Small organic molecule disruptors of Cav3.2 - USP5 interactions reverse inflammatory and neuropathic pain.

BACKGROUND: Cav3.2 channels facilitate nociceptive transmission and are upregulated in DRG neurons in response to nerve injury or peripheral inflammation. We reported that this enhancement of Cav3.2 currents in afferent neurons is mediated by deubiquitination of the channels by the deubiquitinase USP5, and that disrupting USP5/Cav3.2 channel interactions protected from inflammatory and neuropathic pain. RESULTS: Here we describe the development of a small molecule screening assay for USP5-Cav3.2 disruptors, and report on two hits of a ~5000 compound screen - suramin and the flavonoid gossypetin. In mouse models of inflammatory pain and neuropathic pain, both suramin and gossypetin produced dose-dependent and long-lasting mechanical anti-hyperalgesia that was abolished or greatly attenuated in Cav3.2 null mice. Suramin and Cav3.2/USP5 Tat-disruptor peptides were also tested in models of diabetic neuropathy and visceral pain, and provided remarkable protection. CONCLUSIONS: Overall, our findings provide proof of concept for a new class of analgesics that target T-type channel deubiquitination.

Alterations in STriatal-Enriched protein tyrosine Phosphatase expression, activation, and downstream signaling in early and late stages of the YAC128 Huntington's disease mouse model.

Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre-symptomatic (excitotoxin-sensitive) transgenic mice expressing full-length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex-NMDAR) localization and signaling. Furthermore, Ex-NMDAR stimulation facilitates excitotoxicity in wild-type cortical neurons via calpain-mediated cleavage of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen-activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain-mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal-regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6-week-old, excitotoxin-sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA-induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR-post-synaptic density protein-95 (PSD-95) binding but not by calpain inhibition. In 1-year-old excitotoxin-resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR-PSD-95 interactions contributes to elevated p38 signaling in early, excitotoxin-sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin-resistant stages. The YAC128 Huntington's disease mouse model shows early, enhanced susceptibility to NMDA receptor-mediated striatal apoptosis, progressing to late-stage excitotoxicity resistance. This study shows that elevated NMDA receptor-PSD-95 interactions as well as decreased extrasynaptic STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61) activation may contribute to early enhanced apoptotic signaling. In late-stage YAC128 mice, reduced STEP61 levels and activity correlate with elevated MAPK signaling, consistent with excitotoxicity resistance. Solid and dotted arrows indicate conclusions drawn from the current study and other literature, respectively.

Chronic blockade of extrasynaptic NMDA receptors ameliorates synaptic dysfunction and pro-death signaling in Huntington disease transgenic mice.

In the YAC128 mouse model of Huntington disease (HD), elevated extrasynaptic NMDA receptor (Ex-NMDAR) expression contributes to the onset of striatal dysfunction and atrophy. A shift in the balance of synaptic-extrasynaptic NMDAR signaling and localization is paralleled by early stage dysregulation of intracellular calcium signaling pathways, including calpain and p38 MAPK activation, that couple to pro-death cascades. However, whether aberrant calcium signaling is a consequence of elevated Ex-NMDAR expression in HD is unknown. Here, we aimed to identify calcium-dependent pathways downstream of Ex-NMDARs in HD. Chronic (2-month) treatment of YAC128 and WT mice with memantine (1 and 10mg/kg/day), which at a low dose selectively blocks Ex-NMDARs, reduced striatal Ex-NMDAR expression and current in 4-month old YAC128 mice without altering synaptic NMDAR levels. In contrast, calpain activity was not affected by memantine treatment, and was elevated in untreated YAC128 mice at 1.5months but not 4months of age. In YAC128 mice, memantine at 1mg/kg/day rescued CREB shut-off, while both doses suppressed p38 MAPK activation to WT levels. Taken together, our results indicate that Ex-NMDAR activity perpetuates increased extrasynaptic NMDAR expression and drives dysregulated p38 MAPK and CREB signaling in YAC128 mice. Elucidation of the pathways downstream of Ex-NMDARs in HD could help provide novel therapeutic targets for this disease.

Mitigation of augmented extrasynaptic NMDAR signaling and apoptosis in cortico-striatal co-cultures from Huntington's disease mice.

We recently reported evidence for disturbed synaptic versus extrasynaptic NMDAR transmission in the early pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin. Studies in glutamatergic cells indicate that synaptic NMDAR transmission increases phosphorylated cyclic-AMP response element binding protein (pCREB) levels and drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation reduces pCREB and promotes cell death. By generating striatal and cortical neuronal co-cultures to investigate the glutamatergic innervation of striatal neurons, we demonstrate that dichotomous synaptic and extrasynaptic NMDAR signaling also occurs in GABAergic striatal medium-sized spiny neurons (MSNs), which are acutely vulnerable in HD. Further, we show that wild-type (WT) and HD transgenic YAC128 MSNs co-cultured with cortical cells have similar levels of glutamatergic synapses, synaptic NMDAR currents and synaptic GluN2B and GluN2A subunit-containing NMDARs. However, NMDAR whole-cell, and especially extrasynaptic, current is elevated in YAC128 MSNs. Moreover, GluN2B subunit-containing NMDAR surface expression is markedly increased, irrespective of whether or not the co-cultured cortical cells express mutant huntingtin. The data suggest that MSN cell-autonomous increases in extrasynaptic NMDARs are driven by the HD mutation. Consistent with these results, we find that extrasynaptic NMDAR-induced pCREB reductions and apoptosis are also augmented in YAC128 MSNs. Moreover, both NMDAR-mediated apoptosis and CREB-off signaling are blocked by co-application of either memantine or the GluN2B subunit-selective antagonist ifenprodil in YAC128 MSNs. GluN2A-subunit-selective concentrations of the antagonist NVP-AAM077 did not reduce cell death in either genotype. Cortico-striatal co-cultures provide an in vitro model system in which to better investigate striatal neuronal dysfunction in disease than mono-cultured striatal cells. Results from the use of this system, which partially recapitulates the cortico-striatal circuit and is amenable to acute genetic and pharmacological manipulations, suggest that pathophysiological NMDAR signaling is an intrinsic frailty in HD MSNs that can be successfully targeted by pharmacological interventions.

Calpain and STriatal-Enriched protein tyrosine phosphatase (STEP) activation contribute to extrasynaptic NMDA receptor localization in a Huntington's disease mouse model.

In Huntington's disease (HD), the mutant huntingtin (mhtt) protein is associated with striatal dysfunction and degeneration. Excitotoxicity and early synaptic defects are attributed, in part, to altered NMDA receptor (NMDAR) trafficking and function. Deleterious extrasynaptic NMDAR localization and signalling are increased early in yeast artificial chromosome mice expressing full-length mhtt with 128 polyglutamine repeats (YAC128 mice). NMDAR trafficking at the plasma membrane is regulated by dephosphorylation of the NMDAR subunit GluN2B tyrosine 1472 (Y1472) residue by STriatal-Enriched protein tyrosine Phosphatase (STEP). NMDAR function is also regulated by calpain cleavage of the GluN2B C-terminus. Activation of both STEP and calpain is calcium-dependent, and disruption of calcium homeostasis occurs early in the HD striatum. Here, we show increased calpain cleavage of GluN2B at both synaptic and extrasynaptic sites, and elevated extrasynaptic total GluN2B expression in the YAC128 striatum. Calpain inhibition significantly reduced extrasynaptic GluN2B expression in the YAC128 but not wild-type striatum. Furthermore, calpain inhibition reduced whole-cell NMDAR current and the surface/internal GluN2B ratio in co-cultured striatal neurons, without affecting synaptic GluN2B localization. Synaptic STEP activity was also significantly higher in the YAC128 striatum, correlating with decreased GluN2B Y1472 phosphorylation. A substrate-trapping STEP protein (TAT-STEP C-S) significantly increased VGLUT1-GluN2B colocalization, as well as increasing synaptic GluN2B expression and Y1472 phosphorylation. Moreover, combined calpain inhibition and STEP inactivation reduced extrasynaptic, while increasing synaptic GluN2B expression in the YAC128 striatum. These results indicate that increased STEP and calpain activation contribute to altered NMDAR localization in an HD mouse model, suggesting new therapeutic targets for HD.

P38 MAPK is involved in enhanced NMDA receptor-dependent excitotoxicity in YAC transgenic mouse model of Huntington disease.

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.

Mechanisms underlying NMDA receptor synaptic/extrasynaptic distribution and function.

Research over the last few decades has shaped our understanding of the crucial involvement of the N-methyl-D-aspartate receptor (NMDAR) in mediating excitatory synaptic neurotransmission, neuronal development and learning and memory. The complexity of NMDAR modulation has escalated with the knowledge that receptors can traffic between synaptic and extrasynaptic sites, and that location on the plasma membrane profoundly affects the physiological function of NMDARs. Moreover, mechanisms that regulate NMDAR subcellular localization and function, such as protein-protein interactions, phosphorylation, palmitoylation, ubiquitination and receptor proteolytic cleavage, may differ for synaptic and extrasynaptic NMDARs. Recent studies suggest that NMDAR mislocalization is a dominant contributing factor to glutamatergic dysfunction and pathogenesis in neurological disorders such as Huntington's disease, Alzheimer's disease and ischemia. Therapeutic approaches that specifically rectify receptor mislocalization or target resulting downstream apoptotic signaling could be beneficial for preventing disease onset or progression across many disorders that are commonly caused by NMDAR dysfunction. This review will summarize the molecular mechanisms that regulate synaptic and extrasynaptic NMDAR localization in both physiologic and pathogenic states.

Early increase in extrasynaptic NMDA receptor signaling and expression contributes to phenotype onset in Huntington's disease mice.

N-methyl-D-aspartate receptor (NMDAR) excitotoxicity is implicated in the pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder. However, NMDARs are poor therapeutic targets, due to their essential physiological role. Recent studies demonstrate that synaptic NMDAR transmission drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation promotes cell death. We report specifically increased extrasynaptic NMDAR expression, current, and associated reductions in nuclear CREB activation in HD mouse striatum. The changes are observed in the absence of dendritic morphological alterations, before and after phenotype onset, correlate with mutation severity, and require caspase-6 cleavage of mutant huntingtin. Moreover, pharmacological block of extrasynaptic NMDARs with memantine reversed signaling and motor learning deficits. Our data demonstrate elevated extrasynaptic NMDAR activity in an animal model of neurodegenerative disease. We provide a candidate mechanism linking several pathways previously implicated in HD pathogenesis and demonstrate successful early therapeutic intervention in mice.

Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms.

The ability to modify synaptic transmission between neurons is a fundamental process of the nervous system that is involved in development, learning, and disease. Thus, synaptic plasticity is the ability to bidirectionally modify transmission, where long-term potentiation and long-term depression (LTD) represent the best characterized forms of plasticity. In the hippocampus, two main forms of LTD coexist that are mediated by activation of either N-methyl-d-aspartic acid receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). Compared with NMDAR-LTD, mGluR-LTD is less well understood, but recent advances have started to delineate the underlying mechanisms. mGluR-LTD at CA3:CA1 synapses in the hippocampus can be induced either by synaptic stimulation or by bath application of the group I selective agonist (R,S)-3,5-dihydroxyphenylglycine. Multiple signaling mechanisms have been implicated in mGluR-LTD, illustrating the complexity of this form of plasticity. This review provides an overview of recent studies investigating the molecular mechanisms underlying hippocampal mGluR-LTD. It highlights the role of key molecular components and signaling pathways that are involved in the induction and expression of mGluR-LTD and considers how the different signaling pathways may work together to elicit a persistent reduction in synaptic transmission.

Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

The tyrosine phosphatase STEP mediates AMPA receptor endocytosis after metabotropic glutamate receptor stimulation.

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. DHPG-induced 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.

Characterization of the delta2 glutamate receptor-binding protein delphilin: Splicing variants with differential palmitoylation and an additional PDZ domain.

The glutamate receptor delta2 (GluRdelta2) is predominantly expressed at parallel fiber-Purkinje cell postsynapses and plays crucial roles in synaptogenesis and synaptic plasticity. Although the mechanism by which GluRdelta2 functions remains unclear, its lack of channel activity and its role in controlling the endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors have suggested that GluRdelta2 may convey signals by interacting with intracellular signaling molecules. Among several proteins that interact with GluRdelta2, delphilin is unique in that it is selectively expressed at parallel fiber-Purkinje cell synapses and that, in addition to a single PDZ domain, it contains a formin homology domain that is thought to regulate actin dynamics. Here, we report a new isoform of delphilin, designated as L-delphilin, that has alternatively spliced N-terminal exons encoding an additional PDZ domain. Although original delphilin, designated S-delphilin, was palmitoylated at the N terminus, this region was spliced out in L-delphilin. As a result, S-delphilin was associated with plasma membranes in COS cells and dendritic spines in hippocampal neurons, whereas L-delphilin formed clusters in soma and dendritic shafts. In addition, S-delphilin, but not L-delphilin, facilitated the expression of GluRdelta2 on the cell surface. These results indicate that, like PSD-95 and GRIP/ABP, delphilin isoforms with differential palmitoylation and clustering capabilities may provide two separate intracellular and surface GluRdelta2 pools and may control GluRdelta2 signaling in Purkinje cells.

Tyrosine phosphatases regulate AMPA receptor trafficking during metabotropic glutamate receptor-mediated long-term depression.

Two forms of long-term depression (LTD), triggered by activation of NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), respectively, can be induced at CA1 synapses in the hippocampus. Compared with NMDAR-LTD, relatively little is known about mGluR-LTD. Here, we show that protein tyrosine phosphatase (PTP) inhibitors, orthovanadate and phenylarsine oxide, selectively block mGluR-LTD induced by application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG-LTD), because NMDAR-LTD is unaffected by these inhibitors. Furthermore, DHPG-LTD measured using whole-cell recording is similarly blocked by either bath-applied or patch-loaded PTP inhibitors. These inhibitors also block the changes in paired-pulse facilitation and coefficient of variation that are associated with the expression of DHPG-LTD. DHPG treatment of hippocampal slices was associated with a decrease in the level of tyrosine phosphorylation of GluR2 AMPA receptor (AMPAR) subunits, an effect blocked by orthovanadate. Finally, in dissociated hippocampal neurons, orthovanadate blocked the ability of DHPG to reduce the number of AMPA receptor clusters on the surface of dendrites. Again, the effects of PTP blockade were selective, because NMDA-induced decreases in surface AMPAR clusters was unaffected by orthovanadate. Together, these data suggest that activation of postsynaptic PTP results in tyrosine dephosphorylation of AMPARs and their removal from the synapse.