Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis

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1 Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis Marietta Zille, PhD Burke Medical Research Institute Weill Cornell Medicine White Plains, NY May 01, 2017

2 Therapeutic interventions for primary injury have been limited. 2

3 Secondary Injury Includes Edema Formation, Inflammation, and Cell Death Edema Inflammation Secondary Injury Cell death 3

4 Why is studying cell death relevant for ICH patients? 4

5 Why Is Studying Cell Death Relevant for ICH patients? Death is not passive response of the cell Cells actively engage in cell death signaling in response to specific stimuli Studying the rich biology of these pathways gives rise to thereapeutics 5

6 How does lysed blood induce cell death after ICH?

7 The Lab s Approach In vitro models In vivo models Hemorrhagic Stroke 7

8 Cell Survival [%] Modeling ICH In Vitro: Hemoglobin and Hemin Toxicity Hemin [µm]

9 Modeling ICH In Vivo: Collagenase Infusion 5 minute long injection (ca. 0.8µL) 5 minute long diffusion Sham ICH

10 Traditional Approach

11 Unbiased Approach

12 How Does Lysed Blood Induce Cell Death After ICH? Pharmacology Biochemistry/Molecular Morphology

13 How Does Lysed Blood Induce Cell Death After ICH? Cell Death Mechanism Autophagy Subcategory Cell Death Inhibitor Target Conc. Macroautophagy Mitophagy 3-Methyladenine Bafilomycin A1 Chloroquine diphosphate salt Rapamycin Mitochondrial division inhibitor 1 Phosphoinositide 3-kinase (PI3K), autophagosome formation Endosomal acidification Lysosomal function Mechanistic target of rapamycin (mtor), autophagy inducer µM µM µM 0.1-5µM GTPase activity in dynamin-related protein Drp-1, abnormal mitophagy µM z-vad-fmk Caspases µM 100% Caspase-dependent apoptosis Cycloheximide Protein synthesis µM Cyclosporine A SB Cyclophilin D (mitoch. permeability transition pore) p38 mitogen-activated protein (MAP) kinase (p38) µM 1-30µM SP c-jun N-terminal kinase (JNK) µM Cycloheximide Protein synthesis µM Actinomycin D mrna synthesis µM Regulated Necrosis Ferroptosis Parthanatos Necroptosis Ferrostatin-1 Deferoxamine N-Acetylcysteine Trolox, vitamin E analog U0126 PARP inhibitor III Olaparib (AZD-2281, trade name Lynparza) Necrostatin-1 Canonical ferroptosis inhibitor, reactive lipid species (RLS) Iron, hypoxia-inducible factor (HIF) prolyl hydroxylase domaincontaining (PHD) inhibition Reactive oxygen species (ROS), RLS RLS Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) Poly(ADP-ribose) polymerase 1 and 2 (PARP1 and 2) PARP1 and 2 Receptor-interacting protein kinase 1 (RIP1) µM µM µM µM 1-20µM µM 1-20µM µM 50% 13 Zille et al., Stroke, 2017

14 Cell Death After ICH In Vitro Shares Features of Ferroptosis & Necroptosis Cell Death Mechanism Autophagy Subcategory Cell Death Inhibitor Target Conc. %Viability Macroautophagy Mitophagy Caspase-dependent apoptosis Regulated Necrosis Ferroptosis Parthanatos Necroptosis 3-Methyladenine Bafilomycin A1 Chloroquine diphosphate salt Rapamycin Mitochondrial division inhibitor 1 Phosphoinositide 3-kinase (PI3K), autophagosome formation Endosomal acidification Lysosomal function Mechanistic target of rapamycin (mtor), autophagy inducer µM µM µM 0.1-5µM ± (500µM) ± (10nM) ± 9.06 (5µM) ± (1µM) GTPase activity in dynamin-related protein Drp-1, abnormal mitophagy µM ± 5.59 (50µM) z-vad-fmk Caspases µM Cycloheximide Protein synthesis µM Cyclosporine A SB Cyclophilin D (mitoch. permeability transition pore) p38 mitogen-activated protein (MAP) kinase (p38) µM 1-30µM SP c-jun N-terminal kinase (JNK) µM Cycloheximide Protein synthesis µM Actinomycin D mrna synthesis µM Ferrostatin-1 Deferoxamine N-Acetylcysteine Trolox, vitamin E analog U0126 PARP inhibitor III Olaparib (AZD-2281, trade name Lynparza) Necrostatin-1 Canonical ferroptosis inhibitor, µM reactive lipid species (RLS) Iron, hypoxia-inducible factor (HIF) prolyl hydroxylase domaincontaining (PHD) inhibition µM Reactive oxygen species (ROS), µM RLS RLS Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) Poly(ADP-ribose) polymerase 1 and 2 (PARP1 and 2) PARP1 and 2 Receptor-interacting protein kinase 1 (RIP1) * p<0.05 compared to 100µM hemin, # p<0.05 U0126 vs. U0124 same concentrations µM 1-20µM µM 1-20µM µM ± ± ± (500nM) ± 7.15 (5µM) ± 5.78 (30µM) ± 2.78 (0.1µM) ± (1µM) ± * (1µM) ± 8.53 * ± 7.08 * (1mM) 88.3 ± * ± * # (10µM) ± (50µM) ± (20µM) 77.4 ± * 100% 50% 14 Zille et al., Stroke, 2017

15 Ferroptosis (Operational Definition) Iron-dependent form of non-apoptotic cell death Accumulation of lipid peroxidation products Activation of MAP kinases Lack of rupture and blebbing of the plasma membrane Shrunken mitochondria Reduction of mitochondrial cristae number Dixon et al., Cell, 2012 MAP - Mitogen-activated protein kinase 15

16 Ferroptosis As a Therapeutic Target for ICH Patients? Cell Death Mechanism Regulated Necrosis Subcategory Cell Death Inhibitor Target Conc. %Viability Ferroptosis Cycloheximide Protein synthesis µM Actinomycin D mrna synthesis µM Ferrostatin-1 Deferoxamine N-Acetylcysteine Trolox, vitamin E analog U0126 Canonical ferroptosis inhibitor, reactive lipid species (RLS) Iron, hypoxia-inducible factor (HIF) prolyl hydroxylase domaincontaining (PHD) inhibition Reactive oxygen species (ROS), RLS RLS Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) µM µM µM µM 1-20µM ± 2.78 (0.1µM) ± (1µM) ± * (1µM) ± 8.53 * ± 7.08 * (1mM) 88.3 ± * ± * # (10µM) 100% 50% * p<0.05 compared to 100µM hemin, # p<0.05 U0126 vs. U0124 same concentrations Stroke, Zille et al., Stroke, 2017

17 Ferroptosis As a Therapeutic Target for ICH Patients? Cell Death Mechanism Regulated Necrosis Subcategory Cell Death Inhibitor Target Conc. %Viability Ferroptosis Cycloheximide Protein synthesis µM Actinomycin D mrna synthesis µM Ferrostatin-1 Deferoxamine N-Acetylcysteine Trolox, vitamin E analog U0126 Canonical ferroptosis inhibitor, reactive lipid species (RLS) Iron, hypoxia-inducible factor (HIF) prolyl hydroxylase domaincontaining (PHD) inhibition Reactive oxygen species (ROS), RLS RLS Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) µM µM µM µM 1-20µM ± 2.78 (0.1µM) ± (1µM) ± * (1µM) ± 8.53 * ± 7.08 * (1mM) 88.3 ± * ± * # (10µM) 100% 50% * p<0.05 compared to 100µM hemin, # p<0.05 U0126 vs. U0124 same concentrations JCI Insight, April Zille et al., Stroke, 2017

18 Ferroptosis As a Therapeutic Target for ICH Patients? Cell Death Mechanism Regulated Necrosis Subcategory Cell Death Inhibitor Target Conc. %Viability Ferroptosis Cycloheximide Protein synthesis µM Actinomycin D mrna synthesis µM Ferrostatin-1 Deferoxamine N-Acetylcysteine Trolox, vitamin E analog U0126 Canonical ferroptosis inhibitor, reactive lipid species (RLS) Iron, hypoxia-inducible factor (HIF) prolyl hydroxylase domaincontaining (PHD) inhibition Reactive oxygen species (ROS), RLS RLS Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) µM µM µM µM 1-20µM ± 2.78 (0.1µM) ± (1µM) ± * (1µM) ± 8.53 * ± 7.08 * (1mM) 88.3 ± * ± * # (10µM) 100% 50% OP-026 N-acetylcysteine targets nuclear envelope-derived, toxic lipids to improve outcomes following hemorrhagic stroke in mice Presenter: Saravanan S. Karuppagounder * p<0.05 compared to 100µM hemin, # p<0.05 U0126 vs. U0124 same concentrations 18 Zille et al., Stroke, 2017

19 Necroptosis (Operational Definition) Activation of receptor-interacting protein 1 and 3 (RIP1 and RIP3) Rupture of plasma membrane Cytoplasmic swelling Swelling of cytoplasmic organelles Int J Cell Biol, 2014 FADD Fas-associated protein with death cflip Cellular FLICE-like inhibitory protein MLKL Mixed lineage kinase domain-like Modified from from Vanden Berghe et al, Nat Rev, 2014; Linkermann, Kidney International,

20 Summary 20 Zille et al., Stroke, 2017

21 Ratan Lab Rajiv Ratan Amit Kumar Ishraq Alim Joseph Caulfield Megan Bourassa Saravanan Karuppagounder Yingxin Chen Acknowledgements EM Core, Weill Cornell Teresa Milner June Chan Elizabeth Jonas (Yale) Peter J. Gough (GSK) John Bertin (GSK) Joshua Finger (GSK) Funding German Research Foundation, DFG Zi 1613/1-1 Adelson Foundation