Preconditioning the Brain for Stroke Prevention

Transcription

1 Preconditioning the Brain for Stroke Prevention The Peritz Scheinberg Cerebral Vascular Disease Research Laboratories; Department of Neurology Miguel A. Perez-Pinzon, Ph.D., FAHA

2 Dirnagl et al., TINS; 2003: 26(5): 248

3 The main goal in this field is to define the pathophysiological mechanisms of cerebral ischemiainduced cell death with the goal of finding therapies to ameliorate the consequences of the insult

4 Strategies for Neuroprotection Cocktail of drugs that target all these pathological mechanisms Or, the use of a drug that have pleiotropic properties that can protect against most of these pathological phases

5 Strategies for Neuroprotection with Pleiotropic Properties Ischemic Conditioning

6 Ischemic Preconditioning That which does not kill us makes us stronger Friedrich Nietzsche, Ecce Homo (1908) Ischemic preconditioning refers to the ability of a brief ( sublethal ) ischemic episode, or mild stress insult followed by a period of reperfusion, to increase an organ s resistance to injury (ischemic tolerance)

7 Sham Ischemia IPC + Ischemia A B C RACK IPC R1 Ischemia R2 D CA1 CA2 DG CA3 Dave et al. J Neurosci Res. 2005, 82, E Number of normal neurons / mm CA * * # Sham Ischemia IPC + Ischemia

8 Other models of conditioning Figure 4 Number of normal neurons Sham CA CA Sham IPC + CA IPC + CA 15 min RPC + CA 30 min RPC + CA

9 IPC DPCPX Pre-synaptic Post-synaptic MK-801 U73122 A1 Receptor Phospholipase-C NMDA Calcium BAPTA-AM BAPTA OAG DAG PKC Translocation to its RACKs RACK V1-2 ERK activation PD NEUROPROTECTION

10 Protein Kinase C epsilon (PKCε) activation is necessary for IPC neuroprotection 100 a % of PI fluorescence b A,c b 0 Sham OGD IPC V1-2 + IPC RACK Raval et al., 2003, J Neurosci. 23(2):

11 IPC DPCPX Pre-synaptic Post-synaptic MK-801 U73122 A1 Receptor Phospholipase-C NMDA Calcium BAPTA-AM BAPTA OAG DAG PKC Translocation to its RACKs RACK V1-2 ERK activation PD HIPPOCAMPAL CA1 NEUROPROTECTION

12 Preconditioning enhances GABA release Glutamate * GABA % baseline % baseline Dave et al. J Neurosci Res. 2005, 82, Ischemia Ischemia min Baseline Ischemia Reperfusion # * Sham Ischemia IPC + ischemia Sham Ischemia IPC + ischemia

13 Whole Cell Recording + Axon

14 Preconditioning alters GABA mpsc Defazio et al. JCBFM. 2009, 29, 375

15 Preconditioning alters GABA mpsc Defazio et al. JCBFM. 2009, 29, 375

16 PKC preconditioning alters action potential properties in CA1 hippocampal neurons ** -40 Tat Rack Action potential Amplitude (mv) After-Hyperpolarization Amplitude (mv) Action Potential Threshold (mv) 80 * Tat Rack Neumann et al. JCBFM Jan;35(1): ** Tat Rack

17 Membrane Potential (mv) PKC preconditioning induces a delay in membrane depolarization during OGD Tat εrack Time (min) OGD Onset Neumann et al. JCBFM Jan;35(1): * n=9 *p<0.05

18 Ischemic preconditioning Presynaptic NF- B PKC ERK1/2 CREB GABAergic BDNF p75 Glial cells camp TrkB Glutamate BDNF NMDA receptor p75 TrkB Glial cells Ca ++ TrkB Postsynaptic CaMKII

19 TrkB activation is necessary for ψεrack mediated protection % CA1 Cell Death (PI Fluorescence) * *** TAT ψεrack ψεrack + DMSO ** ψεrack + ANA-12

20 SUMMARY ψεrack increases BDNF expression and TrkB phosphorylation BDNF expression and TrkB phosphorylation alter action potential properties ψεrack mediated arc expression triggers a decrease in AMPAR mepscs ψεrack delays the onset of anoxic depolarization TrkB activation and arc expression are necessary for ψεrack mediated neuroprotection

21 MITOCHONDRIAL DYSFUNCTION Perez-Pinzon et al. Journal of Cerebral Blood Flow & Metabolism (2012), 1 15

22 PKCε Enhances Maximal NADH Fluorescence ΨεPC (ΨεRACK 1h) 48h NADH Autofluorescence Tat ΨεPC Baseline Maximal Fluorescence Increase [%] *** Morris Blanco et al. (2014) J Cereb. Blood Flow & Metab

23 Major Biosynthetic Pathway for NAD + Production Nampt= nicotinamide phosphoriboslytransferase NAM= nicotinamide NMNAT NMN Nampt NMN=Nicotinamide mononucleotide NAD + NAM Nmnat=NMN adenyl transferase

24 PKCε Enhances Nampt Fluorescence in the Rat Cortex 0.2 mg/kg i.p. ΨεRACK 48h Fixed Cortex Hoechst Nampt CoxIV Merge Tat ΨεRACK Nampt Fluorescence (Arbitrary Units) ** Morris Blanco et al. (2014) J Cereb. Blood Flow & Metab

25 Nampt= nicotinamide phosphoriboslytransferase NAM= nicotinamide NMNAT NMN Nampt NMN=Nicotinamide mononucleotide NAD + NAM Nmnat=NMN adenyl transferase Sirtuins

26 Mitochondrial activity Promotes mitochondrial biogenesis and activity by activation of PGC-1α PGC 1α Ac PGC 1α Inflammatory response Inhibition of NF-ĸB transcriptional activity p50 p65 Ac SIRT1 Global gene repression Histone deacetylation p50 p65 Ac FOXO FOXO Ac p53 p53 Oxidative stress Shift in FOXO regulated gene expression from cell death to survival Apoptosis Inactivation of p53-dependent transcription of pro-apoptotic proteins

27 Resveratrol Caloric Restriction SIRT1 Decreased Reactive Oxygen Species Preserved Mitochondrial Function Insulin Growth Factor Signaling Pathway Enhanced Lifespan

28 Resveratrol preconditioning (RPC) decreases CA1 cell death in organotypic slices after OGD 100 % of PI fluorescence % of PI fluorescence OGD 48 h 96 h 75 M 100 M 200 M 500 M 0 OGD IPC 10 M 50 M 100 M IPC Resveratrol treatment for 1 h Sirtinol treatment after IPC for 48 h Raval et al. JCBFM Sep;26(9):1141-7

29 Resveratrol preconditioning (RPC) reduces infarct after MCAo in mice Veh RPC Veh RPC Narayanan et al. Stroke (6):

30 1600 RPC decreases CA1 cell death after cardiac arrest in rats Number of normal neurons # # # Sham Vehicle IPC DMSO DMSO sirtinol # Resveratrol (mg/kg) Resveratrol (10 mg/kg) Della-Morte et al. Neuroscience. 2009

31 CAN YOU DRINK YOUR WAY TO PRECONDITIONING??? Images courtesy of Google Images

32 A SINGLE APPLICATION OF RPC INDUCES ISCHEMIC TOLERANCE AGAINST FOCAL ISCHEMIA FOR TWO WEEKS RPC (10 mg/kg) or Veh (DMSO-Saline) Koronowski et al Stroke.

33 A B Sirt1-Promoter Binding C BDNF D ** ** * * * Sirt1 80 kda UCP2 70 kda β-actin 45 kda VDAC 32 kda

34 Transcriptomic changes induced by RPC within the long-term window of IPC Nathalie Khoury et al. Mol Neurobiol Oct 20

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36 CONCLUSIONS 1. Signaling pathways activated by Ischemic Preconditioning exhibit pleiotropic properties that lead to neuroprotection 2. PKC and Resveratrol are two pharmacological agents that emulate IPC and have pleiotropic properties 3. Pharmacological agents that emulate IPC could be used in a prophylactic manner to enhance ischemic tolerance in patients prone to cerebral ischemia

37 Clinical Scenarios for IPC Implementation a)carotid endarectomy and coronary artery bypass graft (CABG) which both result in high incidence of strokes and cognitive deficits b)transient ischemic attacks (TIA) patients, from which 10% will have a stroke within a month c) Sub-arachnoid hemorrhage (SAH) patients from which 20-30% undergo delayed cerebral ischemia (DCI) d)neurosurgical procedures e)all stroke patients have a higher incidence of subsequent strokes

38 Precondition able population Stroke Survivors ~ 660,000 Carotid Endarterectomy ~ 132,000 Coronary Artery Bypass Graft ~ 500,000 TIA ~ 240,000 Sub-arachnoid Hemorrhage ~ 55,650 Neurosurgical Procedures

39 Acknowledgements ISCHEMIC PRECONDITIONING: MECHANISMS OF NEUROPROTECTION: 2R01NS METABOLIC MASTER REGULATORS FOR ISCHEMIC NEUROPROTECTION: 5R01NS Two F31: 5F31NS (Narayanan) and 5F31NS (Koronowski) Post- and Pre-doctoral Fellowships: Kahlilia Morris- Blanco, Nathalie Khoury, Jake Neumann, John Thompson

40 Dave, K.R. Raval, A.P. DeFazio, R.A. Juan Young Koch, S. Sick, T. ACKNOWLEDGEMENTS The Pertiz Scheinberg Cerebral Vascular Disease Research Laboratories Kim, E.J. Lange-Asschenfeldt, C. Saul, I. Della Morte, D. Thompson, J. Morris-Blanco, K.M. Narayanan S. Neumann J. Cohan C. Koronowski K.B. Khoury N. Stradecki H.

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