RESUMO
SLC30A9 encodes a ubiquitously zinc transporter (ZnT9) and has been consistently suggested as a candidate for positive selection in humans. However, no direct adaptive molecular phenotype has been demonstrated. Our results provide evidence for directional selection operating in two major complementary haplotypes in Africa and East Asia. These haplotypes are associated with differential gene expression but also differ in the Met50Val substitution (rs1047626) in ZnT9, which we show is found in homozygosis in the Denisovan genome and displays accompanying signatures suggestive of archaic introgression. Although we found no significant differences in systemic zinc content between individuals with different rs1047626 genotypes, we demonstrate that the expression of the derived isoform (ZnT9 50Val) in HEK293 cells shows a gain of function when compared with the ancestral (ZnT9 50Met) variant. Notably, the ZnT9 50Val variant was found associated with differences in zinc handling by the mitochondria and endoplasmic reticulum, with an impact on mitochondrial metabolism. Given the essential role of the mitochondria in skeletal muscle and since the derived allele at rs1047626 is known to be associated with greater susceptibility to several neuropsychiatric traits, we propose that adaptation to cold may have driven this selection event, while also impacting predisposition to neuropsychiatric disorders in modern humans.
Assuntos
Hominidae , Animais , Humanos , Células HEK293 , Hominidae/genética , Homeostase/genética , Zinco , Genética Humana , Seleção Genética , Haplótipos , Genoma HumanoRESUMO
Alzheimer's disease (AD) is tightly linked to oxidative stress since amyloid beta-peptide (Aß) aggregates generate free radicals. Moreover, the aggregation of Aß is increased by oxidative stress, and the neurotoxicity induced by the oligomers and fibrils is in part mediated by free radicals. Interestingly, it has been reported that oxidative stress can also induce BACE1 transcription and expression. BACE1 is the key enzyme in the cleavage of the amyloid precursor protein to produce Aß, and the expression of this enzyme has been previously shown to be enhanced in the brains of Alzheimer's patients. Here, we have found that BACE1 expression is increased in the hippocampi from AD patients at both the early (Braak stage II) and late (Braak stage VI) stages of the disease as studied by immunohistochemistry and western blot. To address the role of Aß and oxidative stress in the regulation of BACE1 expression, we have analyzed the effect of subtoxic concentrations of Aß oligomers (0.25 µM) and H2O2 (10 mM) on a human neuroblastoma cell line. Firstly, our results show that Aß oligomers and H2O2 induce an increase of BACE1 mRNA as we studied by qPCR. Regarding BACE1 translation, it is dependent on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), since BACE1 mRNA bears a 5'UTR that avoids its translation under basal conditions. BACE1 5'UTR contains four upstream initiating codons (uAUGs), and its translation is activated when eIF2α is phosphorylated. Consistently, we have obtained that Aß oligomers and H2O2 increase the levels of BACE1 and p-eIF2α assayed by western blot and confocal microscopy. Our results suggest that Aß oligomers increase BACE1 translation by phosphorylating eIF2α in a process that involves oxidative stress and conforms a pathophysiological loop, where the Aß once aggregated favors its own production continuously by the increase in BACE1 expression as observed in AD patients.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação para Cima/efeitos dos fármacos , Regiões 5' não Traduzidas , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , FosforilaçãoRESUMO
Human life unfolds not only in time and space, but also in the recollection and interweaving of memories. Therefore, individual human identity depends fully on a proper access to the autobiographical memory. Such access is hindered under pathological conditions such as Alzheimer's disease, which affects millions of people worldwide. Unfortunately, no effective cure exists to prevent this disorder, the impact of which will rise alarmingly within the next decades. While Alzheimer's disease is largely considered to be the outcome of amyloid-ß (Aß) peptide accumulation in the brain, conceiving this complex disorder strictly as the result of Aß-neurotoxicity is perhaps a too straight-line simplification. Instead, complementary to this view, the tableau of molecular disarrangements in the Alzheimer's disease brain may be reflecting, at least in part, a loss of function phenotype in memory processing. Here we take BACE1 translation and degradation as a gateway to study molecular mechanisms putatively involved in the transition between memory and neurodegeneration. BACE1 participates in the excision of Aß-peptide from its precursor holoprotein, but plays a role in synaptic plasticity too. Its translation is governed by eIF2α phosphorylation: a hub integrating cellular responses to stress, but also a critical switch in memory consolidation. Paralleling these dualities, the eIF2α-kinase HRI has been shown to be a nitric oxide-dependent physiological activator of hippocampal BACE1 translation. Finally, beholding BACE1 as a representative protease active in the CNS, we venture a new perspective on the cellular basis of memory, which may incorporate neurodegeneration in itself as a drift in memory consolidating systems.
RESUMO
Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.
Assuntos
Vesículas Extracelulares/metabolismo , Interleucina-4/metabolismo , Esclerose Múltipla/metabolismo , Animais , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/ultraestrutura , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Vesículas Extracelulares/ultraestrutura , Feminino , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The activation of N-Methyl D-Aspartate Receptor (NMDAR) by glutamate is crucial in the nervous system function, particularly in memory and learning. NMDAR is composed by two GluN1 and two GluN2 subunits. GluN2B has been reported to participate in the prevalent NMDAR subtype at synapses, the GluN1/2A/2B. Here we studied the regulation of GluN2B expression in cortical neurons finding that glutamate up-regulates GluN2B translation through the action of nitric oxide (NO), which induces the phosphorylation of the eukaryotic translation initiation factor 2 α (eIF2α). It is a process mediated by the NO-heme-regulated eIF2α kinase (HRI), as the effect was avoided when a specific HRI inhibitor or a HRI small interfering RNA (siHRI) were used. We found that the expressed GluN2B co-localizes with PSD-95 at the postsynaptic ending, which strengthen the physiological relevance of the proposed mechanism. Moreover the receptors bearing GluN2B subunits upon NO stimulation are functional as high Ca2+ entry was measured and increases the co-localization between GluN2B and GluN1 subunits. In addition, the injection of the specific HRI inhibitor in mice produces a decrease in memory retrieval as tested by the Novel Object Recognition performance. Summarizing our data suggests that glutamatergic stimulation induces HRI activation by NO to trigger GluN2B expression and this process would be relevant to maintain postsynaptic activity in cortical neurons.
Assuntos
Córtex Cerebelar/patologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/genética , Fármacos Atuantes sobre Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/metabolismo , Heme/metabolismo , Humanos , Memória , Camundongos , Camundongos Endogâmicos , Neurônios/patologia , Óxido Nítrico/metabolismo , Fosforilação , Biossíntese de Proteínas , RNA Interferente Pequeno/genética , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
AIMS: Hippocampus is the brain center for memory formation, a process that requires synaptogenesis. However, hippocampus is dramatically compromised in Alzheimer's disease due to the accumulation of amyloid ß-peptide, whose production is initiated by ß-site APP Cleaving Enzyme 1 (BACE1). It is known that pathological stressors activate BACE1 translation through the phosphorylation of the eukaryotic initiation factor-2α (eIF2α) by GCN2, PERK, or PKR kinases, leading to amyloidogenesis. However, BACE1 physiological regulation is still unclear. Since nitric oxide (NO) participates directly in hippocampal glutamatergic signaling, we investigated the neuronal role of the heme-regulated eukaryotic initiation factor eIF2α kinase (HRI), which can bind NO by a heme group, in BACE1 translation and its physiological consequences. RESULTS: We found that BACE1 is expressed on glutamate activation with NO being the downstream effector by triggering eIF2α phosphorylation, as it was obtained by Western blot and luciferase assay. It is due to the activation of HRI by NO as assayed by Western blot and immunofluorescence with an HRI inhibitor and HRI siRNA. BACE1 expression was early detected at synaptic spines, contributing to spine growth and consolidating the hippocampal memory as assayed with mice treated with HRI or neuronal NO synthase inhibitors. INNOVATION: We provide the first description that HRI and eIF2α are working in physiological conditions in the brain under the control of nitric oxide and glutamate signaling, and also that BACE1 has a physiological role in hippocampal function. CONCLUSION: We conclude that BACE1 translation is controlled by NO through HRI in glutamatergic hippocampal synapses, where it plays physiological functions, allowing the spine growth and memory consolidation.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Sinapses/metabolismo , eIF-2 Quinase/metabolismo , Animais , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/metabolismo , Ácido Glutâmico/farmacologia , Hipocampo/embriologia , Hipocampo/metabolismo , Humanos , Consolidação da Memória , Camundongos , Neurônios/citologia , Fosforilação , Biossíntese de Proteínas , RatosRESUMO
Ischemic stroke is an acute vascular event that compromises neuronal viability, and identification of the pathophysiological mechanisms is critical for its correct management. Ischemia produces increased nitric oxide synthesis to recover blood flow but also induces a free radical burst. Nitric oxide and superoxide anion react to generate peroxynitrite that nitrates tyrosines. We found that fibrinogen nitrotyrosination was detected in plasma after the initiation of ischemic stroke in human patients. Electron microscopy and protein intrinsic fluorescence showed that in vitro nitrotyrosination of fibrinogen affected its structure. Thromboelastography showed that initially fibrinogen nitrotyrosination retarded clot formation but later made the clot more resistant to fibrinolysis. This result was independent of any effect on thrombin production. Immunofluorescence analysis of affected human brain areas also showed that both fibrinogen and nitrotyrosinated fibrinogen spread into the brain parenchyma after ischemic stroke. Therefore, we assayed the toxicity of fibrinogen and nitrotyrosinated fibrinogen in a human neuroblastoma cell line. For that purpose we measured the activity of caspase-3, a key enzyme in the apoptotic pathway, and cell survival. We found that nitrotyrosinated fibrinogen induced higher activation of caspase 3. Accordingly, cell survival assays showed a more neurotoxic effect of nitrotyrosinated fibrinogen at all concentrations tested. In summary, nitrotyrosinated fibrinogen would be of pathophysiological interest in ischemic stroke due to both its impact on hemostasis - it impairs thrombolysis, the main target in stroke treatments - and its neurotoxicity that would contribute to the death of the brain tissue surrounding the infarcted area.
Assuntos
Apoptose , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Fibrinogênio/metabolismo , Fibrinólise , Neurônios/metabolismo , Acidente Vascular Cerebral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Isquemia Encefálica/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/patologia , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-ß peptide (Aß) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aß aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.
Assuntos
Albuminas/metabolismo , Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Tirosina/análogos & derivados , Idoso , Albuminas/efeitos dos fármacos , Albuminas/farmacologia , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/patologia , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Feminino , Glicosilação , Humanos , Masculino , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Neurônios/efeitos dos fármacos , Agregados Proteicos/fisiologia , Tripsina/farmacologia , Tirosina/metabolismo , Proteínas tau/metabolismoRESUMO
Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.
Assuntos
Estresse Oxidativo/efeitos dos fármacos , Proteínas/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Tirosina/análogos & derivados , Tirosina/efeitos dos fármacosRESUMO
Local control of protein synthesis at synapses is crucial for synaptic plasticity and memory formation. However, little is known about the signals coupling neurotransmitter release with the translational machinery during these processes. Here, we studied the involvement of heme-regulated inhibitor (HRI) kinase, a kinase activated by nitric oxide that phosphorylates eukaryotic initiation factor 2α (eIF2α), in object recognition (OR) memory consolidation. Phosphorylated eIF2α mediates two opposing effects upon translation: translational arrest of most mRNAs and translational activation of selected mRNAs bearing specific features in their 5'untranslated regions (5'UTRs). We found that HRI kinase activation in the CA1 region of the dorsal hippocampus is necessary for retention of OR memory in rats. Accordingly, learning induced a transient increase in the phosphorylation state of eIF2α in dorsal CA1 which was abolished by the HRI kinase inhibitor N-(2,6-dimethylbenzyl)-6,7-dimethoxy-2H-[1]benzofuro[3,2-c]pyrazol-3-amine hydrochloride (AMI). The increase in p-eIF2α was associated with increased expression of BACE1 and activating transcription factor 4, two proteins containing eIF2α-responsive 5'UTRs in their mRNAs that play a key role in synaptic plasticity. Our data suggests that learning promotes the transient phosphorylation of eIF2α to allow for translation of specific 5'UTR-mRNAs through a process requiring HRI kinase activation.
Assuntos
Hipocampo/enzimologia , Reconhecimento Psicológico/fisiologia , eIF-2 Quinase/metabolismo , Animais , Masculino , Memória/fisiologia , Fosforilação/fisiologia , Ratos , Ratos WistarRESUMO
Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aß42 versus Aß40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aß) generation as well. Here we demonstrate that the secretion of Aß is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO(-)), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aß towards Aß(42) and increased the Aß(42) /Aß(40) ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Senescência Celular/efeitos dos fármacos , Neurônios/metabolismo , Ácido Peroxinitroso/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Domínio Catalítico , Células Cultivadas , Humanos , Camundongos , Neurônios/citologia , Fragmentos de Peptídeos/metabolismo , Presenilina-1/química , Presenilina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Risco , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer's disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5'untranslated region (5'UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5'UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5'UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (Iâ¶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5'UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Herpesvirus Humano 1/fisiologia , eIF-2 Quinase/metabolismo , Idoso , Secretases da Proteína Precursora do Amiloide/genética , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Feminino , Células HeLa , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , eIF-2 Quinase/genéticaRESUMO
Different mechanisms including oxidative stress are proposed for amyloid-ß peptide (Aß) neurotoxicity, and here we contribute to demonstrate that nitro-oxidative stress is playing a key role. Yeasts are a well-known model for H2O2 toxicity. Interestingly, yeast cell wall prevents interaction of Aß fibrils with membrane receptors or calcium channels and we found a significant viability reduction in yeasts when challenged with Aß fibrils. Furthermore, iron and copper chelators, as well as the antioxidants glutathione and trolox, were neuroprotective on neuroblastoma cells and mouse hippocampal neurons challenged with Aß fibrils. Glutathione prevents the oxidation, glycation and nitrotyrosination of cell proteins induced by Aß. Trolox protected neurons in cell viability studies, maintaining the vesicular transport integrity and preventing the trigger of apoptotic mechanisms. Interestingly, we have also found that brain derived neuronal factor (BDNF) and neurotrophin-3 (NT-3) were able to protect mouse hippocampal and cortical neurons against H2O2 and Aß fibrils. Considering that superoxide anion, produced by Aß cell damage, and nitric oxide, whose production is altered in AD, react to form the highly reactive peroxynitrite anion, we studied the role of trolox to ameliorate the peroxynitrite cell damage. Finally, one of the major proteins to be nitrotyrosinated in AD, the triose phosphate isomerase (TPI) was assayed searching for a denitrase activity that could reverse intracellular nitrotyrosination. We have found that human neuroblastoma SH-SY5Y cells express a constitutive denitrase activity that partially denitrated nitro-TPI. Altogether, our results support a key role of nitro-oxidative stress in the neuronal damage induced by Aß fibrils.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Amiloide/metabolismo , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Idoso , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Cromanos/farmacologia , Desferroxamina/farmacologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Glutationa/metabolismo , Hipocampo/citologia , Humanos , Peróxido de Hidrogênio/farmacologia , Imunoprecipitação/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Masculino , Camundongos , Modelos Biológicos , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Fragmentos de Peptídeos/farmacologia , Ratos , Sideróforos/farmacologia , Extratos do Timo/metabolismoRESUMO
Alzheimer's disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Lobo Frontal/metabolismo , Modelos Moleculares , Triose-Fosfato Isomerase/metabolismo , Tirosina/análogos & derivados , Peptídeos beta-Amiloides/análise , Animais , Western Blotting , Estudos de Casos e Controles , Linhagem Celular , Linhagem Celular Tumoral , Lobo Frontal/química , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica , Neuroblastoma , Emaranhados Neurofibrilares/metabolismo , Estresse Oxidativo , Ácido Peroxinitroso/análise , Ácido Peroxinitroso/metabolismo , Fosforilação , Triose-Fosfato Isomerase/análise , Tirosina/metabolismo , Proteínas tau/análise , Proteínas tau/metabolismoRESUMO
Cerebral amyloid angiopathy, associated to most cases of Alzheimer's disease (AD), is characterized by the deposition of amyloid ss-peptide (Ass) in brain vessels, although the origin of the vascular amyloid deposits is still controversial: neuronal versus vascular. In the present work, we demonstrate that primary cultures of human cerebral vascular smooth muscle cells (HC-VSMCs) have all the secretases involved in amyloid ss-protein precursor (APP) cleavage and produce Ass(1-40) and Ass(1-42). Oxidative stress, a key factor in the etiology and pathophysiology of AD, up-regulates ss-site APP cleaving enzyme 1 (BACE1) expression, as well as Ass(1-40) and Ass(1-42) secretion in HC-VSMCs. This process is mediated by c-Jun N-terminal Kinase and p38 MAPK signaling and appears restricted to BACE1 regulation as no changes in the other secretases were observed. In conclusion, oxidative stress-mediated up-regulation of the amyloidogenic pathway in human cerebral vascular smooth muscle cells may contribute to the overall cerebrovascular amyloid angiopathy observed in AD patients.