Corrigendum: Editorial: Metabolic modulation of cellular function.

[This corrects the article DOI: 10.3389/fcell.2024.1395922.].

Characterization of metabolic alterations in the lean metabolically unhealthy alpha defensin transgenic mice.

Inflammation is consistently linked to dysmetabolism. In transgenic mice (Def+/+) model the neutrophilic peptide, alpha defensin, proved atherogenic. This phenotype occurred despite favorable cholesterol and glucose levels, and lower body weight and blood pressure. In this study, integration of metabolic&behavioral phenotyping system, endocrine, biochemical and mitochondrial assessment, pathological and immunohistochemical tests, and multiple challenge tests was established to explore the metabolic impact of alpha defensin. Compared to the control group, Def+/+ mice exhibited lower total energy expenditure and carbohydrate utilization, and higher fat oxidation. Their ACTH-cortisol and thyroid profiles were intact. Intriguingly, they had low levels of glucagon, with high ammonia, uric acid, triglyceride, and lactate. Mitochondrial evaluations were normal. Overall, defensin-induced hypoglucagonemia is associated with lipolysis, restricted glucose oxidation, and enhanced wasting. Def+/+ mice may be a useful model for studying the category of lean, apparently metabolically healthy, and atherosclerotic phenotype, with insight into a potential inflammatory-metabolic link.

Mitochondrial derived vesicles- Quo Vadis?

Mitochondria are dynamic, intracellular organelles with a separate genome originating from prokaryotes. They perform numerous functions essential for cellular metabolism and energy production. Mitochondrial-derived vesicles (MDVs) are single or double membrane-enclosed vesicles, formed and released from the mitochondrial sub-compartments into the cytosol, in response to various triggers. MDVs interact with other organelles such as lysosomes and peroxisomes or may be incorporated and excreted via extracellular vesicles (EVs). MDVs selectively incorporate diverse protein and lipid cargoes and are involved in various functions such as mitochondrial quality control, immunomodulation, energy complementation, and compartmentalization and transport. This review aims to provide a summary of the current knowledge of MDVs biogenesis, release, cargoes, and roles.

The Beneficial Effect of Mitochondrial Transfer Therapy in 5XFAD Mice via Liver-Serum-Brain Response.

We recently reported the benefit of the IV transferring of active exogenous mitochondria in a short-term pharmacological AD (Alzheimer's disease) model. We have now explored the efficacy of mitochondrial transfer in 5XFAD transgenic mice, aiming to explore the underlying mechanism by which the IV-injected mitochondria affect the diseased brain. Mitochondrial transfer in 5XFAD ameliorated cognitive impairment, amyloid burden, and mitochondrial dysfunction. Exogenously injected mitochondria were detected in the liver but not in the brain. We detected alterations in brain proteome, implicating synapse-related processes, ubiquitination/proteasome-related processes, phagocytosis, and mitochondria-related factors, which may lead to the amelioration of disease. These changes were accompanied by proteome/metabolome alterations in the liver, including pathways of glucose, glutathione, amino acids, biogenic amines, and sphingolipids. Altered liver metabolites were also detected in the serum of the treated mice, particularly metabolites that are known to affect neurodegenerative processes, such as carnosine, putrescine, C24:1-OH sphingomyelin, and amino acids, which serve as neurotransmitters or their precursors. Our results suggest that the beneficial effect of mitochondrial transfer in the 5XFAD mice is mediated by metabolic signaling from the liver via the serum to the brain, where it induces protective effects. The high efficacy of the mitochondrial transfer may offer a novel AD therapy.

Early Infiltration of Innate Immune Cells to the Liver Depletes HNF4α and Promotes Extrahepatic Carcinogenesis.

Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.

Mitochondrial-derived vesicles retain membrane potential and contain a functional ATP synthase.

Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.

Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

High Sucrose Diet-Induced Subunit I Tyrosine 304 Phosphorylation of Cytochrome c Oxidase Leads to Liver Mitochondrial Respiratory Dysfunction in the Cohen Diabetic Rat Model.

The mitochondrial oxidative phosphorylation process generates most of the cellular energy and free radicals in mammalian tissues. Both factors play a critical role in numerous human diseases that could be affected by reversible phosphorylation events that regulate the function and activity of the oxidative phosphorylation complexes. In this study, we analyzed liver mitochondria of Cohen diabetes-sensitive (CDs) and Cohen diabetes-resistant (CDr) rats, using blue native gel electrophoresis (BN-PAGE) in combination with mitochondrial activity measurements and a site-specific tyrosine phosphorylation implicated in inflammation, a known driver of diabetes pathology. We uncovered the presence of a specific inhibitory phosphorylation on tyrosine 304 of catalytic subunit I of dimeric cytochrome c oxidase (CcO, complex IV). Driven by a high sucrose diet in both CDr and CDs rats, Y304 phosphorylation, which occurs close to the catalytic oxygen binding site, correlates with a decrease in CcO activity and respiratory dysfunction in rat liver tissue under hyperglycemic conditions. We propose that this phosphorylation, specifically seen in dimeric CcO and induced by high sucrose diet-mediated inflammatory signaling, triggers enzymatic activity decline of complex IV dimers and the assembly of supercomplexes in liver tissue as a molecular mechanism underlying a (pre-)diabetic phenotype.

Variable effects of omaveloxolone (RTA408) on primary fibroblasts with mitochondrial defects.

Omaveloxolone (RTA408) is a second-generation oleanane triterpenoid Nrf2 inducer with antioxidant and anti-inflammatory properties and was reported to improve mitochondrial bioenergetics. It is currently being tested in medical trials for Friedrich ataxia, a genetic, multi-organ disease involving mitochondrial dysfunction. Thus, omaveloxolone could potentially be beneficial for additional disorders involving mitochondrial dysfunction. To this end, we investigated its effect on primary fibroblasts derived from patients with mitochondrial complex I deficiency, mitochondrial cytochrome oxidase deficiency, and two recessive forms of Parkinson's disease. Patients and control cells were incubated in the presence or absence of 50 nM omaveloxolone for 72 h prior to measurements. Generally, growth on galactose medium and ATP production were unaltered. Mitochondrial membrane potential was slightly but significantly decreased, while reactive oxygen species (ROS) production was variably decreased. Mitochondrial mass and mitochondrial DNA (mtDNA) contents were significantly increased in the patient's cells. These results were partially confirmed by the results of oxygen consumption studies which disclosed increased maximal oxygen consumption rates in most cells and increased energy status in all treated cells. Further investigation is required to explore the precise effect of omaveloxolone on mitochondrial function in disease.

Replicative Stress Coincides with Impaired Nuclear DNA Damage Response in COX4-1 Deficiency.

Cytochrome c oxidase (COX), a multimeric protein complex, is the final electron acceptor in the mitochondrial electron transfer chain. Primary COX deficiency, caused by mutations in either mitochondrial DNA or nuclear-encoded genes, is a heterogenous group of mitochondrial diseases with a wide range of presentations, ranging from fatal infantile to subtler. We previously reported a patient with primary COX deficiency due to a pathogenic variant in COX4I1 (encoding the common isoform of COX subunit 4, COX4-1), who presented with bone marrow failure, genomic instability, and short stature, mimicking Fanconi anemia (FA). In the present study, we demonstrated that accumulative DNA damage coincided primarily with proliferative cells in the patient's fibroblasts and in COX4i1 knockdown cells. Expression analysis implicated a reduction in DNA damage response pathways, which was verified by demonstrating impaired recovery from genotoxic insult and decreased DNA repair. The premature senescence of the COX4-1-deficient cells prevented us from undertaking additional studies; nevertheless, taken together, our results indicate replicative stress and impaired nuclear DNA damage response in COX4-1 deficiency. Interestingly, our in vitro findings recapitulated the patient's presentation and present status.

Cytochrome c Oxidase Activity as a Metabolic Regulator in Pancreatic Beta-Cells.

Pancreatic ß-cells couple glucose-stimulated insulin secretion (GSIS) with oxidative phosphorylation via cytochrome c oxidase (COX), a mitochondrial respiratory-chain enzyme. The Cohen diabetic-sensitive (CDs) rats exhibit hyperglycemia when fed a diabetogenic diet but maintain normoglycemia on a regular diet. We have previously reported a decreased COX activity in CDs rats and explored its relevance for type 2 diabetes (T2D). In this study, we investigated the relation between COX activity in islets, peripheral-blood mononuclear cells (PBMCs), and GSIS during diabetes development in CDs rats fed a diabetogenic diet for 4, 11, 20, and 30 days and during reversion to normoglycemia in hyperglycemic CDs rats fed a reversion diet for 7, 11, and 20 days. An oral glucose-tolerance test was performed at different periods of the diets measuring blood glucose and insulin concentrations. COX activity was determined in islets and PBMCs isolated from rats at the different periods of the diets. We demonstrated a progressive reduction in COX activity in CDs-islets that correlated positively with the decreasing GSIS (R2 = 0.9691, p < 0.001) and inversely with the elevation in blood glucose levels (R2 = 0.8396, p < 0.001). Hyperglycemia was initiated when islet COX activity decreased below 46%. The reversion diet restored >46% of the islet COX activity and GSIS while re-establishing normoglycemia. Interestingly, COX activity in PBMCs correlated significantly with islet COX activity (R2 = 0.8944, p < 0.001). Our data support islet COX activity as a major metabolic regulator of ß-cells function. The correlation between COX activity in PBMCs and islets may serve as a noninvasive biomarker to monitor ß-cell dysfunction in diabetes.

Multifaceted Analyses of Isolated Mitochondria Establish the Anticancer Drug 2-Hydroxyoleic Acid as an Inhibitor of Substrate Oxidation and an Activator of Complex IV-Dependent State 3 Respiration.

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) has been extensively investigated as a cancer therapy mainly based on its regulation of membrane lipid composition and structure, activating various cell fate pathways. We discovered, additionally, that 2OHOA can uncouple oxidative phosphorylation, but this has never been demonstrated mechanistically. Here, we explored the effect of 2OHOA on mitochondria isolated by ultracentrifugation from U118MG glioblastoma cells. Mitochondria were analyzed by shotgun lipidomics, molecular dynamic simulations, spectrophotometric assays for determining respiratory complex activity, mass spectrometry for assessing beta oxidation and Seahorse technology for bioenergetic profiling. We showed that the main impact of 2OHOA on mitochondrial lipids is their hydroxylation, demonstrated by simulations to decrease co-enzyme Q diffusion in the liquid disordered membranes embedding respiratory complexes. This decreased co-enzyme Q diffusion can explain the inhibition of disjointly measured complexes I-III activity. However, it doesn't explain how 2OHOA increases complex IV and state 3 respiration in intact mitochondria. This increased respiration probably allows mitochondrial oxidative phosphorylation to maintain ATP production against the 2OHOA-mediated inhibition of glycolytic ATP production. This work correlates 2OHOA function with its modulation of mitochondrial lipid composition, reflecting both 2OHOA anticancer activity and adaptation to it by enhancement of state 3 respiration.

What Can We Learn from the Parents of Children Affected with Mucopolysaccharidosis Type III-A in Israel?

Sanfilippo Syndrome, or mucopolysaccharidosis type III (MPS III), is a group of autosomal-recessive lysosomal storage disorders leading to tissue accumulation of heparan sulfate. MPS III is caused by deficiency in one of 4 enzymes involved in lysosomal degradation of heparan sulfate. Based on the relevant enzyme deficiency, 4 types have been recognized. MPS III constitutes a progressive neurodegenerative and systemic disorder. Parents of children diagnosed with MPS III were interviewed using a retrospective questionnaire based on the known clinical manifestations of MPS III. Eight patients from 4 unrelated families of varied ethnic origin were included. All children were diagnosed with MPS type III-A. Average age at diagnosis was 6.1 years. The most common early clinical manifestations leading to parental suspicion of illness were speech delay and coarse facial features. All children were reported to have global developmental delay, sleep disorders, recurrent infections, hyperactivity, and decreased hearing. The time from first medical inquiry until diagnosis was over 2 years on average, consistent with the delay in diagnosis described in the literature. MPS III children frequently undergo early and repeated ear, nose and throat surgeries, thus we suggest that a high index of suspicion is warranted in relevant clinical circumstances.

Infantile SOD1 deficiency syndrome caused by a homozygous SOD1 variant with absence of enzyme activity.

Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.

Comparing anti-aging hallmark activities of Metformin and Nano-PSO in a mouse model of genetic Creutzfeldt-Jakob Disease.

Advanced age is the main risk factor for the manifestation of late onset neurodegenerative diseases. Metformin, an anti-diabetic drug, was shown to extend longevity, and to ameliorate the activity of recognized aging hallmarks. Here, we compared the clinical, pathologic and biochemical effects of Metformin to those of Nano-PSO (Granagard), a brain targeted anti-oxidant shown by us to delay disease advance in transgenic mice mimicking for genetic Creutzfeldt Jacob disease (CJD) linked to the E200KPrP mutation. We demonstrate that both Metformin and Nano-PSO reduced aging hallmarks activities such as activated AMPK, the main energy sensor of cells as well as Nrf2 and COX IV1, regulators of oxidation, and mitochondrial activity. Both compounds reduced inflammation and increased stem cells production, however did not decrease PrP accumulation. As opposed to Nano-PSO, Metformin neither delayed clinical disease advance in these mice nor reduced the accumulation of sulfated glycosaminoglycans, a pathologic feature of prion disease. We conclude that elevation of anti-aging markers may not be sufficient to delay the fatal advance of genetic CJD.

Levodopa-responsive dystonia caused by biallelic PRKN exon inversion invisible to exome sequencing.

Biallelic pathogenic variants in PRKN (PARK2), encoding the E3 ubiquitin ligase parkin, lead to early-onset Parkinson's disease. Structural variants, including duplications or deletions, are common in PRKN due to their location within the fragile site FRA6E. These variants are readily detectable by copy number variation analysis. We studied four siblings with levodopa-responsive dystonia by exome sequencing followed by genome sequencing. Affected individuals developed juvenile levodopa-responsive dystonia with subsequent appearance of parkinsonism and motor fluctuations that improved by subthalamic stimulation. Exome sequencing and copy number variation analysis were not diagnostic, yet revealed a shared homozygous block including PRKN. Genome sequencing revealed an inversion within PRKN, with intronic breakpoints flanking exon 5. Breakpoint junction analysis implicated non-homologous end joining and possibly replicative mechanisms as the repair pathways involved. Analysis of cDNA indicated skipping of exon 5 (84 bp) that was replaced by 93 bp of retained intronic sequence, preserving the reading frame yet altering a significant number of residues. Balanced copy number inversions in PRKN are associated with a severe phenotype. Such structural variants, undetected by exome analysis and by copy number variation analysis, should be considered in the relevant clinical setting. These findings raise the possibility that PRKN structural variants are more common than currently estimated.

Multiple Acyl-CoA Dehydrogenase Deficiency with Variable Presentation Due to a Homozygous Mutation in a Bedouin Tribe.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid and amino acid oxidation defect caused by a deficiency of the electron-transfer flavoprotein (ETF) or the electron-transfer flavoprotein dehydrogenase (ETFDH). There are three phenotypes of the disease, two neonatal forms and one late-onset. Previous studies have suggested that there is a phenotype-genotype correlation. We report on six patients from a single Bedouin tribe, five of whom were sequenced and found to be homozygous to the same variant in the ETFDH gene, with variable severity and age of presentation. The variant, NM_004453.3 (ETFDH): c.524G>A, p.(R175H), was previously recognized as pathogenic, although it has not been reported in the literature in a homozygous state before. R175H is located near the FAD binding site, likely affecting the affinity of FAD for EFT:QO. The single homozygous ETFDH pathogenic variant was found to be causing MADD in this cohort with an unexpectedly variable severity of presentation. The difference in severity could partly be explained by early diagnosis via newborn screening and early treatment with the FAD precursor riboflavin, highlighting the importance of early detection by newborn screening.

Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency.

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient's cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.

A novel homozygous MSTO1 mutation in Ashkenazi Jewish siblings with ataxia and myopathy.

MSTO1 is a cytoplasmic protein that modulates mitochondrial dynamics by promoting mitochondrial fusion. Mutations in the MSTO1 gene are responsible for an extremely rare condition characterized by early-onset myopathy and cerebellar ataxia. We report here two siblings from a large Ashkenazi Jewish family, presenting with a progressive neuromuscular disease characterized by ataxia and myopathy. By whole exome sequencing, we found a novel homozygous missense mutation (c.1403T>A, p.Leu468Gln) in MSTO1. Studies performed on fibroblasts from the index patient demonstrated the pathogenic role of the identified variant; we found that MSTO1 protein level was reduced and that mitochondrial network was fragmented or formed enlarged structures. Moreover, patient's cells showed reduced mitochondrial DNA amount. Our report confirms that MSTO1 mutations are typically recessive, and associated with clinical phenotypes characterized by early-onset muscle impairment and ataxia, often with upper motor neuron signs and varied cognitive impairment.