ABSTRACT
Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.
ABSTRACT
Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1ß, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.
Subject(s)
Alveolar Bone Loss , Immediate-Early Proteins , Protein Serine-Threonine Kinases , TNF Receptor-Associated Factor 3 , Alveolar Bone Loss/genetics , Animals , Cytokines/metabolism , Genes, jun , Immediate-Early Proteins/metabolism , Immunity , Inflammation , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Porphyromonas gingivalis/pathogenicity , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering , Signal Transduction , TNF Receptor-Associated Factor 2/metabolism , TNF Receptor-Associated Factor 3/metabolismABSTRACT
Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-α, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre-mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage-priming conditions, respectively. Combined with decreased FoxO1 phosphorylation and the subsequent suppressed cytoplasmic translocation observed, SGK1 deficiency robustly enhances FoxO1 activity and drives macrophage to preferential M1 phenotypes. Furthermore, FoxO1 inhibition abrogates M1 phenotypes, and STAT3 overexpression results in a significant increase of M2 phenotypes, indicating that both FoxO1 and STAT3 are involved in SGK1-mediated macrophage polarization. Additionally, SGK1 differentially regulates the expression of M1 and M2 molecular markers, including CD68 and F4/F80 and CD163 and CD206, respectively, and protects against Porphyromonas gingivalis-induced alveolar bone loss in a mouse model. Taken together, these results have demonstrated that SGK1 is critical for macrophage polarization and periodontal bone loss, and for the first time, to our knowledge, we elucidated a bifurcated signaling circuit by which SGK1 promotes alternative, while suppressing inflammatory, macrophage polarization.
Subject(s)
Forkhead Box Protein O1/immunology , Immediate-Early Proteins/immunology , Inflammation/immunology , Macrophages/immunology , Protein Serine-Threonine Kinases/immunology , STAT3 Transcription Factor/immunology , Animals , Macrophage Activation/immunology , Mice , Signal Transduction/immunologyABSTRACT
Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.
Subject(s)
Directed Molecular Evolution , Machine Learning , Serotonin/metabolism , Algorithms , Amino Acid Sequence , Amygdala/physiology , Animals , Behavior, Animal , Binding Sites , Brain/metabolism , HEK293 Cells , Humans , Kinetics , Linear Models , Mice , Mice, Inbred C57BL , Photons , Protein Binding , Serotonin Plasma Membrane Transport Proteins/metabolism , Sleep/physiology , Wakefulness/physiologyABSTRACT
Genetically encoded dopamine sensors based on green fluorescent protein (GFP) enable high-resolution imaging of dopamine dynamics in behaving animals. However, these GFP-based variants cannot be readily combined with commonly used optical sensors and actuators, due to spectral overlap. We therefore engineered red-shifted variants of dopamine sensors called RdLight1, based on mApple. RdLight1 can be combined with GFP-based sensors with minimal interference and shows high photostability, permitting prolonged continuous imaging. We demonstrate the utility of RdLight1 for receptor-specific pharmacological analysis in cell culture, simultaneous assessment of dopamine release and cell-type-specific neuronal activity and simultaneous subsecond monitoring of multiple neurotransmitters in freely behaving rats. Dual-color photometry revealed that dopamine release in the nucleus accumbens evoked by reward-predictive cues is accompanied by a rapid suppression of glutamate release. By enabling multiplexed imaging of dopamine with other circuit components in vivo, RdLight1 opens avenues for understanding many aspects of dopamine biology.
Subject(s)
Behavior, Animal/physiology , Biosensing Techniques/methods , Brain/metabolism , Dopamine/metabolism , Neurons/metabolism , Animals , Cues , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Receptors, Dopamine/genetics , Receptors, Dopamine/metabolism , RewardABSTRACT
Multiple aspects of neural activity, from neuronal firing to neuromodulator release and signaling, underlie brain function and ultimately shape animal behavior. The recently developed and constantly growing toolbox of genetically encoded sensors for neural activity, including calcium, voltage, neurotransmitter and neuromodulator sensors, allows precise measurement of these signaling events with high spatial and temporal resolution. Here, we describe the engineering, characterization and application of our recently developed dLight1, a suite of genetically encoded dopamine (DA) sensors based on human inert DA receptors. dLight1 offers high molecular specificity, requisite affinity and kinetics and great sensitivity for measuring DA release in vivo. The detailed workflow described in this protocol can be used to systematically characterize and validate dLight1 in increasingly intact biological systems, from cultured cells to acute brain slices to behaving mice. For tool developers, we focus on characterizing five distinct properties of dLight1: dynamic range, affinity, molecular specificity, kinetics and interaction with endogenous signaling; for end users, we provide comprehensive step-by-step instructions for how to leverage fiber photometry and two-photon imaging to measure dLight1 transients in vivo. The instructions provided in this protocol are designed to help laboratory personnel with a broad range of experience (at the graduate or post-graduate level) to develop and utilize novel neuromodulator sensors in vivo, by using dLight1 as a benchmark.
Subject(s)
Neurotransmitter Agents/metabolism , Optogenetics/methods , Receptors, Dopamine/metabolism , Animals , Dopamine/metabolism , Genetic Engineering/methods , Humans , Luminescent Proteins/genetics , Neurons/metabolism , WorkflowABSTRACT
Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.
Subject(s)
Biosensing Techniques , Cerebral Cortex/metabolism , Dopamine/metabolism , Neuroimaging/methods , Neurotransmitter Agents/metabolism , Optogenetics , Animals , Calcium/analysis , Calcium/metabolism , Cerebral Cortex/chemistry , Corpus Striatum , Dopamine/analysis , Genetic Engineering , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Humans , Learning , Mice , Neurons/physiology , Neurotransmitter Agents/analysis , Receptors, Dopamine D1/chemistry , Receptors, Dopamine D1/genetics , Serotonin/analysis , Serotonin/metabolismABSTRACT
Intercellular genetic communication is an essential requirement for coordination of cell proliferation and differentiation and has an important role in many cellular processes. Gap junction channels possess large pore allowing passage of ions and small molecules between cells. MicroRNAs (miRNAs) are small regulatory RNAs that can regulate gene expression broadly. Here, we report that miRNAs can pass through gap junction channels in a connexin-dependent manner. Connexin43 (Cx43) had higher permeability, whereas Cx30 showed little permeability to miRNAs. In the tested connexin cell lines, the permeability to miRNAs demonstrated: Cx43 > Cx26/30 > Cx26 > Cx31 > Cx30 = Cx-null. However, consistent with a uniform structure of miRNAs, there was no significant difference in permeability to different miRNAs. The passage is efficient; the miRNA level in the recipient cells could be up to 30% of the donor level. Moreover, the transferred miRNA is functional and could regulate gene expression in neighboring cells. Connexin mutation and gap junctional blockers could eliminate this miRNA intercellular transfer and gene regulation. These data reveal a novel mechanism for intercellular genetic communication. Given that connexin expression is cell-specific, this connexin-dependent, miRNA intercellular genetic communication may play an important role in synchronizing and coordinating proliferation and differentiation of specific cell types during multicellular organ development.
Subject(s)
Gap Junctions/metabolism , Gene Expression Regulation , MicroRNAs/genetics , MicroRNAs/metabolism , RNA Transport , Animals , Cell Line , Connexins/genetics , Connexins/metabolism , Gene Silencing , Humans , Intracellular Space , Mice , Mutation , RNA Transport/drug effectsABSTRACT
A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.
Subject(s)
Cochlea/enzymology , Proteomics/methods , Animals , Cattle , Electrophoresis, Polyacrylamide Gel , Liver/metabolism , Mass Spectrometry , Mice , Multienzyme Complexes/metabolism , NAD/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidases/metabolism , Oxidation-Reduction , Proteome/metabolism , Red MeatABSTRACT
A major challenge in neuroscience is to decipher the logic of neural circuitry and to link it to learning, memory, and behavior. Synaptic transmission is a critical event underlying information processing within neural circuitry. In the extracellular space, the concentrations and distributions of excitatory, inhibitory, and modulatory neurotransmitters impact signal integration, which in turn shapes and refines the function of neural networks. Thus, the determination of the spatiotemporal relationships between these chemical signals with synaptic resolution in the intact brain is essential to decipher the codes for transferring information across circuitry and systems. Here, we review approaches and probes that have been employed to determine the spatial and temporal extent of neurotransmitter dynamics in the brain. We specifically focus on the design, screening, characterization, and application of genetically encoded indicators directly probing glutamate, the most abundant excitatory neurotransmitter. These indicators provide synaptic resolution of glutamate dynamics with cell-type specificity. We also discuss strategies for developing a suite of genetically encoded probes for a variety of neurotransmitters and neuromodulators.
Subject(s)
Brain/metabolism , Diagnostic Imaging , Luminescent Proteins , Neurotransmitter Agents/metabolism , Animals , Glutamic Acid/metabolism , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Models, Molecular , Neurotransmitter Agents/geneticsABSTRACT
Recent developments in genetically encoded indicators of neural activity (GINAs) have greatly advanced the field of systems neuroscience. As they are encoded by DNA, GINAs can be targeted to genetically defined cellular populations. Combined with fluorescence microscopy, most notably multi-photon imaging, GINAs allow chronic simultaneous optical recordings from large populations of neurons or glial cells in awake, behaving mammals, particularly rodents. This large-scale recording of neural activity at multiple temporal and spatial scales has greatly advanced our understanding of the dynamics of neural circuitry underlying behavior-a critical first step toward understanding the complexities of brain function, such as sensorimotor integration and learning. Here, we summarize the recent development and applications of the major classes of GINAs. In particular, we take an in-depth look at the design of available GINA families with a particular focus on genetically encoded calcium indicators (GCaMPs), sensors probing synaptic activity, and genetically encoded voltage indicators. Using the family of the GCaMP as an example, we review established sensor optimization pipelines. We also discuss practical considerations for end users of GINAs about experimental methods including approaches for gene delivery, imaging system requirements, and data analysis techniques. With the growing toolbox of GINAs and with new microscopy techniques pushing beyond their current limits, the age of light can finally achieve the goal of broad and dense sampling of neuronal activity across time and brain structures to obtain a dynamic picture of brain function.
ABSTRACT
T-type calcium channels are expressed in many diverse tissues, including neuronal, cardiovascular, and endocrine. T-type calcium channels are known to play roles in the development, maintenance, and repair of these tissues but have also been implicated in disease when not properly regulated. Calcium channel blockers have been developed to treat various diseases and their use clinically is widespread due to both their efficacy as well as their safety. Aside from their established clinical applications, recent studies have suggested neuroprotective effects of T-type calcium channel blockers. Many of the current T-type calcium channel blockers could act on other molecular targets besides T-type calcium channels making it uncertain whether their neuroprotective effects are solely due to blocking of T-type calcium channels. In this review, we discuss these drugs as well as newly developed chemical compounds that are designed to be more selective for T-type calcium channels. We review in vitro and in vivo evidence of neuroprotective effects by these T-type calcium channel blockers. We conclude by discussing possible molecular mechanisms underlying the neuroprotective effects by T-type calcium channel blockers.
Subject(s)
Calcium Channel Blockers/metabolism , Calcium Channels, T-Type/metabolism , Nervous System Diseases/metabolism , Nervous System Diseases/prevention & control , Neuroprotective Agents/metabolism , Animals , Calcium Channel Blockers/therapeutic use , Humans , Neuroprotective Agents/therapeutic useABSTRACT
Noise is the most common occupational and environmental hazard. Noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit, after age-related hearing loss (presbycusis). Although promising approaches have been identified for reducing NIHL, currently there are no effective medications to prevent NIHL. Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL. We turned this difficulty into an advantage by asking whether NIHL could be effectively prevented by targeting multiple signaling pathways with a combination of drugs already approved by U.S. Food and Drug Administration (FDA). We previously found that antiepileptic drugs blocking T-type calcium channels had both prophylactic and therapeutic effects for NIHL. NIHL can also be reduced by an up-regulation of glucocorticoid (GC) signaling pathways. Based on these findings, we tested a combination therapy for NIHL that included ethosuximide and zonisamide (anticonvulsants) and dexamethasone and methylprednisolone (synthetic GCs) in mice under exposure conditions typically associated with dramatic permanent threshold shifts (PTS). We first examined possible prophylactic effects for each drug when administered alone 2 h before noise, and calculated the median effective dose (ED50). We then tested for synergistic effects of two-drug combinations (anticonvulsant + GC), and identified combinations with the strongest synergy against NIHL, based on a previously established combination index (CI) metric. We repeated similar tests to determine their therapeutic effects when administered the same drugs 24 h after the noise exposure. Our study shows the feasibility of developing pharmacological intervention in multiple pathways, and discovering drug combinations with optimal synergistic effects in preventing permanent NIHL.
Subject(s)
Anticonvulsants/administration & dosage , Glucocorticoids/administration & dosage , Hearing Loss, Noise-Induced/drug therapy , Hearing Loss, Noise-Induced/prevention & control , Animals , Calcium Channel Blockers/administration & dosage , Dexamethasone/administration & dosage , Drug Combinations , Drug Synergism , Drug Therapy, Combination , Ethosuximide/administration & dosage , Evoked Potentials, Auditory, Brain Stem/drug effects , Female , Hearing Loss, Noise-Induced/physiopathology , Isoxazoles/administration & dosage , Male , Methylprednisolone/administration & dosage , Mice , Mice, Inbred C57BL , ZonisamideABSTRACT
Complexity in higher animals derives in part from various modalities of protein-coding gene expression regulation, including microRNA repression by binding to 3'-untranslated regions (UTRs) of specific genes. Reporter constructs containing candidate microRNA target sites are a popular approach of functional studies, and full-length 3'-UTR sequences are preferred because they contain all regulatory elements and preserve higher order structure as much as possible. However, this approach is often handicapped by the extreme length of the 3'-UTR. Here, we present a rapid and accurate cloning procedure to generate full-length 3'-UTR reporter constructs by recombinogenic engineering (recombineering) in vivo cloning. The approach includes making retrieval constructs by sequence- and ligation-independent cloning (SLIC) and retrieving the full-length 3'-UTR in one exon to the retrieval construct from a bacterial artificial chromosome (BAC) by recombineering to generate the final full-length 3'-UTR reporter construct for the gene of interest. This method is successfully implemented with mouse full-length 3'-UTRs of Igf1 (6.5 kb), Igf1r (7.5 kb), and Sp1 (5.5 kb). Expansion of this method is adaptable to retrieve 3'-UTRs encoded in more than one exon by removing the introns from the BAC first with recombineering. This method will advance functional studies of regulation of gene expression at the post-transcriptional level through microRNA suppression.
Subject(s)
3' Untranslated Regions/genetics , Cloning, Molecular/methods , Gene Expression Regulation , Genes, Reporter , Genetic Engineering/methods , Animals , Chromosomes, Artificial, Bacterial/genetics , Exons , Insulin-Like Growth Factor I/genetics , Introns , Mice , MicroRNAs/genetics , Plasmids/genetics , Sp1 Transcription Factor/geneticsABSTRACT
The expression of 350 microRNAs (miRNAs) in epididymis of rat from postnatal development to adult (from postnatal days 7-70) was profiled with home-made miRNA microarray. Among them, 48 miRNAs changed significantly, in which the expression of miR-200a increased obviously with time, in a good agreement with that obtained from northern blot analysis. The real-time quantitative-polymerase chain reaction result indicated that temporal expression of rat ß-catenin was exactly inversed to that of miR-200a during rat epididymal development, implying that miR-200a might also target ß-catenin mRNA in rat epididymis as reported by Saydam et al. in humans. The bioinformatic analysis indicated that 3' untranslated region of rat ß-catenin mRNA did contain a putative binding site for miR-200a. Meanwhile, it was found that the sequence of this binding site was different from that of human ß-catenin mRNA with a deletion of two adjacent nucleotides (U and C). But the results of luciferase targeting assay in HEK 293T cells and the overexpression of miR-200a in rat NRK cells demonstrated that miR-200a did target rat ß-catenin mRNA and cause the suppression of its expression. All these results show that miR-200a should be involved in rat epididymal development by targeting ß-catenin mRNA of rat and suppressing its expression.
Subject(s)
Epididymis/metabolism , Gene Expression Regulation, Developmental , MicroRNAs/biosynthesis , beta Catenin/metabolism , Animals , Base Sequence , Cloning, Molecular , Cluster Analysis , Computational Biology/methods , Gene Expression Profiling , HEK293 Cells , Humans , Male , Models, Genetic , Molecular Sequence Data , RatsABSTRACT
Long-lived mutant mice, both Ames dwarf and growth hormone receptor gene-disrupted or knockout strains, exhibit heightened cognitive robustness and altered IGF1 signaling in the brain. Here, we report, in both these long-lived mice, that three up-regulated lead microRNAs, miR-470, miR-669b, and miR-681, are involved in posttranscriptional regulation of genes pertinent to growth hormone/IGF1 signaling. All three are most prominently localized in the hippocampus and correspond to reduced expression of key IGF1 signaling genes: IGF1, IGF1R, and PI3 kinase. The decline in these genes' expression translates into decreased phosphorylation of downstream molecules AKT and FoxO3a. Cultures transfected with either miR-470, miR-669b, or miR-681 show repressed endogenous expression of all three genes of the IGF1 signaling axis, most significantly IGF1R, while other similarly up-regulated microRNAs, including let-7g and miR-509, do not induce the same levels of repression. Transduction study in IGF1-responsive cell cultures shows significantly reduced IGF1R expression, and AKT to some extent, most notably by miR-681. This is accompanied by decreased levels of downstream phosphorylated forms of AKT and FoxO3a upon IGF1 stimulation. Suppression of IGF1R by the three microRNAs is further validated by IGF1R 3'UTR reporter assays. Taken together, our results suggest that miR-470, miR-669b, and miR-681 are all functionally able to suppress IGF1R and AKT, two upstream genes controlling FoxO3a phosphorylation status. Their up-regulation in growth hormone signaling-deficient mutant mouse brain suggests reduced IGF1 signaling at the posttranscriptional level, for numerous gains of neuronal function in these long-lived mice.
Subject(s)
Gene Expression Regulation , Growth Hormone/deficiency , Hippocampus/metabolism , Longevity , MicroRNAs , Receptor, IGF Type 1/deficiency , Signal Transduction/genetics , Animals , Cell Proliferation , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Deletion , Growth Hormone/genetics , Hippocampus/cytology , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Mice , Mice, Transgenic , MicroRNAs/genetics , MicroRNAs/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptor, IGF Type 1/genetics , TransfectionABSTRACT
The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-ß converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-ß. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-ß associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-ß. Inhibition of S6K1 abrogated the phosphorylation of GSK3-ß while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin's ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3.
Subject(s)
Glycogen Synthase Kinase 3/immunology , Immunity, Innate/immunology , Proteins/immunology , Signal Transduction/immunology , Blotting, Western , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Separation , Cytokines/biosynthesis , Dendritic Cells/immunology , Dendritic Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression , Gene Expression Profiling , Gene Expression Regulation/immunology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Immunoprecipitation , Inflammation/immunology , Inflammation/metabolism , Macrophages/immunology , Macrophages/metabolism , Mechanistic Target of Rapamycin Complex 1 , Monocytes/immunology , Monocytes/metabolism , Multiprotein Complexes , Proteins/metabolism , TOR Serine-Threonine KinasesABSTRACT
The decline in cognitive robustness with aging can be attributed to complex genetic pathways involving many cellular dysfunctions, cumulative over time, precipitating in frailty and loss of wellness in the elderly brain. The size and health of the neuronal cell population determines cognitive robustness in mammals. A transgenic mouse model over-expressing Bcl-2 has been shown to rescue neurons from naturally occurring cell death (NOCD). Here we show that in the brain of calorie-restricted (CR) mice, there is an age-dependent decreased expression of microRNAs mmu-miR-181a-1*, mmu-miR-30e and mmu-miR-34a, with a corresponding gain in Bcl-2 expression, and decreases in pro-apoptosis genes such as Bax and cleavage of Caspases. Functional characterization shows that these miRNAs repress Bcl-2 expression by the 3'UTR reporter assays, accompanied by loss of this gene's endogenous expression, and a gain in pro-apoptosome-specific proteins. Over-expression of these miRNAs increases the rate of apoptosis, accompanied by a decline in Bcl-2 expression in miRNA-transfected mouse and human cell lines. We report here that down-regulation of miR-34a, -30e, and -181a permits their shared target gene expression (Bcl-2) to remain at a high level without post-transcriptional repression, accompanied by concomitant low levels of Bax expression and Caspase cleaving; this chain event may be a part of the underlying mechanism contributing to the gain in neuronal survival in long-lived CR-fed mice.
Subject(s)
Brain/metabolism , Caloric Restriction , MicroRNAs/genetics , 3' Untranslated Regions , Aging/genetics , Aging/metabolism , Aging/pathology , Animals , Apoptosis/genetics , Brain/pathology , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line , Cell Survival , Down-Regulation , Gene Expression , Genes, bcl-2 , Humans , Mice , Mice, Transgenic , Models, Biological , Signal Transduction , bcl-2-Associated X Protein/metabolismABSTRACT
Age-dependent loss of oxidative defense is well recognized in rodent models, although the control mechanism is still obscure; a few studies have shown how microRNAs, a non-coding RNA species, regulate the expression of their target genes at the post-transcriptional level. In the current study, miR-34a and miR-93 are observed to increase in middle- and old-age rat liver, compared to young rats; the up-regulation of these two miRNAs is determined by qPCR through a grind-and-find approach, and histochemical in situ hybridization. Three commonly used miRNA target prediction programs suggest four candidate targets of miR-34a and miR-93: Sp1, Nrf2 (Nfe2l2), Sirt1 and Mgst1; their expression is found to be reduced inversely to the up-regulation of the two miRNAs by Western blotting of protein extracts, as well as immunofluorescence staining of intact liver tissues. Furthermore, the suppression of the four proteins by miR-34a/miR-93 is examined in HEK 293 cells by transfection and co-transfection; miR-34a represses all four proteins' expression, whereas miR-93 affects only Sp1, Sirt1 and Mgst1. Taken together, our study suggests a model of post-transcriptional repression, not only of genes involved in oxidative stress regulation and oxidative stress defense proteins, such as Sirt1 and Mgst1, but also of upstream transcription factors (TFs) regulating their activation, since Sp1 is the TF for both Sirt1 and Mgst1, and Nrf2 is the TF of Mgst1. Thus, up-regulation of both miR-34a and miR-93 constitutes an inescapable repression of two vital oxidative defense genes, by targeting not only the targets, but also transcription factors controlling their activation, a double dampening regulation at the post-transcriptional level.
Subject(s)
Aging/physiology , Glutathione Transferase/biosynthesis , Liver/metabolism , MicroRNAs/biosynthesis , Oxidative Stress/physiology , Sirtuin 1/biosynthesis , Animals , Glutathione Transferase/genetics , HEK293 Cells , Humans , Mice , MicroRNAs/genetics , Rats , Rats, Inbred F344 , Sirtuin 1/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Up-RegulationABSTRACT
Among non-coding RNAs, microRNAs may be one of the best known subgroups, due to their unique function of negatively controlling gene expression, by either degrading target messages or binding to their 3'-untranslated region to inhibit translation. Thus gene expression can be repressed through post-transcriptional regulation, implemented as a 'dimmer switch', in contrast to the all-or-none mode of suppression. Work from our laboratory and others shows that during aging, dysregulated expression of microRNAs generally occurs in groups, suggesting that their actions may be functionally coordinated as a 'pack' by common transcriptional regulators; the accumulation of these 'pack' disorganizations may be the underlying culprit contributing to the pathoetiology of many age-dependent disease states. The fact that many microRNAs are coordinated in their expression, due to either the close proximity of their genomic locations or sharing the same transcriptional regulation, suggests that future strategies for correcting age-dependent microRNA disorganization may need to involve a system biology, rather than a reductionist, approach. Therefore, understanding age-dependent changes of microRNA expression in 'packs' may open an entirely new frontier, i.e. how particular groups of non-coding RNAs, functioning together, contribute to mechanisms regulating aging and longevity.