The striatal-enriched phosphatase (STEP) is a component of the NMDA-receptor-mediated excitotoxic signaling pathway, which plays a key role in ischemic brain injury. Using neuronal cultures and a rat model of ischemic stroke, we show that STEP plays an initial role in neuroprotection, during the insult, by disrupting the p38 MAPK pathway. Degradation of active STEP during reperfusion precedes ischemic brain damage and is associated with secondary activation of p38 MAPK. Application of a cell-permeable STEP-derived peptide that is resistant to degradation and binds to p38 MAPK protects cultured neurons from hypoxia-reoxygenation injury and reduces ischemic brain damage when injected up to 6 h after the insult. Conversely, genetic deletion of STEP in mice leads to sustained p38 MAPK activation and exacerbates brain injury and neurological deficits after ischemia. Administration of the STEP-derived peptide at the onset of reperfusion not only prevents the sustained p38 MAPK activation but also reduces ischemic brain damage in STEP KO mice. The findings indicate a neuroprotective role of STEP and suggest a potential role of the STEP-derived peptide in stroke therapy.
Edited by Roger J. Colbran Homocysteine, a metabolite of the methionine cycle, is a known agonist of N-methyl-D-aspartate receptor (NMDAR), a glutamate receptor subtype and is involved in NMDAR-mediated neurotoxicity. Our previous findings have shown that homocysteine-induced, NMDAR-mediated neurotoxicity is facilitated by a sustained increase in phosphorylation and activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK). In the current study, we investigated the role GluN1/GluN2A-containing functional NMDAR (GluN2A-NMDAR) and GluN1/GluN2B-containing functional NMDAR (GluN2B-NMDAR) in homocysteine-induced neurotoxicity. Our findings revealed that exposing primary cortical neuronal cultures to homocysteine leads to a sustained low-level increase in intracellular Ca 2؉ . We also showed that pharmacological inhibition of GluN2A-NMDAR or genetic deletion of the GluN2A subunit attenuates homocysteine-induced increase in intracellular Ca 2؉ . Our results further established the role of GluN2A-NMDAR in homocysteine-mediated sustained ERK MAPK phosphorylation and neuronal cell death. Of note, the preferential role of GluN2A-NMDAR in homocysteine-induced neurotoxicity was distinctly different from glutamate-NMDAR-induced excitotoxic cell death that involves overactivation of GluN2B-NMDAR and is independent of ERK MAPK activation. These findings indicate a critical role of GluN2A-NMDAR-mediated signaling in homocysteine-induced neurotoxicity.
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.
Excessive release of Zn 2؉ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn 2؉ -induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn 2؉ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn 2؉ -mediated phosphorylation of STEP 61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP 61 at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP 61 toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn 2؉ -induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn 2؉ -dependent neurotoxicity. The strong correlation between the temporal profile of STEP 61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP 61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn 2؉ . The study provides further insight into the mechanisms of regulation of STEP 61 and also offers a molecular basis for the Zn 2؉ -induced sustained activation of ERK2.A growing body of evidence indicates that Zn 2ϩ , the second most abundant transition metal in the brain, is released at high concentrations during excitotoxic neurological conditions and can contribute to neuronal injury (1-7). The proposed mechanisms associated with Zn 2ϩ -dependent neurotoxicity include alteration of activity of a diverse group of post-synaptic receptors like ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 3 N-methyl-D-aspartic acid (NMDA), voltagegated calcium channels and neurotrophic receptors (7-12). Receptor activation in turn modulates the function of several intracellular kinases including protein kinase C, mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (ERK MAPK), and the Src family of tyrosine kinases resulting in impaired energy production, excitability and oxidative stress (8,9,(13)(14)(15). However, the role of phosphatases in Zn 2ϩ -induced neurotoxicity is currently not well understood.The intracellular tyrosine phosphatase, STEP (striatalenriched tyrosine phosphatase, also known as PTPN5) is expressed exclusively in the central nervous system (16,17) and is emerging as a key regulator of neuronal survival and death. The STEP-family of PTPs includes both membrane-assoc...
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 ¶
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