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1.
Nat Commun ; 15(1): 356, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38191621

RESUMO

Neurodegeneration is the primary driver of disease progression in multiple sclerosis (MS) resulting in permanent disability, creating an urgent need to discover its underlying mechanisms. Herein, we establish that dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) results in differential of binding to RNA targets causing alternative RNA splicing, which contributes to neurodegeneration in MS and its models. Using RNAseq of MS brains, we discovered differential expression and aberrant splicing of hnRNP A1 target RNAs involved in neuronal function and RNA homeostasis. We confirmed this in vivo in experimental autoimmune encephalomyelitis employing CLIPseq specific for hnRNP A1, where hnRNP A1 differentially binds and regulates RNA, including aberrantly spliced targets identified in human samples. Additionally, dysfunctional hnRNP A1 expression in neurons caused neurite loss and identical changes in splicing, corroborating hnRNP A1 dysfunction as a cause of neurodegeneration. Collectively, these data indicate hnRNP A1 dysfunction causes altered neuronal RNA splicing, resulting in neurodegeneration in MS.


Assuntos
Ribonucleoproteína Nuclear Heterogênea A1 , Esclerose Múltipla , Humanos , Processamento Alternativo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Esclerose Múltipla/genética , RNA , Splicing de RNA/genética
2.
Neurobiol Dis ; 170: 105775, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35618205

RESUMO

Neurodegeneration, the progressive loss or damage to neurons and axons, underlies permanent disability in multiple sclerosis (MS); yet its mechanisms are incompletely understood. Recent data indicates autoimmunity to several intraneuronal antigens, including the RNA binding protein (RBP) heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), as contributors to neurodegeneration. We previously showed that addition of anti-hnRNP A1 antibodies, which target the same immunodominant domain of MS IgG, to mice with experimental autoimmune encephalomyelitis (EAE) worsened disease and resulted in an exacerbation of hnRNP A1 dysfunction including cytoplasmic mislocalization of hnRNP A1, stress granule (SG) formation and neurodegeneration in the chronic stages of disease. Because this previous study focused on a singular timepoint during EAE, it is unclear whether anti-hnRNP A1 antibody induced hnRNP A1 dysfunction caused neurodegeneration or was result of it. In the present study, we analyzed in vivo and in vitro models of anti-hnRNP A1 antibody-mediated autoimmunity for markers of hnRNP A1 dysfunction and neurodegeneration over a time course to gain a better understanding of the connection between hnRNP A1 dysfunction and neurodegeneration. Anti-hnRNP A1 antibody treatment resulted in increased neuronal hnRNP A1 mislocalization and nuclear depletion temporally followed by altered RNA expression and SG formation, and lastly an increase in necroptotic signalling and neuronal cell death. Treatment with necrostatin-1s inhibited necroptosis and partially rescued anti-hnRNP A1 antibody-mediated neurodegeneration while clathrin knockdown specifically inhibited anti-hnRNP A1 antibody uptake into neurons. This data identifies a novel antibody-mediated mechanism of neurodegeneration, which may be targeted to inhibit neurodegeneration and prevent permanent neurological decline in persons living with MS.


Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Autoimunidade , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Camundongos , Esclerose Múltipla/metabolismo , Degeneração Neural , Neurônios/metabolismo , Ribonucleoproteínas
3.
eNeuro ; 8(6)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34697074

RESUMO

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA binding protein (RBP) that is localized within neurons and plays crucial roles in RNA metabolism. Its importance in neuronal functioning is underscored from the study of its pathogenic features in many neurodegenerative diseases where neuronal hnRNP A1 is mislocalized from the nucleus to the cytoplasm resulting in loss of hnRNP A1 function. Here, we model hnRNP A1 loss-of-function by siRNA-mediated knock-down in differentiated Neuro-2a cells. Through RNA sequencing (RNA-seq) followed by gene ontology (GO) analyses, we show that hnRNP A1 is involved in important biological processes, including RNA metabolism, neuronal function, neuronal morphology, neuronal viability, and stress granule (SG) formation. We further confirmed several of these roles by showing that hnRNP A1 knock-down results in a reduction of neurite outgrowth, increase in cell cytotoxicity and changes in SG formation. In summary, these findings indicate that hnRNP A1 loss-of-function contributes to neuronal dysfunction and cell death and implicates hnRNP A1 dysfunction in the pathogenesis of neurodegenerative diseases.


Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/genética , Neuritos , Neurônios , Grânulos de Estresse , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Camundongos , Neuritos/patologia , Neurônios/patologia
4.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809384

RESUMO

Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.


Assuntos
Esclerose Lateral Amiotrófica/genética , Ribonucleoproteína Nuclear Heterogênea A1/genética , Esclerose Múltipla/genética , Degeneração Neural/genética , Esclerose Lateral Amiotrófica/patologia , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Esclerose Múltipla/patologia , Mutação/genética
5.
Ann Clin Transl Neurol ; 7(7): 1214-1224, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32608162

RESUMO

OBJECTIVE: Neurodegeneration is thought to be the primary cause of neurological disability in multiple sclerosis (MS). Dysfunctional RNA-binding proteins (RBPs) including their mislocalization from nucleus to cytoplasm, stress granule formation, and altered RNA metabolism have been found to underlie neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia. Yet, little is known about the role of dysfunctional RBPs in the pathogenesis of neurodegeneration in MS. As a follow-up to our seminal finding of altered RBP function in a single case of MS, we posited that there would be evidence of RBP dysfunction in cortical neurons in MS. METHODS: Cortical neurons from 12 MS and six control cases were analyzed by immunohistochemistry for heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and TAR-DNA-binding protein-43 (TDP-43). Seven distinct neuronal phenotypes were identified based on the nucleocytoplasmic staining of these RBPs. Statistical analyses were performed by analyzing each phenotype in relation to MS versus controls. RESULTS: Analyses revealed a continuum of hnRNP A1 and TDP-43 nucleocytoplasmic staining was found in cortical neurons, from neurons with entirely nuclear staining with little cytoplasmic staining in contrast to those with complete nuclear depletion of RBPs concurrent with robust cytoplasmic staining. The neuronal phenotypes that showed the most nucleocytoplasmic mislocalization of hnRNP A1 and TDP-43 statistically distinguished MS from control cases (P < 0.01, P < 0.001, respectively). INTERPRETATION: The discovery of hnRNP A1 and TDP-43 nucleocytoplasmic mislocalization in neurons in MS brain demonstrate that dysfunctional RBPs may play a role in neurodegeneration in MS, as they do in other neurological diseases.


Assuntos
Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Esclerose Múltipla/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Neurônios/classificação
6.
J Comp Neurol ; 528(10): 1704-1724, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31872424

RESUMO

Neurodegeneration, including loss of neurons and axons, is a feature of progressive forms of multiple sclerosis (MS). The mechanisms underlying neurodegeneration are mostly unknown. Research implicates autoimmunity to nonmyelin self-antigens as important contributors to disease pathogenesis. Data from our lab implicate autoimmunity to the RNA binding protein (RBP) heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a possible mechanism of neurodegeneration in MS. MS patients make antibodies to hnRNP A1, which have been shown to lead to neuronal dysfunction in vitro. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we show here that injection of anti-hnRNP A1 antibodies, in contrast to control antibodies, resulted in worsened disease and increased neurodegeneration. We found a reduction of NeuN+ neuronal cell bodies in areas of the ventral gray matter of the spinal cord where anti-hnRNP A1 antibodies localized. Neurons displayed increased levels of hnRNP A1 nucleocytoplasmic mislocalization and stress granule formation, both markers of neuronal injury. Anti-hnRNP A1 antibodies were found to surround neuronal cell bodies and interact with CD68+ immune cells via Fc receptors. Additionally, anti-hnRNP A1 antibodies were found within neuronal cell bodies including those of the ventral spinocerebellar tract (VSCT), a tract previously shown to undergo neurodegeneration in anti-hnRNP A1 antibody injected EAE mice. Finally, both immune cells and neurons showed increased levels of inducible nitric oxide synthase, another indicator of cell damage. These findings suggest that autoimmunity to RBPs, such as hnRNP A1, play a role in neurodegeneration in EAE with important implications for the pathogenesis of MS.


Assuntos
Autoanticorpos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Ribonucleoproteína Nuclear Heterogênea A1/imunologia , Degeneração Neural/imunologia , Neurônios/patologia , Animais , Autoantígenos/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla , Neurônios/imunologia
7.
J Neuropathol Exp Neurol ; 78(4): 348-364, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30863858

RESUMO

Luman/CREB3 is an important early retrograde axotomy signal regulating acute axon outgrowth in sensory neurons through the adaptive unfolded protein response. As the injury response is transcriptionally multiphasic, a spatiotemporal analysis of Luman/CREB3 localization in rat dorsal root ganglion (DRG) with unilateral L4-L6 spinal nerve injury was conducted to determine if Luman/CREB3 expression was similarly regulated. Biphasic alterations in Luman/CREB3 immunofluorescence and nuclear localization occurred in neurons ipsilateral to 1-hour, 1-day, 2-day, 4-day, and 1-week injury, with a largely parallel, but less avid response contralaterally. This biphasic response was not observed at the transcript level. To assess whether changes in neuronal Luman expression corresponded with an altered intrinsic capacity to grow an axon/neurite in vitro, injury-conditioned and contralateral uninjured DRG neurons underwent a 24-hour axon growth assay. Two-day injury-conditioned neurons exhibited maximal outgrowth capacity relative to naïve, declining at later injury-conditioned timepoints. Only neurons contralateral to 1-week injury exhibited significantly higher axon growth capacity than naïve. In conclusion, alterations in neuronal injury-associated Luman/CREB3 expression support that a multiphasic cell body response occurs and reveal a novel contralateral plasticity in axon growth capacity at 1-week post-injury. These adaptive responses have the potential to inform when repair or therapeutic intervention may be most effective.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Lateralidade Funcional/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axônios/metabolismo , Axotomia , Gânglios Espinais/metabolismo , Masculino , Neuritos/metabolismo , Ratos , Ratos Wistar
8.
J Neuroimmunol ; 324: 149-156, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30190085

RESUMO

Dysfunction of the RNA binding protein (RBP) heterogeneous nuclear ribonuclear protein A1 (hnRNP A1) has been shown to contribute to the pathogenesis of neurodegenerative diseases, but its involvement in multiple sclerosis (MS) is largely unknown. In a neuronal cell line, interferon-γ caused hnRNP A1 nucleocytoplasmic mislocalization; colocalization of hnRNP A1 in stress granules (SGs); and inhibition of translation. Neurons in the brain of a MS patient showed pathogenic RBP dysfunction, including nuclear depletion of hnRNP A1, its mislocalization to the cytoplasm, and its colocalization in SGs. These data indicate a role for dysfunctional hnRNP A1 in the pathogenesis of MS.


Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Esclerose Múltipla/metabolismo , Estresse Oxidativo/fisiologia , Linhagem Celular Tumoral , Ribonucleoproteína Nuclear Heterogênea A1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
9.
Biochemistry ; 52(45): 7999-8011, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24128008

RESUMO

Protein kinase C-related kinases (PRKs) are members of the protein kinase C superfamily of serine-threonine kinases and can be activated by binding to members of the Rho family of GTPases via a Rho-binding motif known as an HR1 domain. Three tandem HR1 domains reside at the N-terminus of the PRKs. We have assessed the ability of the HR1a and HR1b domains from the three PRK isoforms (PRK1, PRK2, and PRK3) to interact with the three Rho isoforms (RhoA, RhoB, and RhoC). The affinities of RhoA and RhoC for a construct encompassing both PRK1 HR1 domains were similar to those for the HR1a domain alone, suggesting that these interactions are mediated solely by the HR1a domain. The affinities of RhoB for both the PRK1 HR1a domain and the HR1ab didomain were higher than those of RhoA or RhoC. RhoB also bound more tightly to the didomain than to the HR1a domain alone, implicating the HR1b domain in the interaction. As compared with PRK1 HR1 domains, PRK2 and PRK3 domains bind less well to all Rho isoforms. Uniquely, however, the PRK3 domains display a specificity for RhoB that requires both the C-terminus of RhoB and the PRK3 HR1b domain. The thermal stability of the HR1a and HR1b domains was also investigated. The PRK2 HR1a domain was found to be the most thermally stable, while PRK2 HR1b, PRK3 HR1a, and PRK3 HR1b domains all exhibited lower melting temperatures, similar to that of the PRK1 HR1a domain. The lower thermal stability of the PRK2 and PRK3 HR1b domains may impart greater flexibility, driving their ability to interact with Rho isoforms.


Assuntos
Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Dicroísmo Circular , Humanos , Ligação Proteica , Isoformas de Proteínas/genética , Proteína Quinase C/genética , Estabilidade Proteica , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/genética , Proteína de Ligação a GTP rhoC
10.
Biochemistry ; 50(14): 2860-9, 2011 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-21351730

RESUMO

Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.


Assuntos
Mutação , Proteína Quinase C/genética , Proteína rhoA de Ligação ao GTP/genética , Algoritmos , Sítios de Ligação/genética , Dicroísmo Circular , Análise Mutacional de DNA , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluções/química , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/metabolismo
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