Ranjana Poddar

Homocysteine–NMDA receptor-mediated activation of extracellular signal-regulated kinase leads to neuronal cell death

Hyperhomocysteinemia is an independent risk factor for stroke and neurological abnormalities. However, the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor‐mediated activation of the extracellular signal‐regulated kinase‐mitogen‐activated protein (ERK‐MAP) kinase pathway in homocysteine‐dependent neurotoxicity. The study demonstrates that in neurons l‐homocysteine‐induced cell death was mediated through activation of NMDA receptors. The study also shows that homocysteine‐dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK‐MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine‐mediated neuronal cell death thereby demonstrating that activation of ERK‐MAP kinase signaling pathway is an intermediate step that couples homocysteine‐mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response‐element binding protein (CREB), a pro‐survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine‐mediated neurotoxicity.

Intranuclear matrix metalloproteinases promote DNA damage and apoptosis induced by oxygen-glucose deprivation in neurons.

Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood-brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen-glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons during acute stroke injury.

NR2B‐NMDA receptor mediated modulation of the tyrosine phosphatase STEP regulates glutamate induced neuronal cell death

J. Neurochem. (2010) 115, 1350–1362.

Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine-NMDA receptor mediated neuronal cell death

Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor‐mediated activation of extracellular signal‐regulated kinase (ERK)‐MAPK in homocysteine‐induced neuronal cell death. In this study, we examined the involvement of the stress‐induced MAPK, p38 in homocysteine‐induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine‐mediated NMDA receptor stimulation and subsequent Ca2+ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine‐induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine‐NMDA receptor‐induced neuronal cell death.

Oxidative stress-induced oligomerization inhibits the activity of the non-receptor tyrosine phosphatase STEP61.

J. Neurochem. (2011) 116, 1097–1111.

Neuroprotective Role of a Brain-Enriched Tyrosine Phosphatase, STEP, in Focal Cerebral Ischemia

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.

Dephosphorylation of specific sites in the kinase-specificity sequence domain leads to ubiquitin-mediated degradation of the tyrosine phosphatase STEP.

STEP (striatal-enriched phosphatase) is a non-receptor tyrosine phosphatase that is specifically expressed in the neurons of the central nervous system. STEP regulates the activity of several effector molecules involved in synaptic plasticity and neuronal cell survival, including MAPKs (mitogen-activated protein kinases), Src family kinases and NMDA (N-methyl-D-aspartic acid) receptors. The critical role of STEP in regulating these effectors requires that its activity be tightly regulated. Previous studies have demonstrated that the activity of STEP is regulated through reversible phosphorylation of a serine residue within the KIM (kinase-interacting motif), by cAMP-dependent PKA (protein kinase A). In the present paper we show that STEP is endogenously phosphorylated at two additional sites located within the KISs (kinase-specificity sequences). The basal activity of ERK (extracellular-signal-regulated kinase) and p38 MAPKs plays an important role in the phosphorylation of these two sites. Dephosphorylation of these two sites leads to polyubiquitination and proteolytic degradation of STEP. Conversely, the proteasome inhibitors MG-132 and epoxomicin can stabilize STEP. The active form of STEP is more susceptible to degradation than the inactive form. Taken together the results of the present paper establish that ubiquitin-dependent proteolysis could be a novel mechanism for irreversibly terminating the activity of STEP.

Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP: MOLECULAR BASIS FOR ZN2+-INDUCED ERK MAPK ACTIVATION.

Excessive release of Zn2+ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn2+-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 Zn2+ 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. Zn2+-mediated phosphorylation of STEP61 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 STEP61 at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP61 toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn2+-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn2+-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 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 Zn2+. The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn2+-induced sustained activation of ERK2.

Role of AMPA receptors in homocysteine-NMDA receptor-induced crosstalk between ERK and p38 MAPK

Homocysteine, a metabolite of the methionine cycle has been reported to play a role in neurotoxicity through activation of N‐methyl‐d‐aspartate receptors (NMDAR)‐mediated signaling pathway. The proposed mechanisms associated with homocysteine‐NMDAR‐induced neurotoxicity involve a unique signaling pathway that triggers a crosstalk between extracellular signal‐regulated kinase (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. This study investigates whether α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (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 have 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 1‐napthyl acetyl spermine trihydrochloride (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 2,3‐dioxo‐6‐nitro‐1,2,3,4,tetrahydrobenzoquinoxaline‐7‐sulfonamide (NBQX)/6‐cyano‐7‐nitroquinoxaline‐2,3, ‐dione (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.

A peptide mimetic of tyrosine phosphatase STEP as a potential therapeutic agent for treatment of cerebral ischemic stroke

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.