RESUMO
Spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease) is one of nine neurodegenerative disorders that are caused by expansion of polyglutamine-encoding CAG repeats. Intracellular accumulation of abnormal proteins in these diseases, a pathological hallmark, is associated with defects in protein homeostasis. Enhancement of the cellular proteostasis capacity with small molecules has therefore emerged as a promising approach to treatment. Here, we characterize a novel curcumin analog, ASC-JM17, as an activator of central pathways controlling protein folding, degradation and oxidative stress resistance. ASC-JM17 acts on Nrf1, Nrf2 and Hsf1 to increase the expression of proteasome subunits, antioxidant enzymes and molecular chaperones. We show that ASC-JM17 ameliorates toxicity of the mutant androgen receptor (AR) responsible for SBMA in cell, fly and mouse models. Knockdown of the Drosophila Nrf1 and Nrf2 ortholog cap 'n' collar isoform-C, but not Hsf1, blocks the protective effect of ASC-JM17 on mutant AR-induced eye degeneration in flies. Our observations indicate that activation of the Nrf1/Nrf2 pathway is a viable option for pharmacological intervention in SBMA and potentially other polyglutamine diseases.
Assuntos
Atrofia Bulboespinal Ligada ao X/genética , Curcumina/análogos & derivados , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Transtornos Musculares Atróficos/genética , Fator 1 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/genética , Receptores Androgênicos/genética , Fatores de Transcrição/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Atrofia Bulboespinal Ligada ao X/tratamento farmacológico , Atrofia Bulboespinal Ligada ao X/patologia , Curcumina/administração & dosagem , Curcumina/química , Modelos Animais de Doenças , Drosophila melanogaster/genética , Técnicas de Silenciamento de Genes , Fatores de Transcrição de Choque Térmico , Humanos , Camundongos , Transtornos Musculares Atróficos/tratamento farmacológico , Transtornos Musculares Atróficos/patologia , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Agregação Patológica de Proteínas/genética , Dobramento de Proteína/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/administração & dosagemRESUMO
The cellular microenvironment plays a critical role in shaping and directing the process of communication between the cells. Soluble signals are responsible for many cellular behaviors such as cell survival, proliferation and differentiation. Despite the importance of soluble signals, canonical methods are not well suited to the study of soluble factor interactions between multiple cell types. Macro-scale technology often puts cells into a convective environment that can wash away and dilute soluble signals from their targets, minimizing local concentrations of important factors. In addition, current methods such as transwells, require large numbers of cells and are limited to studying just two cell types. Here, we present data supporting the use of microchannels to study soluble factor signaling providing improved sensitivity as well as the ability to move beyond existing co-culture and conditioned medium paradigms. In addition, we present data suggesting that microculture can be used to unmask effects of population demographics. In this example the data support the hypothesis that a growth promoting subpopulation of cells exists in the mouse mammary gland.
Assuntos
Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Macrófagos/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Comunicação Parácrina , Neoplasias da Próstata/metabolismo , Linhagem Celular , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Macrófagos/citologia , Masculino , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%). Stem cell activity can be enriched by >200 fold (over 80% of activity), based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22%) and Lrp5-/- mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16(Ink4a) and TA-p63. CONCLUSIONS/SIGNIFICANCE: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function.