Publications by authors named "Andreas S Reichert"

92 Publications

Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet-Mediated Amyloid Aggregation in Alzheimer's Disease.

Int J Mol Sci 2021 Sep 6;22(17). Epub 2021 Sep 6.

Department of Vascular and Endovascular Surgery, Experimental Vascular Medicine, Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany.

Background: Alzheimer's disease (AD) is characterized by an accumulation of amyloid β (Aβ) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aβ to form Aβ aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein.

Methods: The effects of Aβ-mediated mitochondrial dysfunction in platelets were investigated in vitro.

Results: Aβ40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aβ40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αIIβ activation during synergistic stimulation from ADP and Aβ40. Aβ40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C.

Conclusions: Mitochondrial dysfunction contributes to platelet-mediated Aβ aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.
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http://dx.doi.org/10.3390/ijms22179633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431787PMC
September 2021

Linking transport and translation of mRNAs with endosomes and mitochondria.

EMBO Rep 2021 10 17;22(10):e52445. Epub 2021 Aug 17.

Institute of Microbiology, Cluster of Excellence on Plant Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

In eukaryotic cells, proteins are targeted to their final subcellular locations with precise timing. A key underlying mechanism is the active transport of cognate mRNAs, which in many systems can be linked intimately to membrane trafficking. A prominent example is the long-distance endosomal transport of mRNAs and their local translation. Here, we describe current highlights of fundamental mechanisms of the underlying transport process as well as of biological functions ranging from endosperm development in plants to fungal pathogenicity and neuronal processes. Translation of endosome-associated mRNAs often occurs at the cytoplasmic surface of endosomes, a process that is needed for membrane-assisted formation of heteromeric protein complexes and for accurate subcellular targeting of proteins. Importantly, endosome-coupled translation of mRNAs encoding mitochondrial proteins, for example, seems to be particularly important for efficient organelle import and for regulating subcellular mitochondrial activity. In essence, these findings reveal a new mechanism of loading newly synthesised proteins onto endocytic membranes enabling intimate crosstalk between organelles. The novel link between endosomes and mitochondria adds an inspiring new level of complexity to trafficking and organelle biology.
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http://dx.doi.org/10.15252/embr.202152445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490996PMC
October 2021

Rapid metabolic and bioenergetic adaptations of astrocytes under hyperammonemia - a novel perspective on hepatic encephalopathy.

Biol Chem 2021 Aug 30;402(9):1103-1113. Epub 2021 Jul 30.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Universitätsstraße 1, D-40225 Düsseldorf, Germany.

Hepatic encephalopathy (HE) is a well-studied, neurological syndrome caused by liver dysfunctions. Ammonia, the major toxin during HE pathogenesis, impairs many cellular processes within astrocytes. Yet, the molecular mechanisms causing HE are not fully understood. Here we will recapitulate possible underlying mechanisms with a clear focus on studies revealing a link between altered energy metabolism and HE in cellular models and . The role of the mitochondrial glutamate dehydrogenase and its role in metabolic rewiring of the TCA cycle will be discussed. We propose an updated model of ammonia-induced toxicity that may also be exploited for therapeutic strategies in the future.
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http://dx.doi.org/10.1515/hsz-2021-0172DOI Listing
August 2021

Conserved GxxxG and WN motifs of MIC13 are essential for bridging two MICOS subcomplexes.

Biochim Biophys Acta Biomembr 2021 Dec 13;1863(12):183683. Epub 2021 Jul 13.

Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Düsseldorf, Medical Faculty and University Hospital Düsseldorf, Düsseldorf, Germany. Electronic address:

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.
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http://dx.doi.org/10.1016/j.bbamem.2021.183683DOI Listing
December 2021

Emerging Roles of the MICOS Complex in Cristae Dynamics and Biogenesis.

Biology (Basel) 2021 Jun 29;10(7). Epub 2021 Jun 29.

Institute of Biochemistry and Molecular Biology I, Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany.

Mitochondria are double membrane-enclosed organelles performing important cellular and metabolic functions such as ATP generation, heme biogenesis, apoptosis, ROS production and calcium buffering. The mitochondrial inner membrane (IM) is folded into cristae membranes (CMs) of variable shapes using molecular players including the 'mitochondrial contact site and cristae organizing system' (MICOS) complex, the dynamin-like GTPase OPA1, the FF ATP synthase and cardiolipin. Aberrant cristae structures are associated with different disorders such as diabetes, neurodegeneration, cancer and hepato-encephalopathy. In this review, we provide an updated view on cristae biogenesis by focusing on novel roles of the MICOS complex in cristae dynamics and shaping of cristae. For over seven decades, cristae were considered as static structures. It was recently shown that cristae constantly undergo rapid dynamic remodeling events. Several studies have re-oriented our perception on the dynamic internal ambience of mitochondrial compartments. In addition, we discuss the recent literature which sheds light on the still poorly understood aspect of cristae biogenesis, focusing on the role of MICOS and its subunits. Overall, we provide an integrated and updated view on the relation between the biogenesis of cristae and the novel aspect of cristae dynamics.
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http://dx.doi.org/10.3390/biology10070600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301002PMC
June 2021

High-throughput screening for natural compound-based autophagy modulators reveals novel chemotherapeutic mode of action for arzanol.

Cell Death Dis 2021 05 31;12(6):560. Epub 2021 May 31.

Institute of Molecular Medicine I, Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.

Autophagy is an intracellular recycling pathway with implications for intracellular homeostasis and cell survival. Its pharmacological modulation can aid chemotherapy by sensitizing cancer cells toward approved drugs and overcoming chemoresistance. Recent translational data on autophagy modulators show promising results in reducing tumor growth and metastasis, but also reveal a need for more specific compounds and novel lead structures. Here, we searched for such autophagy-modulating compounds in a flow cytometry-based high-throughput screening of an in-house natural compound library. We successfully identified novel inducers and inhibitors of the autophagic pathway. Among these, we identified arzanol as an autophagy-modulating drug that causes the accumulation of ATG16L1-positive structures, while it also induces the accumulation of lipidated LC3. Surprisingly, we observed a reduction of the size of autophagosomes compared to the bafilomycin control and a pronounced accumulation of p62/SQSTM1 in response to arzanol treatment in HeLa cells. We, therefore, speculate that arzanol acts both as an inducer of early autophagosome biogenesis and as an inhibitor of later autophagy events. We further show that arzanol is able to sensitize RT-112 bladder cancer cells towards cisplatin (CDDP). Its anticancer activity was confirmed in monotherapy against both CDDP-sensitive and -resistant bladder cancer cells. We classified arzanol as a novel mitotoxin that induces the fragmentation of mitochondria, and we identified a series of targets for arzanol that involve proteins of the class of mitochondria-associated quinone-binding oxidoreductases. Collectively, our results suggest arzanol as a valuable tool for autophagy research and as a lead compound for drug development in cancer therapy.
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http://dx.doi.org/10.1038/s41419-021-03830-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167120PMC
May 2021

Common Principles and Specific Mechanisms of Mitophagy from Yeast to Humans.

Int J Mol Sci 2021 Apr 22;22(9). Epub 2021 Apr 22.

Institute of Biochemistry and Molecular Biology I, Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.

Mitochondria are double membrane-bound organelles in eukaryotic cells essential to a variety of cellular functions including energy conversion and ATP production, iron-sulfur biogenesis, lipid and amino acid metabolism, and regulating apoptosis and stress responses. Mitochondrial dysfunction is mechanistically linked to several neurodegenerative diseases, cancer, and ageing. Excessive and dysfunctional/damaged mitochondria are degraded by selective autophagic pathways known as mitophagy. Both budding yeast and mammals use the well-conserved machinery of core autophagy-related genes () to execute and regulate mitophagy. In mammalian cells, the PINK1-PARKIN mitophagy pathway is a well-studied pathway that senses dysfunctional mitochondria and marks them for degradation in the lysosome. PINK1-PARKIN mediated mitophagy relies on ubiquitin-binding mitophagy adaptors that are non-ATG proteins. Loss-of-function mutations in and are linked to Parkinson´s disease (PD) in humans, and defective mitophagy is proposed to be a main pathomechanism. Despite the common view that yeast cells lack PINK1- and PARKIN-homologs and that mitophagy in yeast is solely regulated by receptor-mediated mitophagy, some studies suggest that a ubiquitination-dependent mitophagy pathway also exists. Here, we will discuss shared mechanisms between mammals and yeast, how mitophagy in the latter is regulated in a ubiquitin-dependent and -independent manner, and why these pathways are essential for yeast cell survival and fitness under various physiological stress conditions.
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http://dx.doi.org/10.3390/ijms22094363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122514PMC
April 2021

Autophagy promotes mitochondrial respiration by providing serine for one-carbon-metabolism.

Autophagy 2021 Apr 18:1-4. Epub 2021 Apr 18.

Institute of Biochemistry and Molecular Biology I, Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.

Whether macroautophagy/autophagy is physiologically relevant to regulate mitochondrial function for a rapid and dynamic adaptation of yeast cells to respiratory growth was not fully understood until recently. May . (2020. ) report that bulk autophagy provides serine as a one-carbon (1C) metabolite that controls respiratory growth onset by initiating mitochondrial initiator tRNA modification and mitochondrial translation linking autophagy mechanistically to mitochondrial function. We discuss the mechanistic interplay between autophagy, one-carbon-metabolism, and mitochondrial function and the possible implications in neurodegeneration, aging, and carcinogenesis.
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http://dx.doi.org/10.1080/15548627.2021.1909408DOI Listing
April 2021

Protease OMA1 modulates mitochondrial bioenergetics and ultrastructure through dynamic association with MICOS complex.

iScience 2021 Feb 29;24(2):102119. Epub 2021 Jan 29.

Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA.

Remodeling of mitochondrial ultrastructure is a process that is critical for organelle physiology and apoptosis. Although the key players in this process-mitochondrial contact site and cristae junction organizing system (MICOS) and Optic Atrophy 1 (OPA1)-have been characterized, the mechanisms behind its regulation remain incompletely defined. Here, we found that in addition to its role in mitochondrial division, metallopeptidase OMA1 is required for the maintenance of intermembrane connectivity through dynamic association with MICOS. This association is independent of OPA1, mediated via the MICOS subunit MIC60, and is important for stability of MICOS and the intermembrane contacts. The OMA1-MICOS relay is required for optimal bioenergetic output and apoptosis. Loss of OMA1 affects these activities; remarkably it can be alleviated by MICOS-emulating intermembrane bridge. Thus, OMA1-dependent ultrastructure support is required for mitochondrial architecture and bioenergetics under basal and stress conditions, suggesting a previously unrecognized role for OMA1 in mitochondrial physiology.
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http://dx.doi.org/10.1016/j.isci.2021.102119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892988PMC
February 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

The relevance of mitochondrial morphology for human disease.

Int J Biochem Cell Biol 2021 05 18;134:105951. Epub 2021 Feb 18.

Department of General Pediatrics, Neonatology and Pediatric Cardiology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany. Electronic address:

Mitochondria are highly dynamic organelles, which undergo frequent structural and metabolic changes to fulfil cellular demands. To facilitate these processes several proteins are required to regulate mitochondrial shape and interorganellar communication. These proteins include the classical mitochondrial fusion (MFN1, MFN2, and OPA1) and fission proteins (DRP1, MFF, FIS1, etc.) as well as several other proteins that are directly or indirectly involved in these processes (e.g. YME1L, OMA1, INF2, GDAP1, MIC13, etc.). During the last two decades, inherited genetic defects in mitochondrial fusion and fission proteins have emerged as an important class of neurodegenerative human diseases with variable onset ranging from infancy to adulthood. So far, no causal treatment strategies are available for these disorders. In this review, we provide an overview about the current knowledge on mitochondrial dynamics under physiological conditions. Moreover, we describe human diseases, which are associated with genetic defects in these pathways.
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http://dx.doi.org/10.1016/j.biocel.2021.105951DOI Listing
May 2021

Cristae Membrane Dynamics - A Paradigm Change.

Trends Cell Biol 2020 12 23;30(12):923-936. Epub 2020 Sep 23.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany. Electronic address:

Mitochondria are dynamic organelles that have essential metabolic and regulatory functions. Earlier studies using electron microscopy (EM) revealed an immense diversity in the architecture of cristae - infoldings of the mitochondrial inner membrane (IM) - in different cells, tissues, bioenergetic and metabolic conditions, and during apoptosis. However, cristae were considered to be largely static entities. Recently, advanced super-resolution techniques have revealed that cristae are independent bioenergetic units that are highly dynamic and remodel on a timescale of seconds. These advances, coupled with mechanistic and structural studies on key molecular players, such as the MICOS (mitochondrial contact site and cristae organizing system) complex and the dynamin-like GTPase OPA1, have changed our view on mitochondria in a fundamental way. We summarize these recent findings and discuss their functional implications.
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http://dx.doi.org/10.1016/j.tcb.2020.08.008DOI Listing
December 2020

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner.

Dis Model Mech 2020 11 4;13(10). Epub 2020 Nov 4.

Institute for Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany

Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of N-labeled ammonia showed rapid incorporation of N into glutamate and glutamate-derived amino acids. Downregulating human [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.
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http://dx.doi.org/10.1242/dmm.047134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657470PMC
November 2020

Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein.

Cells 2020 08 24;9(9). Epub 2020 Aug 24.

Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.
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http://dx.doi.org/10.3390/cells9091950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564595PMC
August 2020

MIC26 and MIC27 cooperate to regulate cardiolipin levels and the landscape of OXPHOS complexes.

Life Sci Alliance 2020 10 11;3(10). Epub 2020 Aug 11.

Institute of Biochemistry and Molecular Biology I, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany

Homologous apolipoproteins of MICOS complex, MIC26 and MIC27, show an antagonistic regulation of their protein levels, making it difficult to deduce their individual functions using a single gene deletion. We obtained single and double knockout (DKO) human cells of and and found that DKO show more concentric onion-like cristae with loss of CJs than any single deletion indicating overlapping roles in formation of CJs. Using a combination of complexome profiling, STED nanoscopy, and blue-native gel electrophoresis, we found that MIC26 and MIC27 are dispensable for the stability and integration of the remaining MICOS subunits into the complex suggesting that they assemble late into the MICOS complex. MIC26 and MIC27 are cooperatively required for the integrity of respiratory chain (super) complexes (RCs/SC) and the FF-ATP synthase complex and integration of F subunits into the monomeric FF-ATP synthase. While cardiolipin was reduced in DKO cells, overexpression of cardiolipin synthase in DKO restores the stability of RCs/SC. Overall, we propose that MIC26 and MIC27 are cooperatively required for global integrity and stability of multimeric OXPHOS complexes by modulating cardiolipin levels.
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http://dx.doi.org/10.26508/lsa.202000711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425215PMC
October 2020

Endogenous Carbon Monoxide Signaling Modulates Mitochondrial Function and Intracellular Glucose Utilization: Impact of the Heme Oxygenase Substrate Hemin.

Antioxidants (Basel) 2020 Jul 23;9(8). Epub 2020 Jul 23.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, D-40001 Düsseldorf, Germany.

Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.
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http://dx.doi.org/10.3390/antiox9080652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465082PMC
July 2020

Distinct influence of the anthracycline derivative doxorubicin on the differentiation efficacy of mESC-derived endothelial progenitor cells.

Biochim Biophys Acta Mol Cell Res 2020 07 26;1867(7):118711. Epub 2020 Mar 26.

Institute of Toxicology, Medical Faculty, Heinrich-Heine-University Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany. Electronic address:

Cardiotoxicity is a highly relevant, because often life-threatening, adverse effect of doxorubicin (Doxo)-based anticancer therapy. Here, we investigated the Doxo-response of cardiovascular stem/progenitor cells employing a mouse embryonic stem cell (mESC)-based in vitro differentiation model. Endothelial progenitor cells revealed a pronounced Doxo sensitivity as compared to mESC, differentiated endothelial-like (EC) and cardiomyocyte-like cells (CM) and CM progenitors, which rests on the activation of senescence. Doxo treatment of EC progenitors altered protein expression of individual endothelial markers, actin cytoskeleton morphology, mRNA expression of genes related to mitochondrial functions, autophagy, apoptosis, and DNA repair as well as mitochondrial DNA content, respiration and ATP production in the surviving differentiated EC progeny. By contrast, LDL uptake, ATP-stimulated Ca release, and cytokine-stimulated ICAM-1 expression remained unaffected by the anthracycline treatment. Thus, exposure of EC progenitors to Doxo elicits isolated and persistent dysfunctions in the surviving EC progeny. In conclusion, we suggest that Doxo-induced injury of EC progenitors adds to anthracycline-induced cardiotoxicity, making this cell-type a preferential target for pharmacoprotective and regenerative strategies.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118711DOI Listing
July 2020

Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner.

EMBO Rep 2020 03 18;21(3):e49776. Epub 2020 Feb 18.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

The mitochondrial inner membrane can reshape under different physiological conditions. How, at which frequency this occurs in living cells, and the molecular players involved are unknown. Here, we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that neighbouring crista junctions (CJs) dynamically appose and separate from each other in a reversible and balanced manner in human cells. Staining of cristae membranes (CM), using various protein markers or two lipophilic inner membrane-specific dyes, further revealed that cristae undergo continuous cycles of membrane remodelling. These events are accompanied by fluctuations of the membrane potential within distinct cristae over time. Both CJ and CM dynamics depended on MIC13 and occurred at similar timescales in the range of seconds. Our data further suggest that MIC60 acts as a docking platform promoting CJ and contact site formation. Overall, by employing advanced imaging techniques including fluorescence recovery after photobleaching (FRAP), single-particle tracking (SPT), live-cell STED and high-resolution Airyscan microscopy, we propose a model of CJ dynamics being mechanistically linked to CM remodelling representing cristae membrane fission and fusion events occurring within individual mitochondria.
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http://dx.doi.org/10.15252/embr.201949776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054676PMC
March 2020

CNP mediated selective toxicity on melanoma cells is accompanied by mitochondrial dysfunction.

PLoS One 2020 17;15(1):e0227926. Epub 2020 Jan 17.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells. Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties. The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage. Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line. Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor. We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes. Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation. Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death. CNP-induced cell death is abolished by administration of PEG-conjugated catalase. Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227926PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968876PMC
April 2020

Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria.

Plant Physiol 2020 02 9;182(2):692-706. Epub 2019 Dec 9.

Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Heinrich Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis () with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis mutant deficient in the mitochondrial Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.
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http://dx.doi.org/10.1104/pp.19.00732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997695PMC
February 2020

Hepatic encephalopathy is linked to alterations of autophagic flux in astrocytes.

EBioMedicine 2019 Oct 21;48:539-553. Epub 2019 Oct 21.

Institute of Biochemistry and Molecular Biology I, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany. Electronic address:

Background: Hepatic encephalopathy (HE) is a severe neuropsychiatric syndrome caused by various types of liver failure resulting in hyperammonemia-induced dysfunction of astrocytes. It is unclear whether autophagy, an important pro-survival pathway, is altered in the brains of ammonia-intoxicated animals as well as in HE patients.

Methods: Using primary rat astrocytes, a co-culture model of primary mouse astrocytes and neurons, an in vivo rat HE model, and post mortem brain samples of liver cirrhosis patients with HE we analyzed whether and how hyperammonemia modulates autophagy.

Findings: We show that autophagic flux is efficiently inhibited after administration of ammonia in astrocytes. This occurs in a fast, reversible, time-, dose-, and ROS-dependent manner and is mediated by ammonia-induced changes in intralysosomal pH. Autophagic flux is also strongly inhibited in the cerebral cortex of rats after acute ammonium intoxication corroborating our results using an in vivo rat HE model. Transglutaminase 2 (TGM2), a factor promoting autophagy, is upregulated in astrocytes of in vitro- and in vivo-HE models as well as in post mortem brain samples of liver cirrhosis patients with HE, but not in patients without HE. LC3, a commonly used autophagy marker, is significantly increased in the brain of HE patients. Ammonia also modulated autophagy moderately in neuronal cells. We show that taurine, known to ameliorate several parameters caused by hyperammonemia in patients suffering from liver failure, is highly potent in reducing ammonia-induced impairment of autophagic flux. This protective effect of taurine is apparently not linked to inhibition of mTOR signaling but rather to reducing ammonia-induced ROS formation.

Interpretation: Our data support a model in which autophagy aims to counteract ammonia-induced toxicity, yet, as acidification of lysosomes is impaired, possible protective effects thereof, are hampered. We propose that modulating autophagy in astrocytes and/or neurons, e.g. by taurine, represents a novel strategy to treat liver diseases associated with HE.

Funding: Supported by the DFG, CRC974 "Communication and Systems Relevance in Liver Injury and Regeneration", Düsseldorf (Project number 190586431) Projects A05 (DH), B04 (BG), B05 (NK), and B09 (ASR).
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http://dx.doi.org/10.1016/j.ebiom.2019.09.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838440PMC
October 2019

Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent.

EMBO J 2019 11 14;38(22):e101056. Epub 2019 Oct 14.

Department of Medicine (Endocrinology), Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.

The mitochondrial membrane potential (ΔΨ ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨ . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨ at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨ , corresponding to cristae and IBM. ΔΨ was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨ heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨ . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.
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http://dx.doi.org/10.15252/embj.2018101056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856616PMC
November 2019

In vitro selective cytotoxicity of the dietary chalcone cardamonin (CD) on melanoma compared to healthy cells is mediated by apoptosis.

PLoS One 2019 25;14(9):e0222267. Epub 2019 Sep 25.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.

Malignant melanoma is an aggressive type of cancer and the deadliest form of skin cancer. Even though enormous efforts have been undertaken, in particular the treatment options against the metastasizing form are challenging and the prognosis is generally poor. A novel therapeutical approach is the application of secondary plant constituents occurring in food and food products. Herein, the effect of the dietary chalcone cardamonin, inter alia found in Alpinia species, was tested using human malignant melanoma cells. These data were compared to cardamonin treated normal melanocytes and dermal fibroblasts representing healthy cells. To investigate the impact of cardamonin on tumor and normal cells, it was added to monolayer cell cultures and cytotoxicity, proliferation, tumor invasion, and apoptosis were studied with appropriate cell biological and biochemical methods. Cardamonin treatment resulted in an apoptosis-mediated increase in cytotoxicity towards tumor cells, a decrease in their proliferation rate, and a lowered invasive capacity, whereas the viability of melanocytes and fibroblasts was hardly affected at such concentrations. A selective cytotoxic effect of cardamonin on melanoma cells compared to normal (healthy) cells was shown in vitro. This study along with others highlights that dietary chalcones may be a valuable tool in anticancer therapies which has to be proven in the future in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222267PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760786PMC
March 2020

Functional Interplay between Cristae Biogenesis, Mitochondrial Dynamics and Mitochondrial DNA Integrity.

Int J Mol Sci 2019 Sep 3;20(17). Epub 2019 Sep 3.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.

Mitochondria are vital cellular organelles involved in a plethora of cellular processes such as energy conversion, calcium homeostasis, heme biogenesis, regulation of apoptosis and ROS reactive oxygen species (ROS) production. Although they are frequently depicted as static bean-shaped structures, our view has markedly changed over the past few decades as many studies have revealed a remarkable dynamicity of mitochondrial shapes and sizes both at the cellular and intra-mitochondrial levels. Aberrant changes in mitochondrial dynamics and cristae structure are associated with ageing and numerous human diseases (e.g., cancer, diabetes, various neurodegenerative diseases, types of neuro- and myopathies). Another unique feature of mitochondria is that they harbor their own genome, the mitochondrial DNA (mtDNA). MtDNA exists in several hundreds to thousands of copies per cell and is arranged and packaged in the mitochondrial matrix in structures termed mt-nucleoids. Many human diseases are mechanistically linked to mitochondrial dysfunction and alteration of the number and/or the integrity of mtDNA. In particular, several recent studies identified remarkable and partly unexpected links between mitochondrial structure, fusion and fission dynamics, and mtDNA. In this review, we will provide an overview about these recent insights and aim to clarify how mitochondrial dynamics, cristae ultrastructure and mtDNA structure influence each other and determine mitochondrial functions.
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http://dx.doi.org/10.3390/ijms20174311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747513PMC
September 2019

Carbon monoxide releasing molecule 401 (CORM-401) modulates phase I metabolism of xenobiotics.

Toxicol In Vitro 2019 Sep 17;59:215-220. Epub 2019 Apr 17.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Postfach 10 10 07, D-40001 Düsseldorf, Germany.

Next to its well-studied toxicity, carbon monoxide (CO) is recognized as a signalling molecule in various cellular processes. Thus, CO-releasing molecules (CORMs) are of considerable interest for basic research and drug development. Aim of the present study was to investigate if CO, released from CORMs, inhibits cytochrome P450-dependent monooxygenase (CYP) activity and modulates xenobiotic metabolism. CORM-401 was used as a model CO delivering compound; inactive CORM-401 (iCORM-401), unable to release CO, served as control compound. CO release from CORM-401, but not from iCORM-401, was validated using the cell free myoglobin assay. CO-dependent inhibition of CYP activity was shown by 7-ethoxyresorufin-O-deethylation (EROD) with recombinant CYP and HepG2 cells. Upon CORM-401 exposure EROD activity of recombinant CYP decreased concentration dependently, while iCORM-401 had no effect. Treatment with CORM-401 decreased EROD activity in HepG2 cells at concentrations higher than 50 μM CORM-401, while iCORM-401 showed no effect. At the given concentrations cell viability was not affected. Amitriptyline was selected as a model xenobiotic and formation of its metabolite nortriptyline by recombinant CYP was determined by HPLC. CORM-401 treatment inhibited the formation of nortriptyline whereas iCORM-401 treatment did not. Overall, we demonstrate CO-mediated inhibitory effects on CYP activity when applying CORMs. Since CORMs are currently under drug development, the findings emphasize the importance to take into account that this class of compounds may interfere with xenobiotic metabolism.
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http://dx.doi.org/10.1016/j.tiv.2019.04.018DOI Listing
September 2019

Selectively Addressing Mitochondrial Glutathione and Thioredoxin Redox Systems.

Cell Chem Biol 2019 03;26(3):316-318

Institute of Biochemistry and Molecular Biology I, Faculty of Medicine, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.

The mitochondrial matrix is a prominent place in redox biology. It contains mitochondrial glutathione (mGSH), pivotal for maintaining redox homeostasis. In this issue of Cell Chemical Biology, Booty et al., (2019) introduce mitoCDNB, which depletes the mGSH pool, creating new avenues of organellar redox research.
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http://dx.doi.org/10.1016/j.chembiol.2019.02.017DOI Listing
March 2019

The BH3 mimetic compound BH3I-1 impairs mitochondrial dynamics and promotes stress response in addition to its pro-apoptotic key function.

Toxicol Lett 2018 Oct 18;295:369-378. Epub 2018 Jul 18.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich Heine University Düsseldorf, Postfach 10 10 07, D-40001 Düsseldorf, Germany. Electronic address:

BH3 mimetics, such as BH3I-1, act as Bcl-2 antagonists, promote apoptosis and are used in basic research studies on apoptotic signaling and are currently tested as experimental anti-tumor agents. The present study addresses time- and dose-dependent responses of BH3I-1 on apoptosis, cellular stress defense mediated by heme oxygenase-1 (HO-1), and mitochondrial morphology. As expected, treatment of normal human dermal fibroblasts with BH3I-1 induced apoptosis as determined by typical markers including cytochrome c release, loss of procaspase-3, and PARP cleavage. Induction of the cellular stress response marker HO-1 precedes apoptosis induction whereas fragmentation of the mitochondrial network was triggered even more rapidly. No difference in apoptosis induction was found upon depletion of HO-1 by siRNA compared to controls suggesting that apoptosis induction by BH3I-1 is not affected by HO-1. To evaluate the functional interplay between mitochondrial fragmentation and HO-1 induction, murine embryonic fibroblasts lacking the fission factor Drp1 were used. In Drp1 knock out cells, HO-1 levels were low compared to wild type cells, both in untreated controls as well as after BH3I-1 exposure, demonstrating that Drp1 is at least in part required for determining basal and inducible HO-1 levels. Considering the sequence of events, it was shown here that BH3I-1 dependent apoptosis is a rather late event, while effects on mitochondrial morphology and cellular stress response (HO-1 induction) are observed rapidly after exposure of cells to the compound. We propose that BH3I-1 is a valuable tool for studying cellular stress responses as well as mitochondrial dynamics in future studies. Since BH3 mimetics are promising experimental anticancer drugs, our data further imply that additional biological effects such as upregulation of detoxifying systems or changes in mitochondrial dynamics could interfere, in combination therapy, with selective drug toxicity and thus need to be taken into account for drug development.
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http://dx.doi.org/10.1016/j.toxlet.2018.07.017DOI Listing
October 2018

Nanotherapy and Reactive Oxygen Species (ROS) in Cancer: A Novel Perspective.

Antioxidants (Basel) 2018 Feb 22;7(2). Epub 2018 Feb 22.

Institute of Biochemistry and Molecular Biology I, Medical Faculty, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.

The incidence of numerous types of cancer has been increasing over recent years, representing the second-most frequent cause of death after cardiovascular diseases. Even though, the number of effective anticancer drugs is increasing as well, a large number of patients suffer from severe side effects (e.g., cardiomyopathies) caused by these drugs. This adversely affects the patients' well-being and quality of life. On the molecular level, tumor cells that survive treatment modalities can become chemotherapy-resistant. In addition, adverse impacts on normal (healthy, stromal) cells occur concomitantly. Strategies that minimize these negative impacts on normal cells and which at the same time target tumor cells efficiently are needed. Recent studies suggest that redox-based combinational nanotherapies may represent one option in this direction. Here, we discuss recent advances in the application of nanoparticles, alone or in combination with other drugs, as a promising anticancer tool. Such novel strategies could well minimize harmful side effects and improve patients' health prognoses.
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http://dx.doi.org/10.3390/antiox7020031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836021PMC
February 2018
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