RESUMO
In pathology, the deployment of artificial intelligence (AI) in clinical settings is constrained by limitations in data collection and in model transparency and interpretability. Here we describe a digital pathology framework, nuclei.io, that incorporates active learning and human-in-the-loop real-time feedback for the rapid creation of diverse datasets and models. We validate the effectiveness of the framework via two crossover user studies that leveraged collaboration between the AI and the pathologist, including the identification of plasma cells in endometrial biopsies and the detection of colorectal cancer metastasis in lymph nodes. In both studies, nuclei.io yielded considerable diagnostic performance improvements. Collaboration between clinicians and AI will aid digital pathology by enhancing accuracies and efficiencies.
RESUMO
We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-µm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.
Assuntos
DNA/genética , Congelamento , Genoma , Manejo de Espécimes/métodos , Animais , Encéfalo , Linhagem Celular , Eritrócitos , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Queratinócitos , Camundongos , Replicação de Sequência Autossustentável , Neoplasias da Glândula Tireoide , Transposases/metabolismoRESUMO
Three key elements to precision medicine are stratification by risk, detection of pathophysiological processes as early as possible (even before clinical presentation), and alignment of mechanism of action of intervention(s) with an individual's molecular driver(s) of disease. Used for decades in the management of some rare diseases and now gaining broad currency in cancer care, a precision medicine approach is beginning to be adapted to cognitive impairment and dementia. This review focuses on the application of precision medicine to address the clinical and biological complexity of two common neurodegenerative causes of dementia: Alzheimer disease and Parkinson disease.
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Demência/fisiopatologia , Doença de Parkinson/fisiopatologia , Medicina de Precisão , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Doença de Alzheimer/terapia , Transtornos Cognitivos/genética , Transtornos Cognitivos/fisiopatologia , Demência/genética , Humanos , Mutação , Doença de Parkinson/genética , Doença de Parkinson/terapiaRESUMO
The goal of precision medicine is to deliver optimally targeted and timed interventions tailored to an individual's molecular drivers of disease. This concept has wide currency in cancer care and in some diseases caused by monogenetic mutations, such as cystic fibrosis, and recently has been endorsed by the White House Office of Science and Technology for more widespread application in medicine. Here we describe our vision of how precision medicine can bring greater clarity to the clinical and biological complexity of the two most common neurodegenerative diseases, Alzheimer's disease and Parkinson's disease.
Assuntos
Doença de Alzheimer/terapia , Doença de Parkinson/terapia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Humanos , Mutação , Doença de Parkinson/genética , Doença de Parkinson/patologiaRESUMO
Apolipoprotein E4 (APOE4) genotype is the strongest genetic risk factor for late-onset Alzheimer disease and confers a proinflammatory, neurotoxic phenotype to microglia. Here, we tested the hypothesis that bone marrow cell APOE genotype modulates pathological progression in experimental Alzheimer disease. We performed bone marrow transplants (BMT) from green fluorescent protein-expressing human APOE3/3 or APOE4/4 donor mice into lethally irradiated 5-month-old APPswe/PS1ΔE9 mice. Eight months later, APOE4/4 BMT-recipient APPswe/PS1ΔE9 mice had significantly impaired spatial working memory and increased detergent-soluble and plaque Aß compared with APOE3/3 BMT-recipient APPswe/PS1ΔE9 mice. BMT-derived microglia engraftment was significantly reduced in APOE4/4 recipients, who also had correspondingly less cerebral apoE. Gene expression analysis in cerebral cortex of APOE3/3 BMT recipients showed reduced expression of tumor necrosis factor-α and macrophage migration inhibitory factor (both neurotoxic cytokines) and elevated immunomodulatory IL-10 expression in APOE3/3 recipients compared with those that received APOE4/4 bone marrow. This was not due to detectable APOE-specific differences in expression of microglial major histocompatibility complex class II, C-C chemokine receptor (CCR) type 1, CCR2, CX3C chemokine receptor 1 (CX3CR1), or C5a anaphylatoxin chemotactic receptor (C5aR). Together, these findings suggest that BMT-derived APOE3-expressing cells are superior to those that express APOE4 in their ability to mitigate the behavioral and neuropathological changes in experimental Alzheimer disease.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Comportamento Animal , Transplante de Medula Óssea , Doença de Alzheimer/imunologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Quimera/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/metabolismo , Habituação Psicofisiológica , Hematopoese , Hipocampo/patologia , Humanos , Imunidade Inata , Imunomodulação/imunologia , Memória de Curto Prazo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Monócitos/patologia , Fenótipo , Placa Amiloide/metabolismo , Placa Amiloide/patologiaRESUMO
Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of the brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2 Gy irradiation, and 5.5 Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5 Gy irradiation resulted in significant engraftment of BMT-derived cells in the brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5 Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in the optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms that MyA, but not NMyA, BMT is able to overcome.
Assuntos
Transplante de Medula Óssea/métodos , Sistema Nervoso Central/citologia , Condicionamento Pré-Transplante/métodos , Animais , Ciclofosfamida/farmacologia , Raios gama , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Monócitos/citologia , Agonistas Mieloablativos/farmacologia , Retina/citologia , Retina/transplante , Resultado do Tratamento , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have neuroinflammatory pathologic component, such as AD. We hypothesized that PGE(2) receptor subtype 1 (EP1) signaling (linked to intracellular Ca(2+) release) regulates Aß peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aß(1-40) through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aß(1-42). In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aß neurotoxicity ~50 % without altering high molecular weight Aß immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aß(1-42) neurotoxicity. Nimodipine, a Ca(2+) channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aß peptide neurotoxicity.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Dinoprostona/metabolismo , Síndromes Neurotóxicas/prevenção & controle , Antagonistas de Prostaglandina/farmacologia , Receptores de Prostaglandina E Subtipo EP1/efeitos dos fármacos , Animais , Western Blotting , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Corantes , Humanos , Hidrazinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Nimodipina/farmacologia , Oxazepinas/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/toxicidade , Sais de Tetrazólio , TiazóisRESUMO
BACKGROUND: Inheritance of the human ε4 allele of the apolipoprotein (apo) E gene (APOE) significantly increases the risk of developing Alzheimer's disease (AD), in addition to adversely influencing clinical outcomes of other neurologic diseases. While apoE isoforms differentially interact with amyloid ß (Aß), a pleiotropic neurotoxin key to AD etiology, more recent work has focused on immune regulation in AD pathogenesis and on the mechanisms of innate immunomodulatory effects associated with inheritance of different APOE alleles. APOE genotype modulates expression of proximal genes including APOC1, which encodes a small apolipoprotein that is associated with Aß plaques. Here we tested the hypothesis that APOE-genotype dependent innate immunomodulation may be mediated in part by apoC-I. METHODS: ApoC-I concentration in cerebrospinal fluid from control subjects of differing APOE genotypes was quantified by ELISA. Real-time PCR and ELISA were used to analyze apoC-I mRNA and protein expression, respectively, in liver, serum, cerebral cortex, and cultured primary astrocytes derived from mice with targeted replacement of murine APOE for human APOE ε3 or ε4. ApoC-I direct modulation of innate immune activity was investigated in cultured murine primary microglia and astrocytes, as well as human differentiated macrophages, using specific toll-like receptor agonists LPS and PIC as well as Aß. RESULTS: ApoC-I levels varied with APOE genotype in humans and in APOE targeted replacement mice, with ε4 carriers showing significantly less apoC-I in both species. ApoC-I potently reduced pro-inflammatory cytokine secretion from primary murine microglia and astrocytes, and human macrophages, stimulated with LPS, PIC, or Aß. CONCLUSIONS: ApoC-I is immunosuppressive. Our results illuminate a novel potential mechanism for APOE genotype risk for AD; one in which patients with an ε4 allele have decreased expression of apoC-I resulting in increased innate immune activity.
Assuntos
Apolipoproteína C-I/metabolismo , Regulação da Expressão Gênica/genética , Neuroglia/metabolismo , Idoso , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Apolipoproteína C-I/líquido cefalorraquidiano , Apolipoproteína C-I/genética , Apolipoproteína C-I/farmacologia , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Poli I-C/farmacologiaRESUMO
The Adult Changes in Thought (ACT) study is a longitudinal population-based prospective cohort study of brain aging and incident dementia in the Seattle metropolitan area. Observational studies using autopsies from ACT indicate that dementia is a convergent syndrome that commonly derives from Alzheimer's disease (AD), microvascular brain injury (mVBI), and Lewy body disease (LBD), and that these diseases have prevalent subclinical forms that also are commonly co-morbid. The existence of subclinical diseases highlights potential opportunities to intervene before the development of clinically apparent impairments. Our observations suggest that some such interventions already may exist to suppress processes of AD (statin therapy) or mVBI (treatment of hypertension). Reduced burden of LBD is associated with cigarette smoking; although smoking is not recommended as an intervention, these exposure data may provide clues to alternative neuroprotective mechanisms. Self reported anti-oxidant supplementation was without apparent effect in this cohort on indices of AD, mVBI, or LBD. Continued observational studies of brain aging will provide further insight into the convergent complexity of the dementia syndrome and its subclinical forms as well as highlight potential interventions that will require validation in clinical trials.
Assuntos
Doença de Alzheimer/patologia , Transtornos Cerebrovasculares/patologia , Demência/epidemiologia , Demência/patologia , Doença por Corpos de Lewy/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/complicações , Doença de Alzheimer/epidemiologia , Encéfalo/patologia , Transtornos Cerebrovasculares/complicações , Transtornos Cerebrovasculares/epidemiologia , Estudos de Coortes , Comorbidade , Demência/etiologia , Feminino , Humanos , Doença por Corpos de Lewy/complicações , Doença por Corpos de Lewy/epidemiologia , Estudos Longitudinais , Masculino , PrevalênciaRESUMO
A complex therapeutic challenge for Alzheimer's disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid beta (Abeta) peptides. One potential target is selective suppression of microglial prostaglandin E(2) receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2(-/-)) into lethally irradiated 5-month-old wild-type or APPswe-PS1DeltaE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2(-/-) microglia was more efficient in APPswe-PS1DeltaE9 than in wild-type mice, and APPswe-PS1DeltaE9 mice that received EP2(-/-) BMT had increased cortical microglia compared with APPswe-PS1DeltaE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Abeta plaques and potentially more neurotoxic soluble Abeta species. An additional 25% reduction in cerebral cortical Abeta burden was achieved in mice that received EP2(-/-) BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Abeta peptide and plaque accumulation in the cerebrum of patients with AD.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/patologia , Camundongos Transgênicos , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Animais , Transplante de Medula Óssea/métodos , Córtex Cerebral/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Prostaglandina E Subtipo EP2/genéticaRESUMO
Cigarette smoking has been associated repeatedly in observational studies with decreased risk of Parkinson's disease (PD), but its relationship to the risk of dementia or Alzheimer's disease (AD) is inconsistent. All of these studies have used clinical diagnoses of disease. We tested the hypothesis that lifetime cigarette use might be associated with reduced risk of neuropathologic changes of Lewy-related pathology (LRP) in multiple brain regions or with reduced risk of consensus neuropathologic changes of AD in a prospective community-based study of brain aging and dementia, the Adult Changes in Thought (ACT) study. We observed that heavy lifetime cigarette smoking (>50 pack years) was associated with significantly reduced relative risk (RR) for LRP, but not AD-type pathologic changes, after correcting for selection bias, and with significantly reduced frequency of LRP in the substantia nigra. These findings are the first of which we are aware to associate reduced LRP in human brain with any exposure, and substantiate observational studies that have associated cigarette smoking with reduced risk of PD. Although cigarette smoking is too toxic to suggest as a treatment, if confirmed, these findings may guide future therapeutic strategies that attempt to suppress LRP in human brain by other means.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Corpos de Lewy/patologia , Fumar/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Probabilidade , Estudos Prospectivos , Risco , Fatores de TempoRESUMO
Oxidative stress, protein misfolding, protein complex formation, and detergent insolubility are biochemical features of Alzheimer's disease (AD). We tested the cause-and-effect relationships among these using MC65 human neuroblastoma cells that exhibit toxicity upon conditional expression of carboxy-terminal fragments (CTFs) of the human amyloid precursor protein (APP). Treatments with three different antioxidants (alpha-tocopherol, N-acetyl cysteine, and alpha-lipoic acid) or three different compounds (glycerol, trimethylamine-N-oxide, and 4-phenylbutyric acid) that have been described to have a "chemical chaperone" function in promoting protein folding all had a protective effect on MC65 cells and decreased markers of oxidative damage and accumulation of high molecular weight amyloid (A) beta-immunoreactive (IR) species. However, chaperones partially reduced detergent insolubility of the remaining Abeta-IR species, while antioxidants did not. These results suggest that protein misfolding associated with overexpression of APP CTFs promotes oxidative stress and cytotoxicity and contributes to formation of detergent-insoluble species that appear unrelated to cytotoxicity.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Chaperonas Moleculares/metabolismo , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/efeitos dos fármacos , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Antioxidantes/farmacologia , Linhagem Celular , Detergentes/química , Detergentes/farmacologia , Humanos , Chaperonas Moleculares/farmacologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Placa Amiloide/química , Placa Amiloide/metabolismo , Dobramento de Proteína , Solubilidade/efeitos dos fármacosRESUMO
Abstract Alterations in glutathione (GSH) metabolism are associated with neurodegeneration in Alzheimer's disease (AD), and GSH depletion follows application of exogenous fibrillar amyloid beta (Abeta) peptides in experimental systems; these results are commonly cited as evidence of oxidative damage in AD. We used MC65 human neuroblastoma cells that conditionally express carboxy-terminal fragments of the Abeta precursor protein (Abeta/CTFs) to directly test the hypothesis that GSH is part of the cellular response to stressors associated with Abeta/CTF accumulation and not simply a marker of oxidative damage. Our data showed that Abeta/CTFs accumulated by post-translational processes and were associated with progressive increases in oxidative damage and cytotoxicity. Ethycrinic acid (EA) or diethyl maleate (DEM), reagents that deplete GSH through non-specific thiol adduction, gave rise to dose-dependent cytotoxicity that was independent of Abeta/CTF expression and minimally responsive to alpha-tocopherol (AT). In contrast, buthionine sulfoximine (BSO), a selective inhibitor of GSH synthase, not only augmented Abeta/CTF-associated cell death but unexpectedly potentiated Abeta/CTF accumulation; both outcomes were completely suppressed by AT. These data suggest that antioxidants may serve as 'Abeta targeting' therapies that suppress toxic protein aggregation rather than simply acting as downstream radical scavengers.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Espaço Extracelular/metabolismo , Glutationa/metabolismo , Fragmentos de Peptídeos/metabolismo , alfa-Tocoferol/análogos & derivados , Precursor de Proteína beta-Amiloide/química , Análise de Variância , Antioxidantes/farmacologia , Western Blotting/métodos , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cromatografia Líquida de Alta Pressão/métodos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/efeitos dos fármacos , F2-Isoprostanos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neuroblastoma , Fragmentos de Peptídeos/química , Inibidores da Síntese de Proteínas/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tetraciclina/farmacologia , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Tocoferóis , alfa-Tocoferol/farmacologiaRESUMO
To identify the proteins associated with soluble alpha-synuclein (AS) that might promote AS aggregation, a key event leading to neurodegeneration, we quantitatively compared protein profiles of AS-associated protein complexes in MES cells exposed to rotenone, a pesticide that produces parkinsonism in animals and induces Lewy body (LB)-like inclusions in the remaining dopaminergic neurons, and to vehicle. We identified more than 250 proteins associated with Nonidet P-40 soluble AS, and demonstrated that at least 51 of these proteins displayed significant differences in their relative abundance in AS complexes under conditions where rotenone was cytotoxic and induced formation of cytoplasmic inclusions immunoreactive to anti-AS. Overexpressing one of these proteins, heat shock protein (hsp) 70, not only protected cells from rotenone-mediated cytotoxicity but also decreased soluble AS aggregation. Furthermore, the protection afforded by hsp70 transfection appeared to be related to suppression of rotenone-induced oxidative stress as well as mitochondrial and proteasomal dysfunction.
Assuntos
Proteínas do Tecido Nervoso/química , Proteômica/métodos , Sequência de Aminoácidos , Animais , Western Blotting , Sobrevivência Celular , Cromatografia Líquida , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Citometria de Fluxo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Espectrometria de Massas , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Neurônios/metabolismo , Nêutrons , Estresse Oxidativo , Testes de Precipitina , Complexo de Endopeptidases do Proteassoma , Proteoma , Ratos , Rotenona/farmacologia , Transdução de Sinais , Sinucleínas , Fatores de Tempo , Transfecção , alfa-SinucleínaRESUMO
Ischemic injury to brain is associated with both disruption of the blood-brain barrier and increased oxidative stress. Given the neurotoxicity associated with exposure to oxidized low-density lipoprotein (oxLDL) in vitro, we tested the hypothesis that oxLDL may be present in parenchymal cells of cerebrum after infarction and that oxLDL may influence the pathophysiology of cerebral infarction. Our results showed that the subacute phase of cerebral infarction in patients was characterized by the appearance of oxLDL epitopes in astrocytes, but not neurons or microglia, in the perinecrotic zone. We further demonstrated that minimally oxLDL was most effectively internalized by primary cultures of rat astrocytes, and that exposure to minimal oxLDL stimulated astrocyte interleukin-6 secretion but did not alter nitric oxide production. These results demonstrate for the first time that oxLDL is present in brain parenchyma of patients with ischemic infarction and suggest a potential mechanism by which oxLDL may activate innate immunity and thereby indirectly influence neuronal survival.
Assuntos
Astrócitos/metabolismo , Encéfalo/patologia , Infarto Cerebral/fisiopatologia , Interleucina-6/metabolismo , Lipoproteínas LDL/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Encéfalo/metabolismo , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Lactente , Lipoproteínas LDL/farmacologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , RatosRESUMO
4-Hydroxy-2-nonenal (HNE), a potent toxin formed in the brain from oxidation of polyunsaturated fatty acids, is increased in Alzheimer disease (AD), where it is a proposed effector of amyloid beta peptide-mediated neurotoxicity. Detoxification of HNE via the mercapturic acid pathway (MAP) is the primary means by which other organs, such as liver, limit its toxic effects. Here we examined the distribution and activity of MAP detoxification for HNE in cerebrum. Our results showed that rat cerebral cortex and especially synaptosomes were less well equipped to detoxify HNE via the MAP than liver. Glutathione transferases (GSTs) catalyze the committed step in the MAP; GST-mu and GST-pi, but not OST-alpha, were detected in neurons and astrocytes in cerebrum from AD patients and controls. MAP activity in frontal cortex of AD patients was modestly but significantly increased compared to controls. These data suggest that lipid peroxidation may present a greater toxic burden to cerebrum than to other organs, and that a component of response to injury in late stage AD is a slight increase in MAP activity.
Assuntos
Acetilcisteína/metabolismo , Aldeídos/metabolismo , Doença de Alzheimer/metabolismo , Córtex Cerebral/metabolismo , Peroxidação de Lipídeos/fisiologia , Estresse Oxidativo/fisiologia , Terminações Pré-Sinápticas/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Córtex Cerebral/fisiopatologia , Ácidos Graxos Insaturados/metabolismo , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Sinaptossomos/metabolismoRESUMO
Activation of glial innate immunity is widely proposed to contribute to a number of degenerative and destructive diseases of brain. However, the precise role of activated innate immunity has been difficult to define in vivo because of multiple simultaneous pathogenic processes and responses to injury that confound interpretation of results from complex models of disease. Here, we used the model of intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) to test the hypothesis that directly activated glial innate immunity leads to neurodegeneration in cerebrum and to establish the molecular determinants of and neuroprotectants from such innate immunity-mediated neuronal damage. Our results showed that ICV LPS induced delayed, reversible oxidative damage to cerebral neuronal membranes as measured by F4-neuroprostanes that was coincident with degeneration of the hippocampal pyramidal neuron dendritic system, but not neuron death, in adult mice. Both neuronal oxidative damage and dendritic degeneration were NF-kappaB and iNOS dependent and were completely suppressed by ibuprofen and alpha-tocopherol, but not naproxen or gamma-tocopherol. These results prove that activation of glial innate immunity can lead to neurodegeneration independent of other pathologic processes, closely associate oxidative damage to neuronal membranes with degeneration of the dendritic system, and provide a possible explanation for the varying efficacy of neuroprotectants that have been suggested in observational studies of dementia.
Assuntos
Dendritos/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Imunidade Inata , Degeneração Neural/prevenção & controle , Telencéfalo/efeitos dos fármacos , Análise de Variância , Animais , Biomarcadores , Temperatura Corporal/efeitos dos fármacos , Contagem de Células , Óxidos N-Cíclicos , Dendritos/metabolismo , Dinoprostona/metabolismo , Ácidos Docosa-Hexaenoicos/farmacocinética , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Interações Medicamentosas , Feminino , Imuno-Histoquímica/métodos , Injeções Intraventriculares , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Degeneração Neural/induzido quimicamente , Neuroglia/imunologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxidos de Nitrogênio/farmacologia , Oxirredução/efeitos dos fármacos , Peptídeos/genética , Peptídeos/metabolismo , Coloração pela Prata/métodos , Telencéfalo/citologia , Telencéfalo/imunologia , Fatores de Tempo , Tocoferóis/farmacologiaRESUMO
Carbonyl stress from products of lipid peroxidation, such as 4-hydroxynonenal (HNE), and products of sugars in diabetes mellitus, such as methylglyoxal (MG) and glyoxal (G), may contribute to neurodegeneration in Alzheimer's disease (AD). We tested the hypothesis that these carbonyls alter the proposed central pathogenic mechanism of AD, intracellular amyloid-beta (A beta)-mediated cytotoxicity, using a human neuroblastoma cell line that conditionally expresses carboxy-terminal fragments (CTFs) of the amyloid precursor protein. HNE was a potent cytotoxin, whereas G was mildly cytotoxic; cytotoxicity from each was independent of A beta/CTF expression and not altered by alpha-tocopherol. In contrast, MG cytotoxicity was enhanced by the induced expression of A beta/CTFs and suppressed by alpha-tocopherol. alpha-tocopherol cytoprotection was accompanied by decreased A beta/CTF aggregation. G also promoted beta/CTF aggregation but by mechanisms unaffected by alpha-tocopherol treatment. Our findings showed that A beta/CTF aggregation and cytotoxicity may be profoundly altered by aldehydes associated with diabetes and that in the case of MG, this process is suppressed by alpha-tocopherol. Moreover, our results suggest that while intracellular aggregation of A beta/CTFs may be necessary for the development of toxicity attributable to their expression in this model, the presence of high-molecular weight aggregated A beta/CTFs does not invariably lead to cytotoxicity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Transporte/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/complicações , Precursor de Proteína beta-Amiloide/biossíntese , Anticorpos Monoclonais/imunologia , Western Blotting , Citotoxinas/metabolismo , Complicações do Diabetes , Eletroforese , Glioxal/metabolismo , Humanos , Espaço Intracelular , Aldeído Pirúvico/metabolismo , Tioléster Hidrolases , alfa-Tocoferol/metabolismoRESUMO
Dithiocarbamate (DTC)-based pesticides have been implicated in Parkinson's disease (PD) through epidemiological links to increased risk of PD, clinical reports of parkinsonism following occupational exposure to the DTC-based pesticide maneb, and experimental studies showing dopaminergic neurodegeneration with combined exposure of rats to maneb and paraquat. We hypothesize that the manganese-ethylene-bis-dithiocarbamate (MnEBDC) complex in maneb may produce oxidative stress by catalyzing catechol oxidation. We tested this hypothesis by performing a structure-function analysis of metal-EBDC and metal-diethyldithiocarbamate (DEDC) complexes of Mn2+, Zn2+, and Cu2+ to catalyze oxidation of N-acetyldopamine (NA-DA) and 3,4-dihydroxyphenylacetic acid (DP) in the presence and absence of N-acetylcysteine (NAC), a model of glutathione. Both Mn-DTCs retained the capacity of the parent ion to catalyze one-electron oxidation of NA-DA, but lost the ability to catalyze DP oxidation. Strikingly, while Zn2+ did not catalyze catechol oxidation, both Zn-DTCs catalyzed one-electron oxidation of NA-DA but not DP. While Cu2+ catalyzed oxidation of both catechols, Cu-DTCs were inert. Similar results were obtained with MnEBDC and dopamine or norepinephrine; however, zinc-ethylene-bis-dithiocarbamate was less efficient at catalyzing oxidation of these catechols. Our results point to the potential for manganese- and zinc-containing EBDC pesticides to promote oxidative stress in catecholaminergic regions of the brain.
Assuntos
Carbamatos/química , Catecóis/química , Praguicidas/química , Catálise , Oxidantes/química , Oxirredução , Estresse Oxidativo , Oxigênio/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/química , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Recent studies of cerebrospinal fluid (CSF) have shown increased oxidation of CSF lipoproteins in Alzheimer's disease (AD) patients, and neurotoxicity from oxidized CSF lipoproteins in culture. Since inheritance of different alleles of the apolipoprotein (apo) E gene is a risk factor for AD and apoE is the major lipoprotein trafficking molecule in brain, we hypothesized that apoE may modify the pathogenesis of AD by directing the delivery of oxidized CSF lipoproteins to neurons. To test this hypothesis, we adapted a method previously used with isolated plasma lipoproteins to specifically label lipid particles in situ in native CSF and quantified their delivery to human SK-N-BE(2)C neuroblastoma cells. CSF lipoproteins were delivered to neuronal cells largely through apoE-dependent processes. Importantly, CSF lipoproteins from AD patients were delivered more efficiently than CSF lipoproteins from age-matched controls; this effect was not associated with apoE genotype or degree of CSF lipoprotein oxidation but was associated with apoE monomer concentration that tended to be lower in AD patients. The inverse relationship between apoE monomer concentration and CSF lipoprotein delivery was duplicated in SK-N-BE(2)C cells, but not human astrocytoma cells, using artificial lipid particles and purified human apoE. These results suggest that lipoproteins in CSF from AD patients are delivered more efficiently to neurons than are CSF lipoproteins from controls, and that this abnormality may be explained largely by variations in CSF apoE concentration.