NMDA receptor (NMDAR)-mediated excitotoxicity plays an important role in several CNS disorders, including epilepsy, stroke, and ischemia. Here we demonstrate the involvement of striatal-enriched protein tyrosine phosphatase (STEP) in this critical process. STEP 61 is an alternatively spliced member of the family that is present in postsynaptic terminals. In an apparent paradox, STEP 61 regulates extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, two proteins with opposing functions; activated p38 promotes cell death, whereas activated ERK1/2 promotes cell survival. We found that synaptic stimulation of NMDARs promoted STEP 61 ubiquitination and degradation, concomitant with ERK1/2 activation. In contrast, extrasynaptic stimulation of NMDARs invoked calpain-mediated proteolysis of STEP 61 , producing the truncated cleavage product STEP 33 and activation of p38. The calpain cleavage site on STEP was mapped to the kinase interacting motif, a domain required for substrate binding. As a result, STEP 33 neither interacts with nor dephosphorylates STEP substrates. A synthetic peptide spanning the calpain cleavage site efficiently reduced STEP 61 degradation and attenuated p38 activation and cell death in slice models. Furthermore, this peptide was neuroprotective when neurons were subjected to excitotoxicity or cortical slices were exposed to ischemic conditions. These findings suggest a novel mechanism by which differential NMDAR stimulation regulates STEP 61 to promote either ERK1/2 or p38 activation and identifies calpain cleavage of STEP 61 as a valid target for the development of neuroprotective therapy.

Amyloid ␤ (A␤) is involved in the etiology of Alzheimer's disease (AD) and may contribute to cognitive deficits by increasing internalization of ionotropic glutamate receptors. Striatal-enriched protein tyrosine phosphatase 61 (STEP 61 ), which is targeted in part to the postsynaptic terminal, has been implicated in this process. Here we show that STEP 61 levels are progressively increased in the cortex of Tg2576 mice over the first year, as well as in prefrontal cortex of human AD brains. The increased STEP 61 was associated with greater STEP activity, dephosphorylation of phospho-tyr 1472 of the NR2B subunit, and decreased NR1 and NR2B subunits on neuronal membranes. Treatment with A␤-enriched medium also increased STEP 61 levels and decreased NR1/NR2B abundance in mouse cortical cultures as determined by biotinylation experiments. In STEP knock-out cultures, A␤ treatment failed to induce NMDA receptor internalization. The mechanism for the increase in STEP 61 levels appears to involve the ubiquitin proteasome system. Blocking the proteasome resulted in elevated levels of STEP 61 . Moreover, STEP 61 -ubiquitin conjugates were increased in wild-type cortical slices upon A␤ treatment as well as in 12 month Tg2576 cortex. These findings reveal a novel mechanism by which A␤-mediated accumulation of STEP 61 results in increased internalization of NR1/NR2B receptor that may contribute to the cognitive deficits in AD.

Although it is well established that AMPA receptor (AMPAR) trafficking is a central event in several forms of synaptic plasticity, the mechanisms that regulate the surface expression of AMPARs are poorly understood. Previous work has shown that striatal-enriched protein tyrosine phosphatase (STEP) mediates NMDAR endocytosis. This protein tyrosine phosphatase is enriched in the synapses of the striatum, hippocampus, cerebral cortex, and other brain regions. In the present investigation, we have explored whether STEP also regulates AMPAR internalization. We found that (RS)-3,5-dihydroxyphenylglycine (DHPG) stimulation triggered a dose-dependent increase in STEP translation in hippocampal slices and synaptoneurosomes, a process that requires stimulation of mGluR5 (metabotropic glutamate receptor 5) and activation of mitogen-activated protein kinases and phosphoinositide-3 kinase pathways. DHPGinduced AMPAR internalization and tyrosine dephosphorylation of GluR2 (glutamate receptor 2) was blocked by a substrate-trapping TAT-STEP [C/S] protein in hippocampal slices and cultures. Moreover, DHPG-triggered AMPAR internalization was abolished in STEP knock-out mice and restored after replacement of wild-type STEP. These results suggest a role for STEP in the regulation of AMPAR trafficking.

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder. Early in the pathophysiology of AD, synaptic function is disrupted by soluble Aβ oligomers, possibly through Aβ-mediated internalization of NMDA receptors. Striatal-enriched phosphatase (STEP) is a tyrosine phosphatase that regulates the internalization of NMDA receptors. Recent work shows that STEP is elevated in the prefrontal cortex of human AD patients and in animal models of AD. Here, we use genetic manipulations to reduce STEP activity in a triple transgenic AD mouse model and show that a decrease in STEP levels reverses cognitive and cellular deficits observed in these mice. Our results suggest that STEP inhibitors may prove therapeutic for this devastating disorder.β amyloid | glutamate receptor trafficking | protein tyrosine phosphatase | long-term potentiation A lzheimer's disease (AD) is the most common form of dementia. Considerable evidence implicates β amyloid (Aβ) peptides in the pathophysiology of AD (1). Aβ 1-42 is derived from sequential cleavage of amyloid procursor protein (APP) by β-and γ-secretases. A recent hypothesis suggests that soluble Aβ oligomers disrupt synaptic and cognitive function early in the disease process (2-4). This model is supported by the findings that synaptic function is disrupted and synapses are lost early in the disorder and in animal models, even before amyloid plaques are detected (5-7). Application of soluble Aβ preparations results in synapse loss, blocks long-term potentiation (LTP), and impairs cognitive function in animals (8-11). Furthermore, animals that express high levels of Aβ show impaired synaptic plasticity and learning (6,12,13).Striatal-enriched phosphatase 61 (STEP 61 ; protein tyrosine phosphatase non-receptor 5 [PTPN5]) is a brain-specific tyrosine phosphatase targeted to synaptic compartments in striatum, hippocampus, cortex, and related brain regions (14-16). STEP 61 associates with the NMDA subclass of glutamate receptors, decreases NMDA receptor (NMDAR) activity, and opposes the induction of LTP through dephosphorylation of Y 1472 on the NR2B receptor subunit, leading to endocytosis of NR1/NR2B receptors (17, 18). Acute reduction in STEP expression by RNAi leads to increased surface expression and function of NR1/NR2B receptors (19), and STEP knockout (STEP −/− ) mice have enhanced hippocampal LTP (SI Appendix, Fig. S1). In addition, STEP 61 dephosphorylates a regulatory tyrosine within the activation loop of two enzymes critical for the development of synaptic strengthening, ERK1/2 and Fyn, thereby inactivating them (20,21). Together, these findings support the current model that STEP activity opposes the development of synaptic strengthening (22).Elevated levels of STEP 61 are found in several transgenic AD mouse models as well as human AD prefrontal cortex (Tg-2576 and J20) (23, 24). The increase in STEP 61 levels and activity contributes to the removal of NR1/NR2B complexes from synapses (24). Given that STEP 61 regulates Aβ-induced internalization of NR1/...

STriatal Enriched protein tyrosine Phosphatase (STEP) is a brain-specific protein that is thought to play a role in synaptic plasticity. This hypothesis is based on previous findings demonstrating a role for STEP in the regulation of the extracellular signal-regulated kinase1/2 (ERK1/2). We have now generated a STEP knockout mouse and investigated the effect of knocking out STEP in the regulation of ERK1/2 activity. Here, we show that the STEP knockout mice are viable and fertile and have no detectable cytoarchitectural abnormalities in the brain. The homozygous knockout mice lack the expression of all STEP isoforms, whereas the heterozygous mice have reduced STEP protein levels when compared with the wild-type mice. The STEP knockout mice show enhanced phosphorylation of ERK1/2 in the striatum, CA2 region of the hippocampus, as well as central and lateral nuclei of the amygdala. In addition, the cultured neurons from KO mice showed significantly higher levels of pERK1/2 following synaptic stimulation when compared with wild-type controls. These data demonstrate more conclusively the role of STEP in the regulation of ERK1/2 activity.