Surojit Paul

Regulation of NMDA receptor trafficking by amyloid-|[beta]|

Amyloid-β peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-β reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-β promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-β by treating neurons with a γ-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-β application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-β–dependent endocytosis of NMDA receptors required the α-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-β can cause synaptic dysfunction and contribute to Alzheimer disease pathology.

NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling

The intracellular mechanism(s) by which a cell determines the duration of extracellular signal–regulated kinase (ERK) activation is not well understood. We have investigated the role of STEP, a striatal-enriched tyrosine phosphatase, in the regulation of ERK activity in rat neurons. Glutamate-mediated activation of NMDA receptors leads to the rapid but transient phosphorylation of ERK in cultured neurons. Here we show that activation of NMDA receptors led to activation of STEP, which limited the duration of ERK activity as well as its translocation to the nucleus and its subsequent downstream nuclear signaling. In neurons, STEP is phosphorylated and inactive under basal conditions. NMDA-mediated influx of Ca2+, but not increased intracellular Ca2+ from other sources, leads to activation of the Ca2+-dependent phosphatase calcineurin and the dephosphorylation and activation of STEP. We have identified an important mechanism involved in the regulation of ERK activity in neurons that highlights the complex interplay between serine/threonine and tyrosine kinases and phosphatases.

Tyrosine phosphatase STEP is a tonic brake on induction of long-term potentiation.

The functional roles of protein tyrosine phosphatases (PTPs) in the developed CNS have been enigmatic. Here we show that striatal enriched tyrosine phosphatase (STEP) is a component of the N-methyl-D-aspartate receptor (NMDAR) complex. Functionally, exogenous STEP depressed NMDAR single-channel activity in excised membrane patches. STEP also depressed NMDAR-mediated synaptic currents whereas inhibiting endogenous STEP enhanced these currents. In hippocampal slices, administering STEP into CA1 neurons did not affect basal glutamatergic transmission evoked by Schaffer collateral stimulation but prevented tetanus-induced long-term potentiation (LTP). Conversely, inhibiting STEP in CA1 neurons enhanced transmission and occluded LTP induction through an NMDAR-, Src-, and Ca(2+)-dependent mechanism. Thus, STEP acts as a tonic brake on synaptic transmission by opposing Src-dependent upregulation of NMDARs.

Antibodies against neural, nuclear, cytoskeletal, and streptococcal epitopes in children and adults with Tourette’s syndrome, Sydenham’s chorea, and autoimmune disorders

BACKGROUND Some cases of Tourettes syndrome (TS) are hypothesized to be caused by autoantibodies that develop in response to a preceding group A beta hemolytic streptococcal infection. METHODS To test this hypothesis, we looked for the presence ot total and IgG antibodies against neural, nuclear, cytoskeletal and streptococcal epitopes using indirect immunofluorescent assays and Western blot techniques in three patient groups: TS (n = 81), SC (n = 27), and a group of autoimmune disorders (n = 52) and in normal controls (n = 67). Subjects were ranked after titrations of autoantibodies from 0 to 227 according to their level of immunoreactivity. RESULTS TS patients had a significantly higher mean rank for total antineural and antinuclear antibodies, as well as antistreptolysin O titers. However, among children and adolescents, only the total antinuclear antibodies were increased in TS patients compared to age matched controls. Compared to SC patients, TS patients had a significantly lower mean rank for total and IgG class antineural antibodies, significantly lower IgG class anticytoskeletal antibodies, and a significantly higher rank for total antinuclear antibodies. Compared to a mixed group of autoimmune disorders, the TS patients had a significantly lower mean rank for total and IgG class antineural antibodies, total and IgG class antinuclear antibodies, IgG class anticytoskeletal antibodies, and a significantly higher rank for antistreptococcal antibodies. CONCLUSIONS TS patients had significantly higher levels of total antineural and antinuclear antibodies than did controls. Their relation to IgG class antineural and antinuclear antibodies, markers for prior streptococcal infection, and other clinical characteristics, especially chronological age, was equivocal.

The Dopamine/D1 Receptor Mediates the Phosphorylation and Inactivation of the Protein Tyrosine Phosphatase STEP via a PKA-Dependent Pathway

The striatal-enriched protein tyrosine phosphatase (STEP) family is expressed within dopaminoceptive neurons of the CNS and is particularly enriched within the basal ganglia and related structures. Alternative splicing produces several isoforms that are found in a number of subcellular compartments, including postsynaptic densities of medium spiny neurons. The variants include STEP61, a membrane-associated protein, and STEP46, a cytosolic protein. The C terminals of these two isoforms are identical, whereas the N-terminal domain of STEP61 contains a novel 172 amino acid sequence that includes several structural motifs not present in STEP46. Amino acid sequencing revealed a number of potential phosphorylation sites in both STEP isoforms. Therefore, we investigated the role of phosphorylation in regulating STEP activity. Both STEP61 and STEP46 are phosphorylated on seryl residues by a cAMP-dependent protein kinase (PKA)-mediated pathway in striatal homogenates. The specific residues phosphorylated in STEP61 were identified by site-directed mutagenesis and tryptic phosphopeptide mapping as Ser160 and Ser221, whereas the major site of phosphorylation in STEP46 was shown to be Ser49. Ser160 is located within the unique N terminal of STEP61. Ser221 and Ser49 are equivalent residues present in STEP61 and STEP46, respectively, and are located at the center of the kinase-interacting motif that has been implicated in protein–protein interactions. Phosphorylation at this site decreases the activity of STEP in vitro by reducing its affinity for its substrate. In vivo studies using striatal slices demonstrated that the neurotransmitter dopamine leads to the phosphorylation of STEP via activation of D1 receptors and PKA.

Synaptic plasticity: one STEP at a time

Striatal enriched tyrosine phosphatase (STEP) has recently been identified as a crucial player in the regulation of synaptic function. It is restricted to neurons within the CNS and acts by downregulating the activity of MAP kinases, the tyrosine kinase Fyn and NMDA receptors. By modulating these substrates, STEP acts on several parallel pathways that impact upon the progression of synaptic plasticity. Here, we review recent advances that demonstrate the importance of STEP in normal cognitive function, and its possible involvement in cognitive disorders such as Alzheimers disease.

The Striatal-Enriched Protein Tyrosine Phosphatase Gates Long-Term Potentiation and Fear Memory in the Lateral Amygdala

BACKGROUND Formation of long-term memories is critically dependent on extracellular-regulated kinase (ERK) signaling. Activation of the ERK pathway by the sequential recruitment of mitogen-activated protein kinases is well understood. In contrast, the proteins that inactivate this pathway are not as well characterized. METHODS Here we tested the hypothesis that the brain-specific striatal-enriched protein tyrosine phosphatase (STEP) plays a key role in neuroplasticity and fear memory formation by its ability to regulate ERK1/2 activation. RESULTS STEP co-localizes with the ERKs within neurons of the lateral amygdala. A substrate-trapping STEP protein binds to the ERKs and prevents their nuclear translocation after glutamate stimulation in primary cell cultures. Administration of TAT-STEP into the lateral amygdala (LA) disrupts long-term potentiation (LTP) and selectively disrupts fear memory consolidation. Fear conditioning induces a biphasic activation of ERK1/2 in the LA with an initial activation within 5 minutes of training, a return to baseline levels by 15 minutes, and an increase again at 1 hour. In addition, fear conditioning results in the de novo translation of STEP. Inhibitors of ERK1/2 activation or of protein translation block the synthesis of STEP within the LA after fear conditioning. CONCLUSIONS Together, these data imply a role for STEP in experience-dependent plasticity and suggest that STEP modulates the activation of ERK1/2 during amygdala-dependent memory formation. The regulation of emotional memory by modulating STEP activity may represent a target for the treatment of psychiatric disorders such as posttraumatic stress disorder (PTSD), panic, and anxiety disorders.

Knockout of STriatal enriched protein tyrosine phosphatase in mice results in increased ERK1/2 phosphorylation

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. Synapse 63:69–81, 2009.

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.

NR2B-NMDA receptor-mediated increases in intracellular Ca2+ concentration regulate the tyrosine phosphatase, STEP, and ERK MAP kinase signaling

J. Neurochem. (2010) 114, 1107–1118.