Novel Neuroprotective Targets of Adenosine and Glutamate Signaling in an Animal Model of Ischemic Stroke

Abstract

Stroke is a major cause of death and disability worldwide. The role of adenosine as an endogenous neuromodulator is well established; however, adenosine plays a crucial pathological role in neurodegenerative disorders including ischemic stroke. The extracellular levels of adenosine are elevated following cerebral hypoxia or ischemia. The adenosine A1 receptor (A1R) is the most abundant adenosine receptor in the brain and mediates short-term neuroprotective effects during ischemia through inhibition of presynaptic glutamate release and mediating internalization of GluA1 and GluA2 subunits of AMPA receptors (AMPARs) through protein phosphatases including protein phosphatase 2A (PP2A). However, prolonged stimulation of the inhibitory A1Rs results in desensitization of A1R with subsequent elevation of surface expression of the excitatory adenosine A2A receptor (A2ARs). On the other hand, A2AR promotes presynaptic release of glutamate, which contributes to elevation of extracellular glutamate and subsequent glutamate toxicity and neuronal death following cerebral ischemia. This study examines the role of A2AR, some regulatory molecules involved in A1R downstream signaling, and AMPAR in cerebral hypoxia. Ischemic stroke not only causes elevation of extracellular adenosine, but also increases levels of glutamate, the major excitatory neurotransmitter in the brain, which can cause glutamate-induced excitotoxicity. In fact, several glutamate receptor antagonists and inhibitors of glutamate signaling have been examined as neuroprotective agents in ischemic stroke with little to no success in clinical trials. This thesis examines the potential neuroprotective effects of perampanel, an FDA-approved anti-epileptic drug, which blocks AMPARs in a non-competitive manner. Using in vivo and ex vivo models of ischemic damage, I also investigated the neuroprotective potential of perampanel. This thesis provides evidence of neuroprotective potential of perampanel in focal cortical ischemia. Treatment with perampanel prevented hippocampal cell and neuronal death. In addition, perampanel improved behavioural outcomes following cerebral ischemia and attenuated memory and motor deficits. Moreover, perampanel prevented anxiety and depressive-like behaviours. In this work, I examined the potential neuroprotective effects of istradefylline, a selective A2AR antagonist, that was recently approved by FDA for the treatment of Parkinson’s disease (PD). Indirect inhibition of glutamate toxicity using istradefylline showed similar results in both ex vivo and in vivo models of cerebral ischemia. In addition, I investigated the role of serine/threonine protein kinase casein kinase II (CK2) and the protein phosphatase 2A (PP2A) in hypoxia-induced neuronal injury. I used the new investigational drug LB-100 (a PP2A inhibitor) currently used in clinical trial for various solid tumors to examine the role of PP2A in mediating adenosine-induced neuronal death during hypoxia. Pial vessel disruption (PVD) surgery was used as an in vivo model of small vessel stroke to examine the potential neuroprotective effects of istradefylline, perampanel, and several Tat-interfering peptides designed to prevent AMPAR endocytosis or reduce CK2-mediated activation of PP2A, which are two signaling molecules activated downstream of A1R stimulation. In addition, several behavioural tests were performed to assess the levels of motor and memory deficits following induction of ischemic stroke. This thesis provides novel evidence that both clinically approved A2AR and AMPAR antagonists prevented hippocampal neuronal death and behavioural abnormalities, including cognitive and motor deficits and anxiety-like and depressive-like behaviours in rats subjected to the PVD stroke model. Moreover, this study adds further support for our model that the A1R downstream target CK2 contributes to ischemia-induced neuronal injury and hypoxia-induced synaptic potentiation. Therefore, the data suggest that reducing the impact of altered function of A1R, CK2/PP2A and A2AR with currently available drugs used in the clinic, may represent an effective neuroprotective therapeutic strategy for ischemic strokes.