SOX10 and GATA3 in Adenoid Cystic Carcinoma and Polymorphous Adenocarcinoma.

Differentiating between adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAC) can be difficult on small biopsies and cytologic specimens. As such, further characterization of their immunophenotype may aid in distinction. Previous studies have found AdCC to be SOX10+/GATA3 variable and PAC to be GATA3 negative. SOX10 expression in PAC has, as yet, not been established. We performed GATA3 and SOX10 immunohistochemistry on whole sections of 25 cases each of AdCC and PAC (including both classic PAC and the cribriform variant) to assess whether these markers are of diagnostic utility in distinguishing between these entities. SOX10 was found to be positive in 100% of PAC and AdCC whereas GATA 3 was immunoreactive in 45% of AdCCs and 20% of PAC. While this is the first series to compare SOX10 and GATA3 staining in these two tumor types, their frequent expression and similar staining patterns render them of limited value in discriminating between these neoplasms.

Impairment and Restoration of Homeostatic Plasticity in Cultured Cortical Neurons From a Mouse Model of Huntington Disease.

Huntington disease (HD) is an inherited neurodegenerative disorder caused by a mutation in the huntingtin gene. The onset of symptoms is preceded by synaptic dysfunction. Homeostatic synaptic plasticity (HSP) refers to processes that maintain the stability of networks of neurons, thought to be required to enable new learning and cognitive flexibility. One type of HSP is synaptic scaling, in which the strength of all of the synapses onto a cell increases or decreases following changes in the cell's level of activity. Several pathways implicated in synaptic scaling are dysregulated in HD, including brain-derived neurotrophic factor (BDNF) and calcium signaling. Here, we investigated whether HSP is disrupted in cortical neurons from an HD mouse model. We treated cultured cortical neurons from wild-type (WT) FVB/N or YAC128 HD mice with tetrodotoxin (TTX) for 48 h to silence action potentials and then recorded miniature excitatory postsynaptic currents. In WT cultures, these increased in both amplitude and frequency after TTX treatment, and further experiments showed that this was a result of insertion of AMPA receptors and formation of new synapses, respectively. Manipulation of BDNF concentration in the culture medium revealed that BDNF signaling contributed to these changes. In contrast to WT cortical neurons, YAC128 cultures showed no response to action potential silencing. Strikingly, we were able to restore the TTX-induced changes in YAC128 cultures by treating them with pridopidine, a drug which enhances BDNF signaling through stimulation of the sigma-1 receptor (S1R), and with the S1R agonist 3-PPP. These data provide evidence for disruption of HSP in cortical neurons from an HD mouse model that is restored by stimulation of S1R. Our results suggest a potential new direction for developing therapy to mitigate cognitive deficits in HD.

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.

Synapse maturation by activity-dependent ectodomain shedding of SIRPα.

Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of signal regulatory protein-α (SIRPα) mediates presynaptic maturation. Two target-derived molecules, fibroblast growth factor 22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain in turn promotes the maturation of the presynaptic terminal. This process involves calcium/calmodulin-dependent protein kinase, matrix metalloproteinases and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has an impact on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses.

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.

N-methyl-D-aspartate receptor subunit- and neuronal-type dependence of excitotoxic signaling through post-synaptic density 95.

NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.

Interaction of postsynaptic density protein-95 with NMDA receptors influences excitotoxicity in the yeast artificial chromosome mouse model of Huntington's disease.

Evidence suggests that NMDA-type glutamate receptors contribute to degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). Previously, we demonstrated that NMDA receptor (NMDAR)-mediated current and/or toxicity is increased in MSNs from the yeast artificial chromosome (YAC) transgenic mouse model expressing polyglutamine (polyQ)-expanded (mutant) full-length human huntingtin (htt). Others have shown that membrane-associated guanylate kinases (MAGUKs), such as PSD-95 and SAP102, modulate NMDAR surface expression and excitotoxicity in hippocampal and cortical neurons and that htt interacts with PSD-95. Here, we tested the hypothesis that an altered association between MAGUKs and NMDARs in mutant huntingtin-expressing cells contributes to increased susceptibility to excitotoxicity. We show that htt coimmunoprecipitated with SAP102 in HEK293T cells and striatal tissue from wild-type and YAC transgenic mice; however, the association of SAP102 with htt or the NMDAR NR2B subunit was unaffected by htt polyQ length, whereas association of PSD-95 with NR2B in striatal tissue was enhanced by increased htt polyQ length. Treatment of cultured MSNs with Tat-NR2B9c peptide blocked binding of NR2B with SAP102 and PSD-95 and reduced NMDAR surface expression by 20% in both YAC transgenic and wild-type MSNs, and also restored susceptibility to NMDAR excitoxicity in YAC HD MSNs to levels observed in wild-type MSNs; a similar effect on excitotoxicity was observed after knockdown of PSD-95 by small interfering RNA. Unlike previous findings in cortical and hippocampal neurons, rescue of NMDA toxicity by Tat-NR2B9c occurred independently of any effect on neuronal nitric oxide synthase activity. Our results elucidate further the mechanisms underlying enhanced excitotoxicity in HD.

Polyglutamine-modulated striatal calpain activity in YAC transgenic huntington disease mouse model: impact on NMDA receptor function and toxicity.

Huntington disease (HD), caused by CAG expansion in the ubiquitously expressed huntingtin gene, is characterized by early dysfunction and death of striatal medium-sized spiny neurons (MSNs). Previous work has shown MSN-specific alterations in NMDA receptor (NMDAR) expression and cell death signaling. Furthermore, studies in HD human brain tissue and a knock-in mouse model demonstrate increases in calpain activity, which can be stimulated by NMDARs and contribute to excitotoxicity. Here, we report increased calpain activity in MSNs from the yeast artificial chromosome (YAC) transgenic mouse model of HD, expressing human full-length huntingtin with 128 polyglutamine repeats (YAC128), compared with wild type. Moreover, the calpain-cleaved product of NMDAR subunit NR2B is increased early, and NR2B expression levels are reduced, in YAC128 striatum. Although steady-state NMDAR surface expression is similar in wild-type and YAC128 MSNs, the rate of loss of NR2B-containing surface receptors is enhanced in YAC128 MSNs, suggesting that NMDAR forward trafficking to the surface is also faster, as previously reported for YAC72 MSNs. Calpain inhibitor-1 treatment normalized the loss rate of surface NMDARs in YAC128 MSNs to that of wild type, and significantly increased surface NMDAR expression in YAC128, but not in wild type or YAC72. With acute NMDAR overstimulation, the increase in calpain activity correlated with polyglutamine length, and calpain inhibitor treatment reduced NMDA-induced apoptosis in YAC72 and YAC128 MSNs to wild-type levels. Thus, the cumulative effect of increasing huntingtin polyglutamine length is to enhance MSN sensitivity to excitotoxicity at least in part by calpain-mediated cell death signaling.

Altered NMDA receptor trafficking in a yeast artificial chromosome transgenic mouse model of Huntington's disease.

Overactivation of NMDA receptors (NMDARs) is believed to play a role in degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). This hereditary disorder is caused by an expansion >35 in the polyglutamine (polyQ) region of the protein huntingtin (htt). Previous work has shown that NMDAR current, calcium signaling, and/or toxicity are enhanced in striatal MSNs in a variety of transgenic mice and cellular models of HD, but whether the enhancement is specific for MSNs or correlated with mutant htt (mhtt) polyQ length is not known. Furthermore, the mechanism underlying the increase in NMDAR activity has not been elucidated. Here we report polyQ length-dependent enhancement of peak NMDAR current density by mhtt in cultured MSNs, but not cortical neurons, from the yeast artificial chromosome (YAC) transgenic HD mouse model. We also observed a shift of NMDAR subunits NR1 and NR2B from internal pools to the plasma membrane and a significantly faster rate of NMDAR insertion to the surface in YAC72 MSNs. In comparing YAC72 with wild-type striatal tissue, subcellular fractionation revealed a relative enrichment of NR1 C2'-containing NMDARs in the vesicle/microsome-enriched fraction, and coimmunoprecipitation experiments demonstrated an increased proportion of NR1 C2' isoforms associated with NR2 subunits, which may contribute to faster forward trafficking of these receptors. Our results suggest that altered NMDAR trafficking may underlie potentiation of NMDAR-mediated current and toxicity in the YAC72 HD mouse model. This polyQ length-dependent, neuronal-specific change in NMDAR activity induced by mhtt may contribute to selective neuronal degeneration in HD.