ABSTRACT
Short sequences that mediate interactions with modular binding domains are ubiquitous throughout eukaryotic proteomes. Networks of short linear motifs (SLiMs) and their corresponding binding domains orchestrate many cellular processes, and the low mutational barrier to evolving novel interactions provides a way for biological systems to rapidly sample selectable phenotypes. Mapping SLiM binding specificity and the rules that govern SLiM evolution is fundamental to uncovering the pathways regulated by these networks and developing the tools to manipulate them. We used high-throughput screening of the human proteome to identify sequences that bind to the Enabled/VASP homology 1 (EVH1) domain of the postsynaptic density scaffolding protein Homer1. This expanded our understanding of the determinants of Homer EVH1 binding preferences and defined a new motif that can facilitate the discovery of additional Homer-mediated interactions. Interestingly, the Homer1 EVH1 domain preferentially binds to sequences containing an N-terminally overlapping motif that is bound by the paralogous family of Ena/VASP actin polymerases, and many of these sequences can bind to EVH1 domains from both protein families. We provide evidence from orthologous EVH1 domains in pre-metazoan organisms that the overlap in human Ena/VASP and Homer binding preferences corresponds to an incomplete divergence from a common Ena/VASP ancestor. Given this overlap in binding profiles, promiscuous sequences that can be recognized by both families either achieve specificity through extrinsic regulatory strategies or may provide functional benefits via multi-specificity. This may explain why these paralogs incompletely diverged despite the accessibility of further diverged isoforms.
Subject(s)
Homer Scaffolding Proteins , Homer Scaffolding Proteins/metabolism , Homer Scaffolding Proteins/chemistry , Homer Scaffolding Proteins/genetics , Humans , Protein Domains , Protein Binding , Amino Acid MotifsABSTRACT
OBJECTIVE: Proinflammatory necroptosis is the main pathological mechanism of ischemic stroke. Homer scaffolding protein 1 (Homer1) is a postsynaptic scaffolding protein that exerts anti-inflammatory effects in most central nervous system diseases. However, the relationship between Homer1 and proinflammatory necroptosis in ischemic stroke remains unclear. AIM: This study aimed to investigate the role of Homer1 in ischemia-induced necroptosis. METHODS: C57BL/6 mice were used to establish a model of permanent middle cerebral artery occlusion model (pMCAO). Homer1 knockdown mice were generated using adeno-associated virus (AAV) infection to explore the role of Homer1 and its impact on necroptosis in pMCAO. Finally, Homer1 protein was stereotaxically injected into the ischemic cortex of Homer1flox/flox/Nestin-Cre +/- mice, and the efficacy of Homer1 was investigated using behavioral assays and molecular biological assays to explore potential mechanisms. RESULTS: Homer1 expression peaked at 8 h in the ischemic penumbral cortex after pMCAO and colocalized with neurons. Homer1 knockdown promoted neuronal death by enhancing necroptotic signaling pathways and aggravating ischemic brain damage in mice. Furthermore, the knockdown of Homer1 enhanced the expression of proinflammatory cytokines. Moreover, injection of Homer1 protein reduced necroptosis-induced brain injury inhibited the expression of proinflammatory factors, and ameliorated the outcomes in the Homer1flox/flox/Nestin-Cre+/- mice after pMCAO. CONCLUSIONS: Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation. These data suggested that Homer1 is a novel regulator of neuronal death and neuroinflammation.
Subject(s)
Brain Ischemia , Ischemic Stroke , Stroke , Mice , Animals , Ischemic Stroke/complications , Ischemic Stroke/metabolism , Ischemic Stroke/pathology , Nestin/metabolism , Nestin/pharmacology , Neuroinflammatory Diseases , Necroptosis , Mice, Inbred C57BL , Infarction, Middle Cerebral Artery/pathology , Neurons/pathology , Brain Ischemia/metabolism , Stroke/complications , Stroke/metabolism , Stroke/pathology , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Homer Scaffolding Proteins/pharmacologyABSTRACT
Fat deposition is critical to pork quality. However, the mechanism of fat deposition remains to be elucidated. Circular RNAs (circRNAs) are ideal biomarkers and are involved in adipogenesis. Here, we investigated the effect and mechanism of circHOMER1 on porcine adipogenesis in vitro and in vivo. Western blotting, Oil red O staining, and HE staining were used to assess the function of circHOMER1 in adipogenesis. The results showed that circHOMER1 inhibited adipogenic differentiation of porcine preadipocytes and suppressed adipogenesis in mice. Dual-luciferase reporter gene, RIP, and pull-down assays demonstrated that miR-23b directly bound to circHOMER1 and the 3'-UTR of SIRT1. Rescue experiments further illustrated the regulatory relationship among circHOMER1, miR-23b, and SIRT1. Conclusively, we demonstrate that circHOMER1 plays an inhibitory role in porcine adipogenesis through miR-23b and SIRT1. The present study revealed the mechanism of porcine adipogenesis, which may be helpful to improve pork quality.
Subject(s)
Adipogenesis , Homer Scaffolding Proteins , MicroRNAs , RNA, Circular , Sirtuin 1 , Animals , Mice , Adipogenesis/genetics , Cell Differentiation , MicroRNAs/genetics , Sirtuin 1/metabolism , Swine , RNA, Circular/genetics , Homer Scaffolding Proteins/geneticsABSTRACT
BACKGROUND: Depression is one of the most common comorbid psychiatric condition associated with epilepsy. It has a negative impact on the patient's quality of life. However, the underlying molecular mechanisms leading to depression are currently unclear. The aim of this study was to determine the hub genes associated with epilepsy and depression. METHODS: Gene expression profiles (GSE47752 and GSE20388) were downloaded from the gene expression omnibus (GEO) database. Differentially expressed genes (DEGs) for epilepsy and depression groups were separately searched. Subsequently, network analyses methods were employed to establish protein-protein interaction (PPI) networks, and to perform Gene Ontology (GO) terms and pathway enrichment analyses for co-expressed DEGs. RESULTS: A total of 772 genes were upregulated in patients with epilepsy whereas 91 genes were up-regulated in patients with depression. In addition, 1304 genes were down-regulated in epilepsy whereas 141 genes were down-regulated in patients with depression. Among co-expressed DEGs, 5 DEGs were up-regulated and 19 were down-regulated. Further analysis revealed that the co-expressed DEGs were involved in regulation of vasculature development, regulation of angiogenesis, glutamate receptor signaling pathway, cellular response to interleukin-1 and positive regulation of protein kinase B signaling. The Arc and Homer1 genes were identified as the common candidate genes involved in the pathogenesis of epilepsy and depression. CONCLUSIONS: Arc and Homer1 may contribute to the comorbidity of epilepsy and depression.
Subject(s)
Cytoskeletal Proteins/metabolism , Epilepsy , Gene Regulatory Networks , Nerve Tissue Proteins/metabolism , Comorbidity , Computational Biology/methods , Data Analysis , Depression/complications , Depression/genetics , Epilepsy/complications , Epilepsy/epidemiology , Epilepsy/genetics , Gene Expression Profiling/methods , Homer Scaffolding Proteins/genetics , Humans , Quality of LifeABSTRACT
BACKGROUND: Inflammation induced by intracerebral hemorrhage (ICH) is one of the main causes of the high mortality and poor prognosis of patients with ICH. A1 astrocytes are closely associated with neuroinflammation and neurotoxicity, whereas A2 astrocytes are neuroprotective. Homer scaffolding protein 1 (Homer1) plays a protective role in ischemic encephalopathy and neurodegenerative diseases. However, the role of Homer1 in ICH-induced inflammation and the effect of Homer1 on the phenotypic conversion of astrocytes remain unknown. METHODS: Femoral artery autologous blood from C57BL/6 mice was used to create an ICH model. We use the A1 phenotype marker C3 and A2 phenotype marker S100A10 to detect astrocyte conversion after ICH. Homer1 overexpression/knock-down mice were constructed by adeno-associated virus (AAV) infection to explore the role of Homer1 and its mechanism of action after ICH. Finally, Homer1 protein and selumetinib were injected into in situ hemorrhage sites in the brains of Homer1flox/flox/Nestin-Cre+/- mice to study the efficacy of Homer1 in the treatment of ICH by using a mouse cytokine array to explore the potential mechanism. RESULTS: The expression of Homer1 peaked on the third day after ICH and colocalized with astrocytes. Homer1 promotes A1 phenotypic conversion in astrocytes in vivo and in vitro. Overexpression of Homer1 inhibits the activation of MAPK signaling, whereas Homer1 knock-down increases the expression of pathway-related proteins. The Homer1 protein and selumetinib, a non-ATP competitive MEK1/2 inhibitor, improved the outcome in ICH in Homer1flox/flox/Nestin-Cre+/- mice. The efficacy of Homer1 in the treatment of ICH is associated with reduced expression of the inflammatory factor TNFSF10 and increased expression of the anti-inflammatory factors activin A, persephin, and TWEAK. CONCLUSIONS: Homer1 plays an important role in inhibiting inflammation after ICH by suppressing the A1 phenotype conversion in astrocytes. In situ injection of Homer1 protein may be a novel and effective method for the treatment of inflammation after ICH.
Subject(s)
Astrocytes , Cerebral Hemorrhage , Animals , Astrocytes/metabolism , Cerebral Hemorrhage/metabolism , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Homer Scaffolding Proteins/pharmacology , Humans , Inflammation/metabolism , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
Neurons encode information by rapidly modifying synaptic protein complexes, which changes the strength of specific synaptic connections. Homer1 is abundantly expressed at glutamatergic synapses, and is known to alter its binding to metabotropic glutamate receptor 5 (mGlu5) in response to synaptic activity. However, Homer participates in many additional known interactions whose activity-dependence is unclear. Here, we used co-immunoprecipitation and label-free quantitative mass spectrometry to characterize activity-dependent interactions in the cerebral cortex of wildtype and Homer1 knockout mice. We identified a small, high-confidence protein network consisting of mGlu5, Shank2 and 3, and Homer1-3, of which only mGlu5 and Shank3 were significantly reduced following neuronal depolarization. We identified several other proteins that reduced their co-association in an activity-dependent manner, likely mediated by Shank proteins. We conclude that Homer1 dissociates from mGlu5 and Shank3 following depolarization, but our data suggest that direct Homer1 interactions in the cortex may be more limited than expected.
Subject(s)
Neurons , Synapses , Animals , Cerebral Cortex/metabolism , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Mice , Mice, Knockout , Microfilament Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Synapses/metabolismABSTRACT
The chemical synapse is one type of cell-adhesion system that transmits information from a neuron to another neuron in the complex neuronal network in the brain. Synaptic transmission is the rate-limiting step during the information processing in the neuronal network and its plasticity is involved in cognitive functions. Thus, morphological and electrophysiological analyses of synapses are of particular importance in neuroscience research. In the current study, we applied super-resolved three-dimensional stimulated emission depletion (3D-STED) microscopy for the morphological analyses of synapses. This approach allowed us to estimate the precise number of excitatory and inhibitory synapses in the mouse hippocampal tissue. We discovered a region-specific increase in excitatory synapses in a model mouse of autism spectrum disorder, Neuroligin-3 KO, with this method. This type of analysis will open a new field in developmental neuroscience in the future.
Subject(s)
Autism Spectrum Disorder/genetics , CA1 Region, Hippocampal/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Membrane Proteins/genetics , Microscopy/methods , Nerve Tissue Proteins/genetics , Neurons/metabolism , Synapses/genetics , Animals , Autism Spectrum Disorder/diagnostic imaging , Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/pathology , CA1 Region, Hippocampal/diagnostic imaging , CA1 Region, Hippocampal/pathology , Cell Adhesion Molecules, Neuronal/deficiency , Cognition/physiology , Disease Models, Animal , Gene Knockout Techniques , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Male , Membrane Proteins/deficiency , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy/instrumentation , Nerve Tissue Proteins/deficiency , Neuroimaging/instrumentation , Neuroimaging/methods , Neurons/pathology , Synapses/metabolism , Synapses/ultrastructure , Synaptic Transmission/physiologyABSTRACT
The HOMER1 gene is involved in synaptic plasticity, learning and memory. Recent studies show that circular RNA derived from HOMER1 (circHOMER1) expression is altered in some Alzheimer's disease (AD) brain regions. In addition, HOMER1 messenger (mRNA) levels have been associated with ß-Amyloid (Aß) deposits in brain cortical regions. Our aim was to measure the expression levels of HOMER1 circRNAs and their linear forms in the human AD entorhinal cortex. First, we showed downregulation of HOMER1B/C and HOMER1A mRNA and hsa_circ_0006916 and hsa_circ_0073127 levels in AD female cases compared to controls by RT-qPCR. A positive correlation was observed between HOMER1B/C, HOMER1A mRNA, and hsa_circ_0073128 with HOMER1B/C protein only in females. Global average area of Aß deposits in entorhinal cortex samples was negatively correlated with HOMER1B/C, HOMER1A mRNA, and hsa_circ_0073127 in both genders. Furthermore, no differences in DNA methylation were found in two regions of HOMER1 promoter between AD cases and controls. To sum up, we demonstrate that linear and circular RNA variants of HOMER1 are downregulated in the entorhinal cortex of female patients with AD. These results add to the notion that HOMER1 and its circular forms could be playing a female-specific role in the pathogenesis of AD.
Subject(s)
Alzheimer Disease/genetics , Entorhinal Cortex/metabolism , Gene Expression Regulation , Homer Scaffolding Proteins/genetics , RNA, Circular/genetics , RNA, Messenger/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Biomarkers , Case-Control Studies , Down-Regulation , Entorhinal Cortex/physiopathology , Female , Humans , Male , Sex FactorsABSTRACT
Tinnitus is deemed as the result of abnormal neural activities in the brain, and Homer proteins are expressed in the brain that convey nociception. The expression of Homer in tinnitus has not been studied. We hypothesized that expression of Homer in the auditory cortex was altered after tinnitus treatment. Mice were injected with sodium salicylate to induce tinnitus. Expression of Homer was detected by quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry assays. We found that Homer1 expression was upregulated in the auditory cortex of mice with tinnitus, while expression of Homer2 or Homer3 exhibited no significant alteration. Effects of two inhibitors of metabolic glutamate receptor 5 (mGluR5), noncompetitive 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) and competitive α-methyl-4-carboxyphenylglycine (MCPG), on the tinnitus scores of the mice and on Homer1 expression were detected. MPEP significantly reduced tinnitus scores and suppressed Homer1 expression in a concentration dependent manner. MCPG had no significant effects on tinnitus scores or Homer1 expression. In conclusion, Homer1 expression was upregulated in the auditory cortex of mice after tinnitus, and was suppressed by noncompetitive mGluR5 inhibitor MPEP, but not competitive mGluR5 inhibitor MCPG.
Subject(s)
Auditory Cortex/metabolism , Homer Scaffolding Proteins/metabolism , Receptor, Metabotropic Glutamate 5/antagonists & inhibitors , Tinnitus/metabolism , Animals , Auditory Cortex/drug effects , Glycine/analogs & derivatives , Glycine/pharmacology , Homer Scaffolding Proteins/genetics , Male , Mice , Pyridines/pharmacologyABSTRACT
Homer is a postsynaptic scaffold protein, which has long and short isoforms. The long form of Homer consists of an N-terminal target-binding domain and a C-terminal multimerization domain, linking multiple proteins within a complex. The short form of Homer only has the N-terminal domain and likely acts as a dominant negative regulator. Homer2a, one of the long form isoforms of the Homer family, expresses with a transient peak in the early postnatal stage of mouse cerebellar granule cells (CGCs); however, the functions of Homer2a in CGCs are not fully understood yet. In this study, we investigated the physiological roles of Homer2a in CGCs using recombinant adenovirus vectors. Overexpression of the Homer2a N-terminal domain construct, which was made structurally reminiscent with Homer1a, altered NMDAR1 localization, decreased NMDA currents, and promoted the survival of CGCs. These results suggest that the Homer2a N-terminal domain acts as a dominant negative protein to attenuate NMDAR-mediated excitotoxicity. Moreover, we identified a novel short form N-terminal domain-containing Homer2, named Homer2e, which was induced by apoptotic stimulation such as ischemic brain injury. Our study suggests that the long and short forms of Homer2 are involved in apoptosis of CGCs.
Subject(s)
Apoptosis , Cerebellum/cytology , Homer Scaffolding Proteins/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Amino Acid Sequence , Animals , Base Sequence , Brain Ischemia/pathology , Homer Scaffolding Proteins/chemistry , Homer Scaffolding Proteins/genetics , Mice, Inbred ICR , Models, Biological , N-Methylaspartate/metabolism , Protein Domains , Protein Isoforms/metabolismABSTRACT
Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding protein (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted gene panel (OTO-NGS-v2). This frameshift mutation produces a premature stop codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is placed close to the previously identified c.840_840dup mutation found in a Chinese family that truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese frameshift mutations might exert a similar effect at the protein level, leading to truncated mutants with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, and also to clarifying the genotype-phenotype correlations of DFNA68 hearing loss.
Subject(s)
Frameshift Mutation , Hearing Loss, Sensorineural , Homer Scaffolding Proteins , Adolescent , Adult , Child , Female , Humans , Male , Codon, Nonsense/genetics , Frameshift Mutation/genetics , Genes, Dominant/genetics , Hearing Loss, Sensorineural/genetics , Homer Scaffolding Proteins/genetics , Pedigree , PhenotypeABSTRACT
Myocardial infarction (MI) is one of most common cardiovascular diseases, and ischemia/reperfusion (I/R) injury is one of the risk factors for severe myocardial injury and dysfunction, even leading to high mortality of myocardial infarction. Liraglutide, a novel glucagon-like peptide 1 (GLP-1) analogue, has been reported to reduce cardiac rupture and infarct size and improve cardiac function in normal and diabetic rodents, however, the mechanisms of liraglutide on cardiomyocytes is not clear. The current research was designed to investigate the hypothesis that liraglutide would protect cardiomyocytes through regulating homer1 expression under hypoxia/reoxygenation (H/R) condition. The results of the present study indicated liraglutide reduced hypoxia-reoxygenation induced cell death and attenuated intracellular calcium overload in H9C2 cardiomyocytes under H/R condition. Moreover, liraglutide significantly increased the Homer1 protein expression, and this protection might be related to Homer1-dependent regulation of endoplasmic reticulum (ER) calcium homeostasis. Taken together, liraglutide protects H9C2 cell against H/R induced cell injury, and this protective effect may inhibit intracellular calcium overload to some extent, through Homer1-dependent regulation of ER calcium homeostasis.
Subject(s)
Homer Scaffolding Proteins/metabolism , Ischemic Preconditioning, Myocardial , Liraglutide/pharmacology , Myocardial Reperfusion Injury/prevention & control , Calcium/metabolism , Cells, Cultured , Homeostasis , Homer Scaffolding Proteins/genetics , Humans , Myocardial Infarction/prevention & control , Myocytes, Cardiac/drug effects , RNA, Messenger/metabolismABSTRACT
Little is known about the cellular signals that organize synapse formation. To explore what signaling pathways may be involved, we employed heterologous synapse formation assays in which a synaptic adhesion molecule expressed in a nonneuronal cell induces pre- or postsynaptic specializations in cocultured neurons. We found that interfering pharmacologically with microtubules or actin filaments impaired heterologous synapse formation, whereas blocking protein synthesis had no effect. Unexpectedly, pharmacological inhibition of c-jun N-terminal kinases (JNKs), protein kinase-A (PKA), or AKT kinases also suppressed heterologous synapse formation, while inhibition of other tested signaling pathways-such as MAP kinases or protein kinase C-did not alter heterologous synapse formation. JNK and PKA inhibitors suppressed formation of both pre- and postsynaptic specializations, whereas AKT inhibitors impaired formation of post- but not presynaptic specializations. To independently test whether heterologous synapse formation depends on AKT signaling, we targeted PTEN, an enzyme that hydrolyzes phosphatidylinositol 3-phosphate and thereby prevents AKT kinase activation, to postsynaptic sites by fusing PTEN to Homer1. Targeting PTEN to postsynaptic specializations impaired heterologous postsynaptic synapse formation induced by presynaptic adhesion molecules, such as neurexins and additionally decreased excitatory synapse function in cultured neurons. Taken together, our results suggest that heterologous synapse formation is driven via a multifaceted and multistage kinase network, with diverse signals organizing pre- and postsynaptic specializations.
Subject(s)
Homer Scaffolding Proteins/genetics , Neurons/metabolism , PTEN Phosphohydrolase/genetics , Synapses/genetics , Actin Cytoskeleton/genetics , Calcium-Binding Proteins/genetics , Cell Adhesion Molecules/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Humans , JNK Mitogen-Activated Protein Kinases/genetics , Microtubules/genetics , Neural Cell Adhesion Molecules/genetics , Phosphatidylinositol Phosphates , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction , Synapses/physiologyABSTRACT
The immunomodulatory function of nitric oxide synthase (NOS2) has been extensively studied. However, some behavioral abnormalities caused by its mutations have been found in a few rodent studies, of which the molecular mechanism remains elusive. In this research, we generated nos2b gene knockout zebrafish (nos2bsou2/sou2 ) using CRISPR/Cas9 approach and investigated their behavioral and molecular changes by doing a series of behavioral detections, morphological measurements, and molecular analyses. We found that, compared with nos2b+/+ zebrafish, nos2bsou2/sou2 zebrafish exhibited enhanced motor activity; additionally, nos2bsou2/sou2 zebrafish were characterized by smaller brain size, abnormal structure of optic tectum, reduced mRNA level of presynaptic synaptophysin and postsynaptic homer1, and altered response to sodium nitroprusside/methylphenidate hydrochloride treatment. These findings will likely contribute to future studies of behavioral regulation.
Subject(s)
Motor Activity , Nitric Oxide Synthase/genetics , Zebrafish Proteins/genetics , Animals , Brain/metabolism , Gene Deletion , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Mutation , Nitric Oxide Synthase/metabolism , Synaptophysin/genetics , Synaptophysin/metabolism , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
Pain sensation is powerfully modulated by signal processing in the brain, and pain becomes chronic with the dysfunction of the pain modulatory system; however, the underlying mechanisms are unclear. We found that the metabotropic glutamate receptor 5 (mGluR5) in the periaqueductal gray (PAG), the key area of endogenous pain modulation, is persistently active in normal conditions to maintain an appropriate sensory perception. In the neuropathic pain condition, Homer1a, an activity-dependent immediate early gene product, disrupted the persistent mGluR5 activity resulting in chronic pain. Remarkably a single-time blockage of the mGluR5 resulted in chronic neuropathic pain-like symptoms even in the absence of nerve injury. The decline of mGluR5 activity induced the pain modulatory dysfunction with a profound reduction of excitability of PAG neurons. These findings uncover the role of the persistent mGluR5 activity in vivo and provide new insight into how pain becomes chronic with the maladaptive coping of the PAG to pain sensation.
Subject(s)
Chronic Pain/physiopathology , Hyperalgesia/physiopathology , Neuralgia/physiopathology , Periaqueductal Gray/pathology , Receptor, Metabotropic Glutamate 5/metabolism , Animals , Chronic Pain/etiology , Chronic Pain/pathology , Disease Models, Animal , Gene Knockdown Techniques , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Humans , Hyperalgesia/etiology , Hyperalgesia/pathology , Male , Neuralgia/etiology , Neuralgia/pathology , Pain Perception/physiology , Periaqueductal Gray/physiopathology , RatsABSTRACT
BACKGROUND: Antipsychotic agents modulate key molecules of the postsynaptic density (PSD), including the Homer1a gene, implicated in dendritic spine architecture. How the antipsychotic receptor profile, dose, and duration of administration may influence synaptic plasticity and the Homer1a pattern of expression is yet to be determined. METHODS: In situ hybridization for Homer1a was performed on rat tissue sections from cortical and striatal regions of interest (ROI) after acute or chronic administration of three antipsychotics with divergent receptor profile: Haloperidol, asenapine, and olanzapine. Univariate and multivariate analyses of the effects of topography, treatment, dose, and duration of antipsychotic administration were performed. RESULTS: All acute treatment regimens were found to induce a consistently higher expression of Homer1a compared to chronic ones. Haloperidol increased Homer1a expression compared to olanzapine in striatum at the acute time-point. A dose effect was also observed for acute administration of haloperidol. CONCLUSIONS: Biological effects of antipsychotics on Homer1a varied strongly depending on the combination of their receptor profile, dose, duration of administration, and throughout the different brain regions. These molecular data may have translational valence and may reflect behavioral sensitization/tolerance phenomena observed with prolonged antipsychotics.
Subject(s)
Antipsychotic Agents/pharmacology , Brain/drug effects , Homer Scaffolding Proteins/genetics , Neuronal Plasticity/drug effects , Animals , Antipsychotic Agents/adverse effects , Brain/metabolism , Brain Mapping , Dibenzocycloheptenes , Dose-Response Relationship, Drug , Duration of Therapy , Haloperidol/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , In Situ Hybridization , Models, Animal , Neuronal Plasticity/genetics , Olanzapine/pharmacology , Post-Synaptic Density/drug effects , Post-Synaptic Density/genetics , Rats , Tissue Distribution/drug effectsABSTRACT
Persistent changes in brain stress and glutamatergic function are associated with post-traumatic stress disorder (PTSD). Rodent exposure to the predator odor trimethylthiazoline (TMT) is an innate stressor that produces lasting behavioral consequences relevant to PTSD. As such, the goal of the present study was to assess early (6 hours and 2 days-Experiment 1) and late (4 weeks-Experiment 2) changes to gene expression (RT-PCR) related to stress and excitatory function following TMT exposure in male, Long-Evans rats. During TMT exposure, rats engaged in stress reactive behaviors, including digging and immobility. Further, the TMT group displayed enhanced exploration and mobility in the TMT-paired context 1 week after exposure, suggesting a lasting contextual reactivity. Gene expression analyses revealed upregulated FKBP5 6 hours post-TMT in the hypothalamus and dorsal hippocampus. Two days after TMT, GRM3 was downregulated in the prelimbic cortex and dorsal hippocampus, but upregulated in the nucleus accumbens. This may reflect an early stress response (FKBP5) that resulted in later glutamatergic adaptation (GRM3). Finally, another experiment 4 weeks after TMT exposure showed several differentially expressed genes known to mediate excitatory tripartite synaptic function in the prelimbic cortex (GRM5, DLG4 and SLC1A3 upregulated), infralimbic cortex (GRM2 downregulated, Homer1 upregulated), nucleus accumbens (GRM7 and SLC1A3 downregulated), dorsal hippocampus (FKBP5 and NR3C2 upregulated, SHANK3 downregulated) and ventral hippocampus (CNR1, GRM7, GRM5, SHANK3 and Homer1 downregulated). These data show that TMT exposure induces stress and excitatory molecular adaptations, which could help us understand the persistent glutamatergic dysfunction observed in PTSD.
Subject(s)
Brain/metabolism , Stress, Psychological/genetics , Animals , Brain/physiology , Disks Large Homolog 4 Protein/genetics , Disks Large Homolog 4 Protein/metabolism , Excitatory Amino Acid Transporter 1/genetics , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Postsynaptic Potentials/genetics , Homer Scaffolding Proteins/genetics , Homer Scaffolding Proteins/metabolism , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Predatory Behavior , Rats , Rats, Long-Evans , Receptors, Metabotropic Glutamate/genetics , Receptors, Metabotropic Glutamate/metabolism , Stress, Psychological/etiology , Stress, Psychological/metabolism , Tacrolimus Binding Proteins/genetics , Tacrolimus Binding Proteins/metabolism , Thiazoles/toxicityABSTRACT
Glutamate is the most excitatory neurotransmitter in the central nervous system and it is involved in the initiation and maintaining of waking and rapid-eye-movement (REM) sleep. Homer proteins act in the trafficking and/or clustering of metabotropic glutamate receptors, and polymorphisms in the HOMER1 gene have been associated with phenotypes related to glutamate signaling dysregulation. In this study, we report the association of a single nucleotide polymorphism (SNP) in the HOMER1 gene (rs3822568) with specific aspects of sleep in a sample of the Brazilian population. To accomplish this, 1,042 individuals were subjected to a full-night polysomnography, and a subset of 983 subjects had rs3822568 genotyping data available. When compared with the A allele carriers, GG genotyped individuals showed higher sleep latency, lower sleep efficiency, reduced number of arousals per hour, lower apnea-hypopnea index (AHI) and lower theta spectral power. In summary, the present findings suggest that the rs3822568 polymorphism in the HOMER1 gene is associated with sleep EEG profiles and might have an impact on sleep quality and sleep structure, with potential to explain inter-individual variation in sleep homeostasis.
Subject(s)
Homer Scaffolding Proteins/physiology , Polymorphism, Single Nucleotide , Sleep Latency/genetics , Brazil , Electroencephalography , Female , Genotype , Homer Scaffolding Proteins/genetics , Humans , Male , Polysomnography , Receptors, Metabotropic Glutamate/metabolismABSTRACT
Metabotropic glutamate receptor 5 (mGluR5) has been reported to contribute to inflammatory pain. The intracellular C-terminal domain has a Homer-binding motif that can form an mGluR5/Homer complex. Phosphorylation of mGluR5 at the Homer binding domain enhances the mGluR5/Homer interaction and modulates intracellular signal transduction. However, the characteristics of this interaction have not been fully elucidated in inflammatory pain. We aimed to evaluate the effects of CFA-induced phosphorylation of mGluR5 at the Homer binding domain on the mGluR5/Homer interaction. Von-frey filaments and thermal latency were used to monitor the development of inflammatory pain. Spinal mGluR5 phosphorylation at Ser1126 and mGluR5/Homer crosslinking were detected. Mutant mGluR5 that could not be phosphorylated at Thr1123 or Ser1126 was evaluated in inflammatory pain. CFA-induced inflammatory pain resulted in obvious phosphorylation at Ser1126 of mGluR5. Moreover, increased phosphorylation at the Homer-binding motif enhanced crosslinking between mGluR5 and Homer. Mutations at Thr1123 and Ser1126 of mGluR5 blocked the development of CFA-induced inflammatory pain. Overall, our findings showed that disruption of the phosphorylation of mGluR5 Thr1123 and Ser1126 alleviated CFA-induced inflammatory pain.
Subject(s)
Freund's Adjuvant/toxicity , Homer Scaffolding Proteins/metabolism , Pain/chemically induced , Pain/metabolism , Receptor, Metabotropic Glutamate 5/metabolism , Animals , Homer Scaffolding Proteins/genetics , Inflammation/chemically induced , Inflammation/genetics , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pain/genetics , Phosphorylation/drug effects , Phosphorylation/physiology , Receptor, Metabotropic Glutamate 5/geneticsABSTRACT
Sleep problems are related to the elevated levels of the Alzheimer's disease (AD) biomarker ß-amyloid (Aß). Hypotheses about the causes of this relationship can be generated from molecular markers of sleep problems identified in rodents. A major marker of sleep deprivation is Homer1a, a neural protein coded by the HOMER1 gene, which has also been implicated in brain Aß accumulation. Here, we tested whether the relationship between cortical Aß accumulation and self-reported sleep quality, as well as changes in sleep quality over 3 years, was stronger in cortical regions with high HOMER1 mRNA expression levels. In a sample of 154 cognitively healthy older adults, Aß correlated with poorer sleep quality cross-sectionally and longitudinally (n = 62), but more strongly in the younger than in older individuals. Effects were mainly found in regions with high expression of HOMER1. The anatomical distribution of the sleep-Aß relationship followed closely the Aß accumulation pattern in 69 patients with mild cognitive impairment or AD. Thus, the results indicate that the relationship between sleep problems and Aß accumulation may involve Homer1 activity in the cortical regions, where harbor Aß deposits in AD. The findings may advance our understanding of the relationship between sleep problems and AD risk.