RESUMO
Identifying causative gene(s) within disease-associated large genomic regions of copy-number variants (CNVs) is challenging. Here, by targeted sequencing of genes within schizophrenia (SZ)-associated CNVs in 1,779 SZ cases and 1,418 controls, we identified three rare putative loss-of-function (LoF) mutations in OTU deubiquitinase 7A (OTUD7A) within the 15q13.3 deletion in cases but none in controls. To tie OTUD7A LoF with any SZ-relevant cellular phenotypes, we modeled the OTUD7A LoF mutation, rs757148409, in human induced pluripotent stem cell (hiPSC)-derived induced excitatory neurons (iNs) by CRISPR-Cas9 engineering. The mutant iNs showed a â¼50% decrease in OTUD7A expression without undergoing nonsense-mediated mRNA decay. The mutant iNs also exhibited marked reduction of dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity. Congruent with the neuronal phenotypes in mutant iNs, our transcriptomic analysis showed that the set of OTUD7A LoF-downregulated genes was enriched for those relating to synapse development and function and was associated with SZ and other neuropsychiatric disorders. These results suggest that OTUD7A LoF impairs synapse development and neuronal function in human neurons, providing mechanistic insight into the possible role of OTUD7A in driving neuropsychiatric phenotypes associated with the 15q13.3 deletion.
Assuntos
Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Variações do Número de Cópias de DNA , Humanos , Neurônios , Esquizofrenia/metabolismo , Sinapses/metabolismoRESUMO
Down syndrome (DS) is a genetic disorder caused by triplication of human chromosome 21. In addition to intellectual disability, DS is defined by a premature aging phenotype and Alzheimer's disease (AD) neuropathology, including septohippocampal circuit vulnerability and degeneration of basal forebrain cholinergic neurons (BFCNs). The Ts65Dn mouse model recapitulates key aspects of DS/AD pathology, namely age-associated atrophy of BFCNs and cognitive decline in septohippocampal-dependent behavioral tasks. We investigated whether maternal choline supplementation (MCS), a well-tolerated treatment modality, protects vulnerable BFCNs from age- and genotype-associated degeneration in trisomic offspring. We also examined the effect of trisomy, and MCS, on GABAergic basal forebrain parvalbumin neurons (BFPNs), an unexplored neuronal population in this DS model. Unbiased stereological analyses of choline acetyltransferase (ChAT)-immunoreactive BFCNs and parvalbumin-immunoreactive BFPNs were conducted using confocal z-stacks of the medial septal nucleus and the vertical limb of the diagonal band (MSN/VDB) in Ts65Dn mice and disomic (2N) littermates at 3-4 and 10-12 months of age. MCS trisomic offspring displayed significant increases in ChAT-immunoreactive neuron number and density compared to unsupplemented counterparts, as well as increases in the area of the MSN/VDB occupied by ChAT-immunoreactive neuropil. MCS also rescued BFPN number and density in Ts65Dn offspring, a novel rescue of a non-cholinergic cell population. Furthermore, MCS prevented age-associated loss of BFCNs and MSN/VDB regional area in 2N offspring, indicating genotype-independent neuroprotective benefits. These findings demonstrate MCS provides neuroprotection of vulnerable BFCNs and non-cholinergic septohippocampal BFPNs, indicating this modality has translational value as an early life therapy for DS, as well as extending benefits to the aging population at large.
Assuntos
Doença de Alzheimer , Prosencéfalo Basal , Síndrome de Down , Humanos , Animais , Camundongos , Idoso , Parvalbuminas , Neurônios GABAérgicos , Colina O-Acetiltransferase , Modelos Animais de Doenças , Degeneração Neural , Suplementos Nutricionais , ColinaRESUMO
BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) play a key role in the rewarding effects of ethanol (EtOH), and while several nAChR subtypes have been implicated, attention has recently shifted to a role for the α3ß4 nAChR. The laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic nucleus that sends excitatory projections to the ventral tegmental area, is an Integral part of the brain reward pathway. Here we investigate a potential role for LDTg α3ß4 nAChRs in EtOH self-administration and reward. METHODS: Sprague-Dawley rats were given ad libitum access to a 20% EtOH solution, as part of a two-bottle choice paradigm. Approximately 1 week after removal of EtOH access, we measured LDTg α3ß4 nAChR current responses to focal application of acetylcholine (ACh), using whole-cell patch clamp electrophysiology recordings in acute brain slices. In addition, we used whole-cell electrophysiology to assess the acute effects of EtOH on the sensitivity of LDTg α3ß4 nAChRs. RESULTS: Focal application of ACh onto LDTg neurons resulted in large α3ß4 nAChR-mediated inward currents, the magnitude of which showed a positive correlation with levels of EtOH self-administration. In addition, using brain slices taken from EtOH-naïve rats, bath application of EtOH resulted in a moderate potentiation of LDTg α3ß4 nAChR sensitivity. CONCLUSIONS: Using a rat model, increased α3ß4 nAChR function was associated with greater EtOH self-administration, with α3ß4 nAChR function also acutely potentiated by EtOH. Assuming that similar findings apply to humans, the α3ß4 nAChR could be a therapeutic target in the treatment of EtOH use disorder.
Assuntos
Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ratos , Ratos Sprague-DawleyRESUMO
The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Glomerulonefrite , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Anticorpos Anticitoplasma de Neutrófilos , Glomerulonefrite/tratamento farmacológico , Humanos , Peroxidase , Ratos , Quinase SykRESUMO
Traditional approaches to studying Alzheimer's disease (AD) using mouse models and cell lines have advanced our understanding of AD pathogenesis. However, with the growing divide between model systems and clinical therapeutic outcomes, the limitations of these approaches are increasingly apparent. Thus, to generate more clinically relevant systems that capture pathological cascades within human neurons, we generated human-induced neurons (HiNs) from AD and non-AD individuals to model cell autonomous disease properties. We selected an AD patient population expressing mutations in presenilin 1 (mPS1), which is linked to increased amyloid production, tau pathology, and calcium signaling abnormalities, among other features. While these AD components are detailed in model systems, they have yet to be collectively identified in human neurons. Thus, we conducted molecular, immune-based, electrophysiological, and calcium imaging studies to establish patterns of cellular pathology in this patient population. We found that mPS1 HiNs generate increased Aß42 and hyperphosphorylated tau species relative to non-AD controls, and exaggerated ER calcium responses that are normalized with ryanodine receptor (RyR) negative allosteric modulators. The inflammasome product, interleukin-18 (IL-18), also increased PS1 expression. This work highlights the potential for HiNs to model AD pathology and validates their role in defining cellular pathogenesis and their utility for therapeutic screening.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Mutação/genética , Neurônios/patologia , Presenilina-1/genética , Regulação Alostérica/fisiologia , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Neurônios/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas tau/metabolismoRESUMO
UNLABELLED: Nicotine and ethanol (EtOH) are among the most widely co-abused substances, and nicotinic acetylcholine receptors (nAChRs) contribute to the behavioral effects of both drugs. Along with their role in addiction, nAChRs also contribute to motor control circuitry. The α7 nAChR subtype is highly expressed in the laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic center that contributes to motor performance through its projections to thalamic motor relay centers, including the mediodorsal thalamus. We demonstrate that EtOH concentrations just above the legal limits for intoxication in humans can inhibit α7 nAChRs in LDTg neurons from rats. This EtOH-induced inhibition is mediated by a decrease in cAMP/PKA signaling. The α7 nAChR-positive allosteric modulator PNU120596 [N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea], which interferes with receptor desensitization, completely eliminated EtOH modulation of these receptors. These data suggest that EtOH inhibits α7 responses through a PKA-dependent enhancement of receptor desensitization. EtOH also inhibited the effects of nicotine at presynaptic α7 nAChRs on glutamate terminals in the mediodorsal thalamus. In vivo administration of PNU120596 either into the cerebral ventricles or directly into the mediodorsal thalamus attenuated EtOH-induced motor impairment. Thus, α7 nAChRs are likely important mediators of the motor impairing effects of moderate EtOH consumption. SIGNIFICANCE STATEMENT: The motor-impairing effects of ethanol contribute to intoxication-related injury and death. Here we explore the cellular and neural circuit mechanisms underlying ethanol-induced motor impairment. Physiologically relevant concentrations of ethanol inhibit activity of a nicotinic receptor subtype that is expressed in brain areas associated with motor control. That receptor inhibition is mediated by decreased receptor phosphorylation, suggesting an indirect modulation of cell signaling pathways to achieve the physiological effects.
Assuntos
Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Transtornos Motores/induzido quimicamente , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Colinérgicos/farmacologia , Colinesterases/metabolismo , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Isoxazóis/farmacologia , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Neurotransmissores/farmacologia , Compostos de Fenilureia/farmacologia , Fosforilação/efeitos dos fármacos , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Potenciais Sinápticos/efeitos dos fármacosRESUMO
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.
Assuntos
Inflamação/imunologia , Inflamação/prevenção & controle , Fatores Inibidores da Migração de Macrófagos/imunologia , Terapia de Alvo Molecular/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Western Blotting , Dexametasona/imunologia , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Enterocolite/imunologia , Enterocolite/metabolismo , Enterocolite/prevenção & controle , Citometria de Fluxo , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Glomerulonefrite/prevenção & controle , Glucocorticoides/imunologia , Glucocorticoides/uso terapêutico , Humanos , Inflamação/metabolismo , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Coelhos , Ratos Endogâmicos WKYRESUMO
Spleen tyrosine kinase (SYK) has an important role in immunoreceptor signaling, and SYK inhibition has accordingly attenuated immune-mediated injury in several in vivo models. However, the effect of SYK inhibition on autoantibody production remains unclear, and SYK inhibition has not been studied in an autoimmune model of renal disease. We, therefore, studied the effect of SYK inhibition in experimental autoimmune GN, a rodent model of antiglomerular basement membrane disease. We show glomerular SYK expression and activation by immunohistochemistry in both experimental and clinical disease, and we show that treatment with fostamatinib, a small molecule kinase inhibitor selective for SYK, completely prevents the induction of experimental autoimmune GN. In established experimental disease, introduction of fostamatinib treatment led to cessation of autoantibody production, reversal of renal injury, preservation of biochemical renal function, and complete protection from lung hemorrhage. B cell ELISpot and flow cytometric analysis suggest that short-term fostamatinib treatment inhibits the generation and activity of antigen-specific B cells without affecting overall B-cell survival. Additionally, fostamatinib inhibited proinflammatory cytokine production by nephritic glomeruli ex vivo and cultured bone marrow-derived macrophages in vitro, suggesting additional therapeutic effects independent of effects on autoantibody production that are likely related to inhibited Fc receptor signaling within macrophages in diseased glomeruli. Given these encouraging results in an in vivo model that is highly applicable to human disease, we believe clinical studies targeting SYK in GN are now warranted.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Doença Antimembrana Basal Glomerular/prevenção & controle , Formação de Anticorpos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxazinas/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Piridinas/uso terapêutico , Aminopiridinas , Animais , Autoanticorpos/sangue , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Morfolinas , Oxazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas , Ratos Endogâmicos WKY , Baço/efeitos dos fármacos , Quinase SykRESUMO
IgA immune complexes are capable of inducing human mesangial cell (HMC) activation, resulting in release of proinflammatory and profibrogenic mediators. The subsequent inflammation, cellular proliferation, and synthesis of extracellular matrix lead to the progression of IgA nephropathy (IgAN). Spleen tyrosine kinase (SYK) is an intracellular protein tyrosine kinase involved in cell signaling downstream of immunoreceptors. In this study, we determined whether SYK is involved in the downstream signaling of IgA1 stimulation in HMC, leading to production of proinflammatory cytokines/chemokines and cell proliferation. Incubation of HMC with IgA1 purified from IgAN patients significantly increased the synthesis of MCP-1 in a dose-dependent manner. There was also significantly increased production of IL-6, IL-8, IFN-γ-inducible protein-10, RANTES, and platelet-derived growth factor-BB. Stimulation of HMC with heat-aggregated IgA1 purified from IgAN patients induced significantly increased HMC proliferation. Both pharmacological inhibition of SYK and knockdown of SYK by small interfering RNA significantly reduced the synthesis of these mediators and inhibited HMC proliferation. Moreover, positive immunostaining for total and phospho-SYK in glomeruli of kidney biopsies from IgAN patients strongly suggests the involvement of SYK in the pathogenesis of IgAN. To our knowledge, we demonstrate, for the first time, the involvement of SYK in the downstream signaling of IgA1 stimulation in HMC and in the pathogenesis of IgAN. Hence, SYK represents a potential therapeutic target for IgAN.
Assuntos
Proliferação de Células , Citocinas/biossíntese , Glomerulonefrite por IGA/enzimologia , Imunoglobulina A/fisiologia , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Células Mesangiais/patologia , Proteínas Tirosina Quinases/fisiologia , Baço/enzimologia , Citocinas/fisiologia , Glomerulonefrite por IGA/imunologia , Glomerulonefrite por IGA/patologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/isolamento & purificação , Mediadores da Inflamação/fisiologia , Células Mesangiais/enzimologia , Células Mesangiais/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , Baço/patologia , Quinase SykRESUMO
Fas ligand is a well-known inducer of apoptosis in cells expressing its receptor Fas; it also prevents autoimmunity by inducing apoptosis of activated T cells. However, Fas ligand also mediates non-apoptotic functions involving inflammatory cell migration and cytokine responses. We sought here to study the role of Fas ligand in nephrotoxic nephritis, a model of crescentic glomerulonephritis, using generalized lymphoproliferative disorder (GLD) mice on a C57BL/6 background, which have defective Fas ligand and display only mild autoimmunity. These mice were significantly protected from glomerular crescent formation, glomerular thrombosis, renal impairment, and albuminuria 15 days after the induction of glomerulonephritis in comparison with wild-type mice. There were a reduced number of apoptotic cells in the glomeruli of nephritic GLD mice but no defect in their antibody responses or splenocyte proliferation at 15 days following the induction of glomerulonephritis. Bone marrow transplantation from wild-type mice restored disease susceptibility to GLD mice; however, wild-type mice were not protected when transplanted with bone marrow from GLD mice. Mesangial cells express Fas ligand in vitro, and these cells isolated from GLD mice produced lower amounts of monocyte chemoattractive protein-1 following interleukin-1 stimulation compared with cells from wild-type mice. Thus, Fas ligand-defective mice are protected from nephrotoxic nephritis, a disease in which both circulating and intrinsic renal cells appear to have a role.
Assuntos
Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Glomerulonefrite/metabolismo , RNA Mensageiro/metabolismo , Albuminúria/genética , Animais , Apoptose/genética , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Transplante de Medula Óssea , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Imunoglobulinas , Imunotoxinas , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Trombose/genéticaRESUMO
Antibody-mediated glomerulonephritis, including that resulting from immune complexes, is an important cause of renal failure and is in need of more specific and effective treatment. Binding of antibody or immune complexes to Fc receptors activates intracellular signal transduction pathways, including spleen tyrosine kinase (Syk), leading to the production of inflammatory cytokines. We examined the effect of R788 (fostamatinib disodium), an oral prodrug of the selective Syk inhibitor R406, in nephrotoxic nephritis in Wistar-Kyoto rats. Treatment with R788 reduced proteinuria, tissue injury, glomerular macrophage and CD8+ cell numbers, and renal monocyte chemoattractant protein-1 (MCP-1) and IL-1beta, even when we started treatment after the onset of glomerulonephritis. When we administered R788 from days 4 to 10, glomerular crescents reduced by 100% (P < 0.01) compared with the vehicle group. When we administered R788 treatment from days 7 to 14, established glomerular crescents reversed (reduced by 21%, P < 0.001), and renal function was better than the vehicle group (P < 0.001). In vitro, R406 downregulated MCP-1 production from mesangial cells and macrophages stimulated with aggregated IgG. These results suggest that Syk is an important therapeutic target for the treatment of glomerulonephritis.
Assuntos
Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Índice de Gravidade de Doença , Aminopiridinas , Animais , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/patologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Morfolinas , Proteínas Tirosina Quinases/metabolismo , Pirimidinas , Ratos , Ratos Endogâmicos WKY , Transdução de Sinais/fisiologia , Quinase SykRESUMO
Traumatic brain injury (TBI), and related diseases such as chronic traumatic encephalopathy (CTE) and Alzheimer's (AD), are of increasing concern in part due to enhanced awareness of their long-term neurological effects on memory and behavior. Repeated concussions, vs. single concussions, have been shown to result in worsened and sustained symptoms including impaired cognition and histopathology. To assess and compare the persistent effects of single or repeated concussive impacts on mediators of memory encoding such as synaptic transmission, plasticity, and cellular Ca2+ signaling, a closed-head controlled cortical impact (CCI) approach was used which closely replicates the mode of injury in clinical cases. Adult male rats received a sham procedure, a single impact, or three successive impacts at 48-hour intervals. After 30 days, hippocampal slices were prepared for electrophysiological recordings and 2-photon Ca2+ imaging, or fixed and immunostained for pathogenic phospho-tau species. In both concussion groups, hippocampal circuits showed hyper-excitable synaptic responsivity upon Schaffer collateral stimulation compared to sham animals, indicating sustained defects in hippocampal circuitry. This was not accompanied by sustained LTP deficits, but resting Ca2+ levels and voltage-gated Ca2+ signals were elevated in both concussion groups, while ryanodine receptor-evoked Ca2+ responses decreased with repeat concussions. Furthermore, pathogenic phospho-tau staining was progressively elevated in both concussion groups, with spreading beyond the hemisphere of injury, consistent with CTE. Thus, single and repeated concussions lead to a persistent upregulation of excitatory hippocampal synapses, possibly through changes in postsynaptic Ca2+ signaling/regulation, which may contribute to histopathology and detrimental long-term cognitive symptoms.
RESUMO
Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)alpha production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNF alpha production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNF alpha production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNF alpha production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNF alpha production. We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.
Assuntos
Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas Tirosina Quinases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/genética , Agamaglobulinemia/metabolismo , Células Cultivadas , Ativação Enzimática , Humanos , Proteínas I-kappa B/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Masculino , Inibidor de NF-kappaB alfa , Proteínas Tirosina Quinases/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in either PKD1 or PKD2 and accounts for 10% of all patients on renal replacement therapy. The kidney disease phenotype is primarily characterized by cyst formation, but there are also prominent interstitial changes (inflammation, apoptosis, proliferation, and fibrosis). Using a model of unilateral ischemia-reperfusion injury, we tested the hypothesis that Pkd2 heterozygous kidneys are more sensitive to injury and that this could lead to interstitial inflammation and fibrosis. Baseline tubular proliferation in heterozygous kidneys was twofold higher than in wild-type kidneys. The magnitude and duration of tubular and interstitial proliferative responses was consistently greater in injured heterozygous compared with wild-type kidneys at all time points. Conversely, tubular p21 expression in heterozygotes was lower at baseline and following injury at all time points. Significantly more neutrophils and macrophages were detected in injured Pkd2 heterozygous kidneys at 2 days, correlating with increased expression of the cytokines interleukin (IL)-1beta and keratinocyte-derived chemokine and resulting in interstitial fibrosis at 28 days. We conclude that Pkd2 dosage influences both susceptibility and nature of the repair responses following injury. Polycystin-2 is therefore likely to play multiple roles in regulating tubular cell viability, repair, and remodeling in the mature kidney.
Assuntos
Dosagem de Genes , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Canais de Cátion TRPP/genética , Cicatrização , Animais , Apoptose , Movimento Celular , Citocinas/metabolismo , Fibroblastos/patologia , Fibrose , Heterozigoto , Inflamação/complicações , Inflamação/enzimologia , Inflamação/patologia , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Macrófagos/patologia , Camundongos , Modelos Biológicos , Rim Policístico Autossômico Dominante/complicações , Rim Policístico Autossômico Dominante/enzimologia , Rim Policístico Autossômico Dominante/patologia , Traumatismo por Reperfusão/complicações , Canais de Cátion TRPP/metabolismoRESUMO
The P2X7 receptor is a ligand-gated cation channel that is normally expressed by a variety of immune cells, including macrophages and lymphocytes. Because it leads to membrane blebbing, release of IL-1beta, and cell death by apoptosis or necrosis, it is a potential therapeutic target for a variety of inflammatory diseases. Although the P2X7 receptor is usually not detectable in normal renal tissue, we previously reported increased expression of both mRNA and protein in mesangial cells and macrophages infiltrating the glomeruli in animal models of antibody-mediated glomerulonephritis. In this study, we used P2X7-knockout mice in the same experimental model of glomerulonephritis and found that P2X7 deficiency was significantly renoprotective compared with wild-type controls, evidenced by better renal function, a striking reduction in proteinuria, and decreased histologic glomerular injury. In addition, the selective P2X7 antagonist A-438079 prevented the development of antibody-mediated glomerulonephritis in rats. These results support a proinflammatory role for P2X7 in immune-mediated renal injury and suggest that the P2X7 receptor is a potential therapeutic target.
Assuntos
Glomerulonefrite/fisiopatologia , Receptores Purinérgicos P2/fisiologia , Animais , Feminino , Glomerulonefrite/prevenção & controle , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Antagonistas do Receptor Purinérgico P2 , Piridinas/farmacologia , Piridinas/uso terapêutico , Ratos , Receptores Purinérgicos P2X7 , Tetrazóis/farmacologia , Tetrazóis/uso terapêuticoRESUMO
Ca2+ homeostasis is essential for multiple neuronal functions and thus, Ca2+ dyshomeostasis can lead to widespread impairment of cellular and synaptic signaling, subsequently contributing to dementia and Alzheimer's disease (AD). While numerous studies implicate Ca2+ mishandling in AD, the cellular basis for loss of cognitive function remains under investigation. The process of synaptic degradation and degeneration in AD is slow, and constitutes a series of maladaptive processes each contributing to a further destabilization of the Ca2+ homeostatic machinery. Ca2+ homeostasis involves precise maintenance of cytosolic Ca2+ levels, despite extracellular influx via multiple synaptic Ca2+ channels, and intracellular release via organelles such as the endoplasmic reticulum (ER) via ryanodine receptor (RyRs) and IP3R, lysosomes via transient receptor potential mucolipin channel (TRPML) and two pore channel (TPC), and mitochondria via the permeability transition pore (PTP). Furthermore, functioning of these organelles relies upon regulated inter-organelle Ca2+ handling, with aberrant signaling resulting in synaptic dysfunction, protein mishandling, oxidative stress and defective bioenergetics, among other consequences consistent with AD. With few effective treatments currently available to mitigate AD, the past few years have seen a significant increase in the study of synaptic and cellular mechanisms as drivers of AD, including Ca2+ dyshomeostasis. Here, we detail some key findings and discuss implications for future AD treatments.
Assuntos
Doença de Alzheimer/metabolismo , Sinalização do Cálcio , Sinapses/fisiologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Neurônios/metabolismo , Estresse Oxidativo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Activation of mu-opioid receptors in the ventral pallidum (VP) is important for the induction of behavioral sensitization to morphine in rats. The present study was designed to ascertain if neurons within the VP demonstrate sensitization at a time when morphine-induced behavioral sensitization occurred (ie 3 or 14 days after five once-daily injections of 10 mg/kg i.p. morphine) in rats. Western blotting was used to evaluate transcription factors altered by opiates, CREB and deltaFosB. CREB levels did not change in the VP, but there was a significant decrease in levels of its active, phosphorylated form (pCREB) at both 3- and 14-days withdrawal. DeltaFosB levels were elevated following a 3-day withdrawal, but returned to normal by 14 days. This profile also was obtained from nucleus accumbens tissue. In a separate group of similarly treated rats, in vivo electrophysiological recordings of VP neuronal responses to microiontophoretically applied ligands were carried out after 14-days withdrawal. The firing rate effects of local applications of morphine were diminished in rats withdrawn from i.p. morphine. Repeated i.p. morphine did not alter GABA-mediated suppression of firing, or the rate enhancing effects of the D1 dopamine receptor agonist SKF82958 or glutamate. However, VP neurons from rats withdrawn from repeated i.p. morphine showed a higher propensity to enter a state of depolarization inactivation to locally applied glutamate. Overall, these findings reveal that decreased pCREB in brain regions such as the VP accompanies persistent behavioral sensitization to morphine and that this biochemical alteration may influence the excitability of neurons in this brain region.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Globo Pálido/efeitos dos fármacos , Morfina/efeitos adversos , Entorpecentes/efeitos adversos , Núcleo Accumbens/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Comportamento Animal , Benzazepinas/farmacologia , Western Blotting/métodos , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Globo Pálido/citologia , Globo Pálido/metabolismo , Ácido Glutâmico/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Núcleo Accumbens/citologia , Núcleo Accumbens/metabolismo , Transtornos Relacionados ao Uso de Opioides/etiologia , Transtornos Relacionados ao Uso de Opioides/metabolismo , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Ethanol (EtOH) and nicotine are the most widely coabused drugs. Tolerance to EtOH intoxication, including motor impairment, results in greater EtOH consumption and may result in a greater likelihood of addiction. Previous studies suggest that cross-tolerance between EtOH and nicotine may contribute to the abuse potential of these drugs. Here we demonstrate that repeated intermittent administration of either EtOH or nicotine in adult male Sprague Dawley rats results in tolerance to EtOH-induced motor impairment and increased EtOH self-administration. These findings suggest that nicotine and EtOH cross-tolerance results in decreased aversive and enhanced rewarding effects of EtOH. Endocannabinoid signaling in the dorsolateral striatum (DLS) has been implicated in both EtOH tolerance and reward, so we investigated whether nicotine or EtOH pretreatment might modulate endocannabinoid signaling in this region. Using similar EtOH and nicotine pretreatment methods resulted in increased paired-pulse ratios of evoked EPSCs in enkephalin-positive medium spiny neurons in DLS slices. Thus, EtOH and nicotine pretreatment may modulate glutamatergic synapses in the DLS presynaptically. Bath application of the CB1 receptor agonist Win 55,2-212 increased the paired-pulse ratio of evoked EPSCs in control slices, while Win 55,2-212 had no effect on paired-pulse ratio in slices from either EtOH- or nicotine-pretreated rats. Consistent with these effects, nicotine pretreatment occluded LTD induction by high-frequency stimulation of the corticostriatal inputs to the dorsolateral striatum. These results suggest that nicotine and EtOH pretreatment modulates striatal synapses to induce tolerance to the motor-impairing effects of EtOH, which may contribute to nicotine and EtOH coabuse.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Depressores do Sistema Nervoso Central/administração & dosagem , Corpo Estriado/efeitos dos fármacos , Etanol/administração & dosagem , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Consumo de Bebidas Alcoólicas/patologia , Animais , Corpo Estriado/patologia , Corpo Estriado/fisiopatologia , Tolerância a Medicamentos , Encefalinas/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Ratos Sprague-Dawley , Autoadministração , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Dopamine contributes to corticostriatal plasticity and motor learning. Dopamine denervation profoundly alters motor performance, as in Parkinson's disease (PD); however, the extent to which these symptoms reflect impaired motor learning is unknown. Here, we demonstrate a D2 receptor blockade-induced aberrant learning that impedes future motor performance when dopamine signaling is restored, an effect diminished by coadministration of adenosine antagonists during blockade. We hypothesize that an inappropriate corticostriatal potentiation in striatopallidal cells of the indirect pathway underlies aberrant learning. We demonstrate synaptic potentiation in striatopallidal neurons induced by D2 blockade and diminished by application of an adenosine antagonist, consistent with behavioral observations. A neurocomputational model of the basal ganglia recapitulates the behavioral pattern and further links aberrant learning to plasticity in the indirect pathway. Thus, D2-mediated aberrant learning may contribute to motor deficits in PD, suggesting new avenues for the development of therapeutics.
Assuntos
Comportamento Animal , Dopamina/metabolismo , Aprendizagem , Doença de Parkinson , Transdução de Sinais , Adenosina/antagonistas & inibidores , Adenosina/metabolismo , Animais , Antagonistas dos Receptores de Dopamina D2 , Camundongos , Camundongos Transgênicos , Atividade Motora , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Doença de Parkinson/terapia , Receptores de Dopamina D2/metabolismoRESUMO
The dopaminergic neurons of the ventral tegmental area (DA VTA neurons) are important for the rewarding and reinforcing properties of drugs of abuse, including ethanol. Ethanol increases the firing frequency of DA VTA neurons from rats and mice. Because of a recent report on block of ethanol excitation in mouse DA VTA neurons with ZD7288, a selective blocker of the hyperpolarization-activated cationic current Ih, we examined the effect of ZD7288 on ethanol excitation in DA VTA neurons from C57Bl/6J and DBA/2J mice and Fisher 344 rats. Ethanol (80 mM) caused only increases in firing rate in mouse DA VTA neurons in the absence of ZD7288, but in the presence of ZD7288 (30 microM), ethanol produced a more transient excitation followed by a decrease of firing. This same biphasic phenomenon was observed in DA VTA neurons from rats in the presence of ZD7288 only at very high ethanol concentrations (160-240 mM) but not at lower pharmacologically relevant concentrations. The longer latency ethanol-induced inhibition was not observed in DA VTA neurons from mice or rats in the presence of barium (100 microM), which blocks G protein-linked potassium channels (GIRKs) and other inwardly rectifying potassium channels. Ethanol may have a direct effect to increase an inhibitory potassium conductance, but this effect of ethanol can only decrease the firing rate if Ih is blocked.