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
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
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
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
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
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
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
Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.
Assuntos
Organoides , Humanos , Animais , Organoides/efeitos dos fármacos , Organoides/metabolismo , Avaliação Pré-Clínica de Medicamentos , Indústria FarmacêuticaRESUMO
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
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.
Assuntos
Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Sistema Nervoso Central/metabolismo , Interleucina-4/biossíntese , Presenilina-1/genética , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Terapia Genética , Hipocampo/metabolismo , Interleucina-4/genética , Interleucina-4/farmacologia , Camundongos , Camundongos Mutantes , Neurônios/química , Neurônios/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
BACKGROUND: Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS: In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin's message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION: We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE: Abnormalities in the fortilin gene may be associated with early pregnancy loss.
Assuntos
Biomarcadores Tumorais/deficiência , Proteínas Morfogenéticas Ósseas/metabolismo , Perda do Embrião/metabolismo , Transdução de Sinais , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Cruzamentos Genéticos , Perda do Embrião/patologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fertilidade , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Saúde , Humanos , Camundongos , Camundongos Mutantes , Neurônios/citologia , Neurônios/metabolismo , Análise de Sobrevida , Proteína Tumoral 1 Controlada por Tradução , Xenopus/embriologia , beta-Galactosidase/metabolismoRESUMO
Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-beta peptides (Abeta) in brain. Although Abeta uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Abeta degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Abeta using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer (125)I-Abeta40 and Abeta42. Initial uptake of fibrillar Abeta40 and Abeta42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Abeta less than 0.5% and saturation of degradation within 24 h. IFN-gamma increased the intracellular retention of fibrillar Abeta40 and Abeta42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-beta1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Abeta degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-gamma and TNF-alpha directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Abeta degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Abeta degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Citocinas/fisiologia , Macrófagos/imunologia , Microglia/imunologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Acetilação , Peptídeos beta-Amiloides/ultraestrutura , Células Cultivadas , Técnicas de Cocultura , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Radioisótopos do Iodo/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/ultraestrutura , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Microglia/metabolismo , Microglia/ultraestrutura , Microscopia de Força Atômica , Fragmentos de Peptídeos/ultraestruturaRESUMO
Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimer's disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Amiloidose/terapia , Quimiocina CCL2/genética , Dependovirus/genética , Gliose/terapia , Aprendizagem em Labirinto/fisiologia , Presenilina-1/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Quimiocina CCL2/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Radial glia cells function as guide cells for neuronal migration and a source of neural progenitor cells, and play a crucial role for the development of the central nervous system. To date, several signals have been demonstrated to promote the formation of radial glia cells and Notch signaling is one such signal. However, the mechanism of the signaling hierarchy of radial glia developmental cascade promoted by Notch signaling still remains incomplete. Here we show that Notch signaling promotes Xenopus radial glia formation and that the Notch activation is sufficient for radial glia formation prior to neural tube closure. Moreover, we have identified Oct-1 (POU2f1), a POU transcription factor, as a downstream target of Notch signaling by microarray based screen. We demonstrate that the expression of Oct-1 in the brain is regulated by Notch signaling and that Oct-1 is sufficient and necessary for radial glia formation. Together, Oct-1 is a downstream effector of Notch signaling during radial glia formation.
Assuntos
Neuroglia/citologia , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Receptores Notch/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/metabolismo , Neuroglia/metabolismo , Fator 1 de Transcrição de Octâmero/antagonistas & inibidores , Oligodesoxirribonucleotídeos Antissenso/genética , Rombencéfalo/citologia , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Transdução de Sinais , Vimentina/genética , Vimentina/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Xenopus laevis/metabolismoRESUMO
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
beta-Amyloid (Abeta) binding affinities and specificities for six bis-styrylbenzenes with multiple magnetically equivalent fluorine atoms in the form of a tetrafluorophenyl core or symmetrical trifluoromethyl and trifluoromethoxy groups were determined by means of fluorescence titrations with amyloid peptide Abeta1-40 and a novel in vitro fluorescence-based assay using APP/PS1 transgenic mouse brain sections. Bis-styrylbenzenes with a tetrafluorophenyl core had increased Abeta binding affinities compared to their monofluorophenyl or phenyl counterparts. Bis-styrylbenzenes with carboxylic acid functional groups had lower Abeta binding affinities than their neutral counterparts. Selected bis-styrylbenzenes were demonstrated to have good blood-brain barrier penetration capabilities. These data extend the SAR of bis-styrylbenzene Abeta binding and provide direction for the development of a noninvasive probe for early detection of Alzheimer's disease using 19F MRI.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Estirenos/síntese química , Peptídeos beta-Amiloides/química , Animais , Barreira Hematoencefálica/metabolismo , Fluorescência , Fluorbenzenos/síntese química , Fluorbenzenos/farmacocinética , Fluorbenzenos/farmacologia , Ligantes , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Estirenos/farmacocinética , Estirenos/farmacologiaRESUMO
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.