RESUMO
Individual differences in the effects of a chronic neuropathic injury on social behaviours characterize both the human experience and pre-clinical animal models. The impacts of these changes to the well-being of the individual are often underappreciated. Earlier work from our laboratory using GeneChip® microarrays identified increased cholecystokinin (CCK) gene expression in the periaqueductal gray (PAG) of rats that showed persistent changes in social interactions during a Resident-Intruder encounter following sciatic nerve chronic constriction injury (CCI). In this study, we confirmed these gene regulation patterns using RT-PCR and identified the anatomical location of the CCK-mRNA as well as the translated CCK peptides in the midbrains of rats with a CCI. We found that rats with persistent CCI-induced changes in social behaviours had increased CCK-mRNA in neurons of the ventrolateral PAG and dorsal raphe nuclei, as well as increased CCK-8 peptide expression in terminal boutons located in the lateral and ventrolateral PAG. The functional significance of these changes was explored by microinjecting small volumes of CCK-8 into the PAG of uninjured rats and observing their Resident-Intruder social interactions. Disturbances to social interactions identical to those observed in CCI rats were evoked when injection sites were located in the rostral lateral and ventrolateral PAG. We suggest that CCI-induced changes in CCK expression in these PAG regions contributes to the disruptions to social behaviours experienced by a subset of individuals with neuropathic injury.
Assuntos
Colecistocinina/biossíntese , Reação de Fuga/fisiologia , Substância Cinzenta Periaquedutal/metabolismo , Neuropatia Ciática/metabolismo , Interação Social , Animais , Reação de Fuga/efeitos dos fármacos , Masculino , Microinjeções/métodos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/psicologia , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/patologia , Neuropatia Ciática/psicologia , Sincalida/administração & dosagemRESUMO
The neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is caused by loss of function of sacsin, a modular protein that is required for normal mitochondrial network organization. To further understand cellular consequences of loss of sacsin, we performed microarray analyses in sacsin knockdown cells and ARSACS patient fibroblasts. This identified altered transcript levels for oxidative phosphorylation and oxidative stress genes. These changes in mitochondrial gene networks were validated by quantitative reverse transcription PCR. Functional impairment of oxidative phosphorylation was then demonstrated by comparison of mitochondria bioenergetics through extracellular flux analyses. Moreover, staining with the mitochondrial-specific fluorescent probe MitoSox suggested increased levels of superoxide in patient cells with reduced levels of sacsin.Key to maintaining mitochondrial health is mitochondrial fission, which facilitates the dynamic exchange of mitochondrial components and separates damaged parts of the mitochondrial network for selective elimination by mitophagy. Fission is dependent on dynamin-related protein 1 (Drp1), which is recruited to prospective sites of division where it mediates scission. In sacsin knockdown cells and ARSACS fibroblasts, we observed a decreased incidence of mitochondrial associated Drp1 foci. This phenotype persists even when fission is induced by drug treatment. Mitochondrial-associated Drp1 foci are also smaller in sacsin knockdown cells and ARSACS fibroblasts. These data suggest a model for ARSACS where neurons with reduced levels of sacsin are compromised in their ability to recruit or retain Drp1 at the mitochondrial membrane leading to a decline in mitochondrial health, potentially through impaired mitochondrial quality control.
Assuntos
Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Espasticidade Muscular/metabolismo , Ataxias Espinocerebelares/congênito , Linhagem Celular Tumoral , Dinaminas , Feminino , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/patologia , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/genética , Espasticidade Muscular/genética , Espasticidade Muscular/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologiaRESUMO
Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.
Assuntos
Colo/metabolismo , Nociceptores/metabolismo , Receptores Purinérgicos P2Y/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Colo/efeitos dos fármacos , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.9/fisiologia , Nucleotídeos de Purina/farmacologia , Nucleotídeos de Pirimidina/farmacologia , Especificidade da EspécieRESUMO
Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid that is essential in brain development and has structural and signaling roles. Acute DHA administration is neuroprotective and promotes functional recovery in animal models of adult spinal cord injury (SCI). However, the mechanisms underlying this recovery have not been fully characterized. Here we investigated the effects of an acute intravenous bolus of DHA delivered after SCI and characterized DHA-induced neuroplasticity within the adult injured spinal cord. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat cervical hemisection SCI model. A mouse pyramidotomy model was used to confirm that this robust sprouting was not species or injury model specific. Furthermore, we demonstrated that corticospinal fibers sprouting to the denervated side of the cord following pyramidotomy contact V2a interneurons. We also demonstrated increased serotonin fibers and synaptophysin in direct contact with motor neurons. DHA also increased synaptophysin in rat cortical cell cultures. A reduction in phosphatase and tensin homolog (PTEN) has been shown to be involved in axonal regeneration and synaptic plasticity. We showed that DHA significantly upregulates miR-21 and downregulates PTEN in corticospinal neurons. Downregulation of PTEN and upregulation of phosphorylated AKT by DHA were also seen in primary cortical neuron cultures and were accompanied by increased neurite outgrowth. In summary, acute DHA induces anatomical and synaptic plasticity in adult injured spinal cord. This study shows that DHA has therapeutic potential in cervical SCI and provides evidence that DHA could exert its beneficial effects in SCI via enhancement of neuroplasticity. SIGNIFICANCE STATEMENT: In this study, we show that an acute intravenous injection of docosahexaenoic acid (DHA) 30 min after spinal cord injury induces neuroplasticity. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat hemisection spinal cord injury model. A mouse pyramidotomy model was used to confirm that the robust sprouting involved V2a interneurons. We show that DHA significantly upregulates miR-21 and phosphorylated AKT, and downregulates phosphatase and tensin homolog (PTEN), which is involved in suppressing anatomical plasticity, in corticospinal neurons and in primary cortical neuron cultures. We conclude that acute DHA can induce anatomical and synaptic plasticity. This provides direct evidence that DHA could exert its beneficial effects in spinal cord injury via neuroplasticity enhancement.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Interneurônios/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Tratos Piramidais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Animais , Células Cultivadas , Vértebras Cervicais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Comportamento Exploratório/efeitos dos fármacos , Feminino , Transtornos Neurológicos da Marcha/tratamento farmacológico , Transtornos Neurológicos da Marcha/etiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intravenosas , Interneurônios/fisiologia , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Neurônios Motores/fisiologia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Plasticidade Neuronal/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tratos Piramidais/lesões , Tratos Piramidais/patologia , Tratos Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Neurônios Serotoninérgicos/fisiologia , Neurônios Serotoninérgicos/ultraestrutura , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation.
Assuntos
Encefalomielite Autoimune Experimental/genética , Células Endoteliais/metabolismo , MicroRNAs/metabolismo , Esclerose Múltipla/genética , NF-kappa B/metabolismo , Linfócitos T/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Adesão Celular/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Inflamação , Microdissecção e Captura a Laser , Masculino , Camundongos , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , TransfecçãoRESUMO
Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9(-/-) mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus-response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9(-/-) afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9(-/-) mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics.
Assuntos
Colo/inervação , Hiperalgesia/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.9/metabolismo , Fibras Aferentes Viscerais/metabolismo , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Adolescente , Adulto , Idoso , Animais , Colo/metabolismo , Colo/fisiopatologia , Dinoprostona/farmacologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Humanos , Hiperalgesia/fisiopatologia , Inflamação/metabolismo , Inflamação/fisiopatologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Estimulação Física , Fibras Aferentes Viscerais/efeitos dos fármacos , Fibras Aferentes Viscerais/fisiopatologia , Adulto JovemRESUMO
Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.
Assuntos
Barreira Hematoencefálica/fisiologia , MicroRNAs/fisiologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Humanos , Masculino , Camundongos , Esclerose Múltipla , Talina/biossíntese , Transcriptoma , Regulação para Cima , Vinculina/biossínteseRESUMO
Peripheral nerve injuries caused by trauma are associated with increased sensory neuron excitability and debilitating chronic pain symptoms. Axotomy-induced alterations in the function of ion channels are thought to largely underlie the pathophysiology of these phenotypes. Here, we characterise the mRNA distribution of Kv2 family members in rat dorsal root ganglia (DRG) and describe a link between Kv2 function and modulation of sensory neuron excitability. Kv2.1 and Kv2.2 were amply expressed in cells of all sizes, being particularly abundant in medium-large neurons also immunoreactive for neurofilament-200. Peripheral axotomy led to a rapid, robust and long-lasting transcriptional Kv2 downregulation in the DRG, correlated with the onset of mechanical and thermal hypersensitivity. The consequences of Kv2 loss-of-function were subsequently investigated in myelinated neurons using intracellular recordings on ex vivo DRG preparations. In naïve neurons, pharmacological Kv2.1/Kv2.2 inhibition by stromatoxin-1 (ScTx) resulted in shortening of action potential (AP) after-hyperpolarization (AHP). In contrast, ScTx application on axotomized neurons did not alter AHP duration, consistent with the injury-induced Kv2 downregulation. In accordance with a shortened AHP, ScTx treatment also reduced the refractory period and improved AP conduction to the cell soma during high frequency stimulation. These results suggest that Kv2 downregulation following traumatic nerve lesion facilitates greater fidelity of repetitive firing during prolonged input and thus normal Kv2 function is postulated to limit neuronal excitability. In summary, we have profiled Kv2 expression in sensory neurons and provide evidence for the contribution of Kv2 dysfunction in the generation of hyperexcitable phenotypes encountered in chronic pain states.
Assuntos
Doenças do Sistema Nervoso Periférico/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Potássio Shab/metabolismo , Animais , Axotomia/métodos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/citologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Dor/etiologia , Dor/metabolismo , Medição da Dor , Doenças do Sistema Nervoso Periférico/complicações , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Células Receptoras Sensoriais/fisiologia , Canais de Potássio Shab/genéticaRESUMO
Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.
Assuntos
Encefalomielite Autoimune Experimental/genética , Técnicas de Inativação de Genes , Esclerose Múltipla/genética , Receptor CB2 de Canabinoide/deficiência , Receptor CB2 de Canabinoide/genética , Receptores de Canabinoides/deficiência , Receptores de Canabinoides/genética , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Deleção de Genes , Imunomodulação/genética , Masculino , Camundongos , Esclerose Múltipla/imunologia , Fenótipo , Especificidade da EspécieRESUMO
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
Assuntos
Regulação para Baixo , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axotomia , Comportamento Animal/fisiologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Fibras Nervosas Mielinizadas/metabolismo , Neuralgia/etiologia , Neuralgia/fisiopatologia , Medição da Dor , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/fisiopatologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Interferente Pequeno , Ratos , Ratos WistarRESUMO
The role of glial cells in Parkinson's disease (PD) is unclear. We have previously reported a striking up-regulation of DnaJB6 heat shock protein in PD substantia nigra astrocytes. Whole genome transcriptome analysis also indicated increased expression of metallothionein genes in substantia nigra and cortex of sporadic PD cases. Metallothioneins are metal-binding proteins in the CNS that are released by astrocytes and associated with neuroprotection. Metallothionein expression was investigated in 18 PD cases and 15 non-PD controls using quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation (ISH) and immunocytochemistry (ICC). We observed a strong increase in the expression of metallothioneins MT1E, MT1F, MT1G, MT1H, MT1M, MT1X and MT2A in both PD nigra and frontal cortex. Expression of LRP2 (megalin), the neuronal metallothionein receptor was also significantly increased. qRT-PCR confirmed metallothionein up-regulation. Astrocytes were found to be the main source of metallothioneins 1 and 2 based on ISH results, and this finding was confirmed by ICC. Our findings demonstrate metallothionein expression by reactive astrocytes in PD nigra and support a neuroprotective role for these cells. The traditional view that nigral astrocytes are non-reactive in PD is clearly incorrect. However, it is possible that astrocytes are themselves affected by the disease process which may explain their comparatively modest and previously overlooked response.
Assuntos
Astrócitos/fisiologia , Metalotioneína/genética , Metalotioneína/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Regulação para Cima/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Astrócitos/citologia , Análise por Conglomerados , Feminino , Humanos , Masculino , Análise em Microsséries , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de SequênciaRESUMO
Retinoic acid receptors (RARs), retinoid X receptors (RXRs), and peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in many cellular processes, such as learning and memory. RAR and RXR mRNA levels decrease with ageing, and the decreases can be reversed by retinoic acid treatment, which also alleviates age-related memory deficits. The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have neuroprotective effects in the aged brain and are endogenous ligands of RXR and PPAR. We investigated whether dietary EPA and DHA supplementation reverses age-related declines in protein levels of these receptors in rat forebrain. Two studies were conducted comparing adult and old rats. In the first, old rats were fed standard or EPA/DHA-enriched (270 mg/kg/day, EPA to DHA ratio 1.5:1) diets for 12 weeks. Analysis by Western blot revealed significant decreases in RARalpha, RXRalpha, RXRbeta, and PPARgamma in the forebrain with ageing, which were reversed by supplementation. Immunohistochemical analysis of the hippocampus showed significant age-related decreases in RARalpha and RXRbeta expression in CA1 and the dentate gyrus, which were restored by supplementation. Decreases in hippocampal doublecortin expression were also partially alleviated, suggesting a positive effect on neurogenesis. We also investigated the effects of DHA supplementation (300 mg/kg/day for 12 weeks) on RARalpha, RXRalpha, and RXRbeta expression in the prefrontal cortex, striatum, and hippocampus. Overall, DHA supplementation appeared to increase receptor expression compared with the untreated old group. These observations illustrate additional mechanisms that might underlie the neuroprotective effects of omega-3 fatty acids in ageing.
Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Ácidos Graxos Ômega-3/metabolismo , Neurogênese/fisiologia , Neurônios/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Dieta , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Proteína Duplacortina , Ácido Eicosapentaenoico/administração & dosagem , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Masculino , Ratos , Ratos WistarRESUMO
C-fiber sensory neurons comprise nociceptors and smaller populations of cells detecting innocuous thermal and light tactile stimuli. Markers identify subpopulations of these cells, aiding our understanding of their physiological roles. The transient receptor potential vanilloid 1 (TRPV1) cation channel is characteristic of polymodal C-fiber nociceptors and is sensitive to noxious heat, irritant vanilloids, and protons. By using immunohistochemistry, in situ hybridization, and retrograde tracing, we anatomically characterize a small subpopulation of C-fiber cells that express high levels of TRPV1 (HE TRPV1 cells). These cells do not express molecular markers normally associated with C-fiber nociceptors. Furthermore, they express a unique complement of neurotrophic factor receptors, namely, the trkC receptor for neurotrophin 3, as well as receptors for neurturin and glial cell line-derived neurotrophic factor. HE TRPV1 cells are distributed in sensory ganglia throughout the neuraxis, with higher numbers noted in the sixth lumbar ganglion. In this ganglion and others of the lumbar and sacral regions, 75% or more of such HE TRPV1 cells express estrogen receptor alpha, suggestive of their regulation by estrogen and a role in afferent sensation related to reproduction. Afferents from these cells provide innervation to the hairy skin of the perineal region and can be activated by thermal stimuli from 38 degrees C, with a maximal response at 42 degrees C, as indicated by induction of extracellular signal-regulated kinase phosphorylation. We hypothesize that apart from participating in normal thermal sensation relevant to thermoregulation and reproductive functions, HE TRPV1 cells may mediate burning pain in chronic pain syndromes with perineal localization.
Assuntos
Temperatura Alta , Períneo/inervação , Células Receptoras Sensoriais/fisiologia , Animais , Regulação da Temperatura Corporal/fisiologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Fibras Nervosas Amielínicas/fisiologia , Ratos , Ratos Wistar , Escroto/inervação , Canais de Cátion TRPV/genética , Bexiga Urinária/inervaçãoRESUMO
Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.
Assuntos
Moduladores de Receptores de Canabinoides/análise , Endocanabinoides , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Neuralgia/tratamento farmacológico , Medula Espinal/patologia , Amidas , Animais , Ácidos Araquidônicos/análise , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Etanolaminas , Glicerídeos/análise , Microglia/metabolismo , Microglia/patologia , Minociclina/uso terapêutico , Neuralgia/patologia , Ácidos Palmíticos/análise , Alcamidas Poli-Insaturadas/análise , RatosRESUMO
An extensive protein-protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin-proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.
Assuntos
Ataxia/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Ataxia/genética , Ataxina-1 , Ataxinas , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Ratos Wistar , Alinhamento de SequênciaRESUMO
Peripheral nerve transections cause much more neuronal death in embryonic and neonatal dorsal root ganglia (DRG) than in adult DRG. Here we used transgenic approaches to examine the hypothesis that NF-kappaB is an important intrinsic factor of adult DRG neurons for their in vivo capacity to survive after nerve injury. We generated transgenic mice expressing the NF-kappaB super-inhibitor (IkappaBalpha-SI), a multi-mutant form of IkappaBalpha, specifically in adult neurons. Adult DRG neurons in these transgenic animals are not abnormally susceptible to apoptosis after peripheral nerve injury, although there is a significant inhibition in the ability of NF-kappaB to translocate into their nucleus. We investigated the observed lack of NF-kappaB neuroprotective function at the level of NF-kappaB transcriptional activity using transgenic NF-kappaB/LacZ reporter mice. We show that the expression of the NF-kappaB reporter transgene is restricted in naïve and injured DRG neurons. However, NF-kappaB transcriptional activity in adult DRG neurons is evident upon exposure to Trichostatin A (TSA) which is a specific inhibitor of histone deacetylases. Taken together our results illustrate that the functions of NF-kappaB are limited in adult primary sensory neurons due to a transcriptional repression mechanism mediated by histone deacetylases, and that intrinsic neuroprotective factors other than NF-kappaB are responsible for the resistance of adult DRG neurons to apoptosis in response to nerve injury.
Assuntos
Gânglios Espinais/citologia , NF-kappa B/metabolismo , Neurônios Aferentes/patologia , Neurônios Aferentes/fisiologia , Animais , Apoptose/fisiologia , Axotomia , Sobrevivência Celular , Células Cultivadas , Gânglios Espinais/fisiologia , Regulação da Expressão Gênica , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Neurônios Aferentes/citologiaRESUMO
We investigated the efficacy of SONU20176289, a member of a group of novel phenylpiperazine derivatives with a mixed dopamine D(2) receptor partial agonist and specific serotonin reuptake inhibitor (SSRI) activity, in a chronic stress model of depression in male tree shrews. Animals were subjected to a 7-day period of psychosocial stress before treatment for 28 days with SONU20176289 (6 mg/kg/day, p.o.), during which stress was maintained. Stress reduced the in vivo brain concentrations of N-acetyl-aspartate, total creatine, and choline-containing compounds, as measured by localized proton magnetic resonance spectroscopy. Post mortem analyses revealed a reduced adult dentate cell proliferation and a decreased GluR2 expression in the prefrontal cortex. All these alterations were prevented by concomitant administration of SONU20176289. The results provide further support to the concept that antidepressant treatments may act by normalizing disturbed neuroplasticity, and indicate that combining dopamine D(2) receptor agonism with SSRI activity may serve as an effective tool in the treatment of depressive/anxiety syndromes.
Assuntos
Depressão/psicologia , Agonistas de Dopamina/farmacologia , Indóis/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Piperazinas/farmacologia , Receptores de Dopamina D2/agonistas , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Química Encefálica/efeitos dos fármacos , Bromodesoxiuridina/farmacologia , Giro Denteado/citologia , Giro Denteado/efeitos dos fármacos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Masculino , Norepinefrina/urina , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptores de AMPA/metabolismo , Meio Social , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/psicologia , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testosterona/metabolismo , TupaiidaeRESUMO
BACKGROUND: We previously found many transient receptor potential vanilloid receptor subtype 1 (TRPV1) axons in the tracheal smooth muscle and epithelium of the guinea pig airway. One report indicates that the number of TRPV1 axons is significantly increased in patients with cough variant asthma. AIM: To determine whether the distribution of TRPV1 in the airways is altered in guinea pigs with an allergic phenotype. METHODS: Ten guinea pigs were assigned to 2 groups in a double-blind study. Five animals were sensitized with ovalbumin and the other 5 underwent sham sensitization. Cryopreserved sections (30 microm) of tracheal tissues removed from each animal were stained with polyclonal serum rabbit anti-TRPV1 antibody (1:30,000) and examined by confocal microscopy. RESULTS: Axons immunoreactive to TRPV1 localized to fine axons within the epithelium and around areas of smooth muscle, were more densely stained and frequent in the ovalbumin than in the sham group. CONCLUSION: The number of TRPV1-immunoreactive axons in the trachea increases under allergic inflammatory conditions.
Assuntos
Asma/imunologia , Canais de Cátion TRPV/biossíntese , Traqueia/imunologia , Animais , Asma/patologia , Axônios/química , Axônios/imunologia , Modelos Animais de Doenças , Método Duplo-Cego , Cobaias , Imuno-Histoquímica , Masculino , Microscopia Confocal , Ovalbumina/imunologia , Canais de Cátion TRPV/análise , Traqueia/inervação , Traqueia/patologiaRESUMO
Stimulation of postsynaptic M(1) muscarinic receptors (M(1)Rs) increases firing rates of both sympathetic and central neurons that underlie increases in vasomotor tone, heart rate, and cognitive memory functioning. At the cellular level, M(1)R stimulation modulates currents through various voltage-gated ion channels, including KCNQ K+ channels (M-current) and both L- and N-type Ca2+ channels (L- and N-current) by a pertussis toxin-insensitive, slow signaling pathway. Depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) during M(1)R stimulation suffices to inhibit M-current. We found previously that following PIP2 hydrolysis by phospholipase C, activation of phospholipase A2 and liberation of a lipid metabolite, most likely arachidonic acid (AA) are necessary for L- and N-current modulation. Here we examined the involvement of a third lipase, diacylglycerol lipase (DAGL), in the slow pathway. We documented the presence of DAGL in superior cervical ganglion neurons, and then tested the highly selective DAGL inhibitor, RHC-80267, for its capacity to antagonize M(1)R-mediated modulation of whole-cell Ca2+ currents. RHC-80267 significantly reduced L- and N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M) but did not affect their inhibition by exogenous AA. Moreover, voltage-dependent inhibition of N-current by Oxo-M remained in the presence of RHC-80267, indicating selective action on the slow pathway. RHC also blocked inhibition of recombinant N-current. In contrast, RHC-80267 had no effect on native M-current inhibition. These data are consistent with a role for DAGL in mediating L- and N-current inhibition. These results extend our previous findings that the signaling pathway mediating L- and N-current inhibition diverges from the pathway initiating M-current inhibition.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/metabolismo , Lipase Lipoproteica/metabolismo , Receptor Muscarínico M1/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo N/genética , Células Cultivadas , Cicloexanonas/metabolismo , Humanos , Hibridização In Situ , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/genética , Agonistas Muscarínicos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oxotremorina/análogos & derivados , Oxotremorina/metabolismo , Técnicas de Patch-Clamp , Toxina Pertussis/metabolismo , Inibidores de Proteases/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptor Muscarínico M1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Gânglio Cervical Superior/citologiaRESUMO
Neuronal pentraxin II (NPTX2) is the most highly upregulated gene in the Parkinsonian substantia nigra based on our whole genome expression profiling results. We show here that it is a novel component of Lewy bodies and Lewy neurites in sporadic Parkinson's disease (PD). NPTX2 is also known as the neuronal activity-regulated protein (Narp), which is secreted and involved in long-term neuronal plasticity. Narp further regulates AMPA receptors which have been found to mediate highly selective non-apoptotic cell death of dopaminergic neurons. NPTX2/Narp is found in close association with alpha-synuclein aggregates in both substantia nigra and cerebral cortex in PD but unlike alpha-synuclein gene expression, which is down-regulated in the Parkinsonian nigra, NPTX2 could represent a driver of the disease process. In view of its profound (>800%) upregulation and its established role in synaptic plasticity as well as dopaminergic nerve cell death, NPTX2 is a very interesting novel player which is likely to be involved in the pathway dysregulation which underlies PD.