Journal: Archives of Toxicology

Article Title: Ferroptosis regulation through Nrf2 and implications for neurodegenerative diseases

doi: 10.1007/s00204-023-03660-8

Figure Lengend Snippet: Characterization of ferroptosis in neurodegenerative diseases (NDs) pathological conditions. There exists a significant correlation between ferroptosis and NDs. Ferroptosis contributes to the advancement of NDs, whereas the expression of Nrf2 and its transcriptionally regulated peptides (GSH, GPX4) decreases with aging. In contrast to the impairment of the antioxidant system, heightened levels of reactive oxygen species (ROS) and increased lipid peroxidation, combined with brain iron deposition, induce ferroptosis. Ferroptosis results in alterations in mitochondrial morphology, neuronal damage and eventual cell death. Drawing by Inkscape

Article Snippet: MitoQ , Radical Trapping Antioxidant (RTA, Targets mitochondrial ROS scavenging → rescues mitochondrial integrity and function) , Oxidative cell death , RSL3-treated neuronal HT22 cells , Mitochondrial ROS, lipid peroxidation↓ , Jelinek et al. ( ) .

Techniques: Expressing

Journal: Archives of Toxicology

Article Title: Ferroptosis regulation through Nrf2 and implications for neurodegenerative diseases

doi: 10.1007/s00204-023-03660-8

Figure Lengend Snippet: Nrf2-regulated iron death mechanisms in neurodegenerative diseases. a Blood–brain barrier (BBB), astrocytes, and neurons. Brain capillary endothelial cells (BCECs) safeguard the function of the BBB. Astrocytes on the abluminal surface of BCECs promote brain iron uptake. Astrocytes are primarily responsible for releasing iron into neurons while attenuating iron toxicity. b the transcytosis model. After Fe 3+ entering the blood circulation forms a complex (holo-Tf) with transferrin (Tf), it binds to TfR1 on the surface of brain microvascular endothelial cells (BMECs), and then the Tf–TfR1 complex enters BMECs through clathrin-mediated endocytosis. One of the two models of iron import across BCEC—the transcytosis model. Holo-Tf is transported directly by vesicles to extraluminal sites for release into the brain. c Nrf2 in astrocytes activates non-cell-autonomous protection of nearby neurons and alleviates oxidative stress (OS) by mediating antioxidant responses. GSH produced in the brain via activated Nrf2 pathway is predominantly derived from astrocytes, whose neuroprotective function depends on the transport of GSH precursors from astrocytes to motor neurons. d Regulation of iron in neurons and mechanisms of Nrf2-regulated ferroptosis in neurodegenerative diseases. The key components of ferroptosis are the lipid and antioxidant systems, as well as iron metabolism. metabolism of iron: The Tf–TfR1 complex carrying Fe 3+ is endocytosed into neurons, Fe 3+ is separated from Tf, and then reduced to Fe 2+ by six transmembrane epithelial antigen-prostate 3 (STEAP3), and Fe 2+ is pumped into the cytoplasm through divalent metal transporter 1 (DMT1), which is also a classic model of iron import across BCEC. Alternatively, Fe 3+ reduced by cellular prion protein (PrPC) is transported into neurons by DMT1. In addition, solute carrier family 39 member 14 (SLC39A14) can also transport Fe 2+ into the cell. Intracellular iron can be stored in two forms: as Fe 3+ stored in ferritin or as active unbound iron called the labile iron pool (LIP). Poly-(rC)-binding protein1/2 (PCBP1/2) are in charge of transferring the iron to ferritin. In some cases, nuclear receptor coactivator 4 (NCOA4) mediates ferritin autophagy, releasing iron. Heme oxygenase 1 (HO1) catalyzes the degradation of heme and can also release Fe 2+ . With the aid of amyloid precursor protein (APP), which is transmitted by soluble tau protein to stabilize FPN1, elevated Fe 2+ can be expelled through Ferroportin1 (FPN1)/copper cyanine (Cp) or FPN1/hephaestin (Heph). Through the IRP–IRE interaction, iron can, when overloaded, increase the expression of ferritin, FPN1, and APP, while blocking the ordinary function of furin, upregulating secretase, and speeding up the deposition of Aβ. By the Fenton reaction, excessive Fe 2+ produces ROS and encourages the oxidation of PUFA on the membranes of cells (PE-PUFA), and finally triggers ferroptosis. Lipid metabolism: Lysophospholipid acyltransferase 3 (LPCAT3), long-chain fatty acid CoA ligase 4 (ACSL4), and other enzyme-sensitize membrane lipids to lipid peroxidation, which, catalyzed by lipoxygenases (LOX), accumulates PUFA-OOH, triggering ferroptosis. Antioxidant system: GSH–GPX4 axis in cytoplasm and mitochondria, ferroptosis suppressor protein 1 (FSP1)–CoQ10 axis in plasma membrane, dihydroorotate dehydrogenase (DHODH)–CoQ 10 H 2 , and GTP cyclohydrolase1–tetrahydrobiopterin (GCH1–BH 4 ) axis in mitochondria. Mitochondrial PTEN-inducible putative kinase 1 (PINK1) expression is regulated by Nrf2 under OS conditions. Keap1–Nrf2–ARE axis: Under OS conditions, Keap1 releases Nrf2, and the increase of Nrf2 levels in the cytoplasm increases its nuclear translocation. Upon nuclear import, Nrf2 forms a heterodimer with the small muscular aponeurotic fibrosarcoma (Maf) protein, enabling Nrf2 to bind to AREs in the upstream promoter regions of a variety of target genes, leading to their transcriptional activation. Drawing by Inkscape

Article Snippet: MitoQ , Radical Trapping Antioxidant (RTA, Targets mitochondrial ROS scavenging → rescues mitochondrial integrity and function) , Oxidative cell death , RSL3-treated neuronal HT22 cells , Mitochondrial ROS, lipid peroxidation↓ , Jelinek et al. ( ) .

Techniques: Produced, Derivative Assay, Binding Assay, Transferring, Expressing, Blocking Assay, Membrane, Clinical Proteomics, Translocation Assay, Activation Assay

Journal: Archives of Toxicology

Article Title: Ferroptosis regulation through Nrf2 and implications for neurodegenerative diseases

doi: 10.1007/s00204-023-03660-8

Figure Lengend Snippet: Signaling molecules associated with ferroptosis and regulated by Nrf2

Article Snippet: MitoQ , Radical Trapping Antioxidant (RTA, Targets mitochondrial ROS scavenging → rescues mitochondrial integrity and function) , Oxidative cell death , RSL3-treated neuronal HT22 cells , Mitochondrial ROS, lipid peroxidation↓ , Jelinek et al. ( ) .

Techniques: Binding Assay, Functional Assay, Clinical Proteomics, Membrane

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: Identification of the protein target of ML405/85 (A) Venn diagram of protein peptide profiles identified by DARTS in DMSO-, ML405-, and 1685-treated cells; 1,016 proteins had essentially the same peptide profile in all three samples, whereas 69 proteins had a different profile from the DMSO control sample. (B) Representative images for the lack of an effect of candidate target protein expression (green) on the cholesterol storage (red) of NPC3 patient cells. (C) Representative images showing the effects of transient expression (left panel) or suppression (right panel) of HSP candidates ERP29, HYOU1, and TRAP1 on cholesterol storage in NPC3 patient cells or mouse NPC1 cells. Left panel: cells expressing candidate proteins are red and cholesterol is green. Right panel: cells in which protein is silenced are green, cholesterol is red. (D) Representative images showing expression of TRAP1 (green) in Fabry disease patient cells decreases the Gb3 storage as determined by VTB staining (red). Likewise, the expression of TRAP1 (red) in Farber and Wolman disease cells decreases the cholesterol storage (green; OlyA) in these cells. Images are representative of at least three independent experiments. Graphs indicate the percentage of transfected cells corrected for lipid storage ( n = 50–75) and are represented as mean ± SD Scale bar, 50 μm.

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques: Expressing, Staining, Transfection

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: List of potential protein targets for the ML405/1685 compounds

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques:

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: ML405/85 mimic TRAP1 actions on mitochondrial respiratory chain enzymes and metabolites (A) Treatment with ML405 inhibits phosphorylation of transiently expressed mitochondria-targeted c-Src at Tyr-416 (p-Y416) but has no effect on total mitochondrial c-Src (Input) in ARPE cells. The blot shown is representative of four independent experiments. (B) Densitometry of phospho- c -Src bands in (A) normalized to total c-Src (Input). (C and D) ML405/85 inhibit complex II (SDH) activity of the mitochondrial respiratory chain in purified ARPE mitochondria (C), which results in the expected increase in cellular succinate levels (D). TTFA: thenoyltrifluoroacetone, complex II inhibitor (positive control). (E) Treatment with ML405 lowers COX activity in purified ARPE mitochondria. (F) Treatment with ML405/85 increase cellular and secreted lactate levels in ARPE cells. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001.

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques: Activity Assay, Purification, Positive Control

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: TRAP1 agonists ameliorate mitochondrial and ER stress (A and B) ML405 decreases the red/green fluorescence ratio in Mitotimer-expressing NPC1 cells, indicating reduced mitochondrial oxidative stress (A, representative image of cell mitochondria quantified in B). (C) Treatment with ML405/85 reduce superoxide levels (red; MitoSox) in isogenic wt and NPC1-null cells but have no effect in isogenic TRAP1-null cells, indicating that the reduction of mitochondrial superoxide is mediated by TRAP1. (D) Treatment with ML405 increases ATP levels in both NPC3 and Fabry patient cells. (E) Treatment with ML405 increases the residual α-galactosidase activity in Fabry patient cells to varying degrees based on the specific α-galactosidase mutation of each cell line. (F–H) Similarly, treatment with ML405 increases the activity of lysosomal acid lipase in Wolman (F), tripeptidyl peptidase 1 in CLNII (G), and galactocerebrosidase in Krabbe (H) patient cells. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001.

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques: Fluorescence, Expressing, Activity Assay, Mutagenesis

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: Mitochondrial dysfunction affects lysosomal function (A) Representative images showing that cell treatment with mitochondrial respiratory chain inhibitors antimycin A (complex III) or atpenin A5 (SDH) induces the lysosomal lipid storage in wt cells and exacerbates the storage in NPC1 cells. (B) Quantitation of the lipid storage seen in wt and NPC1 cells (A). At least 150 cells were quantitated for each sample and each experiment was repeated three times. Images were taken using the same exposure settings. (C) Schematic showing the importance of mTORC1 as a sensor of glucose availability and a master regulator of mitochondria and lysosome/autophagy function. (D) Treatment with 1685 or exogenous TRAP1 expression increases the levels of cellular AMPK and phosphorylated AMPK (pAMPK). GAPDH: glyceraldehyde 3-phosphate dehydrogenase. (E) Effect of ML405/85 on pAMPK levels is rapid, reaching a maximum at about 1 h following treatment. Metform: metformin, known inducer of pAMPK (positive control). (F) Densitometry of pAMPK bands from (E) normalized to GAPDH signal. (G) Treatment with 1685 inhibits phosphorylation of the mTOR target p70S6 kinase in a rapid manner. Graph represents levels of normalized phosphorylated p70S6 kinase relative to total p70S6 kinase protein levels. The blots shown are representative of three independent experiments. Data are represented as the mean ± SD ∗∗∗ p < 0.0005, ∗∗ p < 0.001, ∗ p < 0.05.

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques: Quantitation Assay, Expressing, Positive Control

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet: TRAP1 agonists show efficacy in a mouse Fabry model Age-matched Fabry mice treated with 30 mg/kg ML405 three times/week for four weeks showed a reduced Gb3 storage in the liver (A) plasma (B) kidney (C), and heart (D). Values shown are the average of three replicates, n = 9–10. ∗∗∗ p < 0.0005, ∗∗ p < 0.001, ∗ p < 0.05.

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques:

Journal: iScience

Article Title: Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction

doi: 10.1016/j.isci.2022.104941

Figure Lengend Snippet:

Article Snippet: NPC3 cells were transfected with Flag-tagged expression plasmids: IPO7 (Origene RC215943), PLEK (Origene RC203780), PRDX1 (Origene RC221235), PSMB8 (Genscript), PSMC6 (Origene RC202809), TPM3 (Origene RC209904), ERP29 (Origene RC210918), HYOU1 (GenScript), TRAP1 (Origene RC203439).

Techniques: Recombinant, Cytochrome c Oxidase Assay, Viability Assay, Protein Extraction, BIA-KA, Isolation, Mutagenesis, Variant Assay, Knock-Out, CRISPR, Sequencing, shRNA, Plasmid Preparation, Software, Microscopy