Biology subjects

Pfleger, J.

Publications and source records attributed to Pfleger, J..

4 recordsLinked to original sources

bioRxiv2026

Elevating levels of neuronal MCU in the hippocampus enhances mitochondrial calcium uptake and respiratory efficiency proportional to demand

Mitochondrial calcium signaling integrates energy needs with energy production, amplifying or suppressing mitochondrial respiration in response to activity demand. Neuronal activity is tightly ATPcoupled to increases in mitochondrial calcium uptake, which stimulate the tricarboxylic acid cycle (TCA) and activate calcium-dependent enzymes important for ATP production via oxidative phosphorylation. The mitochondrial calcium uniporter (MCU) is the predominant source of matrix calcium and is differentially expressed across neuronal cell types, suggesting cell-type-specific differences in the coupling of activity-driven calcium levels and mitochondrial respiration. Here, we investigated whether elevating MCU expression enhances mitochondrial calcium uptake and oxidative phosphorylation in the hippocampus. We report that hippocampal mitochondria overexpressing MCU take up calcium at a faster rate without increased sensitivity to calcium overload. By modeling in vivo supply and demand, we found that hippocampal mitochondria overexpressing MCU are more efficient than control mitochondria at responding to increased bioenergetic demand. These findings reveal a role for MCU in modulating mitochondrial calcium uptake and boosting mitochondrial respiration under increasing demand, which contributes to our understanding of how specific cell types may adapt to different bioenergetic demands.

neuroscience↗

bioRxiv2026

Cardiac REDD1 alters glucose and fatty acid metabolic gene expression via an mTORC1-independent, PPAR alpha-dependent mechanism and drives hypertrophic growth

BackgroundRegulated in development and DNA damage 1 (REDD1) is a highly inducible molecule that plays a role in numerous physiological and pathophysiological processes. It is a well-established negative regulator of mammalian target of rapamycin complex 1 (mTORC1), which is critical for maintaining elevated fatty acid-to-glucose oxidation ratio in the heart. In addition, REDD1 deletion results in hyperglycemia, suggesting that REDD1 is critical for tissue glucose metabolism. The role of REDD1 in regulating cardiac glucose and/or fatty acid metabolism in response to physiologic or pathophysiologic cues, however, remains unexplored. MethodsHerein, we utilize AC16 cardiomyocytes with REDD1 deletion, as well as mice with global or cardiomyocyte-specific deletion of Redd1, and their respective controls. We also subject these mice cardiac pressure overload using transverse aortic constriction (TAC) for 2 weeks or sham operation as a control. To examine the molecular regulators of glucose oxidation, we utilized qPCR and western blotting to evaluate pyruvate dehydrogenase (PDH) kinase (PDK) and phospho-PDH (pPDH) levels, respectively. We also directly measured PDH activity and glucose-driven cellular respiration. To investigate the complete REDD1-dependent transcriptome and metabolome, we performed RNA-sequencing (RNA-Seq) and untargeted metabolomics, respectively. To determine if the observed gene expression changes were dependent upon transcription factor peroxisome proliferator-activated receptor alpha (PPAR), we utilized an established pharmacologic PPAR inhibitor, GW6471. Here, we measured PPAR activity directly, as well as the expression of its target genes. In order to determine if our observed effects were mTORC1-dependent, we utilized mTORC1-specific inhibitor, everolimus. Finally, we measured cardiac hypertrophy using gravimetric analyses (heart weight (HW)-to-body weight (BW) or HW-to-tibia length (TL) ratios) and histological analyses of cardiomyocyte cross sectional area (CSA). We also measured mRNA and protein levels of pathological hypertrophic markers Natriuretic Peptide B (Nppb) and Cardiac Ankyrin Repeat Protein (CARP), respectively. ResultsOur data demonstrate that physiological levels of glucose induce REDD1 expression in cardiomyocytes. Further, we show that in cardiomyocytes or the hearts of mice with REDD1 deletion, there is elevated PDK4 expression, as well as increased levels of pPDH (S300 and/or S293) and reduced PDH activity. Interestingly, everolimus treatment has no effect on these alterations. In vitro, we also observe elevated glycolysis and glycolytic capacity, and reduced maximal respiratory capacity (MRC) in the presence of glucose. Interestingly, our RNA-Seq data reveals the upregulation of genes involved in fatty acid catabolism. Further, we demonstrate that PPAR activity is enhanced, and everolimus treatment also has no effect on this parameter. Additionally, we show that treatment of cardiomyocytes with GW6471 normalizes the expression of its target genes (PDK4, ACSL1) and levels of pPDH (S300), that are elevated in cells with REDD1 deletion. Finally, we observe elevated REDD1 in the hearts of mice following TAC. Moreover, we show reduced HW/BW, HW/TL, cardiomyocyte CSA, and levels of cardiac Nppb and CARP in mice with cardiomyocyte Redd1 deletion subjected to TAC versus controls also subjected to TAC. Importantly, TAC-induced reductions in cardiac Pdk4 and pPDH (S293 and S300), are normalized to control levels in mice with Redd1 deletion subjected to TAC. ConclusionsTogether, our findings suggest that physiological glucose-induced and pathological pressure overload-induced REDD1 is required for enhancing glucose oxidation and suppressing fatty acid oxidation in cardiomyocytes. In this way, REDD1 supports cardiac hypertrophic growth. We also outline a mechanism whereby REDD1 inhibits PPAR activity, thereby inhibiting the expression of its target genes, including PDK4 and those involved in fatty acid oxidation. Finally, we demonstrate that these effects are independent of REDD1s ability to inhibit mTORC1.

cell biology↗

bioRxiv2025

Pericyte and Endothelial Cell Responses within Murine Cerebral Capillaries After Blood Flow Cessation

ABSTRACT/SUMMARYBlood flow provides critical inputs for mechanisms governing vascular homeostasis. Altered hemodynamics can therefore trigger a wide range of cellular responses in blood vessels. Endothelial cells (ECs) downstream of atherosclerotic plaques for instance are exposed to turbulent flow, activating inflammatory pathways that promote immune cell infiltration. In conditions like stroke and myocardial infarction, the abrupt loss of blood flow prompts responses in vascular cells such as ECs and pericytes (PCs) to adapt to ischemic or no-flow conditions. To better understand how cerebral capillary ECs and PCs react to the sudden loss of blood flow, we used a murine brain slice model cultured for 12- and 24-hours in artificial cerebrospinal fluid (aCSF) with 95% oxygen supplementation. As expected, inflammation mediators were upregulated in cultured slices compared to non-cultured samples, particularly those associated with leukocyte recruitment. Additionally, transcriptional markers of extracellular matrix (ECM) remodeling and cell-ECM interactions were elevated, consistent with reduced PC coverage along capillaries. We initially presumed these changes reflected blood-brain barrier (BBB) degradation, but instead we found an increase in mRNA transcripts for EC junctions and stable protein levels for junction molecules, with an apparent rearrangement of Claudin5-based tight junctions. Some capillaries also exhibited reduced diameters, suggesting constriction by PCs or a subset thereof. Consistent with these observations, we found an upregulation of the vasoconstrictor Endothelin-1 (ET-1) with its receptors and contractile proteins found in a subpopulation of PCs. Suppressing ET-1 activity prevented Claudin5 upregulation, indicating that ET-1 might regulate microvascular constriction and associated changes in endothelial tight junctions. Overall, these results suggest that in the absence of blood flow, PCs contribute to capillary wall remodeling by (i) potentially mediating a mechanism driven by ET-1 that affects EC Claudin5 dynamics, and (ii) reducing capillary ECM and detaching from microvessel walls.

cell biology↗

bioRxiv2019

H2A.Z-dependent and -independent recruitment of metabolic enzymes to chromatin required for histone modifications

H2A.Z plays a fundamental role in the regulation of transcription and epigenetics, however, the mechanisms that underlie its functions are not fully understood. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with an array of tricarboxylic acid cycle and beta-oxidation enzymes in the mouse heart. Recombinant green florescence fusion proteins combined with mutations of putative nuclear localization signals of select enzymes, including acetyl-CoA acyltransferase 2 (ACAA2), oxoglutarate dehydrogenase (OGDH), and isocitrate dehydrogenase 2 confirmed their nuclear localization and chromatin binding in both rodent and human cells. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the selective association of ACAA2 and OGDH with H2A.Z-occupied transcription start sites. Finally, human H2A.Z-deficient HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, with the exception of pyruvate dehydrogenase, accompanied with reduced posttranslational histone modifications. Thus, the data show that metabolic enzymes are recruited to active promoters for potential site-directed epigenetic modifications.

molecular biology↗