Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture

Alzheimer's disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy

In silico Experimentation of Glioma Microenvironment Development and Anti-tumor Therapy

Tumor cells do not develop in isolation, but co-evolve with stromal cells and tumor-associated immune cells in a tumor microenvironment mediated by an array of soluble factors, forming a complex intercellular signaling network. Herein, we report an unbiased, generic model to integrate prior biochemical data and the constructed brain tumor microenvironment in silico as characterized by an intercellular signaling network comprising 5 types of cells, 15 cytokines, and 69 signaling pathways. The results show that glioma develops through three distinct phases: pre-tumor, rapid expansion, and saturation. We designed a microglia depletion therapy and observed significant benefit for virtual patients treated at the early stages but strikingly no therapeutic efficacy at all when therapy was given at a slightly later stage. Cytokine combination therapy exhibits more focused and enhanced therapeutic response even when microglia depletion therapy already fails. It was further revealed that the optimal combination depends on the molecular profile of individual patients, suggesting the need for patient stratification and personalized treatment. These results, obtained solely by observing the in silico dynamics of the glioma microenvironment with no fitting to experimental/clinical data, reflect many characteristics of human glioma development and imply new venues for treating tumors via selective targeting of microenvironmental components

Aberrant expression of TopBP1 in breast cancer

Aims: The TopBP1 protein includes eight BRCT domains (originally identified in BRCA1) and has homology with BRCA1 over the carboxyl terminal half of the protein. The aim of this study was to determine whether TopBP1 is aberrantly expressed in breast cancer. Methods and results: Sixty-one breast carcinomas from an unselected consecutive patient cohort were studied along with 12 samples of breast tissue from cosmetic breast reduction surgery; these were analysed immunohistochemically for TopBP1 expression using a rabbit polyclonal antibody. This antibody was validated in immunoprecipitation and immunofluorescence experiments. Immunohistochemical analysis demonstrated that TopBP1 was expressed almost exclusively in the nuclei of the normal breast epithelium. However, in a significant number of breast carcinomas TopBP1 was aberrantly expressed, as it was detected in the cytoplasm and nucleus of some tumours and exclusively in the cytoplasm of others. In two out of 61 carcinomas investigated, no TopBP1 expression was detected. Conclusions: For the first time this report demonstrates aberrant expression of the TopBP1 protein in breast carcinoma. We propose TOPBP1 as a breast cancer susceptibility gene

A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2–Dpb11

Phosphorylation often regulates protein–protein interactions to control biological reactions. The Sld2 and Dpb11 proteins of budding yeast form a phosphorylation-dependent complex that is essential for chromosomal DNA replication. The Sld2 protein has a cluster of 11 cyclin-dependent kinase (CDK) phosphorylation motifs (Ser/Thr–Pro), six of which match the canonical sequences Ser/Thr–Pro–X–Lys/Arg, Lys/Arg–Ser/Thr–Pro and Ser/Thr–Pro–Lys/Arg. Simultaneous alanine substitution for serine or threonine in all the canonical CDK-phosphorylation motifs severely reduces complex formation between Sld2 and Dpb11, and inhibits DNA replication. Here we show that phosphorylation of these canonical motifs does not play a direct role in complex formation, but rather regulates phosphorylation of another residue, Thr84. This constitutes a non-canonical CDK-phosphorylation motif within a 28-amino-acid sequence that is responsible, after phosphorylation, for binding of Sld2–Dpb11. We further suggest that CDK-catalysed phosphorylation of sites other than Thr84 renders Thr84 accessible to CDK. Finally, we argue that this novel mechanism sets a threshold of CDK activity for formation of the essential Sld2 to Dpb11 complex and therefore prevents premature DNA replication

Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks

The Cdc45 protein is crucial for the initiation of chromosome replication in eukaryotic cells, as it allows the activation of prereplication complexes (pre-RCs) that contain the MCM helicase. This causes the unwinding of origins and the establishment of DNA replication forks. The incorporation of Cdc45 at nascent forks is a highly regulated and poorly understood process that requires, in budding yeast, the Sld3 protein and the GINS complex. Previous studies suggested that Sld3 is also important for the progression of DNA replication forks after the initiation step, as are Cdc45 and GINS. In contrast, we show here that Sld3 does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, Sld3 is no longer essential for the completion of chromosome replication. Unlike Sld3, GINS is not required for the initial recruitment of Cdc45 to origins and instead is necessary for stable engagement of Cdc45 with the nascent replisome. Like Cdc45, GINS then associates stably with MCM during S-phase