RESUMO
BACKGROUND: A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-ß (Aß) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by ß-, and γ-secretases. There is a great interest in approaches to modulate Aß peptide production and develop therapeutic interventions to reduce Aß levels to halt or slow the progression of neurodegeneration. NEW METHOD: We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aß production, and (2) high-throughput screening assays of new pharmacotherapeutics. RESULTS: In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aß40 and Aß42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. COMPARISON WITH EXISTING METHOD(S): In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. CONCLUSIONS: Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aß production.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Biológicos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ceratolíticos/farmacologia , Neuroblastoma/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de Tempo , Tretinoína/farmacologiaRESUMO
OBJECTIVES/HYPOTHESIS: In contrast to normal epithelium, the desquamating stratified squamous epithelium of temporal bone cholesteatoma characteristically exhibits sustained hyperproliferative growth and a capacity for bone erosion. We conducted genome-wide microarray analyses to determine the molecular nature of cholesteatoma's biological processes and identify disease-associated, altered gene activity. We tested the hypothesis that genes contributing to the pathophysiology of cholesteatoma are differentially expressed compared to control tissue. STUDY DESIGN: Prospective experimental analysis. METHODS: Using new, enhanced microarray platforms and well-annotated human transcriptome probes, we measured global gene expression levels in surgical specimens of cholesteatoma and in the corresponding normal postauricular skin in four patients. Genes of interest were verified by quantitative real time reverse transcriptase polymerase chain reaction analyses using cholesteatoma and postauricular sample pairs (n = 13). External auditory canal skin from six additional patients was also evaluated as a normal control. Immunohistochemistry detected protein expression in tissue sections and the cells involved. RESULTS: DNA chip analyses identified 282 differentially expressed genes in cholesteatoma compared to control samples. Of these, 104 genes were upregulated and 178 were downregulated. Ontological classifications indicate relationships to cellular processes including receptor binding, cell communication and motion, vitamin metabolism, and cytokine-mediated inflammation. Based on potential involvement in disease pathology, 10 genes were selected and independently verified by quantitative polymerase chain reaction. Immunohistochemical detection of transcobalamin-1 and CCL27 implicates cholesteatoma keratinocytes and dermal endothelial cells as contributors in disease processes. CONCLUSIONS: We present a comprehensive, human genome-wide survey of disease-associated gene expression that extends the public database and provides new evidence for molecular mechanisms involved in cholesteatoma pathology. Laryngoscope, 123:S1-S21, 2013.
Assuntos
Quimiocina CCL27/genética , Colesteatoma da Orelha Média/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença/epidemiologia , Transcobalaminas/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Colesteatoma da Orelha Média/patologia , Colesteatoma da Orelha Média/cirurgia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Queratinócitos/patologia , Masculino , Processo Mastoide/cirurgia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Estudos Prospectivos , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Transgenic (Tg) mouse models of Alzheimer's disease (AD) have been extensively used to study the pathophysiology of this dementia and to test the efficacy of drugs to treat AD. The 5XFAD Tg mouse, which contains two presenilin-1 and three amyloid precursor protein (APP) mutations, was designed to rapidly recapitulate a portion of the pathologic alterations present in human AD. APP and its proteolytic peptides, as well as apolipoprotein E and endogenous mouse tau, were investigated in the 5XFAD mice at 3 months, 6 months, and 9 months. AD and nondemented subjects were used as a frame of reference. APP, amyloid-beta (Aß) peptides, APP C-terminal fragments (CT99, CT83, AICD), ß-site APP-cleaving enzyme, and APLP1 substantially increased with age in the brains of 5XFAD mice. Endogenous mouse tau did not show age-related differences. The rapid synthesis of Aß and its impact on neuronal loss and neuroinflammation make the 5XFAD mice a desirable paradigm to model AD.
RESUMO
Alzheimer's disease (AD) dementia impacts all facets of higher order cognitive function and is characterized by the presence of distinctive pathological lesions in the gray matter (GM). The profound alterations in GM structure and function have fostered the view that AD impacts are primarily a consequence of GM damage. However, the white matter (WM) represents about 50% of the cerebrum and this area of the brain is substantially atrophied and profoundly abnormal in both sporadic AD (SAD) and familial AD (FAD). We examined the WM biochemistry by ELISA and Western blot analyses of key proteins in 10 FAD cases harboring mutations in the presenilin genes PSEN1 and PSEN2 as well as in 4 non-demented control (NDC) individuals and 4 subjects with SAD. The molecules examined were direct substrates of PSEN1 such as Notch-1 and amyloid precursor protein (APP). In addition, apolipoproteins, axonal transport molecules, cytoskeletal and structural proteins, neurotrophic factors and synaptic proteins were examined. PSEN-FAD subjects had, on average, higher amounts of WM amyloid-beta (Aß) peptides compared to SAD, which may play a role in the devastating dysfunction of the brain. However, the PSEN-FAD mutations we examined did not produce uniform increases in the relative proportions of Aß42 and exhibited substantial variability in total Aß levels. These observations suggest that neurodegeneration and dementia do not depend solely on enhanced Aß42 levels. Our data revealed additional complexities in PSEN-FAD individuals. Some direct substrates of γ-secretase, such as Notch, N-cadherin, Erb-B4 and APP, deviated substantially from the NDC group baseline for some, but not all, mutation types. Proteins that were not direct γ-secretase substrates, but play key structural and functional roles in the WM, likewise exhibited varied concentrations in the distinct PSEN mutation backgrounds. Detailing the diverse biochemical pathology spectrum of PSEN mutations may offer valuable insights into dementia progression and the design of effective therapeutic interventions for both SAD and FAD.
RESUMO
OBJECTIVES/HYPOTHESIS: The purpose of this study was to evaluate human surgical specimens for cholesteatoma-associated changes in amphiregulin expression and determine potential relations to clinical disease variables. Amphiregulin, an epidermal growth factor receptor ligand, has functions in normal epithelial proliferation and aberrant neoplastic cell growth and is proinflammatory (e.g., rheumatoid arthritis, fibrosis) and active in hyperproliferative cutaneous conditions including psoriasis and wound healing. These known amphiregulin activities and the characteristic epithelial expansion and bone erosion of cholesteatoma pathophysiology prompted testing of the hypothesis that amphiregulin expression levels are altered in cholesteatoma and correlate to the disease state. STUDY DESIGN: Prospective experimental study, cross-sectional analysis. METHODS: Relative changes in amphiregulin gene expression were quantitated by real-time reverse-transcription polymerase chain reaction analyses of cholesteatoma epithelium compared to uninvolved control tissues from patients' postauricular and external auditory canal regions. Western immunoblot assays were performed for qualitative evaluation of amphiregulin protein expression. The t test and Fisher exact test were used for analysis. RESULTS: A statistically significant increase in amphiregulin gene expression was associated with cholesteatoma specimens compared to uninvolved postauricular skin (PAS) and external auditory canal (EAC) skin, P = .004 and P = .002, respectively. From comparisons of 60 sets of skin pairs, the mean ratio of amphiregulin RNA expression for cholesteatoma/PAS is 4.94 (standard error of the mean [SEM] = 1.53, n = 30) and for cholesteatoma/EAC is 7.70 (SEM = 1.57, n = 30). CONCLUSIONS: Amphiregulin is overexpressed in epithelial tissues of human cholesteatoma. Significant relationships were identified between increased amphiregulin expression levels and the extent of cholesteatoma migration and bone erosion. Our study results indicate amphiregulin is a potential biomarker of early cholesteatoma disease processes.
Assuntos
Colesteatoma/genética , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osso Temporal , Adolescente , Adulto , Idoso , Anfirregulina , Biomarcadores/metabolismo , Western Blotting , Criança , Colesteatoma/patologia , Colesteatoma/cirurgia , Colesteatoma da Orelha Média/genética , Colesteatoma da Orelha Média/patologia , Estudos de Coortes , Família de Proteínas EGF , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Adulto JovemRESUMO
Eosinophils are often dominant inflammatory cells present in the lungs of asthma patients. Nonetheless, the role of these leukocytes remains poorly understood. We have created a transgenic line of mice (PHIL) that are specifically devoid of eosinophils, but otherwise have a full complement of hematopoietically derived cells. Allergen challenge of PHIL mice demonstrated that eosinophils were required for pulmonary mucus accumulation and the airway hyperresponsiveness associated with asthma. The development of an eosinophil-less mouse now permits an unambiguous assessment of a number of human diseases that have been linked to this granulocyte, including allergic diseases, parasite infections, and tumorigenesis.