RESUMEN
The pro-inflammatory cytokine interleukin-1ß (IL-1ß), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1ß increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1ß-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R(-/-) mice. Other findings indicate that function-blocking anti-αv ß3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1ß-treated mPCNs, suggesting that established P2Y2R/αv ß3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1ß-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.
Asunto(s)
Corteza Cerebral/citología , Interleucina-1beta/farmacología , Neuronas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Integrina alfaVbeta3/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuritas/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Fosforilación , Cultivo Primario de Células , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/genética , Receptores de Vitronectina/metabolismo , Regulación hacia Arriba , Uridina Trifosfato/farmacologíaRESUMEN
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a "gain-of-function" mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Trans-Empalme , Células Cultivadas , Exones , Terapia Genética/métodos , Células HEK293 , Humanos , Proteína Huntingtina , Lentivirus/genética , Precursores del ARN/genética , ARN Mensajero/genética , Empalmosomas , TransfecciónRESUMEN
Extracellular nucleotides induce cellular responses in the central nervous system (CNS) through the activation of ionotropic P2X and metabotropic P2Y nucleotide receptors. Activation of these receptors regulates a wide range of physiological and pathological processes. In this review, we present an overview of the current literature regarding P2X and P2Y receptors in the CNS with a focus on the contribution of P2X7 and P2Y(2) receptor-mediated responses to neuroinflammatory and neuroprotective mechanisms.
Asunto(s)
Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Inflamación/patología , Nucleótidos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animales , Humanos , Fármacos Neuroprotectores/metabolismoRESUMEN
Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.
Asunto(s)
Fármacos Neuroprotectores , Receptores Purinérgicos P2Y2/fisiología , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiología , Endotelio/fisiología , Humanos , Inflamación/patología , Neuroglía/fisiología , Neuronas/fisiología , Receptores Purinérgicos P2X/fisiología , Transducción de Señal/fisiologíaRESUMEN
Amyloid ß-protein (Aß) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aß. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aß. Treatment of mouse primary microglial cells with fibrillar (fAß(1-42) ) and oligomeric (oAß(1-42) ) Aß(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAß(1-42) and oAß(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAß(1-42) and oAß(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aß(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aß(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aß(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aß phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aß(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aß clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Movimiento Celular/efectos de los fármacos , Microglía/metabolismo , Nucleótidos/metabolismo , Nucleótidos/farmacología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Agonistas del Receptor Purinérgico P2Y , Receptores Purinérgicos P2Y2/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Integrina alfa5/farmacología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microscopía Electrónica de Transmisión , Neurofibrillas/metabolismo , Fagocitosis/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Uridina Trifosfato/farmacología , Proteínas de Unión al GTP rac/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
Alzheimer's disease (AD) is characterized by progressive neurodegeneration associated with the aggregation and deposition of ß-amyloid (Aß(40) and Aß(42) ) peptide in senile plaques. Recent studies suggest that copper may play an important role in AD pathology. Copper concentrations are elevated in amyloid plaques and copper binds with high affinity to the Aß peptide and promotes Aß oligomerization and neurotoxicity. Despite this connection between copper and AD, it is unknown whether the expression of proteins involved in regulating copper homeostasis is altered in this disorder. In this study, we demonstrate that the copper transporting P-type ATPase, ATP7A, is highly expressed in activated microglial cells that are specifically clustered around amyloid plaques in the TgCRND8 mouse model of AD. Using a cultured microglial cell line, ATP7A expression was found to be increased by the pro-inflammatory cytokine interferon-gamma, but not by TNF-α or IL-1ß. Interferon-gamma also elicited marked changes in copper homeostasis, including copper-dependent trafficking of ATP7A from the Golgi to cytoplasmic vesicles, increased copper uptake and elevated expression of the CTR1 copper importer. These findings suggest that pro-inflammatory conditions associated with AD cause marked changes in microglial copper trafficking, which may underlie the changes in copper homeostasis in AD. It is concluded that copper sequestration by microglia may provide a neuroprotective mechanism in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Cobre/metabolismo , Microglía/metabolismo , Adenosina Trifosfatasas/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Transportador de Cobre 1 , ATPasas Transportadoras de Cobre , Vesículas Citoplasmáticas/metabolismo , Homeostasis , Interferón gamma/farmacología , Ratones , Ratones Transgénicos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Transporte de ProteínasRESUMEN
Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G(q) protein-coupled P2Y(2) nucleotide receptor subtype (P2Y(2)R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y(2)R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y(2)R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y(2)R activation increases astrocyte migration. P2Y(2)R activation by UTP increases the expression in astrocytes of alpha(V)beta(3/5) integrins that bind directly to the P2Y(2)R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y(2)R, an interaction required for G(o) and G(12) protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y(2)R expression is increased by stimulation with interleukin-1beta (IL-1beta), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric beta-amyloid peptide (Abeta(1-42)), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y(2)Rs in neurons. Data with rPCNs suggest that P2Y(2)R upregulation by IL-1beta and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1beta-upregulated P2Y(2)Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y(2)Rs in glial cells can promote neuroprotective responses, suggesting that P2Y(2)Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.
Asunto(s)
Encéfalo/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Receptores Purinérgicos P2/metabolismo , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Citoesqueleto/metabolismo , Inflamación/metabolismo , Microvasos/metabolismo , Datos de Secuencia Molecular , Neuroglía/citología , Neuronas/citología , Estructura Secundaria de Proteína , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y2 , Transducción de Señal/fisiologíaRESUMEN
The use of polarized salivary gland cell monolayers has contributed to our understanding of salivary gland physiology. However, these cell models are not representative of glandular epithelium in vivo, and, therefore, are not ideal for investigating salivary epithelial functions. The current study has developed a three-dimensional (3D) cell culture model for rat Par-C10 parotid gland cells that forms differentiated acinar-like spheres on Matrigel. These 3D Par-C10 acinar-like spheres display characteristics similar to differentiated acini in salivary glands, including cell polarization, tight junction (TJ) formation required to maintain transepithelial potential difference, basolateral expression of aquaporin-3 and Na+/K+/2Cl- cotransporter-1, and responsiveness to the muscarinic receptor agonist carbachol that is decreased by the anion channel blocker diphenylamine-2-carboxylic acid or chloride replacement with gluconate. Incubation of the spheres in the hypertonic medium increased the expression level of the water channel aquaporin-5. Further, the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma induced alterations in TJ integrity in the acinar-like spheres without affecting individual cell viability, suggesting that cytokines may affect salivary gland function by altering TJ integrity. Thus, 3D Par-C10 acinar-like spheres represent a novel in vitro model to study physiological and pathophysiological functions of differentiated acini.
Asunto(s)
Diferenciación Celular , Glándula Parótida/citología , Animales , Western Blotting , Carbacol/farmacología , Polaridad Celular , Células Cultivadas , Interferón gamma/farmacología , Microscopía Confocal , Ratas , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , ortoaminobenzoatos/farmacologíaRESUMEN
The heterologous expression and activation of the human P2Y(2) nucleotide receptor (P2Y(2)R) in human 1321N1 astrocytoma cells stimulates alpha-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPPalpha). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y(2)R-mediated production of sAPPalpha occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y(2)R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) up-regulated both P2Y(2)R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y(2)R was abrogated by pre-incubation with Bay 11-7085, an IkappaB-alpha phosphorylation inhibitor, which suggests that P2Y(2)R mRNA transcript levels are regulated through nuclear factor-kappa-B (NFkappaB) signaling. Furthermore, the P2Y(2)R agonist Uridine-5'-triphosphate (UTP) enhanced the release of sAPPalpha in rPCNs treated with IL-1beta or transfected with P2Y(2)R cDNA. UTP-induced release of sAPPalpha from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y(2)R-mediated release of sAPPalpha from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1beta, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y(2)R in neurons.