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
UNLABELLED: Chronic neuropathic pain is often difficult to treat with current pain medications. Gene therapy is presently being explored as a therapeutic approach for the treatment of neuropathic and cancer pain. In this study, we sought to use an injury-specific promoter to deliver the mu-opioid receptor (MOR) transgene such that expression would occur during the injured state only in response to release of injury-specific galanin. To determine whether an injury-specific promoter can produce neuron-specific MOR expression and enhanced antinociception, we compared animals infected with a galanin promoter virus (galMOR) or a human cytomegalovirus promoter virus (cmvMOR). In behavioral assays, we found an earlier onset and a larger magnitude of antinociception in animals infected with galMOR compared with cmvMOR. Immunohistochemical analysis of dorsal root ganglion neurons revealed a significant increase in MOR-positive staining in cmvMOR- and galMOR-treated mice. Spinal cord sections from galMOR-treated mice showed a greater increase in density but not area of MOR-positive staining. These results suggest that using injury-specific promoters to drive gene expression in primary afferent neurons can influence the onset and magnitude of antinociception in a rodent model of neuropathic pain and can be used to upregulate MOR expression in populations of neurons that are potentially injury specific. PERSPECTIVE: An injury-specific promoter (galMOR) was used to drive MOR expression in a population- and injury-specific manner. GalMOR increased antinociception and density of MOR staining in the spinal cord. This article presents evidence that promoter selection is an important component in successful gene expression in an injury- and population-specific manner.
Assuntos
Terapia Genética/métodos , Neuralgia/terapia , Regiões Promotoras Genéticas , Receptores Opioides mu/genética , Simplexvirus/genética , Animais , Citomegalovirus/genética , Modelos Animais de Doenças , Feminino , Galanina/genética , Gânglios Espinais/patologia , Gânglios Espinais/fisiopatologia , Temperatura Alta , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Hiperalgesia/terapia , Imuno-Histoquímica , Vértebras Lombares , Camundongos , Neuralgia/patologia , Neuralgia/fisiopatologia , Neurônios Aferentes/patologia , Neurônios Aferentes/fisiologia , Receptores Opioides mu/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Nervos Espinhais/lesões , TatoRESUMO
Many people worldwide suffer from pain and a portion of these sufferers are diagnosed with a chronic pain condition. The management of chronic pain continues to be a challenge, and despite taking prescribed medication for pain, patients continue to have pain of moderate severity. Current pain therapies are often inadequate, with side effects that limit medication adherence. There is a need to identify novel therapeutic targets for the management of chronic pain. One potential candidate for the treatment of chronic pain is therapies aimed at modulating the vasoactive peptide endothelin-1. In addition to vasoactive properties, endothelin-1 has been implicated in pain transmission in both humans and animal models of nociception. Endothelin-1 directly activates nociceptors and potentiates the effect of other algogens, including capsaicin, formalin, and arachidonic acid. In addition, endothelin-1 has been shown to be involved in inflammatory pain, cancer pain, neuropathic pain, diabetic neuropathy, and pain associated with sickle cell disease. Therefore, endothelin-1 may prove a novel therapeutic target for the relief of many types of chronic pain.