RESUMO
Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.
Assuntos
Axônios , Regeneração Nervosa , Regiões 3' não Traduzidas/genética , Animais , Axônios/metabolismo , Camundongos , Regeneração Nervosa/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismoRESUMO
The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3' untranslated region (3'UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína Semelhante a ELAV 4/genética , Regulação da Expressão Gênica/genética , Córtex Motor/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/genética , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/metabolismo , Linhagem Celular Tumoral , Proteína Semelhante a ELAV 4/metabolismo , Humanos , RNA Mensageiro/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.
Assuntos
Transtorno Bipolar/genética , Cognição , Regulação da Expressão Gênica , RNA Circular/genética , Esquizofrenia/genética , Sinapses/metabolismo , Adulto , Animais , Feminino , Proteínas de Arcabouço Homer/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Córtex Pré-Frontal/metabolismoRESUMO
Identifying genetic factors underlying neuroanatomical variation has been difficult. Traditional methods have used brain regions from predetermined parcellation schemes as phenotypes for genetic analyses, although these parcellations often do not reflect brain function and/or do not account for covariance between regions. We proposed that network-based phenotypes derived via source-based morphometry (SBM) may provide additional insight into the genetic architecture of neuroanatomy given its data-driven approach and consideration of covariance between voxels. We found that anatomical SBM networks constructed on ~ 20 000 individuals from the UK Biobank were heritable and shared functionally meaningful genetic overlap with each other. We additionally identified 27 unique genetic loci that contributed to one or more SBM networks. Both GWA and genetic correlation results indicated complex patterns of pleiotropy and polygenicity similar to other complex traits. Lastly, we found genetic overlap between a network related to the default mode and schizophrenia, a disorder commonly associated with neuroanatomic alterations.
Assuntos
Mapeamento Encefálico/métodos , Encéfalo/fisiopatologia , Estudos de Associação Genética , Rede Nervosa/fisiopatologia , Adulto , Idoso , Transtorno Bipolar/genética , Transtorno Bipolar/fisiopatologia , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/fisiopatologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Componente Principal , Esquizofrenia/genética , Esquizofrenia/fisiopatologiaRESUMO
Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.
Assuntos
MicroRNA Circulante/sangue , Vesículas Extracelulares/metabolismo , Perfilação da Expressão Gênica , Doenças Neurodegenerativas/sangue , Transdução de Sinais , Idoso , Idoso de 80 Anos ou mais , MicroRNA Circulante/genética , Vesículas Extracelulares/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/genéticaRESUMO
The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3'-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4-/- (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3'-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.
Assuntos
Processamento Alternativo/genética , Proteína Semelhante a ELAV 4/genética , Neocórtex/metabolismo , Poliadenilação/genética , Animais , Proteína Semelhante a ELAV 4/metabolismo , Éxons/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Localization and translation of mRNAs within different subcellular domains provides an important mechanism to spatially and temporally introduce new proteins in polarized cells. Neurons make use of this localized protein synthesis during initial growth, regeneration and functional maintenance of their axons. Although the first evidence for protein synthesis in axons dates back to 1960s, improved methodologies, including the ability to isolate axons to purity, highly sensitive RNA detection methods and imaging approaches, have shed new light on the complexity of the transcriptome of the axon and how it is regulated. Moreover, these efforts are now uncovering new roles for locally synthesized proteins in neurological diseases and injury responses. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of how axonal mRNA transport and translation are regulated, and discuss their emerging links to neurological disorders and neural repair.
Assuntos
Axônios/metabolismo , Transporte de RNA/fisiologia , RNA Mensageiro/metabolismoRESUMO
Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic ß cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic ß cells.
Assuntos
Proteínas ELAV/metabolismo , Insulina/genética , Insulina/metabolismo , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Animais , Proteínas ELAV/genética , Proteína Semelhante a ELAV 4 , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Precursores de Proteínas/genéticaRESUMO
HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.
Assuntos
Regiões 3' não Traduzidas , Axônios/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Proteína GAP-43/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Animais , Axônios/ultraestrutura , Sequência de Bases , Ligação Competitiva , Proteína Semelhante a ELAV 4/genética , Proteína GAP-43/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Neuropeptídeos/genética , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Transdução de SinaisRESUMO
There is compelling evidence that epigenetic factors contribute to the manifestation of depression, in which microRNA132 (miR-132) is suggested to play a pivotal role in the pathogenesis and neuronal mechanisms underlying the symptoms of depression. Additionally, several depression-associated genes [MECP2, ARHGAP32 (p250GAP), CREB, and period genes] were experimentally validated as miR-132 targets. However, most studies regarding miR-132 in major depressive disorder are based on post-mortem, animal models or genetic comparisons. This work will be the first attempt to investigate how miR-132 dysregulation may impact covariation of multimodal brain imaging data in 81 unmedicated major depressive patients and 123 demographically-matched healthy controls, as well as in a medication-naïve subset of major depressive patients. MiR-132 values in blood (patients > controls) was used as a prior reference to guide fusion of three MRI features: fractional amplitude of low frequency fluctuations, grey matter volume, and fractional anisotropy. The multimodal components correlated with miR-132 also show significant group difference in loadings. Results indicate that (i) higher miR-132 levels in major depressive disorder are associated with both lower fractional amplitude of low frequency fluctuations and lower grey matter volume in fronto-limbic network; and (ii) the identified brain regions linked with increased miR-132 levels were also associated with poorer cognitive performance in attention and executive function. Using a data-driven, supervised-learning method, we determined that miR-132 dysregulation in major depressive disorder is associated with multi-facets of brain function and structure in fronto-limbic network (the key network for emotional regulation and memory), which deepens our understanding of how miR-132 dysregulation in major depressive disorders contribute to the loss of specific brain areas and is linked to relevant cognitive impairments.
Assuntos
Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/diagnóstico por imagem , MicroRNAs/sangue , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Estudos de Casos e Controles , Cognição/fisiologia , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/metabolismo , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Oxigênio/sangue , Adulto JovemRESUMO
The mammalian embryonic lethal abnormal vision (ELAV)-like protein HuD is a neuronal RNA-binding protein implicated in neuronal development, plasticity, and diseases. Although HuD has long been associated with neuronal development, the functions of HuD in neural stem cell differentiation and the underlying mechanisms have gone largely unexplored. Here we show that HuD promotes neuronal differentiation of neural stem/progenitor cells (NSCs) in the adult subventricular zone by stabilizing the mRNA of special adenine-thymine (AT)-rich DNA-binding protein 1 (SATB1), a critical transcriptional regulator in neurodevelopment. We find that SATB1 deficiency impairs the neuronal differentiation of NSCs, whereas SATB1 overexpression rescues the neuronal differentiation phenotypes resulting from HuD deficiency. Interestingly, we also discover that SATB1 is a transcriptional activator of HuD during NSC neuronal differentiation. In addition, we demonstrate that NeuroD1, a neuronal master regulator, is a direct downstream target of SATB1. Therefore, HuD and SATB1 form a positive regulatory loop that enhances NeuroD1 transcription and subsequent neuronal differentiation. Our results here reveal a novel positive feedback network between an RNA-binding protein and a transcription factor that plays critical regulatory roles in neurogenesis.
Assuntos
Proteína Semelhante a ELAV 4/metabolismo , Retroalimentação Fisiológica , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neurogênese , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Western Blotting , Diferenciação Celular , Células Cultivadas , Proteína Semelhante a ELAV 4/genética , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Ligação Proteica , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Examination of intrinsic functional connectivity using functional MRI (fMRI) has provided important findings regarding dysconnectivity in schizophrenia. Extending these results using a complementary neuroimaging modality, magnetoencephalography (MEG), we present the first direct comparison of functional connectivity between schizophrenia patients and controls, using these two modalities combined. We developed a novel MEG approach for estimation of networks using MEG that incorporates spatial independent component analysis (ICA) and pairwise correlations between independent component timecourses, to estimate intra- and intern-network connectivity. This analysis enables group-level inference and testing of between-group differences. Resting state MEG and fMRI data were acquired from a large sample of healthy controls (n=45) and schizophrenia patients (n=46). Group spatial ICA was performed on fMRI and MEG data to extract intrinsic fMRI and MEG networks and to compensate for signal leakage in MEG. Similar, but not identical spatial independent components were detected for MEG and fMRI. Analysis of functional network connectivity (FNC; i.e., pairwise correlations in network (ICA component) timecourses) revealed a differential between-modalities pattern, with greater connectivity among occipital networks in fMRI and among frontal networks in MEG. Most importantly, significant differences between controls and patients were observed in both modalities. MEG FNC results in particular indicated dysfunctional hyperconnectivity within frontal and temporal networks in patients, while in fMRI FNC was always greater for controls than for patients. This is the first study to apply group spatial ICA as an approach to leakage correction, and as such our results may be biased by spatial leakage effects. Results suggest that combining these two neuroimaging modalities reveals additional disease-relevant patterns of connectivity that were not detectable with fMRI or MEG alone.
Assuntos
Encéfalo/fisiopatologia , Conectoma/métodos , Imageamento por Ressonância Magnética/métodos , Magnetoencefalografia/métodos , Esquizofrenia/fisiopatologia , Adulto , Encéfalo/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esquizofrenia/diagnóstico por imagemRESUMO
Interactions of RNA-binding proteins (RBPs) with their target transcripts are essential for regulating gene expression at the posttranscriptional level including mRNA export/localization, stability, and translation. ZBP1 and HuD are RBPs that play pivotal roles in mRNA transport and local translational control in neuronal processes. While HuD possesses three RNA recognition motifs (RRMs), ZBP1 contains two RRMs and four K homology (KH) domains that either increase target specificity or provide a multi-target binding capability. Here we used isolated cis-element sequences of the target mRNA to examine directly protein-RNA interactions in cell-free systems. We found that both ZBP1 and HuD bind the zipcode element in rat ß-actin mRNA's 3' UTR. Differences between HuD and ZBP1 were observed in their binding preference to the element. HuD showed a binding preference for U-rich sequence. In contrast, ZBP1 binding to the zipcode RNA depended more on the structural level, as it required the proper spatial organization of a stem-loop that is mainly determined by the U-rich element juxtaposed to the 3' end of a 5'-ACACCC-3' motif. On the basis of this work, we propose that ZBP1 and HuD bind to overlapping sites in the ß-actin zipcode, but they recognize different features of this target sequence.
Assuntos
Regiões 3' não Traduzidas , Actinas/genética , Proteína Semelhante a ELAV 4/metabolismo , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Actinas/metabolismo , Animais , Neurônios/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RatosRESUMO
The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the "capture" of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the "tag" is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dendritos/metabolismo , Proteínas ELAV/metabolismo , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Dendritos/enzimologia , Dendritos/genética , Proteínas ELAV/genética , Proteína Semelhante a ELAV 4 , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Neurônios/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Ratos , Sinapses/enzimologia , Sinapses/genética , Sinapses/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
This study investigates the possible mechanism(s) underlying glutathione (GSH) deficiency in the mouse spinal cord during the course of myelin oligodendrocyte glycoprotein35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis. Using the classical enzymatic recycling method and a newly developed immunodot assay, we first demonstrated that total GSH levels (i.e. free GSH plus all its adducts) are reduced in EAE, suggesting an impaired synthesis. The decline in the levels of this essential antioxidant tripeptide in EAE coincides temporally and in magnitude with a reduction in the amount of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis. Other enzymes involved in GSH biosynthesis, whose genes also contain antioxidant-response elements, including glutathione synthetase, cystine/glutamate antiporter, and γ-glutamyl transpeptidase (γ-GT) are diminished in EAE as well. Low levels of γ-glutamylcysteine ligase, glutathione synthetase, and γ-GT are the consequence of reduced mRNA expression, which correlates with diminished expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in both the cytosol and nucleus. Interestingly, the low Nrf2 expression does not seem to be caused by increased degradation via Kelch-like ECH-associated protein 1-dependent or Kelch-like ECH-associated protein 1-independent mechanisms (such as glycogen synthetase kinase-3ß activation), or by reduced levels of Nrf2 mRNA. This suggests that translation of this important transcription factor and/or other still unidentified post-translational processes are altered in EAE. These novel findings are central toward understanding how critical antioxidant and protective responses are lost in inflammatory demyelinating disorders.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Glutationa/deficiência , Fator 2 Relacionado a NF-E2/biossíntese , RNA Mensageiro/biossíntese , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
BACKGROUND: Given the challenges of confirming prenatal alcohol exposure (PAE) during pregnancy using currently established biomarkers of alcohol consumption, we examined whether serum microRNAs (miRNAs) may serve as stable biomarkers for PAE. Alterations in the levels of specific circulating miRNAs have been associated with various disease states and in animal models of fetal alcohol spectrum disorder. METHODS: Pregnant women in this prospective study were recruited from substance abuse and general maternity clinics affiliated with the University of New Mexico. Serum was collected at the time of admission for delivery from 14 subjects who reported ≥1 binge-drinking episode or ≥3 drinks/wk during pregnancy and 16 subjects who reported abstinence during pregnancy and tested negative for 5 ethanol biomarkers. Total RNA was isolated from serum and used for microarray analysis. RESULTS: False discovery rate-corrected analyses of covariance revealed that 55 miRNAs were significantly altered between the 2 groups. Hierarchical clustering using only the significantly altered miRNAs grouped samples into alcohol-consuming and non-alcohol-consuming individuals. Discriminant analysis then identified miRs-122*, -126, -216b, -221*, -3119, -3942-5p, -4704-3p, -4743, -514-5p, and -602 as the top 10 discriminators between the 2 groups. Ingenuity Pathway Analysis of putative miRNA targets illustrated that miRNAs identified in this study are involved in biological pathways that mediate the effects of alcohol, such as brain-derived neurotrophic factor, ERK1/2, and PI3K/AKT signaling. CONCLUSIONS: This is the first report of alterations in serum miRNA expression that are associated with alcohol use during human pregnancy. These results suggest that serum miRNAs could be useful as biomarkers of alcohol exposure.
Assuntos
Consumo de Bebidas Alcoólicas/sangue , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , MicroRNAs/sangue , Adulto , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/genética , Biomarcadores/sangue , Estudos de Coortes , Feminino , Redes Reguladoras de Genes/fisiologia , Humanos , MicroRNAs/genética , Gravidez , Estudos Prospectivos , Adulto JovemRESUMO
Growth-associated protein 43 (GAP-43) is a neuronal phosphoprotein associated with initial axonal outgrowth and synaptic remodeling and recent work also suggests its involvement in cell cycle control. The complex expression of GAP-43 features transcriptional and posttranscriptional components. However, in some conditions, GAP-43 gene expression is controlled primarily by the interaction of stabilizing or destabilizing RNA-binding proteins (RBPs) with adenine and uridine (AU)-rich instability elements (AREs) in its 3'UTR. Like GAP-43, many proteins involved in cell proliferation are encoded by ARE-containing mRNAs, some of which codify cell-cycle-regulating proteins including cyclin D1. Considering that GAP-43 and cyclin D1 mRNA stabilization may depend on similar RBPs, this study evaluated the participation of GAP-43 in cell cycle control and its underlying mechanisms, particularly the possible role of its 3'UTR, using GAP-43-transfected NIH-3T3 fibroblasts. Our results show an arrest in cell cycle progression in the G0/G1 phase. This arrest may be mediated by the competition of GAP-43 3'UTR with cyclin D1 3'UTR for the binding of Hu proteins such as HuR, which may lead to a decrease in cyclin D1 expression. These results might lead to therapeutic applications involving the use of sequences in the B region of GAP-43 3'UTR to slow down cell cycle progression.
Assuntos
Proteína GAP-43/metabolismo , Regiões 3' não Traduzidas , Animais , Adesão Celular , Ciclo Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Ativação Enzimática , Proteína GAP-43/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células NIH 3T3 , Proteínas de Ligação a RNA/metabolismoRESUMO
RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.
RESUMO
Recent microbiome-brain axis findings have shown evidence of the modulation of microbiome community as an environmental mediator in brain function and psychiatric illness. This work is focused on the role of the microbiome in understanding a rarely investigated environmental involvement in schizophrenia (SZ), especially in relation to brain circuit dysfunction. We leveraged high throughput microbial 16s rRNA sequencing and functional neuroimaging techniques to enable the delineation of microbiome-brain network links in SZ. N = 213 SZ and healthy control subjects were assessed for the oral microbiome. Among them, 139 subjects were scanned by resting-state functional magnetic resonance imaging (rsfMRI) to derive brain functional connectivity. We found a significant microbiome compositional shift in SZ beta diversity (weighted UniFrac distance, p = 6 × 10-3; Bray-Curtis distance p = 0.021). Fourteen microbial species involving pro-inflammatory and neurotransmitter signaling and H2S production, showed significant abundance alterations in SZ. Multivariate analysis revealed one pair of microbial and functional connectivity components showing a significant correlation of 0.46. Thirty five percent of microbial species and 87.8 % of brain functional network connectivity from each component also showed significant differences between SZ and healthy controls with strong performance in classifying SZ from healthy controls, with an area under curve (AUC) = 0.84 and 0.87, respectively. The results suggest a potential link between oral microbiome dysbiosis and brain functional connectivity alteration in relation to SZ, possibly through immunological and neurotransmitter signaling pathways and the hypothalamic-pituitary-adrenal axis, supporting for future work in characterizing the role of oral microbiome in mediating effects on SZ brain functional activity.