Biology subjects

Fellman, V.

Publications and source records attributed to Fellman, V..

7 recordsLinked to original sources

bioRxiv2025

Hepatic gene replacement restores energy metabolism and doubles the survival in mouse model of GRACILE syndrome, a neonatal mitochondrial disease

Preclinical gene therapy studies of mitochondrial diseases remain limited due to their multisystemic manifestations and the scarcity of physiologically relevant animal models. Mutations in BCS1L, a nuclear gene encoding an assembly factor for mitochondrial complex III (CIII), are the most common cause of pediatric CIII deficiency. The most severe phenotype, GRACILE syndrome, is caused by a homozygous Finnish founder mutation (c.A232G, p.S78G). The corresponding Bcs1lp.S78G knock-in mouse model effectively recapitulates the human disease, with juvenile-onset hepatopathy, tubulopathy, growth restriction, segmental progeria, and short survival. Here, we performed recombinant adeno-associated virus (rAAV)-mediated gene replacement in this model, which features a postnatal presymptomatic window until weaning. A single intraperitoneal injection of rAAV encoding wild-type Bcs1l restored CIII assembly and activity in the liver, preventing hepatopathy. Hepatocyte-specific correction was sufficient to ameliorate hypoglycemia, improve growth, normalize systemic metabolism, and extend survival by nearly two-fold, despite persistent CIII deficiency in other tissues. Remarkably, liver-directed rescue also prevented skeletal muscle transcriptomic changes, particularly those linked to altered energy substrate utilization. These results underscore the central role of the liver in systemic energy homeostasis and growth regulation in multiorgan mitochondrial diseases and demonstrate the therapeutic potential of hepatocyte-directed gene replacement in phenotypes with prominent hepatopathy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/677965v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b1cf50org.highwire.dtl.DTLVardef@1a1c2e2org.highwire.dtl.DTLVardef@101bdb6org.highwire.dtl.DTLVardef@14e11e3_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

physiology↗

bioRxiv2025

Evolutionarily conserved temperature dependency leads to loss of protein O-GlcNAc in mammalian hypothermia

O-N-acetylglucosaminylation (O-GlcNAcylation) is a conserved, non-canonical glycosylation of intracellular proteins suggested to regulate a wide spectrum of fundamental cell processes and stress responses. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove, respectively, O-GlcNAc on protein serines and threonines. O-GlcNAcylation is thought to be primarily regulated by the availability of its substrate, UDP-GlcNAc, produced by the hexosamine biosynthetic pathway (HBP). We observed a body-wide loss of O-GlcNAcylated proteins in Bcs1l mutant mice, a model of mitochondrial complex III deficiency. UDP-GlcNAc precursors glutamine, UTP and acetyl-CoA were decreased in the mutant liver but, surprisingly, UDP-GlcNAc was not consistently decreased in the organs that showed low O-GlcNAc. Neither N-acetylglucosamine supplementation nor overexpression of the HBP rate-limiting enzyme GFPT1 restored the protein O-GlcNAc levels. The Bcs1l mutant mice become hypothermic and, interestingly, earlier evidence suggest that O-GlcNAcylation depends on ambient temperature in Drosophila embryos. We found that temperature determined O-GlcNAc abundance also in adult flies of tropical and boreal Drosophila species, in the poikilothermic vertebrate zebrafish, and in cultured mammalian cells. In cultured cells, the OGT-OGA protein ratio coincided with temperature and O-GlcNAc level, providing a regulatory mechanism. Pharmacological OGA inhibition decoupled the O-GlcNAc temperature dependency in cultured cells, as did an OGA null allele in Drosophila. In Bcs1l mutant mice, O-GlcNAc levels strongly correlated with body temperature. Increasing the mouse body temperature through transgenic expression of the heat-generating mitochondrial alternative oxidase (AOX) or housing at 35{degrees}C prevented the loss of O-GlcNAc. Pharmacological restoration of O-GlcNAc in the Bcs1l mutant mice produced minimal effects, suggesting that the bulk of O-GlcNAc is dispensable in mild hypothermia. Our findings imply an evolutionarily ancient role of protein O-GlcNAcylation in temperature adaptation and, instead of protein function-specific roles, argue for a global role of O-GlcNAc in temperature control of proteostasis.

physiology↗

bioRxiv2025

Parsing glomerular and tubular structure variability in high-throughput kidney organoid culture

High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four microplate-based high-throughput approaches utilizing spheroid culture steps. Quantitative high-content screening analysis of the nephrin-positive podocytes and ECAD-positive tubular cells revealed that the choice of approach and culture conditions were significantly associated with structure development. The culture approach, iPSC line, experimental replication, and initial cell number explained 35-77% of the variability in the logit-transformed proportion of nephrin and ECAD-positive area, when fitted into multiple linear models. Our study highlights the benefits of high-throughput culture and multivariate techniques to better distinguishing sources of technical and biological variation in morphological analysis of organoids. Our microplate-based high-throughput approach is easily adaptable for other laboratories to combat organoid size variability.

developmental biology↗

bioRxiv2024

Restoration of mitochondrial complex III function in hepatocytes highlights the liver as a key thermogenic organ independent of brown adipocyte activation

Liver is the key hub of systemic energy metabolism and growth regulation, yet its roles in mitochondrial disease pathophysiology remain relatively understudied. Bcs1lp.S78G knock-in mice, carrying a patient mutation causing respiratory complex III (CIII)-deficiency, present juvenile-onset liver and kidney disease, growth restriction, lipodystrophy, and early death. We restored CIII function in the hepatocytes of these mice using recombinant adeno-associated viral vectors (rAAVs) expressing wild-type Bcs1l. A single intraperitoneal injection of rAAVs into presymptomatic juvenile mice prevented liver disease, improved hypoglycemia and growth, normalized hepatic fuel utilization, and doubled the survival. The mutant mice showed hypothermia and brown adipose tissue (BAT) inflammation, and lacked BAT activation basally and upon acute cold challenge. Disrupted foot pad innervation suggested sensory neuropathy and impaired thermosensation as a contributor to the BAT inactivity. Surprisingly, the rAAV-treated mice maintained near-normal body temperature without significant effect on BAT. Increasing cellular respiration via transgenic alternative oxidase (AOX) was sufficient to prevent the hypothermia. The CIII-deficient mice did not reach euthermia until at an ambient temperature of 35{degrees}C, housing at which relieved metabolic stress and ameliorated hepatocyte senescence. We conclude that mitochondrial respiration in hepatocytes is essential for euthermia in mice. Our findings highlight the crucial role of the liver in thermoregulation, hypothermia as a consequence of mitochondrial dysfunction, and the therapeutic potential of rAAV-based gene delivery in a preclinical model of a multiorgan mitochondrial disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=150 HEIGHT=200 SRC="FIGDIR/small/612616v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cc59ecorg.highwire.dtl.DTLVardef@1239b01org.highwire.dtl.DTLVardef@95697eorg.highwire.dtl.DTLVardef@140fa94_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic yet still relatively poorly understood non-canonical glycosylation of intracellular proteins. Several vital branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of this metabolite is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it restrict to specialized chromatographic procedures. To overcome this limitation, we introduce here an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by recombinant O-linked N-acetylglucosamine transferase (OGT) and detection of the modification with a specific antibody. The assay can be performed in dot blot or microplate formats. The key to successful assay was the removal of strong inhibition of OGT by the reaction side product, uridine diphosphate (UDP). We applied the assay to provide the first systematic report of UDP-GlcNAc concentrations in mouse tissues and cultured cells. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

biochemistry↗

bioRxiv2023

Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria

Accumulating evidence suggests mitochondria as key modulators of normal and premature aging, yet whether primary deficiency of oxidative phosphorylation (OXPHOS) can cause progeroid disease remains unclear. Here, we show that mice with severe isolated respiratory complex III (CIII) deficiency display nuclear DNA damage, cell cycle arrest, aberrant mitoses, cellular senescence, and laminopathy-like nuclei in the affected organs such as liver and kidney, and a systemic phenotype strikingly resembling juvenile-onset laminopathic and DNA repair-deficient progeroid syndromes. Mechanistically, CIII deficiency triggered presymptomatic cancer-like c-MYC upregulation followed by excessive anabolic metabolism and illicit cell proliferation against lack of energy and biosynthetic precursors. CIII-independent coenzyme Q oxidation dampened mitochondrial integrated stress response and the c-MYC induction, suppressed the illicit proliferation, and prevented juvenile lethality despite that canonical OXPHOS-linked functions remained uncorrected. Inhibition of c-MYC by expression of a dominant-negative Omomyc protein relieved the DNA damage in CIII-deficient hepatocytes in vivo. Our results unequivocally connect primary OXPHOS deficiency to genomic instability and progeroid disease and suggest that targeting c-MYC and aberrant cell proliferation may provide novel therapeutic strategies in mitochondrial diseases.

pathology↗

bioRxiv2019

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

Deoxyribonucleotide triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to regulation of and disturbances in cellular dNTP concentrations derive from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 10 to 30 mg of biopsy material. In contrast to published assays, this assay is based on long (~200 nucleotides) synthetic single-stranded DNA templates, an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantifies reliably as little as 100 fmol of each of the four dNTPs. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart, and skeletal muscle.

molecular biology↗