid (12-MTD) increased glucose uptake and glycogen synthesis, while 13-methyltetradecanoic acid (13-MTD), 14-methylhexadecanoic acid (14-MHD), and 15-methylhexadecanoic acid (15-MHD) increased oleic acid uptake and 13-MTD and 15-MHD oleic acid oxidation, indicating a more general stimulatory effect on fatty acid than glucose metabolism. Interestingly, the same BCFAs, 13-MTD, 14-MHD, and 15-MHD, appeared to reduce insulin-stimulated glycogen synthesis. Insulin-stimulated phosphorylation of IRS1 was not apparent after exposure to 12-MTD, 13-MTD, and 15-MHD, whereas insulin-stimulated phosphorylation of Akt was unchanged by BCFAs. Incorporation of [14C]leucine into lipids was affected, as 13-MTD increased the total lipid content, and 12-MTD altered the distribution of lipid classes. Metabolic flux analysis indicated that 14-MHD stimulated extracellular acidification. The effects of BCFAs might involve increased mRNA expression of pyruvate dehydrogenase kinase 4. In conclusion, the study demonstrates that different BCFAs have distinct effects on energy metabolism in myotubes, 12-MTD mainly affect glucose metabolism, while 13-MTD, 14-MHD, and 15-MHD modulated oleic acid metabolism. These data suggest that some BCFAs might have therapeutic applications by improving energy metabolism.

PURPOSE:Retinal Pigment Epithelial (RPE) cells perform critical functions in the visual cycle. Their melanin pigmentation, which is organized into specialized compartments - melanosomes, is highly critical for proper vision. A chemical method to induce pigmentation in a non-pigmented model of ARPE-19 cells was applied using L-DOPA as a repurposed drug from the current treatment of Parkinson's disease. METHODS:L-DOPA was optimized for its toxic effect on ARPE-19 cells along with pigmentation development. Gene expression and immunocytochemistry confirmed upregulation of melanogenesis-related genes and proteins. Melanosomes were characterized by TEM. RESULTS:We found 1000 μM L-DOPA to induce pigmentation of ARPE-19 cells by Day 3, and achieve full pigmentation by Day 5. By Day 5, L-DOPA at 1000 μM induced mitochondrial and nuclear DNA damage. However, the gene expression of RPE-specific markers (tyrosinase, TYRP1, CRALBP, PEDF) was significantly different in L-DOPA-treated ARPE-19 cells compared to non-treated ones. Positive expression for Tyrosinase enzyme was confirmed by ICC on both Day 3 and Day 5 of L-DOPA treatment. Transmission electron microscopy showed the de novo melanosome formation with ultrastructural features of various stages of maturity (Stage I to IV), apical-basal polarity and melanosome localization on the apical side of the L-DOPA-treated ARPE-19 cells. CONCLUSION:Our study showed that L-DOPA treatment could induce de novo melanosome formation in amelanotic RPEs. We propose a newer approach of developing an ex vivo model for de novo pigmentation of RPE cells with cell-specific modification and culture condition optimization.

Diagnostics of mitochondrial disease requires a combination of clinical evaluations and biochemical characterization. However, the large normal variation in mitochondrial complex activity limits the precision of biochemical diagnostics. Thus, identifying factors that contribute to such variations could enhance diagnostic accuracy. In comparison, inbred mice demonstrate much less variations in brain mitochondrial activity, but a clear reduction with age. Interestingly, pretreatment of mouse brain mitochondria with the detergent dodecyl maltoside abolishes the reduction. We therefore postulated that DDM pretreatment could be valuable tool for distinguishing between variations caused by posttranslational modifications and those caused by genetic heterogeneity. In this study, we evaluated the effects of age, DDM sensitivity, oxidative damage and single nucleotide polymorphism on biochemical complex activity and the proteome of human muscle mitochondria, which serve as reference standards for mitochondrial diagnostics. Our results indicate that mtDNA variants are the primary contributors to the diversity in biochemical activity in human muscle mitochondria from healthy individuals.

INTRODUCTION:Short-chain fatty acids (SCFAs) are small molecule metabolites mainly produced during microbial fermentation of dietary fibre in the gut and have been shown to have a beneficial impact on human health. The aim of this study was to evaluate the effect of SCFAs on human skeletal muscle energy metabolism. METHODS:Primary human myotubes were analysed for glucose and fatty acid (oleic acid) metabolism, as well as insulin sensitivity and protein synthesis in the presence or absence of SCFAs. RESULTS:The most pronounced effects of SCFAs were observed on 14C-oleic acid uptake and oxidation, as well as 14C-leucine uptake and protein synthesis, following butyrate treatment. Butyrate increased 14C-leucine accumulation twofold, potentially due to protein incorporation. On the other hand, the conversion of 14C-leucine into free fatty acids was reduced by more than 50% by butyrate. Both 14C-acetate and 14C-butyrate were shown to be taken up and utilised by primary human myotubes. None of the SCFAs were found to influence glucose metabolism or insulin effects. CONCLUSION:The results from the current study thus suggest that among the SCFAs, butyrate emerges as the most powerful SCFA in regulating primary human myotube metabolism.

Oxidative phosphorylation involves a complex multi-enzymatic mitochondrial machinery critical for proper functioning of the cell, and defects herein cause a wide range of diseases called “primary mitochondrial disorders” (PMDs). Mutations in about 400 nuclear and 37 mitochondrial genes have been documented to cause PMDs, which have an estimated birth prevalence of 1:5000. Here, we describe a 4-year-old female presenting from early childhood with psychomotor delay and white matter signal changes affecting several brain regions, including the brainstem, in addition to lactic and phytanic acidosis, compatible with Leigh syndrome, a genetically heterogeneous subgroup of PMDs. Whole genome sequencing of the family trio identified a homozygous 12.9 Kb deletion, entirely overlapping the NDUFA4 gene. Sanger sequencing of the breakpoints revealed that the genomic rearrangement was likely triggered by Alu elements flanking the gene. NDUFA4 encodes for a subunit of the respiratory chain Complex IV, whose activity was significantly reduced in the patient’s fibroblasts. In one family, dysfunction of NDUFA4 was previously documented as causing mitochondrial Complex IV deficiency nuclear type 21 (MC4DN21, OMIM 619065), a relatively mild form of Leigh syndrome. Our finding confirms the loss of NDUFA4 function as an ultra-rare cause of Complex IV defect, clinically presenting as Leigh syndrome.

Neoadjuvant chemotherapy (NAT) is increasingly used for the treatment of non‐metastatic pancreatic ductal adenocarcinoma (PDAC) and is established as a standard of care for borderline resectable and locally advanced PDAC. However, full exploitation of its clinical benefits is limited by the lack of biomarkers that assess treatment response. To address this unmet need, global metabolomic profiling was performed on tumor tissue and paired serum samples from patients with treatment‐naïve (TN; n = 18) and neoadjuvant leucovorin calcium (folinic acid), fluorouracil, irinotecan hydrochloride and oxaliplatin (FOLFIRINOX)‐treated (NAT; n = 17) PDAC using liquid chromatography mass spectrometry. Differentially abundant metabolites (DAMs) in TN versus NAT groups were identified and their correlation with various clinical parameters was assessed. Metabolomics profiling identified 40 tissue and five serum DAMs in TN versus NAT PDAC. In general, DAMs associated with amino acid and nucleotide metabolism were lower in NAT compared to TN. Four DAMs—3‐hydroxybutyric acid (BHB), 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid (CMPF), glycochenodeoxycholate and citrulline—were common to both tissue and serum and showed a similar pattern of differential abundance in both groups. A strong positive correlation was observed between serum carbohydrate 19‐9 antigen (CA 19‐9) and tissue carnitines (C12, C18, C18:2) and N8‐acetylspermidine. The reduction in CA 19‐9 following NAT correlated negatively with serum deoxycholate levels, and the latter correlated positively with survival. This study revealed neoadjuvant‐chemotherapy‐induced changes in metabolic pathways in PDAC, mainly amino acid and nucleotide metabolism, and these correlated with reduced CA 19‐9 following neoadjuvant FOLFIRINOX treatment.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in developed countries. The replacement of dysfunctional human retinal pigment epithelium (hRPE) cells by the transplantation of in vitro-cultivated hRPE cells to the affected area emerges as a feasible strategy for regenerative therapy. Synthetic biomimetic membranes arise as powerful hRPE cell carriers, but as biodegradability is a requirement, it also poses a challenge due to its limited durability. hRPE cells exhibit several characteristics that putatively respond to the type of membrane carrier, and they can be used as biomarkers to evaluate and further optimize such membranes. Here, we analyze the pigmentation, transepithelial resistance, genome integrity, and maturation markers of hRPE cells plated on commercial polycarbonate (PC) versus in-house electrospun polylactide-based (PLA) membranes, both enabling separate apical/basolateral compartments. Our results show that PLA is superior to PC-based membranes for the cultivation of hRPEs, and the BEST1/RPE65 maturation markers emerge as the best biomarkers for addressing the quality of hRPE cultivated in vitro. The stability of the cultures was observed to be affected by PLA aging, which is an effect that could be partially palliated by the coating of the PLA membranes.

Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell-based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE derivation, their delivery, and recent clinical studies related to pRPE transplantation in patients. We have also discussed a few limitations with in vitro RPE culture and potential solutions to overcome such problems which can be helpful in developing functional RPE tissue.

Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial–temporal regulation of histone arginine methylation in specific brain regions. This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of the basic biology, including the genetics and molecular pathways of RPE development. It encompasses basic developmental biology and stem cell-based developmental studies to uncover RPE differentiation. Knowledge of the in utero developmental cues provides an inclusive methodology required for deriving RPEs using stem cells.

Representative images of each genotype of PyMT mice with mammary tumors obtained from 12-16-week old female (S1); Histological sections of the lungs from PyMT mice of different OGG1 genotypes (S2); OGG1 activity in mammary tumors isolated from B-nuclear, and D- mitochondrial fractions from PyMT/KO and PyMT/Tg animals (S3); LDH activity in mammary tumors isolated from PyMT/KO, PyMT/WT, PyMT/Tg and PyMT/ KOTg mice (S4).

The branched-chain amino acids (BCAA) leucine, valine, and isoleucine provide precursors for monomethyl branched-chain fatty acids (BCFA). Established reference ranges for BCFAs are lacking. In maple syrup urine disease (MSUD), a rare inborn error of BCAA metabolism, the endogen production is impaired and MSUD patients are treated with a low protein (low BCAA) diet. The protein restriction may affect the dietary intake of BCFA, depending on the dietary choices made. Patients with MSUD are prescribed a more or less protein-restricted diet depending on the severity of the disease. The combination of a protein-restricted diet and subsequent impaired endogenous synthesis may render MSUD patients sensitive to BCFA deficiency, with yet unknown implications. To investigate the possibility of lower circulatory BCFA levels in MSUD that favors dietary BCFA supplementation, we first established fasting-state reference ranges for selected BCFAs and saturated/unsaturated fatty acids in plasma. Then, the effect of fasting on BCFA levels was evaluated by comparing the distribution in a fasting versus a non-fasting cohort. To test the hypothesis that BCFA deficiency could contribute to MSUD pathophysiology, we recruited patients with intermittent, intermediate, and classical form of MSUD and analyzed the corresponding BCFA z-scores. None of the BCFA species had |z-scores| > 2 relative to the reference range. Our findings do not support the requirement of BCFA supplementation in MSUD patients. The origin of BCFAs is discussed. Impaired capacity to synthesize BCFA do not manifest as reduced plasma levels in MSUD, suggesting that endogenous synthesis is dispensable for plasma levels.

Dysfunction of mitochondria has been implicated in Huntington´s disease (HD) pathogenesis. Our previous investigation showed significant impairment of various mitochondrial enzymes and proteins in peripheral blood mononuclear cells (PBMC) of symptomatic HD patients. (Askeland et al., 2019). Aim of present study was to study mitochondrial bioenergetic characteristics in different stages of HD. Material and Methods Study cohort consists of genetically confirmed HD patients: 9 patients in presymptomatic (pre-HD), 12 in early (early-HD) and 21 in symptomatic stage of the disease. 21 age-related controls were included in the study. The CAG repeats were 41.9 ± 1.3 in pre-HD, 45.9 ± 4.3 in early-HD and 45.3 ± 2.9 for the symptomatic HD stage. PBMC were isolated by differential centrifugation, biochemical activities of respiratory chain complexes (RCC) I, II and IV and citrate synthase (CS) were measured by spectrophotometry. Expression of selected mitochondrial proteins were analyzed by SDS/WB. DNA integrity was analyzed by qPCR-based methods. Results Activity of RCC II was significantly increased in all patient groups compared to controls. RCC IV activity (including ratio CIV/CS) was decreased even in pre-HD patients. Protein analysis revealed decreased COX2 subunit of RCC IV and SDH70 subunit of RCC II. No significant changes in nuclear DNA damage, mtDNA damage and mtDNA copy number in any of the carriers, compared to control. Conclusion The results support the hypothesis that biochemical changes in mitochondria occur before the onset of clinical symptoms in HD patients, and prior to impairment in PBMC DNA. Supported by AZV MZ CR NU21-04-00136.