RESUMO
CD40-activated CD40L reverse signaling is a major physiological regulator of axon and dendrite growth from developing hippocampal pyramidal neurons. Here we have studied how CD40L-mediated reverse signaling promotes the growth of these processes. Cultures of hippocampal pyramidal neurons were established from Cd40-/- mouse embryos to eliminate endogenous CD40/CD40L signaling, and CD40L reverse signaling was stimulated by a CD40-Fc chimera. CD40L reverse signaling increased phosphorylation and hence activation of proteins in the PKC, ERK, and JNK signaling pathways. Pharmacological activators and inhibitors of these pathways revealed that whereas activation of JNK inhibited growth, activation of PKC and ERK1/ERK2 enhanced growth. Experiments using combinations of pharmacological reagents revealed that these signaling pathways regulate growth by functioning as an interconnected and interdependent network rather than acting in a simple linear sequence. Immunoprecipitation studies suggested that stimulation of CD40L reverse signaling generated a receptor complex comprising CD40L, PKCß, and the Syk tyrosine kinase. Our studies have begun to elucidate the molecular network and interactions that promote axon and dendrite growth from developing hippocampal neurons following activation of CD40L reverse signaling.
Assuntos
Axônios/metabolismo , Ligante de CD40/metabolismo , Dendritos/fisiologia , Transdução de Sinais , Animais , Butadienos/farmacologia , Antígenos CD40/deficiência , Antígenos CD40/genética , Células Cultivadas , Dendritos/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismoRESUMO
The importance of death receptor (DR) signaling in embryonic development and physiological homeostasis is well established, as is the existence of several molecules that modulate DRs function, among them Fas Apoptotis Inhibitory Molecules. Although FAIM1, FAIM2, and FAIM3 inhibit Fas-induced cell death, they are not structurally related, nor do they share expression patterns. Moreover, they inhibit apoptosis through completely different mechanisms. FAIM1 and FAIM2 protect neurons from DR-induced apoptosis and are involved in neurite outgrowth and neuronal plasticity. FAIM1 inhibits Fas ligand- and tumor necrosis factor alpha-induced apoptosis by direct interaction with Fas receptor and through the stabilization of levels of X-linked inhibitor of apoptosis protein, a potent anti-apoptotic protein that inhibits caspases. Low FAIM1 levels are found in Alzheimer's disease, thus sensitizing neurons to tumor necrosis factor alpha and prompting neuronal loss. FAIM2 protects from Fas by direct interaction with Fas receptor, as well as by modulating calcium release at the endoplasmic reticulum through interaction with Bcl-xL. Several studies prove the role of FAIM2 in diseases of the nervous system, such as ischemia, bacterial meningitis, and neuroblastoma. The less characterized member of the FAIM family is FAIM3, which is expressed in tissues of the digestive and urinary tracts, bone marrow and testes, and restricted to the cerebellum in the nervous system. FAIM3 protects against DR-induced apoptosis by inducing the expression of other DR-antagonists such as CFLAR or through the interaction with the DR-adaptor protein Fas-associated via death domain. FAIM3 null mouse models reveal this protein as an important mediator of inflammatory autoimmune responses such as those triggered in autoimmune encephalomyelitis. Given the differences between FAIMs and the variety of processes in which they are involved, here we sought to provide a concise review about these molecules and their roles in the physiology and pathology of the nervous system. Even though they share name and inhibit Fas-induced cell death, Fas apoptotic inhibitory molecules (FAIMs) are not structurally related and inhibit apoptosis through completely different mechanisms. In this review, we describe FAIM1, FAIM2, and FAIM3 functions in the nervous system, and their implication in diverse pathologies such as neurodegenerative disease, cancer, or autoimmune diseases.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Morte Celular/genética , Sistema Nervoso , Receptor fas/antagonistas & inibidores , Receptor fas/genética , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Humanos , CamundongosRESUMO
BACKGROUND: Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo- and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because ~50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNFα, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFα and FasL in NBL. METHODS: For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNFα, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry. RESULTS: We have found that TNFα is able to increase FasL-induced cell death by a mechanism that involves the NF-κB-mediated induction of the Fas receptor. Moreover, TNFα sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNFα-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNFα. CONCLUSIONS: In summary, our findings reveal that TNFα sensitizes NBL cells to FasL-induced cell death by NF-κB-mediated upregulation of Fas and unveil a new mechanism through which TNFα enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteína Ligante Fas/farmacologia , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/genética , Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Ativação Enzimática/efeitos dos fármacos , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Transcrição GênicaRESUMO
The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.
Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Fármacos Neuroprotetores , Ubiquitinação/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologia , Receptor fas/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Citocromos c/metabolismo , Feminino , Imunoprecipitação , Proteínas Inibidoras de Apoptose/genética , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Células PC12 , Plasmídeos/genética , Ligação Proteica , Conformação Proteica , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteína X Associada a bcl-2/metabolismo , Receptor fas/genéticaRESUMO
Several advances in the field of neurodevelopmental diseases (NDDs) have been reported by 2022. Of course, NDDs comprise a diverse group of disorders, most of which with different aetiologies. However, owing to the development and consolidation of technological approaches, such as proteomics and RNA-sequencing, and to the improvement of brain organoids along with the introduction of artificial intelligence (AI) for biodata analysis, in 2022 new aetiological mechanisms for some NDDs have been proposed. Here, we present hints of some of these findings. For instance, centrioles regulate neuronal migration and could be behind the aetiology of periventricular heterotopia; also, the accumulation of misfolded proteins could explain the neurological effects in COVID-19 patients; and, autism spectrum disorders (ASD) could be the expression of altered cortical arealization. We also cover other interesting aspects as the description of a new NDD characterized by deregulation of genes involved in stress granule (SG) assemblies, or the description of a newly discovered neural progenitor that explains the different phenotypes of tumours and cortical tubers in tuberous sclerosis complex (TSC) disease; and how it is possible to decipher the aetiology of sudden unexplained death in childhood (SUDC) or improve the diagnosis of cortical malformations using formalin-fixed paraffin-embedded samples.
RESUMO
In neurons, it is well established that CREB contributes to learning and memory by orchestrating the translation of experience into the activity-dependent (i.e., driven by neurotransmitters) transcription of plasticity-related genes. The activity-dependent CREB-triggered transcription requires the concerted action of cyclic AMP/protein kinase A and Ca(2+) /calcineurin via the CREB-regulated transcription co-activator (CRTC). It is not known, however, whether a comparable molecular sequence occurs in astrocytes, despite the unquestionable contribution of these cells to brain plasticity. Here we sought to determine whether and how ATP and noradrenaline cause CREB-dependent transcription in rat cortical astrocyte cultures. Both transmitters induced CREB phosphorylation (Western Blots), CREB-dependent transcription (CRE-luciferase reporter assays), and the transcription of Bdnf, a canonical regulator of synaptic plasticity (quantitative RT-PCR). We indentified a Ca(2+) and diacylglycerol-independent protein kinase C at the uppermost position of the cascade leading to CREB-dependent transcription. Notably, CREB-dependent transcription was partially dependent on ERK1/2 and CRTC, but independent of cyclic AMP/protein kinase A or Ca(2+) /calcineurin. We conclude that ATP and noradrenaline activate CREB-dependent transcription in cortical astrocytes via an atypical protein kinase C. It is of relevance that the signaling involved be starkly different to the one described in neurons since there is no convergence of Ca(2+) and cyclic AMP-dependent pathways on CRTC, which, moreover, exerts a modulatory rather than a central role. Our data thus point to the existence of an alternative, non-neuronal, glia-based role of CREB in plasticity.
Assuntos
Trifosfato de Adenosina/farmacologia , Astrócitos/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Norepinefrina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ácido Glutâmico/farmacologia , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Complexos Multiproteicos/análise , Complexos Multiproteicos/química , Linhagem Celular , Sobrevivência Celular , Humanos , Proteínas Luminescentes/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/químicaRESUMO
CD40-activated CD40L-mediated reverse signalling is a major physiological regulator of neurite growth from excitatory and inhibitory neurons in the developing central nervous system (CNS). Whereas in excitatory pyramidal neurons, CD40L reverse signalling promotes the growth and elaboration of dendrites and axons, in inhibitory GABAergic striatal medium spiny neurons (MSNs), it restricts neurite growth and branching. In pyramidal neurons, we previously reported that CD40L reverse signalling activates an interconnected and interdependent signalling network involving protein kinase C (PKC), extracellular regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK) signalling pathways that regulates dendrite and axon growth. Here, we have studied whether these signalling pathways also influence neurite growth from striatal inhibitory MSNs. To unequivocally activate CD40L reverse signalling, we treated MSN cultures from CD40-deficient mice with CD40-Fc. Here, we report that activation of CD40L reverse signalling in these cultures also increased the phosphorylation of PKC, ERK1/2, and JNK. Using pharmacological activators and inhibitors of these signalling pathways singularly and in combination, we have shown that, as in pyramidal neurons, these signalling pathways work in an interconnected and interdependent network to regulate the neurite growth, but their functions, relationships, and interdependencies are different from those observed in pyramidal neurons. Furthermore, immunoprecipitation studies showed that stimulation of CD40L reverse signalling recruits the catalytic fragment of Syk tyrosine kinase, but in contrast to pyramidal neurons, PKC does not participate in this recruitment. Our findings show that distinctive networks of three signalling pathways mediate the opposite effects of CD40L reverse signalling on neurite growth in excitatory and inhibitory neurons.
Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Neuritos/metabolismo , Transdução de Sinais , Animais , Antígenos CD40/deficiência , Proliferação de Células , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurônios GABAérgicos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fosforilação , Mapas de Interação de Proteínas , Proteína Quinase C/metabolismo , Quinase Syk/metabolismoRESUMO
CD40-activated CD40L reverse signaling is a major physiological regulator of neural process growth from many kinds of developing neurons. Here we have investigated whether CD40L-reverse signaling also influences dendrite spine number and morphology in striatal medium spiny neurons (MSNs). Golgi preparations revealed no differences in the spine density, but because the dendrite arbors of MSNs were larger and branched in Cd40 -/- mice, the total number of spines was greater in Cd40 -/- mice. We also detected more mature spines compared with wild-type littermates. Western blot revealed that MSN cultures from Cd40 -/- mice had significantly less PSD-95 and there were changes in RhoA/B/C and Cdc42. Immunocytochemistry revealed that PSD-95 was clustered in spines in Cd40 -/- neurons compared with more diffuse labeling in Cd40 +/+ neurons. Activation of CD40L-reverse signaling with CD40-Fc prevented the changes observed in Cd40 -/- cultures. Our findings suggest that CD40L-reverse signaling influences dendrite spine morphology and related protein expression and distribution.
RESUMO
In vitro results show the ability of the CB(1) receptor agonist CP 55,940 to reduce the affinity of D(2) receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D(2) receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB(1)-D(2) receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB(1)/D(2) receptor-receptor interactions may occur in CB(1)/D(2) formed heteromers. Antagonistic CB(1)/D(2) interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB(1) receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB(1) receptor antagonist rimonobant but also the specific A(2A) receptor antagonist MSX-3 blocked the inhibitory effect of CB(1) receptor agonist on D(2)-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB(1)/D(2) receptor-receptor interactions within CB(1)/D(2) heteromers in which A(2A) receptors may also participate.
Assuntos
Corpo Estriado/metabolismo , Antagonistas dos Receptores de Dopamina D2 , Atividade Motora/fisiologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Comportamento Animal , Linhagem Celular Transformada , Corpo Estriado/efeitos dos fármacos , Cicloexanóis/farmacologia , Dopamina/farmacologia , Agonistas de Dopamina , Interações Medicamentosas , Transferência Ressonante de Energia de Fluorescência/métodos , Guanilil Imidodifosfato/farmacologia , Humanos , Proteínas Luminescentes/metabolismo , Masculino , Ligação Proteica/efeitos dos fármacos , Quimpirol/farmacologia , Radiografia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Canabinoides/metabolismo , Transfecção/métodosRESUMO
Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells.
Assuntos
Receptor A2A de Adenosina/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores de Dopamina D2/metabolismo , Linhagem Celular , Dimerização , Transferência Ressonante de Energia de Fluorescência , Humanos , Rim/embriologia , Medições Luminescentes , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.
Assuntos
Canabinoides/farmacologia , Corpo Estriado/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Receptor A2A de Adenosina/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Agonistas do Receptor A2 de Adenosina , Análise de Variância , Animais , Ácidos Araquidônicos/farmacologia , Comportamento Animal , Canabinoides/agonistas , Canabinoides/antagonistas & inibidores , Linhagem Celular Transformada , Corpo Estriado/metabolismo , AMP Cíclico/metabolismo , Interações Medicamentosas , Humanos , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor A2A de Adenosina/deficiência , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/deficiência , TransfecçãoRESUMO
Dendrite size and morphology are key determinants of the functional properties of neurons and neural circuits. Here we show that CD40, a member of the TNF receptor superfamily, is a major regulator of dendrite growth and elaboration in the developing brain. The dendrites of hippocampal excitatory neurons were markedly stunted in Cd40-/- mice, whereas those of striatal inhibitory neurons were much more exuberant. These striking and opposite phenotypic changes were also observed in excitatory and inhibitory neurons cultured from Cd40-/- mice and were rescued by soluble CD40. The changes in excitatory and inhibitory neurons cultured from Cd40-/- mice were mimicked in neurons of Cd40+/+ mice by treatment with soluble CD40L and were dependent on PKC-ß and PKC-γ, respectively. These results suggest that CD40-activated CD40L reverse signalling has striking and opposite effects on the growth and elaboration of dendrites among major classes of brain neurons by PKC-dependent mechanisms.
Assuntos
Antígenos CD40/metabolismo , Dendritos/fisiologia , Hipocampo/citologia , Neurônios/citologia , Animais , Antígenos CD40/deficiência , Ligante de CD40/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase C/metabolismoRESUMO
The objective of this study was to identify genetic variation in genes encoding death receptors and signals that modulate their activity. After conducting a meta-analysis with five previous genome-wide association studies and aggregated data, the most significant signals, (TNF locus: rs2395488, rs2534672, and rs9267445; and FASLG locus: rs730278), were replicated in 1,046 cases and 372 controls. The rs2395488 and rs2534672 markers showed a modest protective effect (OR = 0.849, p = 0.49780;OR= 0.687, p = 0.11335), in contrast to rs730278 marker (OR = 1.146, p = 0.17212), which did not follow the previous effect direction; in any case it reached the significance level. Final meta-analysis, adding the replication sample, confirmed these observations. We concluded that FASLG marker is not etiologically linked to Alzheimer's disease. However, single nucleotide polymorphisms around TNF locus require further analyses in order to explain the association between Alzheimer's disease and human leukocyte antigen.
Assuntos
Apoptose/genética , Inibidores da Colinesterase/uso terapêutico , Farmacogenética , Polimorfismo Genético/genética , Fator de Necrose Tumoral alfa/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Análise de Variância , Apolipoproteínas E/genética , Apoptose/efeitos dos fármacos , Citocromo P-450 CYP2D6/genética , Donepezila , Proteína Ligante Fas/genética , Feminino , Humanos , Indanos/uso terapêutico , Estudos Longitudinais , Masculino , Entrevista Psiquiátrica Padronizada , Metanálise como Assunto , Piperidinas/uso terapêutico , Valor Preditivo dos Testes , Resultado do TratamentoRESUMO
The use of non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease (AD) is controversial because conclusions from numerous epidemiological studies reporting delayed onset of AD in NSAID users have not been corroborated in clinical trials. The purpose of this personal view is to revise the case for NSAIDs in AD therapeutics in light of: (i) the last report from the only primary prevention trial in AD, ADAPT, which, although incomplete, points to significant protection in long-term naproxen users, and (ii) the recently proposed dynamic model of AD evolution. The model contends that there is a clinical silent phase in AD that can last up to 20 years, the duration depending on life style habits, genetic factors, or cognitive reserve. The failure of many purported disease-modifying drugs in AD clinical trials is forcing the view that treatments will only be efficacious if administered pre-clinically. Here we will argue that NSAIDs failed in clinical trials because they are disease-modifying drugs, and they should be administered in early stages of the disease. A complete prevention trial in cognitively normal individuals is thus called for. Further, the shift of anti-inflammatory treatment to early stages uncovers a knowledge void about the targets of NSAIDs in asymptomatic individuals. AD researchers have mostly relied on post-mortem analysis of Aß plaque-laden brains from demented patients or animal models, thus drawing conclusions about AD pathogenesis based on late symptoms. We will discuss evidence in support that defective, not excessive, inflammation underlies AD pathogenesis, that NSAIDs are multifunctional drugs acting on inflammatory and non-inflammatory targets, and that astrocytes and microglia may play differing roles in disease progression, with an emphasis of ApoEε4 as a key, undervalued target of NSAIDs. According to a meta-analysis of epidemiological data, NSAIDs afford an average protection of 58%. If this figure is true, and translated into patient numbers, NSAID treatment may revive as a worth pursuing strategy to significantly reduce the socio-economical burden imposed by AD.
RESUMO
Profens like ibuprofen, R-flurbiprofen, or CHF5074 are being considered for the treatment of Alzheimer's disease because epidemiological data indicates that non-steroidal anti-inflammatory drugs are protective against neurodegeneration. Rho-GTPases are small G proteins, including RhoA, Cdc42, and Rac1, which control cytoskeleton dynamics. Because ibuprofen promotes axon growth via RhoA in neurons, we examined whether profens modulate astrocyte plasticity via Rho-GTPases. We report that ibuprofen (100-500 µM), R-flurbiprofen (100-500 µM), and CHF5074 (10-30 µM) caused a concentration-dependent stellation of astrocytes in primary cultures, associated with the reorganization of GFAP and actin filaments. The stellation was independent of COX2, α-, ß- or γ-secretase as judged by the lack of effect of inhibitors of these enzymes. RhoA, PAK, and Cdc42, but not Rac1, accounted for the profen-mediated stellation, as concluded from the joint analyses of activities and reversal experiments with adenoviral or pharmacological manipulations. Ibuprofen accelerated migration in a scratch-wound assay, while R-flurbiprofen had no effect and CHF5074 caused deceleration. Cell polarity regulation by Cdc42 and ERK1/2 may underlie the paradoxical effects of profens on migration. We conclude that profens regulate cytoskeleton dynamics in astrocytes via Rho-GTPases, PAK, and ERK1/2. Since migration is a hallmark of astrocyte response during inflammation we propose that, in addition to (or instead of) lowering amyloid-ß42 via secretases, ibuprofen and its derivatives may prevent Alzheimer's disease instead of AD by modulating astrocyte reactivity through Rho-GTPase/PAK/ERK-dependent signaling.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Astrócitos/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/metabolismo , Análise de Variância , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Western Blotting , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Ciclo-Oxigenase 2/metabolismo , Ciclopropanos/metabolismo , Ciclopropanos/farmacologia , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Flurbiprofeno/análogos & derivados , Flurbiprofeno/metabolismo , Flurbiprofeno/farmacologia , Ibuprofeno/metabolismo , Ibuprofeno/farmacologia , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
It has been suggested that L-DOPA-induced hyperhomocysteinemia can increase the risk of stroke, heart disease, and dementia and is an additional pathogenetic factor involved in the progression of Parkinson's disease. In Chinese hamster ovary (CHO) cells stably cotransfected with adenosine A(2A) and dopamine D2 receptors, homocysteine selectively decreased the ability of D2 receptor stimulation to internalize adenosine A(2A)-dopamine D2 receptor complexes. Radioligand-binding experiments in the same cell line demonstrated that homocysteine acts as an allosteric D2 receptor antagonist, by selectively reducing the affinity of D2 receptors for agonists but not for antagonists. Mass spectrometric analysis showed that, by means of an arginine (Arg)-thiol electrostatic interaction, homocysteine forms noncovalent complexes with the two Arg-rich epitopes of the third intracellular loop of the D2 receptor, one of them involved in A(2A)-D2 receptor heteromerization. However, homocysteine was unable to prevent or disrupt A(2A)-D2 receptor heteromerization, as demonstrated with Fluorescence Resonance Energy Transfer (FRET) experiments in stably cotransfected HEK cells. The present results could have implications for Parkinson's disease.