ABSTRACT
Genistein is a kind of isoflavone compounds， also called phytoestrogens， with clinical effects on cardiovascular disease， cancer and postmenopausal-related gynecological diseases， and also has the potentiality in the prevention and treatment of Alzheimer's disease(AD). In this study， the protective effect of genistein on Aβ₂₅₋₃₅-induced PC12 cell injury and effect on CaM-CaMKIV signaling pathway were observed to investigate its mechanism for AD. PC12 cells were cultured and then the safe concentration of genistein and the modeling concentration and optimal time point of administration of Aβ₂₅₋₃₅ were screened by MTT assay. After being pretreated with different concentrations of genistein(25， 50， 100 μmol·L⁻¹) on PC12 cells， the AD model of PC12 cells was induced by Aβ₂₅₋₃₅. Then the survival rate of cells was detected by MTT assay; morphological change of cells was observed under the inverted microscope， and apoptosis of cells was assessed by AO/EB fluorescence staining; the neuroprotective effects of genistein on AD cell model were observed and the optimal concentration of genistein was determined. Expressions of mRNA and protein levels of CaM， CaMKK， CaMKIV and tau were detected by qRT-PCR and Western blot assay， respectively. The results showed that as compared with the blank group， the cell survival rate was decreased; the cell damage and apoptosis were increased; and the expressions of mRNA and protein levels of CaM， CaMKK， CaMKIV and tau were increased in AD model group. Genistein could significantly improve the cell survival rate， reduce the cell damage and apoptosis of AD cell model， and significantly down-regulate the expressions of mRNA and protein levels of CaM， CaMKK， CaMKIV and tau of AD cell model. These results indicated that genistein has obviously neuroprotective effect on the AD cell model induced by Aβ₂₅₋₃₅， and the mechanism may be related to the down-regulation of CaM-CaMKIV signaling pathway and Tau protein expression.
Subject(s)
Animals , Rats , Amyloid beta-Peptides , Apoptosis , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Metabolism , Calmodulin , Metabolism , Cell Survival , Genistein , Pharmacology , PC12 Cells , Peptide Fragments , Protective Agents , Pharmacology , Signal TransductionABSTRACT
Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca concentration with a simultaneous increase in the phosphorylation of Ca/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.
Subject(s)
Humans , Alzheimer Disease , Metabolism , Pathology , Calcium , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Metabolism , Cell Nucleus , Metabolism , Enzyme Activation , Physiology , HEK293 Cells , Neurons , Metabolism , Pathology , Phosphorylation , Signal Transduction , Physiology , tau Proteins , MetabolismABSTRACT
La expresión anormal de moléculas claves en señalización y la función defectuosa de los linfocitos T cumplen un papel significativo en la patogénesis de la enfermedad autoinmune. Las células T muestran numerosas anormalidades en la señalización del complejo TCRζ¹, estas aberraciones resultan en la alteración de la expresión de citoquinas. Mientras algunas de estas anormalidades explican el aumento de la actividad de células B por células T con incremento de los anticuerpos, la disminución en la producción de IL-2 resulta en un aumento en la susceptibilidad a las infecciones, reducción en la activación de las células T, inducción de la muerte celular y prolongada sobrevida de las células T autorreactivas².
The abnormal expression of key molecules in signaling and the malfunction of the T cell T have a significant activity in the pathogenesis of the autoimmune disease. The cells T exhibit numerous abnormalities in the signaling of the complex TCRζ¹, these aberrations result in the alteration of the citoquines. While some of these abnormalities explain the increase of the activity of cells B for cells T with increment of the antibodies production, the decrease in the production of IL-2 induces an increase in the susceptibility to the infections, diminishing in the activation of the cells T, and expansion of the lifespan of the autorreactive cells².