Joan P Forder, PhD October 29th, 2007 Small Concentrations of Peroxynitrite Lead to Neuroprotection Following Stroke Through Inhibition of Synapsin Phosphorylation Joan P Forder, PhD October 29th, 2007

Cerebrovascular Accident (Stroke) Every 10 minutes, someone in Canada has a stroke. 50,000 Canadians will have a stroke this year. Stroke is Canada’s No. 3 killer and a leading cause of severe, long-term disability.  Ischemic Stroke accounts for 70-80% of all strokes.

The mechanism of anoxic neuronal death ONOO - [Ca ] 2+ Ca TRPM7 NMDAR Mitochondria O . 2 NOS PSD-95 Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, Tymianski M. (2003) Cell. 115(7): 863-77.

NeuN (neurons) Nitrotyrosine Overlay Infarct Region 10X Infarct Region 64X Nitrotyrosine expression can be seen outside the nucleus in neurons 24 hrs following stroke surgery

HYPOTHESIS Small amounts of peroxynitrite will protect neurons from oxygen/glucose deprivation (OGD) and translates to a neuroprotective role in vivo

Small Concentrations of Peroxynitrite Did Not Lead To Cell Death primary cortical cell culture with medium containing propidium iodide (PI) add various concentrations of peroxyntrite sequential fluorescence readings over time

Oxygen/Glucose Deprivation (OGD) Experiment anaerobic chamber containing a 5% CO2,10%H2, and 85% N2 (<0.2% O2) atmosphere add concentrations of peroxynitrite or NMDA maintained anoxic for the duration at 37oC Measure cell death Small Concentrations of Peroxynitrite Did Protect Cells From OGD

3 Pial Vessel Occlusion (3PVO) Model of Permanent Ischemia Expose Cortex and Cauterize ECA Cannulation Wait specified time points Slice 2mm thick Stain with TTC Outline and Analyze Contralateral control Ipsialateral infarct Collect Tissue for Immunoprecipitation, Western Blot, Mass Spec, and IHC

Low Concentrations of Peroxynitrite Reduce Infarct Size Following Stroke *

Isolating Nitrated Proteins: Immunoprecipitation Pull down all nitrated proteins in infarct and LTP tissue homogenates using anti-nitrotyrosine antibody Electrophoresis to reveal three distinct bands Process each band by mass spectrometry Confirm each protein using reverse immunoprecipitation Compare results

Cell Signaling Proteins Cytoskeletal/Synaptic Proteins Vesicle Trafficking/Docking Proteins Metabolic Proteins Cell Signaling Proteins Synapsin Neurotransmitter Release Dynamin Endocytosis Na/K ATPase maintaining cell membrane potential maintaining mitochondrial function

HYPOTHESIS Small Concentrations of Peroxynitrite are Neuroprotective Following Stroke Through Inhibition of Synapsin Phosphorylation

Phospho-Synapsin Levels Decrease with Time Following Stroke 0 1 3 6 12 24 48 hrs AR C AR C A B Contra Infarct Anti-phospho-synapsin I Anti-synapsin I C

siRNA knock down of Synapsin I mimics neuroprotective effect of low peroxynitrite concentrations following stroke A B NS 76 77 78 Con PC D C

Proposed Model of Neuroprotection Involving Nitrated Synapsin Illustration created by Ibrahim Khambati

Acknowledgements Dr. Michael Tymianski MD, PhD Hong Sun, MD, PhD and Ibrahim Khambati L Liu, PhD and Yu-Tian Wang, PhD The University of British Columbia And the rest of the Tymianski Lab