1. 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

2. 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.

3. 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):

4. 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

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

6. 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

7. 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

8. 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

9. Low Concentrations of Peroxynitrite Reduce Infarct Size Following Stroke*

10. 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

11. 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

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

13. Phospho-Synapsin Levels Decrease with Time Following Stroke
hrs
AR C
AR C A B
Contra Infarct
Anti-phospho-synapsin I
Anti-synapsin I C

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

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

16. 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