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Background: Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer's disease (AD) pathology. Amyloid beta (Aβ) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aβ activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors., Methods: The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca 2+ ]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aβ 1-42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain., Results: Acute applications of human amylin or Aβ 1-42 resulted in an increase in [Ca 2+ ]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1β, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1β. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls., Conclusion: Microglial amylin receptors mediate Aβ-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Emotional enhancement of memory by noradrenergic mechanisms is well-described, but the long-term consequences of such enhancement are poorly understood. Over time, memory traces are thought to undergo a neural reorganization, that is, a systems consolidation, during which they are, at least partly, transferred from the hippocampus to neocortical networks. This transfer is accompanied by a decrease in episodic detailedness. Here we investigated whether norepinephrine (NE) administration into the basolateral amygdala after training on an inhibitory avoidance discrimination task, comprising two distinct training contexts, alters systems consolidation dynamics to maintain episodic-like accuracy and hippocampus dependency of remote memory. At a 2-d retention test, both saline- and NE-treated rats accurately discriminated the training context in which they had received footshock. Hippocampal inactivation with muscimol before retention testing disrupted discrimination of the shock context in both treatment groups. At 28 d, saline-treated rats showed hippocampus-independent retrieval and lack of discrimination. In contrast, NE-treated rats continued to display accurate memory of the shock-context association. Hippocampal inactivation at this remote retention test blocked episodic-like accuracy and induced a general memory impairment. These findings suggest that the NE treatment altered systems consolidation dynamics by maintaining hippocampal involvement in the memory. This shift in systems consolidation was paralleled by time-regulated DNA methylation and transcriptional changes of memory-related genes, namely Reln and Pkm ζ, in the hippocampus and neocortex. The findings provide evidence suggesting that consolidation of emotional memories by noradrenergic mechanisms alters systems consolidation dynamics and, as a consequence, influences the maintenance of long-term episodic-like accuracy of memory., Competing Interests: The authors declare no conflict of interest.

The identification of genes related to encoding, storage, and retrieval of memories is a major interest in neuroscience. In the current study, we analyzed the temporal gene expression changes in a neuronal mRNA pool during an olfactory long-term associative memory (LTAM) in Caenorhabditis elegans hermaphrodites. Here, we identified a core set of 712 (538 upregulated and 174 downregulated) genes that follows three distinct temporal peaks demonstrating multiple gene regulation waves in LTAM. Compared with the previously published positive LTAM gene set (Lakhina et al., 2015), 50% of the identified upregulated genes here overlap with the previous dataset, possibly representing stimulus-independent memory-related genes. On the other hand, the remaining genes were not previously identified in positive associative memory and may specifically regulate aversive LTAM. Our results suggest a multistep gene activation process during the formation and retrieval of long-term memory and define general memory-implicated genes as well as conditioning-type-dependent gene sets. SIGNIFICANCE STATEMENT The identification of genes regulating different steps of memory is of major interest in neuroscience. Identification of common memory genes across different learning paradigms and the temporal activation of the genes are poorly studied. Here, we investigated the temporal aspects of Caenorhabditis elegans gene expression changes using aversive olfactory associative long-term memory (LTAM) and identified three major gene activation waves. Like in previous studies, aversive LTAM is also CREB dependent, and CREB activity is necessary immediately after training. Finally, we define a list of memory paradigm-independent core gene sets as well as conditioning-dependent genes., (Copyright © 2017 the authors 0270-6474/17/376661-12$15.00/0.)

Increasing age is tightly linked to decreased thickness of the human neocortex. The biological mechanisms that mediate this effect are hitherto unknown. The DNA methylome, as part of the epigenome, contributes significantly to age-related phenotypic changes. Here, we identify an epigenetic signature that is associated with cortical thickness (P=3.86 × 10 -8 ) and memory performance in 533 healthy young adults. The epigenetic effect on cortical thickness was replicated in a sample comprising 596 participants with major depressive disorder and healthy controls. The epigenetic signature mediates partially the effect of age on cortical thickness (P<0.001). A multilocus genetic score reflecting genetic variability of this signature is associated with memory performance (P=0.0003) in 3,346 young and elderly healthy adults. The genomic location of the contributing methylation sites points to the involvement of specific immune system genes. The decomposition of blood methylome-wide patterns bears considerable potential for the study of brain-related traits.

Background: Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression., Methods: We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function., Results: FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems., Conclusion: FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated., Competing Interests: The authors have declared that no competing interests exist.

DNA methylation represents an important link between structural genetic variation and complex phenotypes. The study of genome-wide CpG methylation and its relation to traits relevant to psychiatry has become increasingly important. Here, we analyzed quality metrics of 394,043 CpG sites in two samples of 568 and 319 mentally healthy young adults. For 25% of all CpGs we observed medium to large common epigenetic variation. These CpGs were overrepresented in open sea and shore regions, as well as in intergenic regions. They also showed a strong enrichment of significant hits in association analyses. Furthermore, a significant proportion of common DNA methylation is at least partially genetically driven and thus may be observed similarly across tissues. These findings could be of particular relevance for studies of complex neuropsychiatric traits, which often rely on proxy tissues., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control. In the light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis, and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.

A plastic nervous system requires the ability not only to acquire and store but also to forget. Here, we report that musashi (msi-1) is necessary for time-dependent memory loss in C. elegans. Tissue-specific rescue demonstrates that MSI-1 function is necessary in the AVA interneuron. Using RNA-binding protein immunoprecipitation (IP), we found that MSI-1 binds to mRNAs of three subunits of the Arp2/3 actin branching regulator complex in vivo and downregulates ARX-1, ARX-2, and ARX-3 translation upon associative learning. The role of msi-1 in forgetting is also reflected by the persistence of learning-induced GLR-1 synaptic size increase in msi-1 mutants. We demonstrate that memory length is regulated cooperatively through the activation of adducin (add-1) and by the inhibitory effect of msi-1. Thus, a GLR-1/MSI-1/Arp2/3 pathway induces forgetting and represents a novel mechanism of memory decay by linking translational control to the structure of the actin cytoskeleton in neurons., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The subcellular distribution of the G protein-coupled receptor GPR37 affects cell viability and is implicated in the pathogenesis of parkinsonism. Intracellular accumulation and aggregation of GPR37 cause cell death, whereas GPR37 located in the plasma membrane provides cell protection. We define here a pathway through which the recently identified natural ligand, prosaposin, promotes plasma membrane association of GPR37. Immunoabsorption of extracellular prosaposin reduced GPR37(tGFP) surface density and decreased cell viability in catecholaminergic N2a cells. We found that GPR37(tGFP) partitioned in GM1 ganglioside-containing lipid rafts in the plasma membrane of live cells. This partitioning required extracellular prosaposin and was disrupted by lipid raft perturbation using methyl-β-cyclodextrin or cholesterol oxidase. Moreover, complex formation between GPR37(tGFP) and the GM1 marker cholera toxin was observed in the plasma membrane. These data show functional association between GPR37, prosaposin, and GM1 in the plasma membrane. These results thus tie together the three previously defined components of the cellular response to insult. Our findings identify a mechanism through which the receptor's natural ligand and GM1 may protect against toxic intracellular GPR37 aggregates observed in parkinsonism.

Background: Ethanol affects the signaling of several important neurotransmitter and neuromodulator systems in the CNS. It has been recently proposed that ethanol alters the dynamic lateral organization of proteins and lipids in the plasma membrane, thereby affecting surface receptor-mediated cellular signaling. Our aims are to establish whether pharmacologically relevant levels of ethanol can affect the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscopic level, and investigate the relevance of such perturbations for mu-opioid receptor (MOP) function., Methodology/principal Findings: We used Photoactivated Localization Microscopy with pair-correlation analysis (pcPALM), a quantitative fluorescence imaging technique with high spatial resolution (15-25 nm) and single-molecule sensitivity, to study ethanol effects on protein organization in the plasma membrane. We observed that short (20 min) exposure to 20 and 40 mM ethanol alters protein organization in the plasma membrane of cells that harbor endogenous MOPs, causing a rearrangement of the lipid raft marker glycosylphosphatidylinositol (GPI). These effects could be largely occluded by pretreating the cells with the MOP antagonist naltrexone (200 nM for 3 hours). In addition, ethanol induced pronounced actin polymerization, leading to its partial co-localization with GPI., Conclusions/significance: Pharmacologically relevant levels of ethanol alter the lateral organization of GPI-linked proteins and induce actin cytoskeleton reorganization. Pretreatment with the MOP antagonist naltrexone is protective against ethanol action and significantly reduces the extent to which ethanol remodels the lateral organization of lipid-rafts-associated proteins in the plasma membrane. Super-resolution pcPALM reveals details of ethanol action at the nanoscale level, giving new mechanistic insight on the cellular and molecular mechanisms of its action.

Identifying molecular mechanisms that underlie learning and memory is one of the major challenges in neuroscience. Taken the advantages of the nematode Caenorhabditis elegans, we investigated α-adducin (add-1) in aversive olfactory associative learning and memory. Loss of add-1 function selectively impaired short- and long-term memory without causing acquisition, sensory, or motor deficits. We showed that α-adducin is required for consolidation of synaptic plasticity, for sustained synaptic increase of AMPA-type glutamate receptor (GLR-1) content and altered GLR-1 turnover dynamics. ADD-1, in a splice-form- and tissue-specific manner, controlled the storage of memories presumably through actin-capping activity. In support of the C. elegans results, genetic variability of the human ADD1 gene was significantly associated with episodic memory performance in healthy young subjects. Finally, human ADD1 expression in nematodes restored loss of C. elegans add-1 gene function. Taken together, our findings support a role for α-adducin in memory from nematodes to humans. Studying the molecular and genetic underpinnings of memory across distinct species may be helpful in the development of novel strategies to treat memory-related diseases.

Background: Studies at the behavioral and synaptic level show that effects of ethanol on the central nervous system can involve the opioid signaling system. These interactions may alter the function of a common downstream target. In this study, we examined Ca(2+) channel function as a potential downstream target of interactions between ethanol and μ or κ opioid receptor signaling., Methods: The studies were carried out in a model system, undifferentiated PC12 cells transfected with μ or κ opioid receptors. The PC12 cells express L-type Ca(2+) channels, which were activated by K(+) depolarization. Ca(2+) imaging was used to measure relative Ca(2+) flux during K(+) depolarization and the modulation of Ca(2+) flux by opioids and ethanol., Results: Ethanol, μ receptor activation, and κ receptor activation all reduced the amplitude of the Ca(2+) signal produced by K(+) depolarization. Pretreatment with ethanol or combined treatment with ethanol and μ or κ receptor agonists caused a reduction in the amplitude of the Ca(2+) signal that was comparable to or smaller than that observed for the individual drugs alone, indicating an interaction by the drugs at a downstream target (or targets) that limited the modulation of Ca(2+) flux through L-type Ca(2+) channels., Conclusions: These studies provide evidence for a cellular mechanism that could play an important role in ethanol regulation of synaptic transmission and behavior through interactions with the opioid signaling., (Copyright © 2011 by the Research Society on Alcoholism.)

The genetic basis of phenotypic variation can be partially explained by the presence of copy-number variations (CNVs). Currently available methods for CNV assessment include high-density single-nucleotide polymorphism (SNP) microarrays that have become an indispensable tool in genome-wide association studies (GWAS). However, insufficient concordance rates between different CNV assessment methods call for cautious interpretation of results from CNV-based genetic association studies. Here we provide a cross-population, microarray-based map of copy-number variant regions (CNVRs) to enable reliable interpretation of CNV association findings. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to scan the genomes of 1167 individuals from two ethnically distinct populations (Europe, N=717; Rwanda, N=450). Three different CNV-finding algorithms were tested and compared for sensitivity, specificity, and feasibility. Two algorithms were subsequently used to construct CNVR maps, which were also validated by processing subsamples with additional microarray platforms (Illumina 1M-Duo BeadChip, Nimblegen 385K aCGH array) and by comparing our data with publicly available information. Both algorithms detected a total of 42669 CNVs, 74% of which clustered in 385 CNVRs of a cross-population map. These CNVRs overlap with 862 annotated genes and account for approximately 3.3% of the haploid human genome.We created comprehensive cross-populational CNVR-maps. They represent an extendable framework that can leverage the detection of common CNVs and additionally assist in interpreting CNV-based association studies.

Homeotic (Hox) genes encode transcription factors that confer segmental identity along the anteroposterior axis of the embryo. However the molecular mechanisms underlying Hox-mediated transcription and the differential requirements for specificity in the regulation of the vast number of Hox-target genes remain ill-defined. Here we show that synthetic Sex combs reduced (Scr) genes that encode the Scr C terminus containing the homedomain (HD) and YPWM motif (Scr-HD) are functional in vivo. Synthetic Scr-HD peptides can induce ectopic salivary glands in the embryo and homeotic transformations in the adult fly, act as transcriptional activators and repressors during development, and participate in protein-protein interactions. Their transformation capacity was found to be enhanced over their full-length counterpart and mutations known to transform the full-length protein into constitutively active or inactive variants behaved accordingly in the synthetic peptides. Our results show that synthetic Scr-HD genes are sufficient for homeotic function in Drosophila and suggest that the N terminus of Scr has a role in transcriptional potency, rather than specificity. We also demonstrate that synthetic peptides behave largely in a predictable way, by exhibiting Scr-specific phenotypes throughout development, which makes them an important tool for synthetic biology.

Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to "titrate" the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K(d)) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-d-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. Here, we show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) noncovalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR-gated currents produced by DYNp. They attenuate DYNp-evoked cell death in spinal cord and prevent, as well as reverse, DYNp-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury.

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.