Biology subjects

Harper, M.-E.

Publications and source records attributed to Harper, M.-E..

10 recordsLinked to original sources

bioRxiv2025

Functional and metabolomic analyses of brown adipose tissue during cold-deacclimation reveal rapid adaptations in N-acetylated amino acid metabolism

Non-shivering thermogenesis (NST) in brown adipose tissue (BAT) is rapidly activated in cold environments and is an important thermoregulatory process. Despite the consensus that BAT is inactive under warm ambient temperatures, few studies have sought to examine the metabolic remodelling that occurs when recovering from the cold and re-acclimating to thermoneutral environments (28-32{degrees}C). To elucidate mitochondrial functional and structural aspects involved in BAT metabolic remodelling during cold deacclimation, we acclimated C57BL/6J mice to the cold (4{degrees}C) for 7 days, and subsequently transferred them to thermoneutrality (30{degrees}C) for 3 h, 12 h, 24 h, or 48 h. Comprehensive metabolic phenotyping analyses demonstrated elevated metabolic rates and high food intake during the cold acclimation period, which immediately decreased by [~]40% upon returning to thermoneutrality. High-resolution respirometry of saponin-permeabilized BAT revealed decreases in mitochondrial leak uncoupling by 24 h of cold deacclimation, which corresponded with gradual declines in mitochondrial protein content and UCP1 gene expression. Decreases in BAT mitochondrial content paralleled declines in protein content, as indicated by decreases in the mtDNA/nDNA ratio and mitochondrial surface area by 48 h of cold deacclimation. Metabolomic analysis of BAT from cold-acclimated mice and from mice deacclimated for 48 h at thermoneutrality revealed major changes in pathways related to amino acid metabolism, the tricarboxylic acid cycle (TCA), glutathione, and purine metabolism. Marked decreases in the abundance of N-acetylated amino acids in cold deacclimated mice corresponded with increased aminoacylase 1 (Acy1) expression. Together, these findings highlight the profound metabolic remodelling in BAT during thermogenesis and deactivation.

physiology↗

bioRxiv2025

Purinergic receptor activation rectifies autism-associated endothelial dysfunction

Early cerebrovascular alterations affect brain maturation by impacting trophic support and energy supply. Recent evidence in a 16p11.2 deletion mouse model of autism spectrum disorder (ASD) revealed brain endothelial abnormalities postnatally. Yet, the endothelial alterations eliciting these changes remain unknown. Isolation of brain endothelial cells (ECs) from 14-day old male 16p11.2-deficient and wild-type mice revealed that 16p11.2 deletion-induced endothelial dysfunction is linked to a bioenergetic failure, with reduced intracellular ATP. Intra- or extra-cellular ATP supplementation rescued the function of 16p11.2-deficient ECs in vitro via P2 purinergic receptor activation, specifically P2Y2 receptors. Activating P2Y2 receptors restored cerebrovascular reactivity in 16p11.2-deficient parenchymal arterioles ex vivo and rescued 16p11.2 deletion-associated mouse behaviors. Taken together, this study demonstrates that metabolic reprogramming of brain ECs via purinergic receptor engagement represents a possible therapeutic avenue for ASD.

cell biology↗

bioRxiv2024

OPA1 mediates cardiac function and metabolism: in silico and in vivo evidence

OPA1 is an inner mitochondrial membrane protein that mediates diverse signaling processes. OPA1 is important for cardiac function and protects against cardiac insults such as ischemia reperfusion injury. We sought to further assess OPA1 in human and mouse cardiac pathologies, hypothesizing that OPA1 may also function in a protective manner in chronic heart failure. Bioinformatic analyses of histological and transcript data from the GTEx database indicated that OPA1 expression levels vary in the human heart, where elevated OPA1 transcript levels were correlated with fatty acid, branch chain amino acid and contractile gene signatures. To experimentally assess these correlations, mice with a 1.5-fold whole body OPA1 overexpression (OPA1-OE) were subjected to transverse aortic constriction surgery and displayed improved 2D and 4D cardiac functional parameters compared to WT mice. OPA1-OE mice had no induction of fibrotic transcript markers and displayed sustained transcript levels of fatty acid, branch chain amino acid and contractile markers. Maximal oxidative capacity was sustained in both WT and OPA1-OE cardiac myofibers post-TAC. These results further demonstrate the important role of OPA1 in mediating cardiac function and highlight protective signaling pathways.

biochemistry↗

bioRxiv2023

Mapping relationships between glutathione and SLC25 transporters in cancers using hybrid machine learning models

BackgroundAlterations of metabolism, including changes in mitochondrial and glutathione (GSH) metabolism, are a well appreciated hallmark of many cancers. Mitochondrial GSH (mGSH) transport is a poorly characterized aspect of GSH metabolism, which we investigate in the context of cancer. Existing functional annotation approaches from machine (ML) or deep learning (DL) models based only on protein sequences are unable to annotate functions in biological contexts, meaning new approaches must be developed for this task. ResultsWe develop a flexible ML framework for functional annotation from diverse feature data. This hybrid approach leverages cancer cell line multi-omics data and other biological knowledge data as features, to uncover potential genes involved in mGSH metabolism and membrane transport in cancers. This framework achieves an average AUROC across functional annotation tasks of 0.900 and can be effectively applied to annotate a range of biological functions. For our application, classification models predict the known mGSH transporter SLC25A39 but not SLC25A40 as being highly probably related to GSH metabolism in cancers. SLC25A24 and the orphan SLC25A43 are also predicted to be associated with mGSH metabolism by this approach and structural analysis of these proteins reveal similarities in potential substrate binding regions to the binding residues of SLC25A39. ConclusionThese findings have implications for a better understanding of cancer cell metabolism and novel therapeutic targets with respect to GSH metabolism through potential novel functional annotations of genes. The hybrid ML framework proposed here can be applied to other biological function classifications or multi-omics datasets to generate hypotheses in various biological contexts. Code and a tutorial for generating models and predictions in this framework are available at: https://github.com/lkenn012/mGSH_cancerClassifiers.

bioinformatics↗

bioRxiv2023

Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity

Aims/HypothesisInsulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous; however, this association remains controversial. The aim of this study was to perform an in-depth multi-factorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active), individuals with obesity (Obese) and individuals with Obesity, insulin resistance and type 2 diabetes (T2D). MethodsSkeletal muscle biopsies were obtained from the Vastus Lateralis of individuals who were lean and active (Active- n = 9), individuals with obesity (Obese- n = 9) and individuals with obesity insulin resistance and T2D (T2D- n =22) in this cross-sectional design. Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by BN-PAGE and immunoblot. TCA cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. ResultsActive had greater mitochondrial capacity compared to both Obese and T2D for ex vivo mitochondrial respiration with fatty-acid and glycolytic supported protocols adjusted for mitochondrial content (P < 0.05). Complex IV supercomplex assembley was greater in Active compared to Obese and T2D (P < 0.05) whereas Complex I and III supercomplex assembly was greater in Active compared to T2D only (P < 0.05). TCA cycle intermediates; citrate, succinate, fumarate and malate were all significantly greater in Active compared to Obese and T2D (P < 0.05). Strikingly, Obese and T2D do not differ in any of the skeletal muscle mitochondrial measurements. Active had an upregulation of genes related to respiration/mitochondrial capacity compared to both Obese and T2D. Transcriptional differences between Obese and T2D were not driven by mitochondrial related process. Active had reduced methylation correlated with increased gene expression for important mitochondrial-related genes, including ATP5PD and MFN2. Conclusions/InterpretationsWe reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity) that affect mitochondrial capacity. We highlight that lean, active individuals have enhanced skeletal muscle mitochondrial capacity that is also reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance. Clinical trial numberNCT0191110

molecular biology↗

bioRxiv2022

More than a ligand: PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I interferon pathway.

Targeting the PD-1/PD-L1 axis has transformed the field of immune-oncology. While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced infection with oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 suppressed type I interferon by promoting a metabolic shift characterized by enhanced glucose uptake and glycolysis rate. Lactate generated from glycolysis was the key metabolite responsible for inhibiting type I interferon responses and enhancing oncolytic virus infection in PD-L1-expressing cells. In addition to adding mechanistic insight into PD-L1 intrinsic function and showing that PD-L1 has a broader impact on immunity and cancer biology besides acting as a ligand for PD-1, our results will also help guide the numerous efforts currently ongoing to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials. Once sentence summaryPD-L1 promotes oncolytic virus efficacy.

cancer biology↗

bioRxiv2020

Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role

Srsf3 was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodelling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibres from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating mitochondrial integrity.

physiology↗

bioRxiv2020

A mouse model for spinal muscular atrophy provides insights into non-alcoholic fatty liver disease pathogenesis

Background & aims Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and death in children. SMA patients are more susceptible to dyslipidemia as well as liver steatosis, features reproduced in SMA mouse models. As current pre-clinical models of NAFLD are invariably imperfect and generally take a long time to develop, the rapid development of liver steatosis in SMA mice provides a means to identify molecular markers of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether Smn2B/- mice, a model of severe SMA, display typical features of NAFLD/non-alcoholic steatohepatitis (NASH).Methods Biochemical, histological, electron microscopy, proteomic, and high-resolution respirometry were used.Results The Smn2B/- mice develop steatohepatitis early in life. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in impaired hepatic function including alterations in protein output, complement, coagulation, iron homeostasis, and IGF-1 metabolism. The steatohepatitis is reversible by AAV9-SMN gene therapy. The NAFLD phenotype is likely due to non-esterified fatty acid (NEFA) overload from peripheral lipolysis, subsequent to hyperglucagonemia compounded by reduced muscle use. Mitochondrial β-oxidation contributed to hepatic damage as we observed enhanced hepatic mitochondrial β-oxidation and reactive oxygen species production. Hepatic mitochondrial content, however, was decreased. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight due to their neurological condition, develop hypoglycemia and do not develop hepatic fibrosis.Conclusion The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.Competing Interest StatementMarc-Olivier Deguise received honoraria and travel accommodations from Biogen for speaking engagements at the SMA Summit 2018 held in Montreal, Canada and SMA Academy 2019 held in Toronto, Canada. Rashmi Kothary received honoraria and travel accommodations from Roche as an invited speaker at their global and national board meetings in 2019. RK and the Ottawa Hospital Research Institute have a licensing agreement with Biogen for the Smn2B/- mouse model. Thomas H. Gillingwater has served on global and UK advisory boards for Roche. These COI are outside the scope of this study. All other authors have no competing interests to declare.List of abbreviationsAGCautomatic gain controlALPalkaline phosphataseALTalanine aminotransferaseASTaspartate aminotransferaseBaxBCL2 associated X proteinDAVIDThe Database for Annotation, Visualization and Integrated DiscoveryESEnrichment ScoreFasRFas receptorH&EHematoxylin & eosinHFDhigh fat dietIGF-1insulin-like growth factor 1IGFbp1insulin like growth factor binding protein 1IGF1Rinsulin like growth factor 1 receptorigfalsinsulin like growth factor binding protein acid labile subunitIPAingenuity pathway analysisMCDmethionine and choline deficient dietMCLMarkov Clustering AlgorithmNAFLDnon-alcoholic fatty liver diseaseNASHnon-alcoholic steatohepatitisNEFAnon-esterified fatty acidPpostnatal dayp21cyclin dependent kinase inhibitor 1Ap53tumor protein p53PASPeriodic acid-SchiffSMAspinal muscular atrophySMN1Survival motor neuron 1TMTTandem Mass TaggingTNFR1TNF receptor superfamily member 1AView Full Text

pathology↗

bioRxiv2020

Parkinson Disease-Linked Parkin Mediates Redox Reactions That Lower Oxidative Stress In Mammalian Brain

We recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H2O2) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H2O2 levels were unchanged in adult prkn-/- brain, a parkin-dependent reduction of cytosolic H2O2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn-/- mice. Nevertheless, this rise in oxidative stress led to changes in mitochondrial enzyme activities and the metabolism of glutathione in cells and mammalian brain. In parkins absence reduced glutathione concentrations were increased including in human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H2O2 reduction and glutathione regeneration. These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance.

neuroscience↗

bioRxiv2019

TSC2-deficiency potentiates a catabolic signaling switch that differentiates neural and neural crest lineage development and progressive disease manifestations

Lymphangioleiomyomatosis (LAM) is a rare lung disease marked by cystic destruction caused by invasive LAM cells harboring loss-of-function mutations in TSC2 that exhibit dysregulated mTORC1 signaling. Rapamycin, the only approved treatment, is not curative. Therapeutic discovery has been limited by inadequate cell and animal models due to the unknown LAM cell of origin. We report a novel TSC2--/-- human pluripotent stem cell (hPSC)-derived neural crest cell (NCC) model, which replicates key LAM features. Leveraging this model, temporal RNA sequencing and genome-wide CRISPR knockout screens identified synthetic lethal genes including TWIST1 and SUN4. Antisense oligonucleotides (ASOs) targeting TWIST1 and SUN4 demonstrated selective TSC2 mutant NCC cytotoxicity and inhibited invasion. Using a "test-withdraw-monitor" clinical trial paradigm, TWIST1 and SUN4 ASOs profoundly suppressed tumor viability in a Tsc2--/-- mouse allograft model and eradicated 40% of tumors, outperforming standard of care rapamycin and highlighting their promise as novel cytoablative LAM therapeutics.

cancer biology↗