NMDA Receptors & Stroke Therapies Ajay Chahal Vivian Tang Anushya Vijayaraghevan Chelsea Geen PHM142 Fall 2014 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson

Stroke Caused by lack of blood flow to brain Variety of effects – Partial paralysis, vision loss, dizzyness ect. Risk factors are: – hypertension obesity, diabetes, excessive alcohol consumption, smoking and stress. (Heart and Stroke Foundation, 2014)

NMDA Receptor N-methyl-D-aspartate Receptor Found in synaptic cleft Glutamate-gated ion channel – Calcium – Sodium Activation: ligand binding and depolarization (Li & Tsien, 2009)

NMDA Receptor: Mechanism of Action At resting potential, the channel is blocked by a Mg 2+ (Blanke & VanDongen, 2009)

NMDA Receptor: Mechanism of Action Electrical stimulation of presynaptic neurons releases glutamate into the synaptic cleft. The glutamate binds to the receptor. glutamate (Blanke & VanDongen, 2009)

NMDA Receptor: Mechanism of Action Receptor does not activate because insufficient depolarization (Blanke & VanDongen, 2009)

NMDA Receptor: Mechanism of Action During sufficient depolarization, the Mg 2+ is dislodged from channel and activates Mg 2+ (Blanke & VanDongen, 2009)

NMDA Receptor: Mechanism of Action Activated channel results in influx of Ca 2+ Acts as a secondary messenger to activate intracellular signaling cascade (Blanke & VanDongen, 2009)

Mechanism of Ischemic Cell Death (Bretón and Rodríguez, 2012)

Effects of High Ca 2+ Concentration in Neurons after Ischemia Rama (Bretón and Rodríguez, 2012)

Excitotoxic Signaling by Overstimulation of NMDA Receptor (Bretón and Rodríguez, 2012)

(R&D Systems, 2012)

NMDA & Stroke: Therapeutic Attempts Only therapy currently available: thrombolytic recombinant tissue plasminogen activator (rt-PA) give within 3 hrs – most patients (95%) don’t receive (eg delay to hospital) – increases intracranial haemorrhage risk (Reviewed in Green and Shuaib, 2006) Image

Neuroprotective Agents Another approach! – attempt to interfere with ischaemic cascade mechanisms and decrease tissue damage – have been studied but every one to reach clinical trials… (Reviewed in Green and Shuaib, 2006) Image

Failed Attempts… Table 1: Compounds that have failed clinical trials for acute ischaemic stroke treatment (Green and Shuaib, 2006).

There is still hope! New neuroprotective compounds being investigated – But none currently on market (Reviewed in Green and Shuaib, 2006)

Example: DAPK1 and NMDA DAPK1: death-associated protein kinase 1 – Found to be important part of neuron death signaling cascade downstream of NMDA – Could be a target for neuroprotective drug development! (Reviewed in Martin and Wang, 2010)

Mechanism of Potential Therapeutic Action (Reviewed in Martin and Wang, 2010) Normal synapse: Activate NMDA receptor  neuronal survival signaling complex (SSC)

(Reviewed in Martin and Wang, 2010) Increase Ca 2+ influx through NMDA receptors  activates DAPK  promotes DAPK bind and phosphorylation of NR2B Activated DAPK promotes neuron death signal complex (DSC) that shuts off SSC Using an NR2B interference peptide inhibit aDAPK binding to NR2B  blocks phosphorylation, decrease DSC activation  avoid exitotoxic damage induced by stroke! STROKE CONDITIONS:

Darth Vader = DAPK Lightsaber = Ca 2+ Darth Vader + Lightsaber = activated DAPK Darth Vader + Lightsaber:  gives into anger (phosphorylation) and joins the dark side (binds NR2B)  promotes neuron death signal complex (DSC) shutting off survival signal complex (SSC) Luke Skywalker = NR2B interference peptide Luke Skywalker stops Darth Vader and his lightsaber (aDAPK) from joining the dark side (binding NR2B), blocking Darth Vader’s anger (phosphorylation) Since Darth Vader is no longer angry, death signal complex (DSC) is decreased

Summary STROKE Caused by an event that limits or stops blood flow to the brain – ischemia or hemorrhaging – 2 kinds of ischemic stroke: 1) Thrombotic stroke: blood clot in an artery leading directly to brain 2) Embolic stroke: clot forming somewhere else in the body, travels through blood stream to the brain. Risk factors: hypertension, obesity, diabetes, excessive alcohol consumption, smoking and stress NMDA RECEPTOR A glutamate-gated ion channel - activated when both glutamate is bound to receptor and adequate depolarization occurs Results in Ca 2+ ion influx NMDA receptors excessively activated resulting in increased Ca2+ influx – after ischemia, cytoplasmic Ca2+ levels rise and can trigger many downstream neurotoxic cascades including the activation and overstimulation – the results include activation of several signalling pathways mainly causing an overproduction of free radicals, dysfunction of mitochondria, cell membrane disruption and DNA fragmentation, which together induce neuron death THERAPY Currently only rt-PA for ischaemic stroke (within 3 hours) Neuroprotective agents still being investigated, although most have failed clinical trials

References Blanke, M. L., and VanDongen, A.M.J. (2009). "Activation Mechanisms of the NMDA Receptor." Biology of the NMDA Receptor. Retrieved from Bretón, R.R., and Rodríguez, J.C.G. (2012). Excitotoxicity and oxidative stress in acute ischemic stroke. Stroke, 8, 9. Green, A.R., and Shuaib, A. (2006). Therapeutic strategies for the treatment of stroke. Drug Discovery Today, 11:15/16. doi: /j.drudis Heart and Stroke Foundation. (2014). “Stroke.” Web. 27 Oct Retrieved from Li, F, and Tsien J.Z. (2009). Memory and the NMDA receptors. New England Journal of Medicine, 361(3): doi: /NEJMcibr Martin, H.G.S., and Wang, Y.T. (2010). Blocking the deadly effects of the NDMA receptor in stroke. Cell, 140: DOI /j.cell R&D Systems. “Neuronal Death by Glutamate Excitotoxicity: Protein Mediators & Strategies for Inhibition.” (2012). Retrieved from