RESUMO
Alzheimer's disease (AD) is characterized by progressive decline of cognition and associated neuropsychiatric signs including weight loss, anxiety, depression, agitation, and aggression, which is particularly pronounced in the female gender. Previously, we have shown that a novel ionic co-crystal of lithium salicylate proline (LISPRO) is an improved lithium formulation compared to the carbonate or salicylate form of lithium in terms of safety and efficacy in reducing AD pathology in Alzheimer's mice. The current study is designed to compare the prophylactic effects of LISPRO, lithium carbonate (LC), and lithium salicylate (LS) on cognitive and noncognitive impairments in female transgenic APPswe/PS1dE9 AD mice. Female APPswe/PS1dE9 mice at 4 months of age were orally treated with low-dose LISPRO, LS, or LC for 9 months at 2.25 mmol lithium/kg/day followed by determination of body weight, growth of internal organs, and cognitive and noncognitive behavior. No significant differences in body or internal organ weight, anxiety or locomotor activity were found between lithium treated and untreated APPswe/PS1dE9 cohorts. LISPRO, LC, and LS prevented spatial cognitive decline, as determined by Morris water maze and depression as determined by tail suspension test. In addition, LISPRO treatment was superior in preventing associative memory decline determined by contextual fear conditioning and reducing irritability determined by touch escape test in comparison with LC and LS. In conclusion, low-dose LISPRO, LC, and LS treatment prevent spatial cognitive decline and depression-like behavior, while LISPRO prevented hippocampal-dependent associative memory decline and irritability in APPswe/PS1dE9 mice.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Comportamento/efeitos dos fármacos , Carbonato de Lítio/farmacologia , Compostos de Lítio/farmacologia , Memória/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Medo/efeitos dos fármacos , Feminino , Elevação dos Membros Posteriores , Locomoção/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Prolina , Salicilatos , Memória Espacial/efeitos dos fármacosRESUMO
Amyloid precursor protein (APP) proteolysis is required for production of amyloid-ß (Aß) peptides that comprise ß-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular ß-amyloid deposits as well as levels of various Aß species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, ß-carboxyl-terminal APP fragment and ß-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aß production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloidose/tratamento farmacológico , Encefalopatias/tratamento farmacológico , Cognição/efeitos dos fármacos , Azul de Metileno/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/patologia , Amiloidose/psicologia , Animais , Encefalopatias/patologia , Encefalopatias/psicologia , Células CHO , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , ProteóliseRESUMO
We recently found that sAPPα decreases amyloid-beta generation by directly associating with ß-site amyloid precursor protein (APP)-converting enzyme 1 (BACE1), thereby modulating APP processing. Because inhibition of BACE1 decreases glycogen synthase kinase 3 beta (GSK3ß)-mediated Alzheimer's disease (AD)-like tau phosphorylation in AD patient-derived neurons, we determined whether sAPPα also reduces GSK3ß-mediated tau phosphorylation. We initially found increased levels of inhibitory phosphorylation of GSK3ß (Ser9) in primary neurons from sAPPα over-expressing mice. Further, recombinant human sAPPα evoked the same phenomenon in SH-SY5Y cells. Further, in SH-SY5Y cells over-expressing BACE1, and HeLa cells over-expressing human tau, sAPPα reduced GSK3ß activity and tau phosphorylation. Importantly, the reductions in GSK3ß activity and tau phosphorylation elicited by sAPPα were prevented by BACE1 but not γ-secretase inhibition. In accord, AD mice over-expressing human sAPPα had less GSK3ß activity and tau phosphorylation compared with controls. These results implicate a direct relationship between APP ß-processing and GSK3ß-mediated tau phosphorylation and further define the central role of sAPPα in APP autoregulation and AD pathogenesis.
Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Quinase 3 da Glicogênio Sintase/fisiologia , Transdução de Sinais/fisiologia , Proteínas tau/antagonistas & inibidores , Proteínas tau/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
All reproductively competent adults have gone through puberty. While key genes and signaling pathways that lead to the onset of sexual maturation are known, the molecular mechanisms that determine when an individual enters puberty are only beginning to be understood. Both genetic and environmental factors determine the timing of puberty. New advances in understanding how environmentally sensitive, yet highly heritable developmental processes are regulated have come from the field of epigenetics. Of note, studies investigating the epigenetic control of the onset of puberty suggest that epigenetic repression of key inhibitory loci may play a fundamental role in the initiation of puberty. Current technologies that not only read out the DNA sequence, but also determine how the DNA is modified in response to the environment, promise new insight into how puberty is regulated, including the identification and understanding of gene regulatory networks that control the biological pathways affecting pubertal timing. Here we review the findings to date and discuss how epigenetic investigation can further our understanding of this fundamental aspect of human development.
Assuntos
Epigênese Genética/fisiologia , Epigenômica , Puberdade/fisiologia , Maturidade Sexual/fisiologia , Animais , HumanosRESUMO
To investigate the effect of the luxS gene on the expression of virulence factors in Shiga-like toxin producing and verotoxin-producing Escherichia coli, the luxS gene from E. coli 107/86 (wild type, O139:H1:F18ab, Stx2e) was deleted. The successful deletion of luxS was confirmed by bioluminescence assays. The luxS deletion mutant exhibited changed flagella-related phenotypes, like impaired expression of flagella, decreased flagella motility, reduced biofilm formation, and reduced ability to induce pro-immunity response in host cells, which were restored after complementation with the intact luxS gene. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence.
Assuntos
Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Flagelos/fisiologia , Percepção de Quorum/genética , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/genética , Animais , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Chlorocebus aethiops , Flagelos/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Medições Luminescentes , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed ß-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Transtornos do Comportamento Social/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/farmacologia , Análise de Variância , Animais , Transtorno Autístico/patologia , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Criança , Pré-Escolar , Receptor gp130 de Citocina/metabolismo , Embrião de Mamíferos , Comportamento Exploratório/fisiologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Interleucina-6/metabolismo , Relações Interpessoais , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Agitação Psicomotora/genética , Receptor Notch1/metabolismoRESUMO
Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ-aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABAA modulation for Alzheimer's disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild-type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of ß-amyloid (Aß) by increasing APP α-processing. These effects were blocked by the GABAA antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α-secretase processing, reduced Aß production, and reduced AD-like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aß production and improving cognitive performance, by activating GABAA receptors.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Antioxidantes/uso terapêutico , Flavanonas/uso terapêutico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/fisiologia , Animais , Bicuculina/farmacologia , Células CHO , Cricetulus , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Antagonistas de Receptores de GABA-A/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mutação/genética , TransfecçãoRESUMO
The role of SEF14 fimbriae in virulence remains to be elucidated and in this study, we showed that sefA mutant constructed in the wild-type (WT) Salmonella enteritidis strain 50336 displayed increased invasion to IPEC-J2 cell lines and survival in mouse peritoneal macrophages, and the lethal dose 50% (LD50) in 6-week-old Balb/c mice intra-peritoneally injected with WT S. enteritidis strain decreased significantly upon deletion of sefA indicating their role in virulence. Overall, these results demonstrated that expression of sefA of SEF14 fimbriae enhances S. enteritidis adhesion to epithelial cells and survival in macrophages and contributes to S. enteritidis virulence in mice.
Assuntos
Proteínas de Fímbrias/metabolismo , Salmonella enteritidis/patogenicidade , Fatores de Virulência/metabolismo , Animais , Células Cultivadas , Células Epiteliais/microbiologia , Dose Letal Mediana , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Salmonelose Animal , Suínos , VirulênciaRESUMO
Abnormalities in T-lymphocyte populations and function are observed in autism. Soluble amyloid precursor protein α (sAPP-α) is elevated in some patients with autism and is known to be produced by immune cells. In light of the well-established role of sAPP-α in proliferation, growth, and survival of neurons, we hypothesized an analogous role in the immune system. Thus, we explored whether sAPP-α could modulate immune development and function, especially aspects of the pinnacle cell of the adaptive arm of the immune system: the T cell. To do this, we generated mice overexpressing human sAPP-α and characterized elements of T-cell development, signal transduction, cytokine production, and innate/adaptive immune functions. Here, we report that transgenic sAPP-α-overexpressing (TgsAPP-α) mice displayed increased proportions of CD8(+) T cells, while effector memory T cells were decreased in the thymus. Overall apoptotic signal transduction was decreased in the thymus, an effect that correlated with dramatic elevations in Notch1 activation; while active-caspase-3/total-caspase-3 and Bax/Bcl-2 ratios were decreased. Greater levels of IFN-γ, IL-2, and IL-4 were observed after ex vivo challenge of TgsAPP-α mouse splenocytes with T-cell mitogen. Finally, after immunization, splenocytes from TgsAPP-α mice displayed decreased levels IFN-γ, IL-2, and IL-4, as well as suppressed ZAP70 activation, after recall antigen stimulation. Given elevated levels of circulating sAPP-α in some patients with autism, sAPP-α could potentially drive aspects of immune dysfunction observed in these patients, including dysregulated T-cell apoptosis, aberrant PI3K/AKT signaling, cytokine alterations, and impaired T-cell recall stimulation.
Assuntos
Precursor de Proteína beta-Amiloide/imunologia , Transtorno Autístico/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Precursor de Proteína beta-Amiloide/sangue , Precursor de Proteína beta-Amiloide/genética , Animais , Apoptose/imunologia , Transtorno Autístico/sangue , Western Blotting , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Imunidade/imunologia , Memória Imunológica/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor Notch1/imunologia , Receptor Notch1/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismoRESUMO
Converging lines of evidence indicate dysregulation of the key immunoregulatory molecule CD45 (also known as leukocyte common antigen) in Alzheimer's disease (AD). We report that transgenic mice overproducing amyloid-ß peptide (Aß) but deficient in CD45 (PSAPP/CD45(-/-) mice) faithfully recapitulate AD neuropathology. Specifically, we find increased abundance of cerebral intracellular and extracellular soluble oligomeric and insoluble Aß, decreased plasma soluble Aß, increased abundance of microglial neurotoxic cytokines tumor necrosis factor-α and interleukin-1ß, and neuronal loss in PSAPP/CD45(-/-) mice compared with CD45-sufficient PSAPP littermates (bearing mutant human amyloid precursor protein and mutant human presenilin-1 transgenes). After CD45 ablation, in vitro and in vivo studies demonstrate an anti-Aß phagocytic but proinflammatory microglial phenotype. This form of microglial activation occurs with elevated Aß oligomers and neural injury and loss as determined by decreased ratio of anti-apoptotic Bcl-xL to proapoptotic Bax, increased activated caspase-3, mitochondrial dysfunction, and loss of cortical neurons in PSAPP/CD45(-/-) mice. These data show that deficiency in CD45 activity leads to brain accumulation of neurotoxic Aß oligomers and validate CD45-mediated microglial clearance of oligomeric Aß as a novel AD therapeutic target.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Antígenos Comuns de Leucócito/genética , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Microglia/imunologia , Microglia/patologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Presenilina-1/genética , Multimerização Proteica , Transgenes , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Several prior investigations of Alzheimer's disease (AD) patients have indicated naturally occurring autoantibodies against amyloid-ß (Aß) species are produced. Although many studies have focused on the relative concentrations or binding affinities of autoantibodies against Aß-related proteins in AD and aging, data regarding their functional properties are limited. It is generally believed that these antibodies act to aid in clearance of Aß. However, as antibodies which bind to Aß also typically bind to the parent amyloid precursor protein (APP), we reasoned that certain Aß-targeting autoantibodies may bind to APP thereby altering its conformation and processing. Here we show for the first time, that naturally occurring Aß-reactive autoantibodies isolated from AD patients, but not from healthy controls, promote ß-secretase activity in cultured cells. Furthermore, using monoclonal antibodies to various regions of Aß, we found that antibodies generated against the N-terminal region, especially Aß(1-17) , dose dependently promoted amyloidogenic processing of APP viaß-secretase activation. Thus, this property of certain autoantibodies in driving Aß generation could be of etiological importance in the development of sporadic forms of AD. Furthermore, future passive or active anti-Aß immunotherapies must consider potential off-target effects resulting from antibodies targeting the N-terminus of Aß, as co-binding to the corresponding region of APP may actually enhance Aß generation.
Assuntos
Doença de Alzheimer/imunologia , Secretases da Proteína Precursora do Amiloide/efeitos dos fármacos , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/metabolismo , Autoanticorpos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Linhagem Celular , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Injeções Intraventriculares , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To clone and express fan operon gene clusters of K99 fimbriae in enterotoxigenic Escherichia coli (ETEC) in vitro, and study the activity of the recombinant E. coli expressing K99 fimbriae. METHODS: K99 fimbriae gene clusters were amplified by long-PCR method, using the genomic DNA of K99-fimbriae E. coli C8307 as the DNA template. The 5.7Kb PCR products were inserted into expressing vector pBR322 with restriction endonuclease, then positive clones were screened. The positive recombinant plasmid was transformed into non-fimbriae E. coli SE5000 strains, and pBR322 plasmid was also transformed into SE5000 for negative control strain. RESULTS: The recombination E. coli expressing K99 fimbriae was tested with agglutination assay, using monoclonal antibody serum and brush border vesicles from the piglet small intestinal epithelia cells. The expressed fimbriae on the surface of the recombinant E. coli SE5000 were observed by transmissible electromicroscope. Heat extraction method was employed to isolate and purify K99 fimbriae, which was exerted SDS-PAGE, and 18.5 kDa protein band was detected. The mouse sera produced from recombinant fimbriae was used to test K99-fimbriae strains C83907, C83914, C83260 with positive agglutination results, while negative results were found with E. coli contain other kinds of fimbriae. The assays of SDS-PAGE, Western blot, agglutination assay were used to evaluate antigenicity and biologic activity between C83907 and recombinant strain. Adhesion test with HeLa cell line demonstrated the recombinant strain and wild type have the similar adherence ability, and this adhesion can be inhibited with mouse serum containing polyclonal antibody against recombinant K99 fimbriae. CONCLUSION: This study has laid a good foundation for further study on bioactivity of K99.
Assuntos
Clonagem Molecular , Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Óperon , Animais , Anticorpos Antibacterianos/imunologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Fímbrias Bacterianas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos ICR , SuínosRESUMO
Mutations in the presenilin-1 (PS1) gene are independent causes of familial Alzheimer's disease (AD). AD patients have dysregulated immunity, and PS1 mutant mice exhibit abnormal systemic immune responses. To test whether immune function abnormality caused by a mutant human PS1 gene (mhPS1) could modify AD-like pathology, we reconstituted immune systems of AD model mice carrying a mutant human amyloid precursor protein gene (mhAPP; Tg2576 mice) or both mhAPP and mhPS1 genes (PSAPP mice) with allogeneic bone marrow cells. Here, we report a marked reduction in amyloid-ß (Aß) levels, ß-amyloid plaques and brain inflammatory responses in PSAPP mice following strain-matched wild-type PS1 bone marrow reconstitution. These effects occurred with immune switching from pro-inflammatory T helper (Th) 1 to anti-inflammatory Th2 immune responses in the periphery and in the brain, which likely instructed microglia to phagocytose and clear Aß in an ex vivo assay. Conversely, Tg2576 mice displayed accelerated AD-like pathology when reconstituted with mhPS1 bone marrow. These data show that haematopoietic cells bearing the mhPS1 transgene exacerbate AD-like pathology, suggesting a novel therapeutic strategy for AD based on targeting PS1 in peripheral immune cells.
Assuntos
Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/imunologia , Presenilina-1/genética , Presenilina-1/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Doença de Alzheimer/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Antígeno CD11b/biossíntese , Citocinas/biossíntese , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/metabolismo , Mutação , Placa AmiloideRESUMO
Patients with congenital lymphedema suffer from tissue swelling in part due to mutations in genes regulating lymphatic valve development. Lymphatic valve leaflets grow and are maintained throughout life in response to oscillatory shear stress (OSS), which regulates gene transcription in lymphatic endothelial cells (LECs). Here, we identified the first transcription factor, Foxo1, that repressed lymphatic valve formation by inhibiting the expression of valve-forming genes. We showed that both embryonic and postnatal ablation of Foxo1 in LECs induced additional valve formation in postnatal and adult mice in multiple tissues. Our quantitative analyses revealed that after deletion, the total number of valves in the mesentery was significantly (P < 0.01) increased in the Foxo1LEC-KO mice compared with Foxo1fl/fl controls. In addition, our quantitative real-time PCR (RT-PCR) data from cultured LECs showed that many valve-forming genes were significantly (P < 0.01) upregulated upon knockdown of FOXO1. To confirm our findings in vivo, rescue experiments showed that Foxc2+/- mice, a model of lymphedema-distichiasis, had 50% fewer lymphatic valves and that the remaining valves exhibited backleak. Both valve number and function were completely restored to control levels upon Foxo1 deletion. These findings established FOXO1 as a clinically relevant target to stimulate de novo lymphatic valve formation and rescue defective valves in congenital lymphedema.
Assuntos
Proteína Forkhead Box O1/deficiência , Deleção de Genes , Linfangiogênese , Vasos Linfáticos/metabolismo , Animais , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Glycogen synthase kinase 3 (GSK-3) dysregulation is implicated in the two Alzheimer's disease (AD) pathological hallmarks: beta-amyloid plaques and neurofibrillary tangles. GSK-3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic gamma-secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid-beta (Abeta) peptide generation in both human 'Swedish' mutant APP transgene-bearing neuron-like cells and primary neurons. We also find that luteolin induces changes consistent with GSK-3 inhibition that (i) decrease amyloidogenic gamma-secretase APP processing, and (ii) promote presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Importantly, we find GSK-3alpha activity is essential for both PS1 CTF phosphorylation and PS1-APP interaction. As validation of these findings in vivo, we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Abeta levels, reduces GSK-3 activity, and disrupts PS1-APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Abeta pathology. We suggest that GSK-3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation-dependent regulation of amyloidogenesis.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/biossíntese , Luteolina/farmacologia , Presenilina-1/metabolismo , Administração Oral , Doença de Alzheimer/enzimologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Linhagem Celular , Diosmina/administração & dosagem , Diosmina/química , Diosmina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Luteolina/química , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Apolipoprotein E (apoE) and apoE-mimetic peptides exert prominent anti-inflammatory effects. We determined the anti-inflammatory effects of novel apoE receptor mimetics, composed of the LDL receptor-binding domain of apoE (aa 133-152, ApoEp) or ApoEp with 6 lysines (6KApoEp) or 6 aspartates added at the N-terminus (6DApoEp). BV2 microglia and human THP-1 monocytes were treated with lipopolysaccharide (LPS) in the absence or presence of ApoEp, 6KApoEp or 6DApoEp, followed by determination of pro-inflammatory tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) release by ELISA. As signaling intermediates of inflammation, Signal Transducer and Activator of Transcription 3 (STAT3), Janus-Activated Kinase2 (JAK2) and p38 and p44/42 MAPK phosphorylation levels were determined by Western blot analysis. In addition, we isolated splenocytes from female htau mice treated with 6KApoEp or 6K for 28 weeks, followed by determination of concanavalinA (conA)-mediated interferon gamma (IFNγ) release. 6KApoEp starting at 2.5 µM significantly reduced LPS-mediated TNFα and IL-6 secretion in BV2 and THP-1 cells in a dose-dependent manner. In BV2 cells, 6KApoEp reduced TNFα secretion more effectively than 6DApoEp and ApoEp, which was blocked by PCSK9 treatment, suggesting a role for LDL receptors. 6KApoEp also inhibited LPS-induced p44/42 MAPK, JAK2 and STAT3 phosphorylation, while enhancing p38 MAPK phosphorylation. In addition, conA induced significantly less IFNγ release in splenocytes derived from htau mice treated with 6KApoEp compared with those treated with 6K. Thus, 6KApoEp most effectively reduces LPS-mediated neuroinflammation by interacting with LDL receptors, thus representing a novel anti-inflammatory agent for treatment of neurodegenerative disease.
RESUMO
BACKGROUND: The É4 isoform of apolipoprotein E (apoE4) is a major genetic risk factor for the development of sporadic Alzheimer's disease (AD), and its modification has been an intense focus for treatment of AD during recent years. METHODS: We investigated the binding of apoE, a peptide corresponding to its low-density lipoprotein receptor binding domain (amino acids 133-152; ApoEp), and modified ApoEp to amyloid precursor protein (APP) and their effects on amyloid-ß (Aß) production in cultured cells. Having discovered a peptide (6KApoEp) that blocks the interaction of apoE with N-terminal APP, we investigated the effects of this peptide and ApoEp on AD-like pathology and behavioral impairment in 3XTg-AD and 5XFAD transgenic mice. RESULTS: ApoE and ApoEp, but not truncated apoE lacking the low-density lipoprotein receptor binding domain, physically interacted with N-terminal APP and thereby mediated Aß production. Interestingly, the addition of 6 lysine residues to the N-terminus of ApoEp (6KApoEp) directly inhibited apoE binding to N-terminal APP and markedly limited apoE- and ApoEp-mediated Aß generation, presumably through decreasing APP cellular membrane trafficking and p44/42 mitogen-activated protein kinase phosphorylation. Moreover, while promoting apoE interaction with APP by ApoEp exacerbated Aß and tau brain pathologies in 3XTg-AD mice, disrupting this interaction by 6KApoEp ameliorated cerebral Aß and tau pathologies, neuronal apoptosis, synaptic loss, and hippocampal-dependent learning and memory impairment in 5XFAD mice without altering cholesterol, low-density lipoprotein receptor, and apoE expression levels. CONCLUSIONS: These data suggest that disrupting apoE interaction with N-terminal APP may be a novel disease-modifying therapeutic strategy for AD.
Assuntos
Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E/antagonistas & inibidores , Apolipoproteínas E/metabolismo , Encéfalo/patologia , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Apolipoproteínas E/genética , Células CHO , Cognição/efeitos dos fármacos , Cricetulus , Modelos Animais de Doenças , Feminino , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos TransgênicosRESUMO
Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimer's disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Vasos Sanguíneos/patologia , Terapia Baseada em Transplante de Células e Tecidos , Sangue Fetal/citologia , Sangue Fetal/transplante , Peptídeos beta-Amiloides/sangue , Animais , Astrócitos/patologia , Encéfalo/patologia , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Contagem de Células , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Microglia/patologia , Peptídeos/imunologia , Fagocitose , Placa Amiloide/patologia , Células Th2/imunologiaRESUMO
We previously reported that intraperitoneal (i.p.) injection (20 mg/kg) of (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, decreased beta-amyloid (Abeta) levels and plaques via promotion of the non-amyloidogenic alpha-secretase proteolytic pathway in "Swedish" mutant amyloid precursor protein overexpressing (APPsw, Tg) mice. Here, we find that EGCG administered orally in drinking water (50 mg/kg) similarly reduces Abeta deposition in these mice. Following a six month treatment of an 8 month old cohort, immunohistochemical analysis of coronal sections reveals that plaque burdens were reduced in the cingulate cortex, hippocampus, and entorhinal cortex by 54%, 43%, and 51%, respectively. Congo red plaque burdens were decreased in the cingulate cortex, hippocampus, and entorhinal cortex by 53%, 53%, and 58%, respectively as well. ELISA of brain homogenates of the treatment Tg mice revealed consistent reductions in both Abeta1-40 and 1-42 soluble and insoluble forms. In the present study we also investigated the effect EGCG administration had on tau pathology and cognition in Tg mice. Both i.p. and orally-treated Tg animals were found to have modulated tau profiles, with markedly suppressed sarkosyl-soluble phosphorylated tau isoforms. Radial arm water maze (RAWM) testing for working memory indicated that EGCG provided cognitive benefit to Tg mice with both i.p. and oral administration, although i.p.-treated animals showed a more pronounced benefit because of the greater impairment of their Tg controls at the time of testing. Taken together, these data further the notion of EGCG dietary supplementation as a potentially safe and effective prophylaxis for Alzheimer's disease.
Assuntos
Doença de Alzheimer/complicações , Peptídeos beta-Amiloides/metabolismo , Catequina/análogos & derivados , Transtornos Cognitivos , Fármacos Neuroprotetores/uso terapêutico , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Catequina/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Fosforilação/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
Alzheimer's Disease (AD) is the leading cause of dementia in the elderly. In healthy individuals, amyloid precursor protein (APP) is cleaved by α-secretase, generating soluble α-amyloid precursor protein (sAPPα), which contributes neuroprotective functions in the neuronal environment. In contrast, in the neurodegenerative environment of AD patients, amyloid-ß-peptide (Aß) of either 40 or 42 residues are generated by increased activity of ß- and γ-secretase. These proteins amalgamate in specific regions of the brain, which disrupts neuronal functions and leads to cognitive impairment. Human umbilical cord blood cells (HUCBC) have proven useful as potential immunomodulatory therapies in various models of neurodegenerative diseases, including AD. Our most recent work studied the impact of umbilical cord blood serum (CBS) on modulation of sAPPα production. Heat-sensitive CBS significantly promoted sAPPα production, indicating that heat-sensitive factor(s) play(s) a role in this process. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to determine the molecular source of α-secretase in purified CBS and aged blood serum (AgBS) fraction. Of the proteins identified, the subunits of C1 complex (C1q, C1r, and C1s) and alpha-2-macroglobulin showed significantly greater levels in purified α-CBS fraction (α-CBSF) compared with the AgBS fraction (AgBSF). Specifically, C1 markedly increased sAPPα and alpha-carboxyl-terminal fragment (α-CTF) production in a dose-dependent fashion, whereas C1q alone only minimally increased and C3 did not increase sAPPα production in the absence of sera. Furthermore, C1q markedly increased sAPPα and α-CTF, while decreasing Aß, in CHO/APPwt cells cultured in the presence of whole sera. These results confirm our initial assumption that APP α-secretase activity in human blood serum is mediated by complement C1, opening a potential therapeutic modality for the future of AD.