Homocysteine, a metabolite of the methionine cycle has been reported to play a role in neurotoxicity through activation of NMDAR-mediated signaling pathway. The proposed mechanisms associated with homocysteine-NMDAR induced neurotoxicity involve a unique signaling pathway that triggers a crosstalk between ERK and p38 MAPKs, where activation of p38 MAPK is downstream of and dependent on ERK MAPK. However, the molecular basis of the ERK MAPK mediated p38 MAPK activation is not understood. The current study investigates whether AMPARs play a role in facilitating the ERK MAPK mediated p38 MAPK activation. Using surface biotinylation and immunoblotting approaches we show that treatment with homocysteine leads to a decrease in surface expression of GluA2-AMPAR subunit in neurons, but has no effect on the surface expression of GluA1-AMPAR subunit. Inhibition of NMDAR activation with D-AP5 or ERK MAPK phosphorylation with PD98059 attenuates homocysteine-induced decrease in surface expression of GluA2-AMPAR subunit. The decrease in surface expression of GluA2-AMPAR subunit is associated with p38 MAPK phosphorylation, which is inhibited by NASPM, a selective antagonist of GluA2-lacking Ca2+-permeable AMPARs. These results suggest that homocysteine-NMDAR mediated ERK MAPK phosphorylation leads to a decrease in surface expression of GluA2-AMPAR subunit resulting in Ca2+ influx through the GluA2-lacking Ca2+-permeable AMPARs and p38 MAPK phosphorylation. Cell death assays further show that inhibition of AMPAR activity with NBQX/CNQX or GluA2-lacking Ca2+-permeable AMPAR activity with NASPM attenuates homocysteine-induced neurotoxicity. We have identified an important mechanism involved in homocysteine-induced neurotoxicity that highlights the intermediary role of GluA2-lacking Ca2+-permeable AMPARs in the crosstalk between ERK and p38 MAPKs.

Hyperhomocysteinemia has been implicated in several neurodegenerative disorders including ischemic stroke. However, the pathological consequences of ischemic insult in individuals predisposed to hyperhomocysteinemia and the associated etiology are unknown. In this study, we evaluated the outcome of transient ischemic stroke in a rodent model of hyperhomocysteinemia, developed by subcutaneous implantation of osmotic pumps containing L-homocysteine into male Wistar rats. Our findings show a 42.3% mortality rate in hyperhomocysteinemic rats as compared to 7.7% in control rats. Magnetic resonance imaging of the brain in the surviving rats shows that mild hyperhomocysteinemia leads to exacerbation of ischemic injury within 24h, which remains elevated over time. Behavioral studies further demonstrate significant deficit in sensorimotor functions in hyperhomocysteinemic rats compared to control rats. Using pharmacological inhibitors targeting the NMDAR subtypes, the study further demonstrates that inhibition of GluN2A-containing NMDARs significantly reduces ischemic brain damage in hyperhomocysteinemic rats but not in control rats, indicating that hyperhomocysteinemiamediated exacerbation of ischemic brain injury involves GluN2A-NMDAR signaling. Complementary studies in GluN2A-knockout mice show that in the absence of GluN2A-NMDARs, hyperhomocysteinemia-associated exacerbation of ischemic brain injury is blocked, confirming that GluN2A-NMDAR activation is a critical determinant of the severity of ischemic ¶

Extensive research over the last two decades has advanced our understanding of the pathophysiology of ischemic stroke. However, current pharmacologic therapies are still limited to rapid reperfusion using thrombolytic agents, and neuroprotective approaches that can reduce the consequences of ischemic and reperfusion injury, are still not available. To bridge this gap, we have evaluated the long-term efficacy and therapeutic time window of a novel peptide-based neuroprotectant TAT-STEP, derived from the brain-enriched and neuron-specific tyrosine phosphatase STEP. Using a rat model of transient middle cerebral artery occlusion (90 min), we show that a single intravenous administration of the peptide at the onset of reperfusion (early) or 6 h after the onset of the insult (delayed) reduces mortality rate. In the surviving rats, MRI scans of the brain at days 1, 14 and 28 after the insult show significant reduction in infarct size and improvement of structural integrity within the infarcted area following peptide treatment, regardless of the time of administration. Behavioral assessments show significant improvement in normal gait, motor coordination, sensory motor function and spatial memory following early or delayed peptide treatment. The study establishes for the first time the therapeutic potential of a tyrosine phosphatase in ischemic brain injury.

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