Biology subjects

Carter, R. N.

Publications and source records attributed to Carter, R. N..

10 recordsLinked to original sources

bioRxiv2025

Astrocytes control motor neuronal mitochondrial axonal transport deficits in C9ORF72 ALS.

Disrupted axonal transport and astrocyte dysfunction are implicated in amyotrophic lateral sclerosis (ALS). Here, we show these are intrinsically linked: human induced pluripotent stem cell-derived astrocytes carrying the C9ORF72 mutation disrupt axonal transport and mitochondrial function in motor neurons (MNs). Conversely, either isogenic gene-corrected astrocytes or selectively boosting C9ORF72-astrocyte mitochondrial bioenergetics rescue axonal transport deficits in C9ORF72-MNs. Thus, astrocytes have a dominant effect on axonal mitochondrial transport in the context of ALS.

neuroscience↗

bioRxiv2024

FAK modulates glioblastoma stem cell energetics via actomyosin contractility: regulation of glycolysis and mitochondrial function

Glycolysis and the TCA cycle are reprogrammed in cancer cells to meet bioenergetic and biosynthetic demands, including by engagement with the extracellular matrix (ECM). We show that focal adhesion kinase (FAK), a mediator of integrin-ECM signalling, is driving cellular energetics in a stem cell model of glioblastoma (GBM). FAK gene deletion inhibits both glycolysis and glutamine oxidation, with increased mitochondrial fragmentation and elevated phosphorylation of the mitochondrial protein MTFR1L at S235. Simultaneously, FAK loss causes a mesenchymal to epithelial transition, enhanced acto-myosin contractility as shown by phospho-myosin light chain (p-MLC S19) and impaired cell migration/invasiveness. Rho-kinase (ROCK) inhibitors suppress p-MLC (S19) and restore glutamine oxidation and elongation of mitochondria. Thus, FAK is a key regulator of both glycolysis and glutamine oxidation mediated by acto-myosin contractility that controls both cell and mitochondrial morphology. Moreover, FAK-dependent cellular energetics are coincident properties with GBM stem cell migration, invasiveness and tumour growth in vivo.

cancer biology↗

bioRxiv2023

Mitochondrial dysfunction and mitophagy blockade contribute to renal osteodystrophy in chronic kidney disease-mineral bone disorder

Chronic kidney disease-mineral and bone disorder (CKD-MBD) presents with extra-skeletal calcification and renal osteodystrophy (ROD). The origins of ROD likely lie with elevated uremic toxins and/or an altered hormonal profile but the cellular events responsible remain unclear. Here, we report that stalled mitophagy contributes to mitochondrial dysfunction in bones of a CKD-MBD mouse model, and also human CKD-MBD patients. RNA-seq analysis exposed an altered expression of genes associated with mitophagy and mitochondrial function in tibia of CKD-MBD mice. The accumulation of damaged osteocyte mitochondria and the expression of mitophagy regulators, p62/SQSTM1, ATG7 and LC3 was inconsistent with functional mitophagy, and in mito-QC reporter mice with CKD-MBD, there was a 2.3-fold increase in osteocyte mitolysosomes. Altered expression of mitophagy regulators in human CKD-MBD bones was also observed. To determine if uremic toxins were possibly responsible for these observations, indoxyl sulfate treatment of osteoblasts revealed mitochondria with distorted morphology and whose membrane potential and oxidative phosphorylation were decreased, and oxygen-free radical production increased. The altered p62/SQSTM1 and LC3-II expression was consistent with impaired mitophagy machinery and the effects of indoxyl sulfate were reversible by rapamycin. In conclusion, mitolysosome accumulation from impaired clearance of damaged mitochondria may contribute to the skeletal complications, characteristic of ROD. Targeting mitochondria and the mitophagy process may therefore offer novel routes for intervention to preserve bone health in patients with ROD. Such approaches would be timely as our current armamentarium of anti-fracture medications has not been developed for, or adequately studied in, patients with severe CKD-MBD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/573355v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f23caforg.highwire.dtl.DTLVardef@6e3e94org.highwire.dtl.DTLVardef@1ce6adeorg.highwire.dtl.DTLVardef@ca5558_HPS_FORMAT_FIGEXP M_FIG C_FIG TRANSLATIONAL STATEMENTRenal osteodystrophy (ROD) remains the major skeletal complication of chronic kidney disease-mineral and bone disorder (CKD-MBD). As a disease characterised by biochemical and hormone abnormalities, ROD is exacerbated by osteocyte mitochondrial dysfunction. Advances in our understanding of the mitophagy pathway are vital to improving the clinical management of ROD. The dysregulation of mitophagy in murine and human CKD-MBD bone provided evidence of delayed clearance of damaged mitochondria, which was also observed in uremic toxin-treated-osteoblasts but reversible upon rapamycin treatment. This study reveals the therapeutic potential of managing ROD by restoring defective mitophagy in osteocytes.

genomics↗

bioRxiv2023

Preneoplastic cells switch to Warburg metabolism from their inception exposing multiple vulnerabilities for targeted elimination

Otto Warburg first described tumour cells as displaying enhanced aerobic glycolysis whilst maintaining defective oxidative phosphorylation (OXPHOS) for energy production almost 100 years ago 1,2. Since then, the Warburg effect has been widely accepted as a key feature of rapidly proliferating cancer cells3,4. Targeting cancer metabolism is now being considered as a promising precision oncology therapeutic approach5. What is not clear is how early "Warburg metabolism" initiates during cancer progression and whether changes in energy metabolism might influence tumour progression ab initio. We set out to investigate energy metabolism in HRASG12V driven preneoplastic cell (PNC) at inception, in a zebrafish skin PNC model; and to test how the impact of manipulating energy metabolism in the whole animal may impact PNC initiation. We find that, within 24 hours of HRASG12V induction, PNCs upregulate "Warburg metabolism", and that this is required for their expansion. We show that blocking glycolysis reduces PNC proliferation, whilst increasing available glucose both enhances PNC proliferation and also reduces apoptosis. Impaired OXPHOS of PNCs might be exploited therapeutically since a mild complex I inhibitor, metformin, selectively induces apoptosis and suppresses proliferation of PNCs. In addition, we find mitochondrial fragmentation in PNCs occur prior to metabolic alteration and this is important for their survival since exposure to Drp1/Dnml1 inhibitor, mdivi, which blocks mitochondrial fragmentation leads to enhanced PNC apoptosis. Our data indicate that altered energy metabolism is one of the earliest events upon oncogene activation in somatic cells, which provide a targeted and effective tumour prevention therapy. Key findingsO_LIGlycolysis is upregulated in HRASG12V expressing preneoplastic cells (PNCs) in zebrafish skin, and is required for PNC proliferation C_LIO_LIOXPHOS respiration is impaired in PNCs and metformin complex I inhibition specifically eliminates PNCs C_LIO_LIPNC undergo mitochondrial fragmentation and exhibit reduced membrane potential C_LIO_LIMdivi reverses mitochondrial fragmentation in PNCs and triggers apoptosis C_LI

cancer biology↗

bioRxiv2021

High-content profiling reveals a unified model of copper ionophore dependent cell death in oesophageal adenocarcinoma

Background and AimsOesophageal adenocarcinoma (OAC) is of increasing global concern due to increasing incidence, a lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, lack of driver mutations, and the dominance of large-scale genomic rearrangements. In this work we have characterised three potent and selective hit compounds identified in an innovative high-content phenotypic screening assay. The three hits include two approved drugs; elesclomol and disulfiram, and another small molecule compound, ammonium pyrrolidinedithiocarbamate. We uncover their mechanism of action, discover a targetable vulnerability, and gain insight into drug sensitivity for biomarker-based clinical trials in OAC. MethodsElesclomol, disulfiram, and ammonium pyrrolidinedithiocarbamate were systematically characterised across panels of oesophageal cell lines and patient-derived organoids. Drug treated oesophageal cell lines were morphologically profiled using a high-content, imaging platform. Compounds were assessed for efficacy across patient-derived organoids. Metabolomics and transcriptomics were assessed for the identification of oesophageal-cancer specific drug mechanisms and patient stratification hypotheses. ResultsHigh-content profiling revealed that all three compounds were highly selective for OAC over tissue-matched controls. Comparison of gene expression and morphological signatures unveiled a unified mechanism of action involving the accumulation of copper selectively in cancer cells, leading to dysregulation of proteostasis and cancer cell death. Basal omic analyses revealed proteasome and metabolic markers of drug sensitivity, forming the basis for biomarker-based clinical trials in OAC. ConclusionsIntegrated analysis of high-content imaging, transcriptomic and metabolomic data has revealed a new therapeutic mechanism for the treatment of OAC and represents an alternative target-agnostic drug discovery strategy.

cancer biology↗

bioRxiv2021

Classical macrophage polarisation is limited by human β-defensin 3 via an autocrine IL-4 dependent process.

Human {beta}-defensin 3 (HBD3), is an anti-microbial host-defence peptide, that can rapidly enter macrophages to modulate TLR4 responses to lipopolysaccharide. However, the molecular mechanisms by which HBD3 exerts this anti-inflammatory influence remain unclear. Here, we show mice deleted for the orthologue of HBD3 have an increased acute lipopolysaccharide response in vivo. Furthermore, we found that HBD3 limited the response of macrophages to classical activation, and contemporaneously drove expression of IL-4. An increase in markers of alternative activation, and a change in metabolic flux was also observed. Consistent with these results, HBD3 enhanced the IL-4 mediated polarisation of naive macrophages. Finally, we demonstrate that the ability of HBD3 to limit macrophage classical activation requires IL-4R. These data reveal a previously unrecognised role for HBD3 in influencing the polarisation state of macrophages to enable a state conducive for repair and resolution. SYNOPSIS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/442606v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f81c9aorg.highwire.dtl.DTLVardef@11dca23org.highwire.dtl.DTLVardef@c1e241org.highwire.dtl.DTLVardef@ed46ed_HPS_FORMAT_FIGEXP M_FIG C_FIG The anti-microbial host-defence peptide, Human {beta}-defensin 3 (HBD3), is shown here to modulate the inflammatory response to classical activation by promoting alternative activation through IL-4R, to enable a state conducive for repair and resolution. O_LIKnockout mice for the orthologous gene for HBD3, demonstrate increased acute lipopolysaccharide inflammatory response. C_LIO_LIHBD3 limited the classical activation of macrophages polarised with LPS/IFN{gamma} and drove expression of IL-4. Cells also displayed increase in alternative activation markers and promotion of oxidative phosphorylation. C_LIO_LIHBD3 enhanced the IL-4-mediated activation of naive macrophages. C_LIO_LIThe ability of HBD3 to limit macrophage classical activation and contemporaneously promote alternative activation required IL-4R. C_LI

immunology↗

bioRxiv2021

GLUCOCORTICOIDS REGULATE MITOCHONDRIAL FATTY ACID OXIDATION IN FETAL CARDIOMYOCYTES

The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesised that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes in vivo. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 hours later. Instead, at E17.5, fatty acid oxidation genes were down-regulated by dexamethasone, as was GR itself. PGC-1, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was down-regulated in vivo in fetal hearts at E17.5, 24 hours after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a sheep model of preterm birth, both GR and PGC-1 were down-regulated in fetal heart. These data suggest endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by down-regulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility.

physiology↗

bioRxiv2021

Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy

Little is known about the proteins that direct the highly conserved patterns of spatial genome organisation in fat. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope protein Tmem120a disrupts fat genome organisation, thus causing a novel lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a-/- mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifests upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein laminA mutations. Our data suggest TMEM120A may mediate/instigate novel categories of adipose tissue dysfunction across the adiposity spectrum and provide a new miRNA-based mechanism possibly driving the unexplained muscle hypertrophy in human lipodystrophy.

cell biology↗

bioRxiv2020

The hepatic compensatory response to elevated systemic sulfide impairs medium chain fat oxidation and promotes diabetes

Impaired hepatic glucose and lipid metabolism are hallmarks of type-2 diabetes. Increased sulfide production from cysteine, or sulfide-donor compounds, may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver-enriched mitochondrial SOP enzyme thiosulfate sulfur-transferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia and fatty liver, despite whole-body insulin-sensitisation. Unexpectedly, hepatic sulfide levels were normal in Tst-/- mice, a result of homeostatic induction of mitochondrial sulfide disposal and glutathione excretion associated with net suppression of protein persulfidation and nuclear respiratory factor-2 target proteins. Proteomic and persulfidomic profiling converged on gluconeogenesis and hepatic lipid metabolism and revealed a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role for TST in hepatic metabolism that raises implications for sulfide-donor strategies in the context of liver function and metabolic disease.

physiology↗

bioRxiv2020

Nonalcoholic fatty liver disease is associated with decreased hepatocyte mitochondrial respiration but not mitochondrial number

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term covers a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis (NASH). As the disease progresses, NASH can develop into cirrhosis and hepatocellular carcinoma. However, the underlying mechanisms and the factors which predispose an individual to disease progression remain poorly understood. Whilst NAFLD appears to be associated with mitochondrial dysfunction, it is unclear whether this is due to respiratory impairment, changes in mitochondrial mass, or mitochondrial fragmentation. Using a human pluripotent stem cell-based model of NAFLD we show that exposure to lactate, pyruvate and octanoic acid results in the development of macrovesicular steatosis. We do not observe changes in mitochondrial mass or fragmentation but do find decreases in maximal respiration and reserve capacity, suggesting impairment in the electron transport chain (ETC). Taken together, these findings indicate that the development of macrovesicular steatosis in NAFLD may be linked to the impairment of the ETC in mitochondria.

biochemistry↗