bims-celmim 2024-12-29 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2024–12–29
sixteen papers selected by
Marc Segarra Mondejar

Effects of Aging on Glucose and Lipid Metabolism in Mice.
Metabolic Adaptations To Acute Glucose Uptake Inhibition Converge Upon Mitochondrial Respiration For Leukemia Cell Survival.
Functionally conserved inner mitochondrial membrane proteins CCDC51 and Mdm33 demarcate a subset of fission events.
Light-Inducible Deformation of Mitochondria in Live Cells.
Preventing excessive autophagy protects from the pathology of mtDNA mutations in Drosophila melanogaster.
Alternating Cellular Functions by Optogenetic Control of Organelles.
Pathway Thermodynamic Analysis Postulates Change in Glutamate Metabolism as a Key Factor in Modulating Immune Responses.
Lc-ms-based untargeted metabolomics reveals potential mechanisms of histologic chronic inflammation promoting prostate hyperplasia.
TIPE drives a cancer stem-like phenotype by promoting glycolysis via PKM2/HIF-1α axis in melanoma.
Mitochondria-targeting materials and therapies for regenerative engineering.
Genome-scale modeling identifies dynamic metabolic vulnerabilities during the epithelial to mesenchymal transition.
Pleozymes: Pleiotropic Oxidized Carbon Nanozymes Enhance Cellular Metabolic Flexibility.
A novel mitochondrial-targeted fluorescent probe for ratiometric imaging of nitric oxide in cells and zebrafish.
Simultaneous Imaging of pH and Peroxynitrite in the Endoplasmic Reticulum and Mitochondria: Revealing Organelle Interactions in Alzheimer's Disease Pathogenesis.
Cost-effective and simple flow cytometry quantification of receptor-mediated autophagy using fluorescent tagging.
PKM2 is a key factor to regulate neurogenesis and cognition by controlling lactate homeostasis.

Aging Cell. 2024 Dec 27. e14462

Effects of Aging on Glucose and Lipid Metabolism in Mice.

Evan C Lien, Ngoc Vu, Anna M Westermark, Laura V Danai, Allison N Lau, Yetiş Gültekin, Matthew A Kukurugya, Bryson D Bennett, Matthew G Vander Heiden.

  Aging is accompanied by multiple molecular changes that contribute to aging associated pathologies, such as accumulation of cellular damage and mitochondrial dysfunction. Tissue metabolism can also change with age, in part, because mitochondria are central to cellular metabolism. Moreover, the cofactor NAD+, which is reported to decline across multiple tissues during aging, plays a central role in metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the oxidative synthesis of nucleotides, amino acids, and lipids. To further characterize how tissue metabolism changes with age, we intravenously infused [U-13C]-glucose into young and old C57BL/6J, WSB/EiJ, and diversity outbred mice to trace glucose fate into downstream metabolites within plasma, liver, gastrocnemius muscle, and brain tissues. We found that glucose incorporation into central carbon and amino acid metabolism was robust during healthy aging across these different strains of mice. We also observed that levels of NAD+, NADH, and the NAD+/NADH ratio were unchanged in these tissues with healthy aging. However, aging tissues, particularly brain, exhibited evidence of upregulated fatty acid and sphingolipid metabolism reactions that regenerate NAD+ from NADH. These data suggest that NAD+-generating lipid metabolism reactions may help to maintain the NAD+/NADH ratio during healthy aging.

Keywords:  NAD; aging; metabolic rate; mice

DOI:  https://doi.org/10.1111/acel.14462
bioRxiv. 2024 Nov 22. pii: 2024.11.20.624567. [Epub ahead of print]

Metabolic Adaptations To Acute Glucose Uptake Inhibition Converge Upon Mitochondrial Respiration For Leukemia Cell Survival.

Monika Komza, Jesminara Khatun, Jesse D Gelles, Andrew P Trotta, Ioana Abraham-Enachescu, Juan Henao, Ahmed Elsaadi, Andriana G Kotini, Cara Clementelli, JoAnn Arandela, Sebastian El Ghaity-Beckley, Agneesh Barua, Yiyang Chen, Bridget K Marcellino, Eirini P Papapetrou, Masha V Poyurovsky, Jerry Edward Chipuk.

One hallmark of cancer is the upregulation and dependency on glucose metabolism to fuel macromolecule biosynthesis and rapid proliferation. Despite significant pre-clinical effort to exploit this pathway, additional mechanistic insights are necessary to prioritize the diversity of metabolic adaptations upon acute loss of glucose metabolism. Here, we investigated a potent small molecule inhibitor to Class I glucose transporters, KL-11743, using glycolytic leukemia cell lines and patient-based model systems. Our results reveal that while several metabolic adaptations occur in response to acute glucose uptake inhibition, the most critical is increased mitochondrial oxidative phosphorylation. KL-11743 treatment efficiently blocks the majority of glucose uptake and glycolysis, yet markedly increases mitochondrial respiration via enhanced Complex I function. Compared to partial glucose uptake inhibition, dependency on mitochondrial respiration is less apparent suggesting robust blockage of glucose uptake is essential to create a metabolic vulnerability. When wild-type and oncogenic RAS patient-derived induced pluripotent stem cell acute myeloid leukemia (AML) models were examined, KL-11743 mediated induction of mitochondrial respiration and dependency for survival associated with oncogenic RAS. Furthermore, we examined the therapeutic potential of these observations by treating a cohort of primary AML patient samples with KL-11743 and witnessed similar dependency on mitochondrial respiration for sustained cellular survival. Together, these data highlight conserved adaptations to acute glucose uptake inhibition in diverse leukemic models and AML patient samples, and position mitochondrial respiration as a key determinant of treatment success.

DOI: https://doi.org/10.1101/2024.11.20.624567
J Cell Biol. 2025 Mar 03. pii: e202403140. [Epub ahead of print]224(3):

Functionally conserved inner mitochondrial membrane proteins CCDC51 and Mdm33 demarcate a subset of fission events.

Alia R Edington, Olivia M Connor, Abigail C Love, Madeleine Marlar-Pavey, Jonathan R Friedman.

While extensive work has examined the mechanisms of mitochondrial fission, it remains unclear whether internal mitochondrial proteins in metazoans play a direct role in the process. Previously, the yeast inner membrane protein Mdm33 was shown to be required for normal mitochondrial morphology and has been hypothesized to be involved in mitochondrial fission. However, it is unknown whether Mdm33 plays a direct role, and it is not thought to have a mammalian homolog. Here, we use a bioinformatic approach to identify a structural ortholog of Mdm33 in humans, CCDC51 (also called MITOK), whose depletion phenocopies loss of Mdm33. We find that knockdown of CCDC51 also leads to reduced rates of mitochondrial fission. Further, we spatially and temporally resolve Mdm33 and CCDC51 to a subset of mitochondrial fission events. Finally, we show that CCDC51 overexpression promotes its spatial association with Drp1 and induces mitochondrial fragmentation, suggesting it is a positive effector of mitochondrial fission. Together, our data reveal that Mdm33 and CCDC51 are functionally conserved and suggest that internal mitochondrial proteins are directly involved in at least a subset of mitochondrial fission events in human cells.

DOI: https://doi.org/10.1083/jcb.202403140
Methods Mol Biol. 2025 ;2840 185-200

Light-Inducible Deformation of Mitochondria in Live Cells.

Yutong Song, Peiyuan Huang, Liting Duan.

  Mitochondria are dynamic organelles with constantly changing morphologies. Despite recent reports indicating that mechanical cues modulate mitochondrial morphologies and functions, there is a lack of methods that can exclusively and precisely exert mechanical forces to and deform mitochondria in live cells. Therefore, how mitochondria sense and respond to mechanical forces remains largely elusive. Optogenetic methods open up new venues for remote and precise manipulation of intracellular activities using light, providing an unprecedented opportunity to establish targeted mechano-stimulation toward mitochondria. This chapter describes the development of a novel optogenetic approach to optically mechanostimulate and induce the deformation of mitochondria. In this approach, light-gated protein-protein heterodimerization recruits force-generating molecular motors to the outer mitochondrial membrane, enabling direct exertion of mechanical force on mitochondria. Details for the design, application, and experimental procedures are laid out in this chapter. This method presents a mitochondria-specific mechano-stimulator for studying the correlation between mitochondrial morphology and functions as well as mitochondrial mechanobiology.

Keywords:  Mitochondria; Mitochondrial morphology; Molecular motor; Optogenetics; Organelle mechanobiology

DOI:  https://doi.org/10.1007/978-1-0716-4047-0_14
Nat Commun. 2024 Dec 23. 15(1): 10719

Preventing excessive autophagy protects from the pathology of mtDNA mutations in Drosophila melanogaster.

Najla El Fissi, Florian A Rosenberger, Kai Chang, Alissa Wilhalm, Tom Barton-Owen, Fynn M Hansen, Zoe Golder, David Alsina, Anna Wedell, Matthias Mann, Patrick F Chinnery, Christoph Freyer, Anna Wredenberg.

Aberration of mitochondrial function is a shared feature of many human pathologies, characterised by changes in metabolic flux, cellular energetics, morphology, composition, and dynamics of the mitochondrial network. While some of these changes serve as compensatory mechanisms to maintain cellular homeostasis, their chronic activation can permanently affect cellular metabolism and signalling, ultimately impairing cell function. Here, we use a Drosophila melanogaster model expressing a proofreading-deficient mtDNA polymerase (POLγexo-) in a genetic screen to find genes that mitigate the harmful accumulation of mtDNA mutations. We identify critical pathways associated with nutrient sensing, insulin signalling, mitochondrial protein import, and autophagy that can rescue the lethal phenotype of the POLγexo- flies. Rescued flies, hemizygous for dilp1, atg2, tim14 or melted, normalise their autophagic flux and proteasome function and adapt their metabolism. Mutation frequencies remain high with the exception of melted-rescued flies, suggesting that melted may act early in development. Treating POLγexo- larvae with the autophagy activator rapamycin aggravates their lethal phenotype, highlighting that excessive autophagy can significantly contribute to the pathophysiology of mitochondrial diseases. Moreover, we show that the nucleation process of autophagy is a critical target for intervention.

DOI: https://doi.org/10.1038/s41467-024-55559-2
Methods Mol Biol. 2025 ;2840 175-183

Alternating Cellular Functions by Optogenetic Control of Organelles.

Kangqiang Qiu, Xiuqiong Xu, Kai Zhang, Jiajie Diao.

  Organelles play essential roles in cellular homeostasis and various cellular functions in eukaryotic cells. The current experimental strategy to modulate organelle functions is limited due to the dynamic nature and subcellular distribution of organelles in live cells. Optogenetics utilizes photoactivatable proteins to enable dynamic control of molecular activities through visible light. This modality has been rapidly expanded for the dynamic regulation of organelle functions. This chapter describes a method by optical modulation of the mitochondria-lysosome contacts (MLCs). Detailed procedures of transfection, optogenetic MLCs, mitochondrial morphology, and functional analysis are described. Optogenetic control of organelles in live cells offers an innovative paradigm for cell engineering and synthetic biology.

Keywords:  Mitochondria-lysosome contacts; Mitochondrial fission; Optogenetics; Regulation of cellular functions; Subcellular manipulation

DOI:  https://doi.org/10.1007/978-1-0716-4047-0_13
bioRxiv. 2024 Dec 12. pii: 2024.12.06.627255. [Epub ahead of print]

Pathway Thermodynamic Analysis Postulates Change in Glutamate Metabolism as a Key Factor in Modulating Immune Responses.

Sunayana Malla, Rajib Saha.

Background: Temperature, as seen during fever, plays a pivotal role in modulating immune responses and maintaining cellular homeostasis. Shifts in temperature influence the thermodynamic feasibility of metabolic reactions, with Gibbs free energy (ΔG) serving as a key indicator of the spontaneity of reactions under specific conditions. By altering ΔG in response to temperature changes across various metabolite concentrations and cell types, we can gain insights into the thermodynamic properties of metabolic pathways and identify critical factors involved in metabolism and immune function. Using Max-Min Driving Force (MDF) analysis, we can assess changes in ΔG by varying temperature and metabolite concentrations, allowing for a detailed examination of thermodynamic feasibility at both the pathway and individual reaction levels.
Results: In this study, MDF analysis is applied to measure the changes in the driving force of pathways and the ΔG of each reaction at normal human core temperature (310.15 K) and elevated temperatures (up to 315.15 K). Additionally, we explore how shifts in the thermodynamic feasibility of reactions under immune activation, compared to normal physiological conditions, highlight key metabolic intermediates-such as fructose-1,6-bisphosphate, glucose-6-phosphate, and several steps in glutamate metabolism-as important regulators of metabolic processes and immune responses.
Conclusion: The goal of this study is to underscore the value of thermodynamic parameters such as ΔG, concentration, and temperature in identifying potential therapeutic targets, with the aim of mitigating the detrimental effects of fever while preserving its beneficial aspects.

DOI: https://doi.org/10.1101/2024.12.06.627255
PLoS One. 2024 ;19(12): e0314599

Lc-ms-based untargeted metabolomics reveals potential mechanisms of histologic chronic inflammation promoting prostate hyperplasia.

Jiale Li, Beiwen Wu, Guorui Fan, Jie Huang, Zhiguo Li, Fenghong Cao.

BACKGROUND: Chronic prostatitis may be a risk factor for developing proliferative changes in the prostate, although the underlying mechanisms are not entirely comprehended.
MATERIALS AND METHODS: Fifty individual prostate tissues were examined in this study, consisting of 25 patients diagnosed with prostatic hyperplasia combined with histologic chronic inflammation and 25 patients diagnosed with prostatic hyperplasia alone. We employed UPLC-Q-TOF-MS-based untargeted metabolomics using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to identify differential metabolites that can reveal the mechanisms that underlie the promotion of prostate hyperplasia by histologic chronic inflammation. Selected differential endogenous metabolites were analyzed using bioinformatics and subjected to metabolic pathway studies.
RESULTS: Nineteen differential metabolites, consisting of nine up-regulated and ten down-regulated, were identified between the two groups of patients. These groups included individuals with combined histologic chronic inflammation and those with prostatic hyperplasia alone. Glycerolipids, glycerophospholipids, and sphingolipids were primarily the components present. Metabolic pathway enrichment was conducted on the identified differentially expressed metabolites. Topological pathway analysis revealed the differential metabolites' predominant involvement in sphingolipid, ether lipid, and glycerophospholipid metabolism. The metabolites involved in sphingolipid metabolism were Sphingosine, Cer (d18:1/24:1), and Phytosphingosine. The metabolites involved in ether lipid metabolism were Glycerophosphocholine and LysoPC (O-18:0/0:0). The metabolites involved in glycerophospholipid metabolism were LysoPC (P-18:0/0:0) and Glycerophosphocholine. with Impact > 0. 1 and FDR < 0. 05, the most important metabolic pathway was sphingolipid metabolism.
CONCLUSIONS: In conclusion, our findings suggest that patients with prostate hyperplasia and combined histologic chronic inflammation possess distinctive metabolic profiles. These differential metabolites appear to play a significant role in the pathogenesis of histologic chronic inflammation-induced prostate hyperplasia, primarily through the regulation of sphingolipids and glycerophospholipids metabolic pathways. The mechanism by which histologic chronic inflammation promotes prostate hyperplasia was elucidated through the analysis of small molecule metabolites. These findings support the notion that chronic prostatitis may contribute to an increased risk of prostate hyperplasia.

DOI: https://doi.org/10.1371/journal.pone.0314599
Elife. 2024 Dec 27. pii: RP92741. [Epub ahead of print]13

TIPE drives a cancer stem-like phenotype by promoting glycolysis via PKM2/HIF-1α axis in melanoma.

Maojin Tian, Le Yang, Ziqian Zhao, Jigang Li, Lianqing Wang, Qingqing Yin, Wei Hu, Yunwei Lou, Jianxin Du, Peiqing Zhao.

  TIPE (TNFAIP8) has been identified as an oncogene and participates in tumor biology. However, how its role in the metabolism of tumor cells during melanoma development remains unclear. Here, we demonstrated that TIPE promoted glycolysis by interacting with pyruvate kinase M2 (PKM2) in melanoma. We found that TIPE-induced PKM2 dimerization, thereby facilitating its translocation from the cytoplasm to the nucleus. TIPE-mediated PKM2 dimerization consequently promoted HIF-1α activation and glycolysis, which contributed to melanoma progression and increased its stemness features. Notably, TIPE specifically phosphorylated PKM2 at Ser 37 in an extracellular signal-regulated kinase (ERK)-dependent manner. Consistently, the expression of TIPE was positively correlated with the levels of PKM2 Ser37 phosphorylation and cancer stem cell (CSC) markers in melanoma tissues from clinical samples and tumor bearing mice. In summary, our findings indicate that the TIPE/PKM2/HIF-1α signaling pathway plays a pivotal role in promoting CSC properties by facilitating the glycolysis, which would provide a promising therapeutic target for melanoma intervention.

Keywords:  HIF-1α; PKM2; TIPE; cancer biology; cell biology; glycolysis; human; melanoma; mouse

DOI:  https://doi.org/10.7554/eLife.92741
Biomaterials. 2024 Dec 15. pii: S0142-9612(24)00559-3. [Epub ahead of print]316 123023

Mitochondria-targeting materials and therapies for regenerative engineering.

Hongying Fu, Jingrong Cheng, Le Hu, Boon Chin Heng, Xuehui Zhang, Xuliang Deng, Yang Liu.

  The hemostatic, inflammatory, proliferative, and remodeling phases of healing require precise spatiotemporal coordination and orchestration of numerous biological processes. As the primary energy generators in the cell, mitochondria play multifunctional roles in regulating metabolism, stress reactions, immunity, and cell density during the process of tissue regeneration. Mitochondrial dynamics involves numerous crucial processes, fusion, fission, autophagy, and translocation, which are all necessary for preserving mitochondrial function, distributing energy throughout cells, and facilitating cellular signaling. Tissue regeneration is specifically associated with mitochondrial dynamics due to perturbations of Ca2+, H2O2 and ROS levels, which can result in mitochondrial malfunction. Increasing evidence from multiple models suggests that clinical interventions or medicinal drugs targeting mitochondrial dynamics could be a promising approach. This review highlights significant advances in the understanding of mitochondrial dynamics in tissue regeneration, with specific attention on mitochondria-targeting biomaterials that accelerate multiple tissues' regeneration by regulating mitochondrial metabolism. The innovations in nanomaterials and nanosystems enhance mitochondrial-targeting therapies are critically examined with the prospects of modulating mitochondrial dynamics for new therapies in regenerative engineering.

Keywords:  Mitochondria-targeting materials; Mitochondrial dynamics; Mitochondrial transfer; Regenerative engineering

DOI:  https://doi.org/10.1016/j.biomaterials.2024.123023
Commun Biol. 2024 Dec 27. 7(1): 1704

Genome-scale modeling identifies dynamic metabolic vulnerabilities during the epithelial to mesenchymal transition.

Rupa Bhowmick, Scott Campit, Shiva Krishna Katkam, Venkateshwar G Keshamouni, Sriram Chandrasekaran.

Epithelial-to-mesenchymal transition (EMT) is a conserved cellular process critical for embryogenesis, wound healing, and cancer metastasis. During EMT, cells undergo large-scale metabolic reprogramming that supports multiple functional phenotypes including migration, invasion, survival, chemo-resistance and stemness. However, the extent of metabolic network rewiring during EMT is unclear. In this work, using genome-scale metabolic modeling, we perform a meta-analysis of time-course transcriptomics, time-course proteomics, and single-cell transcriptomics EMT datasets from cell culture models stimulated with TGF-β. We uncovered temporal metabolic dependencies in glycolysis and glutamine metabolism, and experimentally validated isoform-specific dependency on Enolase3 for cell survival during EMT. We derived a prioritized list of metabolic dependencies based on model predictions, literature mining, and CRISPR-Cas9 essentiality screens. Notably, enolase and triose phosphate isomerase reaction fluxes significantly correlate with survival of lung adenocarcinoma patients. Our study illustrates how integration of heterogeneous datasets using a mechanistic computational model can uncover temporal and cell-state-specific metabolic dependencies.

DOI: https://doi.org/10.1038/s42003-024-07408-7
Nanomaterials (Basel). 2024 Dec 15. pii: 2017. [Epub ahead of print]14(24):

Pleozymes: Pleiotropic Oxidized Carbon Nanozymes Enhance Cellular Metabolic Flexibility.

Anh T T Vo, Karthik Mouli, Anton V Liopo, Philip Lorenzi, Lin Tan, Bo Wei, Sara A Martinez, Emily A McHugh, James M Tour, Uffaf Khan, Paul J Derry, Thomas A Kent.

  Our group has synthesized a pleiotropic synthetic nanozyme redox mediator we term a "pleozyme" that displays multiple enzymatic characteristics, including acting as a superoxide dismutase mimetic, oxidizing NADH to NAD+, and oxidizing H2S to polysulfides and thiosulfate. Benefits have been seen in acute and chronic neurological disease models. The molecule is sourced from coconut-derived activated charcoal that has undergone harsh oxidization with fuming nitric acid, which alters the structure and chemical characteristics, yielding 3-8 nm discs with broad redox potential. Prior work showed pleozymes localize to mitochondria and increase oxidative phosphorylation and glycolysis. Here, we measured cellular NAD+ and NADH levels after pleozyme treatment and observed increased total cellular NADH levels but not total NAD+ levels. A 13C-glucose metabolic flux analysis suggested pleozymes stimulate the generation of pyruvate and lactate glycolytically and from the tricarboxylic acid (TCA) cycle, pointing to malate decarboxylation. Analysis of intracellular fatty acid abundances suggests pleozymes increased fatty acid β-oxidation, with a concomitant increase in succinyl- and acetyl-CoA. Pleozymes increased total ATP, potentially via flexible enhancement of NAD+-dependent catabolic pathways such as glycolysis, fatty acid β-oxidation, and metabolic flux through the TCA cycle. These effects may be favorable for pathologies that compromise metabolism such as brain injury.

Keywords:  NADH; fatty acid oxidation; mitochondrial metabolism; nanozyme; oxidized activated carbon nanoparticles; redox mediator

DOI:  https://doi.org/10.3390/nano14242017
Spectrochim Acta A Mol Biomol Spectrosc. 2024 Dec 18. pii: S1386-1425(24)01802-X. [Epub ahead of print]329 125636

A novel mitochondrial-targeted fluorescent probe for ratiometric imaging of nitric oxide in cells and zebrafish.

Bin Lin, Jiaxin Fan, Shuting Li, Yifeng Han.

  Nitric oxide (NO) is a key signaling molecule that regulates energy metabolism, apoptosis, and antioxidant balance within mitochondria. It is closely associated with the development of cardiovascular diseases, neurodegenerative diseases, and cancer. Therefore, developing fluorescent probes capable of accurately detecting NO levels in mitochondria is essential for understanding disease mechanisms and clinical diagnostics. In this study, we developed a novel fluorescent probe based on the isophorone fluorophore. This probe achieves high sensitivity and specific ratiometric detection of NO in mitochondria by regulating the intramolecular charge transfer (ICT) effect. The probe emits red fluorescence before reacting with NO, and the addition of NO triggers an amine-NO addition reaction that inhibits the ICT effect, resulting in a color change to yellow-green fluorescence. This ratiometric fluorescence response provides a new method for quantitatively detecting NO. Additionally, the probe has a significant Stokes shift and good ratiometric wavelength separation, enhancing detection accuracy. It localizes explicitly to mitochondria, directly reflecting changes in mitochondrial NO concentration. Experiments in HeLa cells and zebrafish models have demonstrated the potential application of the probe in diagnosing and studying NO-related diseases. This provides new strategies and tools for researching the biological functions of NO and the early diagnosis of related diseases.

Keywords:  Fluorescent probe; Living cell imaging; Mitochondrial-targeted; Nitric oxide; Zebrafish imaging

DOI:  https://doi.org/10.1016/j.saa.2024.125636
Anal Chem. 2024 Dec 26.

Simultaneous Imaging of pH and Peroxynitrite in the Endoplasmic Reticulum and Mitochondria: Revealing Organelle Interactions in Alzheimer's Disease Pathogenesis.

Lushan Huang, Liyi Ma, Qiaowen Zhao, Qichen Zhu, Guangwei She, Lixuan Mu, Wensheng Shi.

pH and peroxynitrite (ONOO-) are two critical biomarkers to unveil the corresponding status of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are closely related to Alzheimer's disease (AD). Simultaneously monitoring pH and ONOO- fluctuations in the ER and mitochondria during AD progression is pivotal for clarifying the interplay between the disorders of the two organelles and revealing AD pathogenesis. Herein, we designed and synthesized a dual-channel fluorescent probe (DCFP) to visualize pH and ONOO- in the ER and mitochondria. DCFP possessed excellent sensitivity and selectivity to pH and ONOO- without spectral crosstalk and was utilized in monitoring the two analytes within AD model cells and larval zebrafish. Importantly, DCFP could preferentially target mitochondria in normal cells and be enriched in the ER after mitochondrial depolarization. With the aid of DCFP, the slower acidification rate of the ER than that of mitochondria induced by Aβ oligomers (AβOs) was first identified, which could be ascribed to the relief of the AβOs-triggered ER stress through the Ca2+ migration from the ER to mitochondria. Moreover, continuous exposure to AβOs led to mitochondrial Ca2+ overload, accelerating the acidification and ONOO- overproduction within mitochondria. As a result, intracellular oxidative stress levels were elevated, further exacerbating ER stress and aggravating ER acidification in turn. The advanced understanding of the potential interplay between the ER and mitochondria in this work may offer new insights and methodologies for studying AD pathogenesis. The DCFP developed in this work could also be employed to study other diseases related to ER stress and mitochondrial dysfunction.

DOI: https://doi.org/10.1021/acs.analchem.4c03646
FEBS Open Bio. 2024 Dec 23.

Cost-effective and simple flow cytometry quantification of receptor-mediated autophagy using fluorescent tagging.

Mija Marinković, Ana Rožić, Denis Polančec, Ivana Novak.

  Mitophagy, a selective clearance of damaged or superfluous mitochondria via autophagy machinery and lysosomal degradation, is an evolutionarily conserved process essential for various physiological functions, including cellular differentiation and immune responses. Defects in mitophagy are implicated in numerous human diseases, such as neurodegenerative disorders, cancer, and metabolic conditions. Despite significant advancements in mitophagy research over recent decades, novel and robust methodologies are necessary to elucidate its molecular mechanisms comprehensively. In this study, we present a detailed protocol for quantitatively assessing mitophagy through flow cytometry using a mitochondria-targeted fluorescent mitophagy receptor, GFP-BNIP3L/NIX. This method offers a rapid alternative to conventional microscopy or immunoblotting techniques for analyzing mitophagy activity. Additionally, this approach can theoretically be adapted to utilize any fluorescent-tagged selective autophagy receptor, enabling the direct and rapid analysis of various types of receptor-mediated selective autophagy.

Keywords:  BNIP3L/NIX; flow cytometry; fluorescent tagging; receptor‐mediated mitophagy

DOI:  https://doi.org/10.1002/2211-5463.13958
Stem Cell Reports. 2024 Dec 12. pii: S2213-6711(24)00325-4. [Epub ahead of print] 102381

PKM2 is a key factor to regulate neurogenesis and cognition by controlling lactate homeostasis.

Pengyan He, Bingjun Zhang, Wei Jiang, Fan Zhu, Ziqi Liang, Lin Gao, Yuhong Zhang, Yuge Wang, Caixia Wu, Changyong Tang.

  Adult hippocampal neurogenesis (AHN), the process of generating new neurons from adult neural stem/progenitor cells (NSPCs), is crucial for cognitive functions and is influenced by numerous factors, including metabolic processes. Pyruvate kinase M2 (PKM2), a key rate-limiting enzyme in glycolysis, catalyzes the production of pyruvate, which undergoes either oxidative phosphorylation or anaerobic oxidation. We observed that PKM2 is highly expressed in NSPCs, but its significance remains unclear for AHN and cognition. Using knockdown or knockout strategies, we discovered that PKM2 deficiency led to reduced AHN and impaired cognitive functions. Furthermore, we observed that knockout of PKM2 resulted in lower L-lactate levels, and supplementing L-lactate in PKM2 knockout mice improved AHN and cognitive functions. Mechanistically, L-lactate restored neurogenesis via monocarboxylate transporter 2 (MCT2), but not hydroxycarboxylic acid receptor 1. In summary, our findings demonstrate that PKM2 is essential for AHN, and lactate supplementation can restore neurogenesis in an MCT2-dependent manner.

Keywords:  PKM2; cognitive function; lactate homeostasis; neural stem cells

DOI:  https://doi.org/10.1016/j.stemcr.2024.11.011