RESUMO
Complex in vitro models (CIVMs) offer the potential to increase the clinical relevance of preclinical efficacy and toxicity assessments and reduce the reliance on animals in drug development. The European Society of Toxicologic Pathology (ESTP) and Society for Toxicologic Pathology (STP) are collaborating to highlight the role of pathologists in the development and use of CIVM. Pathologists are trained in comparative animal medicine which enhances their understanding of mechanisms of human and animal diseases, thus allowing them to bridge between animal models and humans. This skill set is important for CIVM development, validation, and data interpretation. Ideally, diverse teams of scientists, including engineers, biologists, pathologists, and others, should collaboratively develop and characterize novel CIVM, and collectively assess their precise use cases (context of use). Implementing a morphological CIVM evaluation should be essential in this process. This requires robust histological technique workflows, image analysis techniques, and needs correlation with translational biomarkers. In this review, we demonstrate how such tissue technologies and analytics support the development and use of CIVM for drug efficacy and safety evaluations. We encourage the scientific community to explore similar options for their projects and to engage with health authorities on the use of CIVM in benefit-risk assessment.
Assuntos
Patologistas , Patologia , Toxicologia , Humanos , Toxicologia/métodos , Animais , Bioengenharia , Testes de Toxicidade , Avaliação Pré-Clínica de Medicamentos , Técnicas In VitroRESUMO
ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode-of-action (MoA), thereby supporting discrimination of direct-acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non-genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non-genotoxic chemicals that together cover a broad chemical space. The within-laboratory reproducibility (WLR) was up to 98% (73%-98% across participants) and the overall between-laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays.
Assuntos
Dano ao DNA , Mamíferos , Animais , Humanos , Testes de Mutagenicidade , Reprodutibilidade dos Testes , Genes ReporterRESUMO
Drug-induced proximal tubule (PT) injury remains a serious safety concern throughout drug development. Traditional in vitro 2-dimensional (2D) and preclinical in vivo models often fail to predict drug-related injuries presented in clinical trials. Various 3-dimensional (3D) microphysiological systems (MPSs) have been developed to mimic physiologically relevant properties, enabling them to be more predictive toward nephrotoxicity. To explore the capabilities of an MPS across species, we compared cytotoxicity in hRPTEC/TERT1s and rat primary proximal tubular epithelial cells (rPPTECs) following exposure to zoledronic acid and ibandronate (62.5-500 µM), and antibiotic polymyxin B (PMB) (50 and 250 µM, respectively). For comparison, we investigated cytotoxicity using 2D cultured hRPTEC/TERT1s and rPPTECs following exposure to the same drugs, including overlapping concentrations, as their 3D counterparts. Regardless of the in vitro model, bisphosphonate-exposed rPPTECs exhibited cytotoxicity quicker than hRPTEC/TERT1s. PMB was less sensitive toward nephrotoxicity in rPPTECs than hRPTEC/TERT1s, demonstrating differences in species sensitivity within both 3D and 2D models. Generally, 2D cultured cells experienced faster drug-induced cytotoxicity compared to the MPSs, suggesting that MPSs can be advantageous for longer-term drug-exposure studies, if warranted. Furthermore, ibandronate-exposed hRPTEC/TERT1s and rPPTECs produced higher levels of inflammatory and kidney injury biomarkers compared to zoledronic acid, indicating that ibandronate induces acute kidney injury, but also a potential protective response since ibandronate is less toxic than zoledronic acid. Our study suggests that the MPS model can be used for preclinical screening of compounds prior to animal studies and human clinical trials.
Assuntos
Injúria Renal Aguda , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Ratos , Animais , Ácido Zoledrônico/toxicidade , Ácido Ibandrônico/toxicidade , Difosfonatos/toxicidade , Difosfonatos/uso terapêutico , Túbulos Renais ProximaisRESUMO
BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aß) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model. METHODS: In this report, we developed and characterized cloned lines of amyloid beta (Aß) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aß-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aß T cell epitope loaded haplotype-matched major histocompatibility complex II IAb (MHCII-IAb-KLVFFAEDVGSNKGA) tetramer binding. Aß-Th1 and Aß-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses. RESULTS: The propagated Aß-Th1 and Aß-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aß reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aß-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system. CONCLUSIONS: These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aß reactive Tregs.
Assuntos
Doença de Alzheimer/patologia , Linfócitos T CD4-Positivos/patologia , Presenilina-1/genética , Precursor de Proteína beta-Amiloide/genética , Amiloidose/patologia , Animais , Transtornos Cognitivos/patologia , Transtornos Cognitivos/psicologia , Inflamação/genética , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Drug-induced liver injury (DILI) continues to be a major cause of drug attrition and restrictive labeling. Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of DILI. However, a comprehensive assessment of this phenomenon broadly in the context of DILI is lacking. As such, we used an orthogonal approach comprising a FXR target gene assay in primary human hepatocytes and a commercially available FXR reporter assay to investigate the potential FXR antagonistic effects of an extensive test set of 159 compounds with and without association with clinical DILI. Data were omitted from analysis based on the presence of cytotoxicity to minimize false positive assay signals and other complications in data interpretation. Based on the experimental approaches employed and corresponding data, the prevalence of FXR antagonism was relatively low across this broad DILI test set, with 16-24% prevalence based on individual assay results or combined signals in both assays. Moreover, FXR antagonism was not highly predictive for identifying clinically relevant hepatotoxicants retrospectively, where FXR antagonist classification alone had minimal to moderate predictive value as represented by positive and negative likelihood ratios of 2.24-3.84 and 0.72-0.85, respectively. The predictivity did not increase significantly when considering only compounds with high clinical exposure (maximal or efficacious plasma exposures > 1.0 µM). In contrast, modest gains in predictive value of FXR antagonism were observed considering compounds that also inhibit bile salt export pump. In addition, we have identified novel FXR antagonistic effects of well-studied hepatotoxic drugs, including bosentan, tolcapone and ritonavir. In conclusion, this work represents a comprehensive evaluation of FXR antagonism in the context of DILI, including its overall predictivity and challenges associated with detecting this phenomenon in vitro.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Ácidos e Sais Biliares , Bioensaio , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos , Humanos , Estudos RetrospectivosRESUMO
BACKGROUND: The mixed lineage kinase type 3 inhibitor URMC-099 facilitates amyloid-beta (Aß) clearance and degradation in cultured murine microglia. One putative mechanism is an effect of URMC-099 on Aß uptake and degradation. As URMC-099 promotes endolysosomal protein trafficking and reduces Aß microglial pro-inflammatory activities, we assessed whether these responses affect Aß pathobiogenesis. To this end, URMC-099's therapeutic potential, in Aß precursor protein/presenilin-1 (APP/PS1) double-transgenic mice, was investigated in this model of Alzheimer's disease (AD). METHODS: Four-month-old APP/PS1 mice were administered intraperitoneal URMC-099 injections at 10 mg/kg daily for 3 weeks. Brain tissues were examined by biochemical, molecular and immunohistochemical tests. RESULTS: URMC-099 inhibited mitogen-activated protein kinase 3/4-mediated activation and attenuated ß-amyloidosis. Microglial nitric oxide synthase-2 and arginase-1 were co-localized with lysosomal-associated membrane protein 1 (Lamp1) and Aß. Importatly, URMC-099 restored synaptic integrity and hippocampal neurogenesis in APP/PS1 mice. CONCLUSIONS: URMC-099 facilitates Aß clearance in the brain of APP/PS1 mice. The multifaceted immune modulatory and neuroprotective roles of URMC-099 make it an attractive candidate for ameliorating the course of AD. This is buttressed by removal of pathologic Aß species and restoration of the brain's microenvironment during disease.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Presenilina-1/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Pirróis/farmacologiaRESUMO
We investigated the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on behavioral and pathological outcomes in Alzheimer's disease (AD) and non-transgenic mice. GM-CSF treatment in AD mice reduced brain amyloidosis, increased plasma Aß, and rescued cognitive impairment with increased hippocampal expression of calbindin and synaptophysin and increased levels of doublecortin-positive cells in the dentate gyrus. These data extend GM-CSF pleiotropic neuroprotection mechanisms in AD and include regulatory T cell-mediated immunomodulation of microglial function, Aß clearance, maintenance of synaptic integrity, and induction of neurogenesis. Together these data support further development of GM-CSF as a neuroprotective agent for AD.
Assuntos
Doença de Alzheimer , Encéfalo/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos , Camundongos TransgênicosRESUMO
Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD), dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However, whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end, progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels, increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide , Amiloidose/tratamento farmacológico , Catepsina B/uso terapêutico , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/patologia , Animais , Catepsina B/farmacologia , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Aprendizagem/fisiologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos , Camundongos Transgênicos , Presenilina-1/metabolismoRESUMO
BACKGROUND: Amyloid-ß (Aß)-stimulated microglial inflammatory responses engage mitogen-activated protein kinase (MAPK) pathways in Alzheimer's disease (AD). Mixed-lineage kinases (MLKs) regulate upstream MAPK signaling that include p38 MAPK and c-Jun amino-terminal kinase (JNK). However, whether MLK-MAPK pathways affect Aß-mediated neuroinflammation is unknown. To this end, we investigated if URMC-099, a brain-penetrant small-molecule MLK type 3 inhibitor, can modulate Aß trafficking and processing required for generating AD-associated microglial inflammatory responses. METHODS: Aß1-42 (Aß42) and/or URMC-099-treated murine microglia were investigated for phosphorylated mitogen-activated protein kinase kinase (MKK)3, MKK4 (p-MKK3, p-MKK4), p38 (p-p38), and JNK (p-JNK). These pathways were studied in tandem with the expression of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. Gene expression of the anti-inflammatory cytokines, IL-4 and IL-13, was evaluated by real-time quantitative polymerase chain reaction. Aß uptake and expression of scavenger receptors were measured. Protein trafficking was assessed by measures of endolysosomal markers using confocal microscopy. RESULTS: Aß42-mediated microglial activation pathways were shown by phosphorylation of MKK3, MKK4, p38, and JNK and by expression of IL-1ß, IL-6, and TNF-α. URMC-099 modulated microglial inflammatory responses with induction of IL-4 and IL-13. Phagocytosis of Aß42 was facilitated by URMC-099 with up-regulation of scavenger receptors. Co-localization of Aß and endolysosomal markers associated with enhanced Aß42 degradation was observed. CONCLUSIONS: URMC-099 reduced microglial inflammatory responses and facilitated phagolysosomal trafficking with associated Aß degradation. These data demonstrate a new immunomodulatory role for URMC-099 to inhibit MLK and to induce microglial anti-inflammatory responses. Thus, URMC-099 may be developed further as a novel disease-modifying AD therapy.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Citocinas/metabolismo , Microglia/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Células Cultivadas , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Microscopia Confocal , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estatísticas não Paramétricas , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Modulation of the amyloid-ß (Aß) trafficking pathway heralds a new therapeutic frontier for Alzheimer's disease (AD). As CD74 binds to the amyloid-ß precursor protein (APP) and can suppresses Aß processing, we investigated whether recombinant adeno-associated virus (AAV) delivery of CD74 could reduce Aß production and affect disease outcomes. This idea was tested in a mouse AD model. Cotransduction of AAV-tetracycline-controlled transactivator (tTA) and AAV-tet-response element (TRE)-CD74 resulted in CD74 expression, reduced Aß production in mouse neurons containing the human APP with familial AD-linked mutations. Stereotaxic injection of AAV-TRE-GFP or CD74 into the hippocampi of an AD mouse, defined as a TgCRND8 × calmodulin-dependent protein kinase II derived promoter-tTA double-transgenic, reduced Aß loads and pyramidal neuronal Aß accumulation in the hippocampus. Immunofluorescent studies showed that APP colocalization with Lamp1 was increased in CD74-expressing neurons. Moreover, Morris water maze tasks demonstrated that mice treated with AAV-TRE-CD74 showed improved learning and memory compared to AAV-TRE-GFP control animals. These results support the idea that CD74-induced alteration of Aß processing could improve AD-associated memory deficits as shown in mouse models of human disease.
Assuntos
Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Histocompatibilidade Classe II/genética , Neurônios/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/fisiologia , Amiloidose/metabolismo , Animais , Células Cultivadas , Dependovirus , Modelos Animais de Doenças , Feminino , Terapia Genética , Vetores Genéticos/uso terapêutico , Hipocampo/metabolismo , Hipocampo/cirurgia , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Transgênicos , Microinjeções/métodos , Técnicas EstereotáxicasRESUMO
Cluster of Differentiation-200 (CD200) is an anti-inflammatory glycoprotein expressed in neurons, T cells, and B cells, and its receptor is expressed on glia. Both Alzheimer's disease patients and mouse models display age-related or amyloid-ß peptide (Aß)-induced reductions in CD200. The goal of this study was to determine if neuronal CD200 expression restores hippocampal neurogenesis and reduces Aß in the amyloid precursor protein mouse model. Amyloid precursor protein and wild-type mice were injected at 6 months of age with an adeno-associated virus expressing CD200 into the hippocampus and sacrificed at 12 months. CD200 expression restored neural progenitor cell proliferation and differentiation in the subgranular and granular cell layers of the dentate gyrus and reduced diffuse but not thioflavin-S(+) plaques in the hippocampus. In vitro studies demonstrated that CD200-stimulated microglia increased neural differentiation of neural stem cells and enhanced axon elongation and dendrite number. CD200 also enhanced Aß uptake by microglia. These data indicate that CD200 is capable of enhancing microglia-mediated Aß clearance and neural differentiation and has potential as a therapeutic for Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Antígenos CD/fisiologia , Hipocampo/fisiologia , Neurogênese/genética , Fagocitose/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antígenos CD/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Hipocampo/citologia , Camundongos Transgênicos , Microglia/citologia , Microglia/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/efeitos dos fármacos , Células-Tronco/citologiaRESUMO
Aberrations in hippocampal neurogenesis are associated with learning and memory, synaptic plasticity and neurodegeneration in Alzheimer's disease (AD). However, the linkage between them, ß-amyloidosis and neuroinflammation is not well understood. To this end, we generated a mouse overexpressing familial AD (FAD) mutant human presenilin-1 (PS1) crossed with a knockout (KO) of the CC-chemokine ligand 2 (CCL2) gene. The PS1/CCL2KO mice developed robust age-dependent deficits in hippocampal neurogenesis associated with impairments in learning and memory, synaptic plasticity and long-term potentiation. Neurogliogenesis gene profiling supported ß-amyloid independent pathways for FAD-associated deficits in hippocampal neurogenesis. We conclude that these PS1/CCL2KO mice are suitable for studies linking host genetics, immunity and hippocampal function.
Assuntos
Doença de Alzheimer/fisiopatologia , Quimiocina CCL2/genética , Modelos Animais de Doenças , Hipocampo/fisiopatologia , Neurogênese , Presenilina-1/genética , Doença de Alzheimer/genética , Doença de Alzheimer/psicologia , Animais , Quimiocina CCL2/metabolismo , Hipocampo/metabolismo , Humanos , Potenciação de Longa Duração/genética , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Neurônios/fisiologia , Presenilina-1/metabolismoRESUMO
BACKGROUND: Redox-active metal dyshomeostasis and oxidative stress are associated with mitochondrial dysfunction and amyloid-ß (Aß) neurotoxicity that are linked to both the development of age-related macular degeneration (AMD) and Alzheimer's disease (AD). As potential therapeutic agents, orally active multifunctional antioxidants (MFAOs) possessing two independent functional groups capable of binding redox-active metals and scavenging free radicals have been synthesized. OBJECTIVE: To determine whether MFAOs affect mitochondrial function and reduce the presence of Aß plaque formation. METHODS: The MFAOs were evaluated in cultured SH-SY5Y cells and ARPE-19 cells. MFAO effects on mitochondrial function were investigated using rhodamine 123 staining after 2 hour exposure to MnCl2. MFAO effects on Aß:Zn complex formation were evaluated with Zinquin staining and the ability of the Aß:Zn complex to be degraded by matrix metalloproteinase-2 (MMP-2). The ability of MFAOs to reduce Aß plaque in the brain was determined by orally feeding MFAO for one year to B6;129-Psen1tm1Mpm Tg(AßPPSwe,tauP301L) 1Lfa/Mmjax transgenic mice. Aß levels were determined by ELISA. RESULTS: MFAOs neither adversely affected mitochondrial signaling nor labile cytoplasmic zinc levels. MFAOs protected cells against Mn2+-induced mitochondrial dysfunction. MFAOs also removed zinc from the Aß:Zn complex so that Aß plaque could be degraded by MMP-2. Zinquin staining indicated that the removed zinc was present in the cytoplasm as labile zinc. Orally administered MFAOs reduced the brain levels of both Aß40 and Aß42 isoforms of Aß. CONCLUSION: These studies demonstrate that these MFAOs have metal attenuating properties with therapeutic potential in the treatment of both AMD and AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Mitocôndrias/efeitos dos fármacos , Zinco/metabolismo , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Quinolonas , Rodamina 123 , Compostos de TosilRESUMO
Neuroinflammation affects the pathobiology of Alzheimer's disease (AD). Notably, ß-amyloid (Aß) deposition induces microglial activation and the subsequent production of proinflammatory neurotoxic factors. In maintaining brain homeostasis, microglial plasticity also enables phenotypic transition between toxic and trophic activation states. One important control for such cell activation is through the CC-chemokine ligand 2 (CCL2) and its receptor, the CC-chemokine receptor 2. Both affect microglia and peripheral macrophage immune responses and for the latter, cell ingress across the blood-brain barrier. However, how CCL2-CC-chemokine receptor 2 signaling contributes to AD pathogenesis is not well understood. To this end, we now report that CCL2 deficiency influences behavioral abnormalities and disease progression in Aß precursor protein/presenilin-1 double-transgenic mice. Here, increased cortical and hippocampal Aß deposition is coincident with the formulation of Aß oligomers. Deficits in peripheral Aß clearance and in scavenger, neuroprogenitor, and microglial cell functions are linked to deficient Aß uptake. All serve to accelerate memory dysfunction. Taken together, these data support a role of CCL2 in innate immune functions relevant to AD pathogenesis.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Transtornos Cognitivos/metabolismo , Modelos Animais de Doenças , Doença de Alzheimer/complicações , Amiloidose/complicações , Amiloidose/metabolismo , Animais , Transtornos Cognitivos/complicações , Camundongos , Camundongos TransgênicosRESUMO
Inducible nitric oxide synthase (iNOS) upregulation and consequent NO formation are well-recognized neuroinflammatory responses associated with Parkinson's disease (PD). These contribute to nitrosative protein modifications affecting neuronal injury and cell death. Indeed, a pathobiologic signature for PD is Lewy body formation containing misfolded and aggregated forms of alpha-synuclein (α-syn). Moreover, nitration of α-syn promotes protein aggregation in disease. To model such pathological events, we constructed controllable iNOS and bicistronic α-syn-IRES-tTA adeno-associated virus (AAV) expression vectors. Transduction of iNOS and α-syn AAV constructs led to nitration of α-syn in neurons and overexpression of iNOS promoted protein aggregation. We posit that this AAV system mimics critical protein misfolding events associated with the pathogenesis of PD.
Assuntos
Óxido Nítrico/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Dependovirus/genética , Células HEK293 , Humanos , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Dobramento de Proteína , Transdução Genética , alfa-Sinucleína/genéticaRESUMO
Combined antiretroviral therapy (ART) tremendously improved the lifespan and symptoms associated with AIDS-defining illness in affected individuals. However, chronic ART-treated patients frequently develop age-dependent complications, including dementia, diabetes, and hyperlipidemia: all risk factors of Alzheimer's disease. Importantly, the effect of ART compounds on amyloid generation and clearance has never been systematically examined. Nine prescribed HIV protease inhibitors were tested for their effect on amyloid-ß peptide (Aß) clearance in primary cultured human monocyte-derived macrophages. Atazanavir, ritonavir, and saquinavir modestly inhibited of Aß degradation, while lopinavir, nelfinavir, and ritonavir enhanced secretion of undigested Aß after phagocytosis. Lopinavir, nelfinavir, ritonavir, and saquinavir inhibited endogenous Aß40 production from primary cultured human cortical neurons, which were associated with reduction in Beta-site APP Converting Enzyme 1 (BACE1) and γ-secretase enzyme activities. However, ART compounds showed little inhibition of purified BACE1 activity in vitro, suggesting the indirect effect of ART compounds on BACE1 activity in neurons. Finally, nefinavir or lopinavir/ritonavir (Kaletra) were orally administered for 30 days into APP SCID mice expressing a double mutant form of APP 695 (KM670/671NL + V717F) in homozygosity for the scid allele of Prkdc. There was no difference in beta-amyloidosis by ART drug administration as determined by both immunohistochemistry and ELISA measurements although the therapeutic doses of the ART compounds was present in the brain. These data demonstrated that ART drugs can inhibit Aß clearance in macrophages and Aß production in neurons, but these effects did not significantly alter Aß accumulation in the mouse brain.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Inibidores da Protease de HIV/farmacologia , Doença de Alzheimer/patologia , Animais , Sulfato de Atazanavir , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imuno-Histoquímica , Lopinavir/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Camundongos Transgênicos , Nelfinavir/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligopeptídeos/farmacologia , Piridinas/farmacologia , Ritonavir/farmacologia , Saquinavir/farmacologiaRESUMO
The adult hippocampus plays a central role in memory formation, synaptic plasticity, and neurogenesis. The subgranular zone of the dentate gyrus contains neural progenitor cells with self-renewal and multilineage potency. Transgene expression of familial Alzheimer's disease-linked mutants of ß-amyloid precursor protein (APP) and presenilin-1 leads to a significant inhibition of neurogenesis, which is potentially linked to age-dependent memory loss. To investigate the effect of neurogenesis on cognitive function in a relevant disease model, FGF2 gene is delivered bilaterally to the hippocampi of APP+presenilin-1 bigenic mice via an adenoassociated virus serotype 2/1 hybrid (AAV2/1-FGF2). Animals injected with AAV2/1-FGF2 at a pre- or postsymptomatic stage show significantly improved spatial learning in the radial arm water maze test. A neuropathological investigation demonstrates that AAV2/1-FGF2 injection enhances the number of doublecortin, BrdU/NeuN, and c-fos-positive cells in the dentate gyrus, and the clearance of fibrillar amyloid-ß peptide (Aß) in the hippocampus. AAV2/1-FGF2 injection also enhances long-term potentiation in another APP mouse model (J20) compared with control AAV2/1-GFP-injected littermates. An in vitro study confirmed the enhanced neurogenesis of mouse neural stem cells by direct AAV2/1-FGF2 infection in an Aß oligomer-sensitive manner. Further, FGF2 enhances Aß phagocytosis in primary cultured microglia, and reduces Aß production from primary cultured neurons after AAV2/1-FGF2 infection. Thus, our data indicate that virus-mediated FGF2 gene delivery has potential as an alternative therapy of Alzheimer's disease and possibly other neurocognitive disorders.
Assuntos
Doença de Alzheimer/metabolismo , Transtornos Cognitivos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipocampo/metabolismo , Doenças Neurodegenerativas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células Cultivadas , Transtornos Cognitivos/genética , Transtornos Cognitivos/terapia , Giro Denteado/metabolismo , Giro Denteado/patologia , Dependovirus/genética , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Imuno-Histoquímica , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
The advent and wide introduction of antiretroviral therapy has greatly improved the survival and longevity of HIV-infected patients. Unfortunately, despite antiretroviral therapy treatment, these patients are still afflicted with many complications including cognitive dysfunction. There is a growing body of reports indicating accelerated deposition of amyloid plaques, which are composed of amyloid-ß peptide (Aß), in HIV-infected brains, though how HIV viral infection precipitates Aß accumulation is poorly understood. It is suggested that viral infection leads to increased production and impaired degradation of Aß. Mononuclear phagocytes (macrophages and microglia) that are productively infected by HIV in brains play a pivotal role in Aß degradation through the expression and execution of two endopeptidases, neprilysin (NEP) and insulin-degrading enzyme. In this study, we report that NEP has the dominant endopeptidase activity toward Aß in macrophages. Further, we demonstrate that monomeric Aß degradation by primary cultured macrophages and microglia was significantly impaired by HIV infection. This was accompanied with great reduction of NEP endopeptidase activity, which might be due to the diminished transport of NEP to the cell surface and intracellular accumulation at the endoplasmic reticulum and lysosomes. Therefore, these data suggest that malfunction of NEP in infected macrophages may contribute to acceleration of ß amyloidosis in HIV-inflicted brains, and modulation of macrophages may be a potential preventative target of Aß-related cognitive disorders in HIV-affected patients.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Transtornos Cognitivos/etiologia , Infecções por HIV/complicações , Neprilisina/metabolismo , Fagócitos/virologia , Células Cultivadas , Transtornos Cognitivos/virologia , Endopeptidases/metabolismo , HIV-1/fisiologia , Humanos , Insulisina/metabolismo , Fagócitos/enzimologiaRESUMO
Starting from bisphenolic bis-styrylbenzene DF-9 (4), ß-amyloid (Aß) binding affinity and specificity for phenolic bis-styrylbenzenes, monostyrylbenzenes, and alkyne controls were determined by fluorescence titration with ß-amyloid peptide Aß(1-40) and a fluorescence assay using APP/PS1 transgenic mouse brain sections. Bis-styrylbenzene SAR is derived largely from work on symmetrical compounds. This study is the first to describe Aß binding data for bis-styrylbenzenes unsymmetrical in the outer rings. With one exception, binding affinity and specificity were decreased by adding and/or changing the substitution pattern of phenol functional groups, changing the orientation about the central phenyl ring, replacing the alkene with alkyne bonds, or eliminating the central phenyl ring. The only compound with an Aß binding affinity and specificity comparable to 4 was its 3-hydroxy regioisomer 8. Like 4, 8 crossed the blood-brain barrier and bound to Aß plaques in vivo. By use of a DPPH assay, phenol functional groups with para orientations seem to be a necessary, but insufficient, criterion for good free radical scavenging properties in these compounds.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Sequestradores de Radicais Livres/síntese química , Fenóis/síntese química , Estilbenos/síntese química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/patologia , Desenho de Fármacos , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Técnicas In Vitro , Ligantes , Camundongos , Camundongos Transgênicos , Fenóis/metabolismo , Fenóis/farmacologia , Placa Amiloide/metabolismo , Ligação Proteica , Estereoisomerismo , Estilbenos/metabolismo , Estilbenos/farmacologiaRESUMO
Cytokines play an emerging role as neurotransmitters, neuromodulators, and neurohormones in the brain. This paradigm shift in cytokine function offers a new framework to understand their roles in ameliorating neurodegenerative disorders, such as Alzheimer's disease (AD). Molecular adjuvant therapy of AD animal models with glatiramer acetate induces anti-inflammatory responses and therapeutic effects. Although these effects are potentially mediated through anti-inflammatory cytokine signaling, the exact molecular identities and pathways are poorly understood. Here, we show that virus-mediated expression of the mouse interleukin (IL)-4 gene in beta-amyloid precursor protein + presenilin-1 (APP+PS1) bigenic mice attenuates AD pathogenesis. Introduction of an adeno-associated viral (AAV) vector encoding IL-4 into the hippocampus resulted in sustained expression of IL-4, reduced astro/microgliosis, amyloid-beta peptide (Abeta) oligomerization and deposition, and enhanced neurogenesis. Moreover, increased levels of IL-4 improved spatial learning, promoted phosphorylation of N-methyl-D-aspartate receptor subunit 2B at Tyr 1472, and enhanced its cell surface retention both in vivo and in vitro. Our data suggest that neuronal anti-inflammatory cytokine signaling may be a potential alternative target for non-Abeta-mediated treatment of AD.