Opposing roles of synaptic and extrasynaptic NMDA receptor signaling in cocultured striatal and cortical neurons.

The NMDAR plays a unique and vital role in subcellular signaling. Calcium influx initiates signaling cascades important for both synaptic plasticity and survival; however, overactivation of the receptor leads to toxicity and cell death. This dichotomy is partially explained by the subcellular location of the receptor. NMDARs located at the synapse stimulate cell survival pathways, while extrasynaptic receptors signal for cell death. Thus far, this interplay between synaptic and extrasynaptic NMDARs has been studied exclusively in cortical (CTX) and hippocampal neurons. It was unknown whether other cell types, such as GABAergic medium-sized spiny projection neurons of the striatum (MSNs), which bear the brunt of neurodegeneration in Huntington's disease, follow the same pattern. Here we report synaptic versus extrasynaptic NMDAR signaling in striatal MSNs and resultant activation of cAMP response element binding protein (CREB), in rat primary corticostriatal cocultures. Similarly to CTX, we found in striatal MSNs that synaptic NMDARs activate CREB, whereas extrasynaptic NMDARs dominantly oppose CREB activation. However, MSNs are much less susceptible to NMDA-mediated toxicity than CTX cells and show differences in subcellular GluN2B distribution. Blocking NMDARs with memantine (30 µm) or GluN2B-containing receptors with ifenprodil (3 µm) prevents CREB shutoff effectively in CTX and MSNs, and also rescues both neuronal types from NMDA-mediated toxicity. This work may provide cell and NMDAR subtype-specific targets for treatment of diseases with putative NMDAR involvement, including neurodegenerative disorders and ischemia.

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.

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.

Population-scale analysis of common and rare genetic variation associated with hearing loss in adults.

To better understand the genetics of hearing loss, we performed a genome-wide association meta-analysis with 125,749 cases and 469,497 controls across five cohorts. We identified 53/c loci affecting hearing loss risk, including common coding variants in COL9A3 and TMPRSS3. Through exome sequencing of 108,415 cases and 329,581 controls, we observed rare coding associations with 11 Mendelian hearing loss genes, including additive effects in known hearing loss genes GJB2 (Gly12fs; odds ratio [OR] = 1.21, P = 4.2 × 10-11) and SLC26A5 (gene burden; OR = 1.96, P = 2.8 × 10-17). We also identified hearing loss associations with rare coding variants in FSCN2 (OR = 1.14, P = 1.9 × 10-15) and KLHDC7B (OR = 2.14, P = 5.2 × 10-30). Our results suggest a shared etiology between Mendelian and common hearing loss in adults. This work illustrates the potential of large-scale exome sequencing to elucidate the genetic architecture of common disorders where both common and rare variation contribute to risk.

A Role for the Locus Coeruleus in Hippocampal CA1 Place Cell Reorganization during Spatial Reward Learning.

During spatial learning, hippocampal (HPC) place maps reorganize to represent new goal locations, but little is known about the circuit mechanisms facilitating these changes. Here, we examined how neuromodulation via locus coeruleus (LC) projections to HPC area CA1 (LC-CA1) regulates the overrepresentation of CA1 place cells near rewarded locations. Using two-photon calcium imaging, we monitored the activity of LC-CA1 fibers in the mouse dorsal HPC. We find that the LC-CA1 projection signals the translocation of a reward, predicting behavioral performance on a goal-oriented spatial learning task. An optogenetic stimulation mimicking this LC-CA1 activity induces place cell reorganization around a familiar reward, while its inhibition decreases the degree of overrepresentation around a translocated reward. Our results show that LC acts in conjunction with other factors to induce goal-directed reorganization of HPC representations and provide a better understanding of the role of neuromodulatory actions on HPC place map plasticity.

In-Depth Qualitative Interviews to Explore Healthy Environment Strategies in Food Pantries in the Western United States.

BACKGROUND: Food insecurity and chronic disease are compounded by poverty. A growing number of low-income Americans obtain food regularly from food pantry agencies to combat food insecurity. Evidence demonstrates that food environments may affect healthy dietary behaviors. Food pantry environmental interventions may improve diet quality of low-income, food insecure populations by encouraging healthy food choices. OBJECTIVE: The two a priori exploratory research questions were: What strategies do food pantries and food banks undertake to implement healthy environments affecting the physical space or how clients are treated; and, What challenges do pantries and food banks face during these efforts? DESIGN: Interview data were collected March through May 2016 via in-depth telephone interviews and respondents were recruited by purposive and snowball sampling. PARTICIPANTS AND SETTINGS: Respondents were 43 key informants who represented food bank distributors (n=16), food pantries (n=14), community partners (n=6), and antihunger advocates (n=2) within the 13 western US states. MAIN OUTCOME MEASURES: Strategies to improve the healthfulness of food pantry environments were defined and their corresponding processes described. STATISTICAL ANALYSES PERFORMED: A thematic and descriptive analysis approach was used to elucidate healthy environment strategies. Data were analyzed by inductive modeling and network application of descriptive process codes to address the research question. RESULTS: Seven strategies to address the emotional and physical dimensions of a food pantry environment were identified and described. Associated challenges (n=12), characteristics of initiation (n=9), and evaluation methods (n=7) were revealed. CONCLUSIONS: Findings indicate that these strategies overlap in novel ways and further examination is warranted. Future quantitative research should assess the prevalence of these strategies in a larger sample of food pantries.

Development and Testing of the Healthy Food Pantry Assessment Tool.

OBJECTIVE: To develop and test an observational survey that quantifies food pantry environments (FPE). DESIGN: Best practices in FPE were identified through key informant interviews. The tool was pilot-tested, including a content review, and then field-tested for reliability. SETTING: Key informant phone interviews (n = 41); pilot and field test visits occurred at 45 pantries from multiple states. SUBJECTS: Food bank/pantry staff and nutrition educators were recruited for interviews through purposive and snowball sampling. Pilot and field test survey users (n = 65) were food pantry representatives and matched community partners who both rated the FPE using the tool. VARIABLES MEASURED: Pearson correlation was used to determine test-retest and interrater reliability. ANALYSIS: Qualitative data were coded for healthy FPE strategies. Quantitative data were calculated using descriptive statistics (significant at P < .05). RESULTS: Qualitative data were coded for observable FPE characteristics. Reliability scores were substantial to nearly perfect for 48 of 61 survey items (79%) for test-retest and 49 of 61 (80%) for interrater reliability (Pearson r = .6-1.0). CONCLUSIONS AND IMPLICATIONS: The Healthy Food Pantry Assessment Tool is research-tested and can be used to evaluate and quantify the healthfulness of FPE.

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.