ABSTRACT
Release of testosterone during early development is necessary for masculinization of brain structures in rodents. Wu et al. (2009) now elucidate the neuronal changes caused by aromatase-mediated conversion of testosterone into estrogen in perinatal mice and the ensuing effects on adult behavior.
Subject(s)
Brain/physiology , Estrogens/biosynthesis , Sex Characteristics , Animals , Behavior, AnimalABSTRACT
The male bias in the incidence of autism spectrum disorders (ASDs) is one of the most notable characteristics of this group of neurodevelopmental disorders. The etiology of this sex bias is far from known, but pivotal for understanding the etiology of ASDs in general. Here we investigate whether a "three-hit" (genetic load × environmental factor × sex) theory of autism may help explain the male predominance. We found that LPS-induced maternal immune activation caused male-specific deficits in certain social responses in the contactin-associated protein-like 2 (Cntnap2) mouse model for ASD. The three "hits" had cumulative effects on ultrasonic vocalizations at postnatal day 3. Hits synergistically affected social recognition in adulthood: only mice exposed to all three hits showed deficits in this aspect of social behavior. In brains of the same mice we found a significant three-way interaction on corticotropin-releasing hormone receptor-1 (Crhr1) gene expression, in the left hippocampus specifically, which co-occurred with epigenetic alterations in histone H3 N-terminal lysine 4 trimethylation (H3K4me3) over the Crhr1 promoter. Although it is highly likely that multiple (synergistic) interactions may be at work, change in the expression of genes in the hypothalamic-pituitary-adrenal/stress system (e.g., Crhr1) is one of them. The data provide proof-of-principle that genetic and environmental factors interact to cause sex-specific effects that may help explain the male bias in ASD incidence.
Subject(s)
Autism Spectrum Disorder/genetics , Disease Models, Animal , Gene-Environment Interaction , Social Behavior , Animals , Autism Spectrum Disorder/metabolism , Brain/metabolism , Epigenesis, Genetic , Female , Humans , Hypothalamo-Hypophyseal System/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Pituitary-Adrenal System/metabolism , Receptors, Corticotropin-Releasing Hormone/genetics , Receptors, Corticotropin-Releasing Hormone/metabolism , Sex FactorsABSTRACT
Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells.
Subject(s)
APOBEC-1 Deaminase/metabolism , Epigenesis, Genetic , RNA Editing , Transcriptome , APOBEC-1 Deaminase/genetics , Animals , Cell Movement , Cells, Cultured , Mice , Mice, Inbred C57BL , Monocyte-Macrophage Precursor Cells/cytology , Monocyte-Macrophage Precursor Cells/metabolism , PhagocytosisABSTRACT
Microglia (MG), a heterogeneous population of phagocytic cells, play important roles in central nervous system (CNS) homeostasis and neural plasticity. Under steady-state conditions, MG maintain homeostasis by producing antiinflammatory cytokines and neurotrophic factors, support myelin production, and remove synapses and cellular debris, as well as participating in "cross-correction," a process that supplies neurons with key factors for executing autophagy-lysosomal function. As sentinels for the immune system, MG also detect "danger" signals (pathogenic or traumatic insult), become activated, produce proinflammatory cytokines, and recruit monocytes and dendritic cells to the site of damage through a breached blood-brain barrier or via brain lymphatics. Failure to effectively resolve MG activation can be problematic and can lead to chronic inflammation, a condition proposed to underlie CNS pathophysiology in heritable brain disorders and age-related neurodegenerative and cognitive decline. Here, we show that APOBEC1-mediated RNA editing occurs within MG and is key to maintaining their resting status. Like bone marrow-derived macrophages, RNA editing in MG leads to overall changes in the abundance of edited proteins that coordinate the function of multiple cellular pathways. Conversely, mice lacking the APOBEC1 editing function in MG display evidence of dysregulation, with progressive age-related signs of neurodegeneration, characterized by clustering of activated MG, aberrant myelination, increased inflammation, and lysosomal anomalies that culminate in behavioral and motor deficiencies. Collectively, our study identifies posttranscriptional modification by RNA editing as a critical regulatory mechanism of vital cellular functions that maintain overall brain health.
Subject(s)
APOBEC-1 Deaminase/genetics , Aging/pathology , Brain/metabolism , Microglia/metabolism , RNA Editing , APOBEC-1 Deaminase/metabolism , Aging/metabolism , Animals , Brain/growth & development , Brain/pathology , Lysosomes/metabolism , Lysosomes/ultrastructure , Male , Mice , Microglia/ultrastructure , Myelin Sheath/metabolismABSTRACT
Certain components and functions of the immune system, most notably cytokine production and immune cell migration, are under circadian regulation. Such regulation suggests that circadian rhythms may have an effect on disease onset, progression, and resolution. In the vesicular stomatitis virus (VSV)-induced encephalitis model, the replication, caudal penetration, and survivability of intranasally applied VSV depends on both innate and adaptive immune mechanisms. In the current study, we investigated the effect of circadian time of infection on the progression and outcome of VSV-induced encephalitis and demonstrated a significant decrease in the survival rate in mice infected at the start of the rest cycle, zeitgeber time 0 (ZT0). The lower survival rate in these mice was associated with higher levels of circulating chemokine (C-C motif) ligand 2 (CCL2), a greater number of peripherally derived immune cells accumulating in the olfactory bulb (OB), and increased production of proinflammatory cytokines, indicating an immune-mediated pathology. We also found that the acrophase of molecular circadian clock component REV-ERBα mRNA expression in the OB coincides with the start of the active cycle, ZT12, when VSV infection results in a more favorable outcome. This result led us to hypothesize that REV-ERBα may mediate the circadian effect on survival following VSV infection. Blocking REV-ERBα activity before VSV administration resulted in a significant increase in the expression of CCL2 and decreased survival in mice infected at the start of the active cycle. These data demonstrate that REV-ERBα-mediated inhibition of CCL2 expression during viral-induced encephalitis may have a protective effect.
Subject(s)
Circadian Rhythm/immunology , Encephalitis/metabolism , Nuclear Receptor Subfamily 1, Group D, Member 1/physiology , Vesiculovirus/immunology , Adaptive Immunity , Animals , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Cytokines/genetics , Cytokines/metabolism , Encephalitis/virology , Gene Expression , Male , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/immunology , Mortality , Up-Regulation/immunologyABSTRACT
Stress plays a substantial role in shaping behavior and brain function, often with lasting effects. How these lasting effects occur in the context of a fixed postmitotic neuronal genome has been an enduring question for the field. Synaptic plasticity and neurogenesis have provided some of the answers to this question, and more recently epigenetic mechanisms have come to the fore. The exploration of epigenetic mechanisms recently led us to discover that a single acute stress can regulate the expression of retrotransposons in the rat hippocampus via an epigenetic mechanism. We propose that this response may represent a genomic stress response aimed at maintaining genomic and transcriptional stability in vulnerable brain regions such as the hippocampus. This finding and those of other researchers have made clear that retrotransposons and the genomic plasticity they permit play a significant role in brain function during stress and disease. These observations also raise the possibility that the transposome might have adaptive functions at the level of both evolution and the individual organism.
Subject(s)
Epigenesis, Genetic/physiology , Gene Expression Regulation/physiology , Hippocampus/metabolism , Models, Biological , Retroelements/physiology , Steroids/metabolism , Stress, Physiological/physiology , Animals , Neuronal Plasticity/physiology , RatsABSTRACT
Multiple peptide systems, including neuropeptide Y, leptin, ghrelin, and others, are involved with the control of food intake and body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body weight. In the present study, we used a combination of ligand-binding and receptor-activity assays to characterize a Gαi/o protein-coupled receptor activated by BigLEN in the mouse hypothalamus and Neuro2A cells. We then selected orphan G protein-coupled receptors expressed in the hypothalamus and Neuro2A cells and tested each for activation by BigLEN. G protein-coupled receptor 171 (GPR171) is activated by BigLEN, but not by the C terminally truncated peptide LittleLEN. The four C-terminal amino acids of BigLEN are sufficient to bind and activate GPR171. Overexpression of GPR171 leads to an increase, and knockdown leads to a decrease, in binding and signaling by BigLEN and the C-terminal peptide. In the hypothalamus GPR171 expression complements the expression of BigLEN, and its level and activity are elevated in mice lacking BigLEN. In mice, shRNA-mediated knockdown of hypothalamic GPR171 leads to a decrease in BigLEN signaling and results in changes in food intake and metabolism. The combination of GPR171 shRNA together with neutralization of BigLEN peptide by antibody absorption nearly eliminates acute feeding in food-deprived mice. Taken together, these results demonstrate that GPR171 is the BigLEN receptor and that the BigLEN-GPR171 system plays an important role in regulating responses associated with feeding and metabolism in mice.
Subject(s)
Body Weight/physiology , Feeding Behavior/physiology , Neuropeptides/metabolism , Receptors, G-Protein-Coupled/metabolism , Analysis of Variance , Animals , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Immunohistochemistry , MAP Kinase Signaling System/physiology , Mice , Mice, Inbred C57BL , Phosphorylation , Real-Time Polymerase Chain ReactionABSTRACT
The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB(1)R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB(1)R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB(1)R is upregulated. We found a significant upregulation of AT1R-CB(1)R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB(1)R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.
Subject(s)
Angiotensin II/chemistry , Angiotensin II/metabolism , Receptor, Angiotensin, Type 1/chemistry , Receptor, Angiotensin, Type 1/metabolism , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/metabolism , Angiotensin II/genetics , Animals , Cell Communication/genetics , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/chemistry , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , HEK293 Cells , Humans , Ligands , MAP Kinase Signaling System/genetics , Mice , Mice, Inbred BALB C , Rats , Receptor Cross-Talk/physiology , Receptor, Angiotensin, Type 1/genetics , Receptor, Cannabinoid, CB1/geneticsABSTRACT
This study reveals the presence of dendritic cells (DCs) in the pituitary gland, which play a role in communicating immune activation to the hypothalamic pituitary adrenal (HPA) axis. Using enhanced yellow fluorescent protein (eyfp) expression as a reporter for CD11c, a marker of DCs, we demonstrate anatomically the presence of CD11c/eyfp+ cells throughout the pituitary. Flow cytometric analysis shows that the predominant cellular phenotype of pituitary CD11c/eyfp+ cells resembles that of non-lymphoid DCs. In vivo and in vitro immune challenge with lipopolysaccharide (LPS) stimulates these pituitary CD11c/eyfp+ DCs, but not eyfp(neg) cells, to increase levels of pro-inflammatory cytokines, IL-6, IL-1ß, and TNF-α. In vivo analysis of plasma glucocorticoid (GC) and adrenocorticotropic hormone (ACTH) levels at this early phase of the immune response to LPS suggest that pro-inflammatory cytokine production by DCs within the pituitary may activate the release of GCs from the adrenals via ACTH. Pituitary CD11c/eyfp+ cells also express annexin A1 (ANXA1), indicating a role in GC signal attenuation. In summary, our data demonstrate that a resident DC population of the pituitary gland coordinates GC release in the early phase of systemic immune activation, thereby providing an essential immune signaling sentinel for the initial shaping of the systemic immune response to LPS.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , Encephalitis/immunology , Encephalitis/metabolism , Pituitary Gland/immunology , Pituitary Gland/metabolism , Animals , Annexin A1/metabolism , Bacterial Proteins/metabolism , CD11c Antigen/metabolism , Cytokines/metabolism , Dendritic Cells/cytology , Encephalitis/chemically induced , Female , Lipopolysaccharides , Luminescent Proteins/metabolism , Mice, Transgenic , Pituitary Gland/cytology , Signal TransductionABSTRACT
Elevated levels of thyroid hormones (TH) reduce estradiol (E2)-dependent female sexual behavior. E2 stimulates progesterone receptor (Pgr) and oxytocin receptor (Oxtr) within the ventromedial hypothalamus and preoptic area, critical hypothalamic nuclei for sexual and maternal behavior, respectively. Here, we investigated the impact of TH on E2-dependent transcriptional mechanisms in female mice. First, we observed that triiodothyronine (T3) inhibited the E2 induction of Pgr and Oxtr. We hypothesized that differences in histone modifications and receptor recruitment could explain the influence of TH on E2-responsive Pgr and Oxtr expression. We observed that histone H3 acetylation (H3Ac) and methylation (H3K4me3) was gene and brain-region specific. We then analyzed the recruitment of estrogen receptor α (ERα) and TH receptor α (TRα) on the putative regulatory sequences of Pgr and Oxtr. Interestingly, T3 inhibited E2-induced ERα binding to a specific Pgr enhancer site, whereas TRα binding was not affected, corroborating our theory that the competitive binding of TRα to an ERα binding site can inhibit ERα transactivation and the subsequent E2-responsive gene expression. On the Oxtr promoter, E2 and T3 worked together to modulate ERα and TRα binding. Finally, the E2-dependent induction of cofactors was reduced by hypothyroidism and T3. Thus, we determined that the Pgr and Oxtr promoter regions are responsive to E2 and that T3 interferes with the E2 regulation of Pgr and Oxtr expression by altering the recruitment of receptors to DNA and changing the availability of cofactors. Collectively, our findings provide insights into molecular mechanisms of response to E2 and TH interactions controlling sex behavior in the hypothalamus.
Subject(s)
Estrogen Receptor alpha/metabolism , Hypothalamus/metabolism , Preoptic Area/metabolism , Receptors, Oxytocin/metabolism , Receptors, Progesterone/metabolism , Acetylation/drug effects , Animals , DNA Methylation/drug effects , DNA Methylation/physiology , Estradiol/metabolism , Female , Histones/drug effects , Histones/metabolism , Hormones/pharmacology , Hypothalamus/drug effects , Mice , Preoptic Area/drug effects , Promoter Regions, Genetic , Receptors, Oxytocin/genetics , Receptors, Progesterone/genetics , Thyroid Hormone Receptors alpha/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/physiology , Triiodothyronine/pharmacologyABSTRACT
Expression of sexually dimorphic behaviors critical for reproduction depends on the organizational actions of steroid hormones on the developing brain. We offer the new hypothesis that transcriptional activities in brain regions executing these sexually dimorphic behaviors are modulated by estrogen-induced modifications of histone proteins. Specifically, in preoptic nerve cells responsible for facilitating male sexual behavior in rodents, gene expression is fostered by increased histone acetylation and reduced methylation (Me), and, that the opposite set of histone modifications will be found in females. Conversely, in ventromedial hypothalamic neurons that are responsible for coordinating female sexual behavior, transcriptional activities in genetic females are fostered by increased histone acetylation and reduced Me, and, further, that the opposite set of histone modifications will be found in males. Thus, these epigenetic events will guarantee that effects of sex hormone exposure during the neonatal critical period will be translated into lasting sex differences in adult reproductive behaviors.
Subject(s)
Brain/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Sex Differentiation , Sexual Behavior, Animal/physiology , Sexual Behavior/physiology , Animals , Female , Humans , Male , Models, Biological , Nervous System/metabolism , Sex Characteristics , Transcription, GeneticABSTRACT
Hematopoietic stem cell transplantation (HSCT) is a therapy used for multiple malignant and nonmalignant diseases, with chemotherapy used for pretransplantation myeloablation. The post-HSCT brain contains peripheral engrafted parenchymal macrophages, despite their absence in the normal brain, with the engraftment mechanism still undefined. Here we show that HSCT chemotherapy broadly disrupts mouse brain regenerative populations, including a permanent loss of adult neurogenesis. Microglial density was halved, causing microglial process expansion, coinciding with indicators of broad senescence. Although microglia expressed cell proliferation markers, they underwent cell cycle arrest in S phase with a majority expressing the senescence and antiapoptotic marker p21. In vivo single-cell tracking of microglia after recovery from chemical depletion showed loss of their regenerative capacity, subsequently replaced with donor macrophages. We propose that HSCT chemotherapy causes microglial senescence with a gradual decrease to a critical microglial density, providing a permissive niche for peripheral macrophage engraftment of the brain.
Subject(s)
Hematopoietic Stem Cell Transplantation , Microglia , Animals , Brain , Macrophages , Mice , Transplantation ConditioningABSTRACT
Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS-deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon-optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV-transduced cells showed supraphysiological levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells, and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS-engrafted animals were restored to 10-20% than that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of ex vivo MNDU3-IDS LVV-transduced HSPCs for treatment of MPS II.
Subject(s)
Iduronate Sulfatase , Mucopolysaccharidosis II , Animals , Mice , Humans , Mucopolysaccharidosis II/genetics , Mucopolysaccharidosis II/therapy , Leukocytes, Mononuclear , Iduronate Sulfatase/genetics , Enzyme Replacement Therapy , Disease Models, Animal , Hematopoietic Stem CellsABSTRACT
Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.
Subject(s)
Aspartic Acid Endopeptidases/biosynthesis , Aspartic Acid Endopeptidases/chemistry , Endothelins/chemistry , Enzyme Assays/methods , Fluorescent Dyes/chemistry , Gene Expression Regulation, Enzymologic/physiology , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/chemistry , Peptides/chemistry , Animals , Aspartic Acid Endopeptidases/administration & dosage , Aspartic Acid Endopeptidases/genetics , Endothelin-Converting Enzymes , Endothelins/genetics , Endothelins/metabolism , Enzyme Inhibitors/chemistry , Humans , Metalloendopeptidases/administration & dosage , Metalloendopeptidases/genetics , Mice , Organ Specificity , Peptide Library , Peptides/genetics , Peptides/metabolism , Substrate SpecificityABSTRACT
An increasing body of evidence suggests that endothelin-converting enzyme-2 (ECE-2) is a non-classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE-2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE-2 exhibits a non-classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE-2 cleaves a number of neuropeptides at non-classical sites in vitro; however its role in peptide processing in vivo is poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE-2 knockout (KO) mice might show altered pain and morphine responses compared with wild-type mice. We find that ECE-2 KO mice show decreased response to a single injection of morphine in hot-plate and tail-flick tests. ECE-2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone-precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE-2 KO as compared to wild-type mice. Taken together, our findings suggest a role for ECE-2 in the non-classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE-2 influences morphine tolerance and withdrawal remain unclear.
Subject(s)
Aspartic Acid Endopeptidases/deficiency , Metalloendopeptidases/deficiency , Morphine Dependence/enzymology , Morphine/pharmacology , Neuropeptides/metabolism , Pain/enzymology , Spinal Cord/enzymology , Animals , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Endothelin-Converting Enzymes , Female , Hydrolysis/drug effects , Male , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Morphine Dependence/genetics , Morphine Dependence/metabolism , Neuropeptides/biosynthesis , Neuropeptides/genetics , Pain/genetics , Pain/metabolism , Pain Measurement/methods , Spinal Cord/metabolism , Spinal Cord/pathology , Substance Withdrawal Syndrome/enzymology , Substance Withdrawal Syndrome/genetics , Substance Withdrawal Syndrome/physiopathologyABSTRACT
Endothelin-converting enzyme-2 (ECE-2), a member of M13 family of zinc metallopeptidases, has previously been shown to process a number of neuropeptides including those derived from prodynorphin, proenkephalin, proSAAS, and amyloid precursor protein. ECE-2, unlike ECE-1, exhibits restricted neuroendocrine distribution and acidic pH optimum; it is consistent with a role in the regulation of neuropeptide levels in vivo. Here, we report the generation of a three-dimensional (3D) molecular model of ECE-2 using the crystal structure of neprilysin (EC 3.4.24.11) as a template. On the basis of the predictions made from the molecular model, we mutated and tested two residues, Trp 148 and Tyr 563, in the catalytic site. The mutation of Tyr 563 was found to significantly affect the catalytic activity and inhibitor binding. The molecular model was used to virtually screen a small molecule library of 13 000 compounds. Among the top-scoring compounds three were found to inhibit ECE-2 with high affinity and exhibited specificity for ECE-2 compared to neprilysin. Thus, the model provides a new useful tool to probe the active site of ECE-2 and design additional selective inhibitors of this enzyme.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/chemistry , Models, Molecular , Amino Acid Sequence , Aspartic Acid Endopeptidases/genetics , Catalytic Domain , Conserved Sequence , Endothelin-Converting Enzymes , Glycopeptides/chemistry , Kinetics , Metalloendopeptidases/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Neprilysin/chemistry , Neprilysin/genetics , Protein Conformation , Quinolines/chemistry , Quinoxalines/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Small Molecule Libraries , Structure-Activity Relationship , Tryptophan/genetics , Tyrosine/geneticsABSTRACT
The transfer of genomic information from DNA to mRNA to protein usually occurs with high fidelity, but can also be subverted by a programmed RNA sequence alteration termed 'RNA editing', involving deamination of adenosine to inosine (decoded as guanosine), or of cytosine to uracil. These sequence changes can lead to cellular heterogeneity by generating variable sets of transcripts within otherwise identical cells. Recent studies have demonstrated that editing is most prevalent in cells and tissues with high propensity for plasticity. Within those, RNA editing reproducibly targets transcripts of related function, altering the outcomes of entire pathways at once. In ongoing work, changes in patterns of editing have been correlated with neuronal disease pathogenesis, suggesting that RNA editing harbors diagnostic potential.
Subject(s)
Nervous System Diseases/genetics , Nervous System Diseases/metabolism , RNA Editing/physiology , RNA/metabolism , Animals , Carcinogenesis/genetics , Central Nervous System Diseases/genetics , Central Nervous System Diseases/therapy , Genetic Therapy/methods , Humans , Inflammation/genetics , Inflammation/therapy , Nervous System Diseases/therapyABSTRACT
The level of activity of many animals including humans rises and falls with a period of ~ 24 hours. The intrinsic biological oscillator that gives rise to this circadian oscillation is driven by a molecular feedback loop with an approximately 24 hour cycle period and is influenced by the environment, most notably the light:dark cycle. In addition to the circadian oscillations, behavior of many animals is influenced by multiple oscillations occurring at faster-ultradian-time scales. These ultradian oscillations are also thought to be driven by feedback loops. While many studies have focused on identifying such ultradian oscillations, less is known about how the ultradian behavioral oscillations interact with each other and with the circadian oscillation. Decoding the coupling among the various physiological oscillators may be important for understanding how they conspire together to regulate the normal activity levels, as well in disease states in which such rhythmic fluctuations in behavior may be disrupted. Here, we use a wavelet-based cross-frequency analysis to show that different oscillations identified in spontaneous mouse behavior are coupled such that the amplitude of oscillations occurring at higher frequencies are modulated by the phase of the slower oscillations. The patterns of these interactions are different among different individuals. Yet this variability is not random. Differences in the pattern of interactions are confined to a low dimensional subspace where different patterns of interactions form clusters. These clusters expose the differences among individuals-males and females are preferentially segregated into different clusters. These sex-specific features of spontaneous behavior were not apparent in the spectra. Thus, our methodology reveals novel aspects of the structure of spontaneous animal behavior that are not observable using conventional methodology.
Subject(s)
Behavior, Animal , Animals , Biological Clocks , Female , Male , MiceABSTRACT
General arousal has been operationally defined as an enhanced motor activity and enhanced intensity of response to sensory stimuli. Even though the effects of gonadal hormones on mating behavior have been much studied, their potential effect on generalized arousal, as defined above, has never been evaluated. In the present study we employed a thoroughly validated assay of general arousal to determine the effects of estradiol (E) and testosterone (T) in gonadectomized female and male mice, respectively. The steroids were administered in three different ways: A fast-acting, water soluble preparation given intraperitoneally, an oil solution given subcutaneously, and an oil solution in a subcutaneous Silastic capsule. Motor activity and responses to sensory stimuli were recorded for 24h, 91h, and seven days following hormone administration, respectively. All measures of arousal varied according to the day/night cycle. The water soluble steroid preparation had no reliable effect. When the same doses of estradiol and testosterone were administered subcutaneously in an oil vehicle no effect of either treatment on arousal was observed. The subcutaneously implanted capsule containing estradiol or testosterone had a delayed effect on motor activity in females (four to seven days) but no effect in males. The long time required by the gonadal hormones for affecting arousal would be consistent with, but does not prove, a genomic action. The limited effects of E and T in our arousal assay suggest to us that the strongest actions of these hormones on arousal occur in the context of sequences of responses to sexually relevant stimuli.
Subject(s)
Arousal/physiology , Behavior, Animal/drug effects , Castration , Estradiol/pharmacology , Sex Characteristics , Testosterone/pharmacology , Animals , Arousal/drug effects , Female , Male , Mice , Mice, Inbred C57BL , Motor Activity/drug effects , Physical Stimulation , Smell/drug effects , Time Factors , Touch/drug effectsABSTRACT
Epigenetic alterations are necessary for the establishment of functional and phenotypic diversity in the populations of immune cells of the monocytic lineage. The epigenetic status of individual genes at different time points defines their transcriptional responses throughout development and in response to environmental stimuli. Epigenetic states are defined at the level of DNA modifications, chromatin modifications, as well as at the level of RNA base changes through RNA editing. Drawing from lessons regarding the epigenome and epitranscriptome of cells of the monocytic lineage in the periphery, and from recently published RNAseq data deriving from brain-resident monocytes, we discuss the impact of modulation of these epigenetic states and how they affect processes important for the development of a healthy brain, as well as mechanisms of neurodegenerative disease and aging. An understanding of the varied brain responses and pathologies in light of these novel gene regulatory systems in monocytes will lead to important new insights in the understanding of the aging process and the treatment and diagnosis of neurodegenerative disease.