RESUMO
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.
Assuntos
Nucléolo Celular/metabolismo , Gânglios Espinais/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , Nervo Isquiático/metabolismo , Sequência de Aminoácidos , Animais , Transporte Axonal/genética , Linhagem Celular Tumoral , Nucléolo Celular/ultraestrutura , Gânglios Espinais/citologia , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Neurônios/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Nervo Isquiático/citologia , NucleolinaRESUMO
How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.
Assuntos
Dor Crônica/fisiopatologia , Neuralgia/fisiopatologia , Células Receptoras Sensoriais/fisiologia , alfa Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Benzofenonas/farmacologia , Dor Crônica/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Isoxazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/genética , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , alfa Carioferinas/genéticaRESUMO
Histone deactylase (HDAC) inhibitors show promise as therapeutics for neurodegenerative and psychiatric diseases. Increased expression of brain-derived neurotrophic factor (BDNF) has been associated with memory-enhancing and neuroprotective properties of these drugs, but the mechanism of BDNF induction is not well understood. Here, we compared the effects of a class I/IIb selective HDAC inhibitor SAHA, a class I selective inhibitor MS-275, a class II selective inhibitor MC1568 and a HDAC6 selective inhibitor tubacin on Bdnf mRNA expression in rat primary neurons. We show that inhibition of class II HDACs resulted in rapid upregulation of Bdnf mRNA levels, whereas class I HDAC inhibition produced a markedly delayed Bdnf induction. In contrast to relatively slow upregulation of Bdnf transcripts, histone acetylation at BDNF promoters I and IV was rapidly induced by SAHA. Bdnf induction by SAHA and MS-275 at 24 h was sensitive to protein synthesis inhibition, suggesting that delayed Bdnf induction by HDAC inhibitors is secondary to changed expression of its regulators. HDAC4 and HDAC5 repressed Bdnf promoter IV activity, supporting the role of class II HDACs in regulation of Bdnf expression. In addition, we show a critical role for the cAMP/Ca2+ response element (CRE) in induction of Bdnf promoter IV by MS-275, MC1568, SAHA and sodium valproate. In contrast, MEF2-binding CaRE1 element was not necessary for promoter IV induction by HDAC inhibition. Finally, we show that similarly to Bdnf, the studied HDAC inhibitors differentially induced expression of neuronal activity-regulated genes c-fos and Arc. Together, our findings implicate class II HDACs in transcriptional regulation of Bdnf and indicate that class II selective HDAC inhibitors may have potential as therapeutics for nervous system disorders.