Structure-based classification of tauopathies

Ordered assembly of tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimer’s disease (1,2), Pick’s disease (3), chronic traumatic encephalopathy (CTE) (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a novel three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The AGD fold is also observed in aging-related tau astrogliopathy (ARTAG). Tau protofilament structures from inherited cases with mutations +3 or +16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimer’s disease and primary age-related tauopathy (PART). These findings suggest a hierarchical classification of tauopathies based on their filament folds, which complements clinical diagnosis and neuropathology, and allows identification of new entities, as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of GGT and PSP

Erk1/2 MAP kinases are required for epidermal G2/M progression

Erk1/2 mitogen-activated protein kinases (MAPKs) are often hyperactivated in human cancers, where they affect multiple processes, including proliferation. However, the effects of Erk1/2 loss in normal epithelial tissue, the setting of most extracellular signal-regulated kinase (Erk)–associated neoplasms, are unknown. In epidermis, loss of Erk1 or Erk2 individually has no effect, whereas simultaneous Erk1/2 depletion inhibits cell division, demonstrating that these MAPKs are necessary for normal tissue self-renewal. Growth inhibition caused by Erk1/2 loss is rescued by reintroducing Erk2, but not by activating Erk effectors that promote G1 cell cycle progression. Unlike fibroblasts, in which Erk1/2 loss decreases cyclin D1 expression and induces G1/S arrest, Erk1/2 loss in epithelial cells reduces cyclin B1 and c-Fos expression and induces G2/M arrest while disrupting a gene regulatory network centered on cyclin B1–Cdc2. Thus, the cell cycle stages at which Erk1/2 activity is required vary by cell type, with Erk1/2 functioning in epithelial cells to enable progression through G2/M

Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-rasG12D independent of the gender status. Furthermore, tamoxifen-inducible activation of K-rasG12D promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-rasG12D in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis

Uptake of alkaline earth metals in Alcyonarian spicules (Octocorallia)

Alcyonarian corals (Octocorallia) living in shallow tropical seas produce spicules of high-Mg calcite with ∼13 mol% MgCO3. We cultured the tropical alcyonarian coral Rhythisma fulvum in experiments varying temperature (19–32 °C) and pH (8.15–8.44). Alkalinity depletion caused by spicule formation systematically varied in the temperature experiments increasing from 19 to 29 °C. Spicules were investigated for their elemental ratios (Mg/Ca, Sr/Ca) using ICP-OES, δ44/40Ca using TIMS, as well as δ18O and δ13C by IRMS. Mg/Ca increased with temperature from 146 to 164 mmol/mol, in good agreement with the range observed for marine inorganic calcite. Mg/Ca increased by 1.0 ± 0.4 mmol/mol/°C, similar to the sensitivity of Miliolid foraminifera. The pH experiments revealed a linear relationship between Mg/Ca and carbonate ion concentration of +0.03 ± 0.02 mmol/mol/μMol. Sr/Ca ranges from 2.5 to 2.9 mmol/mol being in good agreement with other high-Mg calcites. Temperature and pH experiments showed linear dependencies of Sr/Ca matching inorganic calcite trends and pointing to a decoupling of crystal precipitation rate and calcification rate. Ca isotopes range between 0.7‰ and 0.9‰ in good agreement with aragonitic scleractinian corals and calcitic coccoliths. Presumably Ca isotopes are fractionated by a biological mechanism that may be independent of the skeletal mineralogy. We observe no temperature trend, but a significant decrease of δ44/40Ca with increasing pH. This inverse correlation may characterise biologically controlled intracellular calcification. Oxygen isotope ratios are higher than expected for isotopic equilibrium with a temperature sensitivity of −0.15 ± 0.03‰/°C. Carbon isotope ratios are significantly lower than expected for equilibrium and positively correlated with temperature with a slope of 0.20 ± 0.04‰/°C. Many of our observations on trace element incorporation in R. fulvum may be explained by inorganic processes during crystal formation, which do not comply with the intracellular mode of calcification in Alcyonarian corals. The observed elemental and isotopic compositions, however, could be explained if the partitioning caused by biological mechanisms mimics the effects of inorganic processes

Probing the roles of LRR RLK genes in Arabidopsis thaliana roots using a custom T-DNA insertion set

Leucine-rich repeat receptor-like protein kinases (LRR RLKs) represent the largest group of Arabidopsis RLKs with approximately 235 members. A minority of these LRR RLKs have been assigned to diverse roles in development, pathogen resistance and hormone perception. Using a reverse genetics approach, a collection of homozygous T-DNA insertion lines for 69 root expressed LRR RLK genes was screened for root developmental defects and altered response after exposure to environmental, hormonal/chemical and abiotic stress. The obtained data demonstrate that LRR RLKs play a role in a wide variety of signal transduction pathways related to hormone and abiotic stress responses. The described collection of T-DNA insertion mutants provides a valuable tool for future research into the function of LRR RLK genes

N-Methyl-D-Aspartate Receptor Link to the MAP Kinase Pathway in Cortical and Hippocampal Neurons and Microglia Is Dependent on Calcium Sensors and Is Blocked by α-Synuclein, Tau, and Phospho-Tau in Non-transgenic and Transgenic APPSw,Ind Mice

N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APPSw,Ind transgenic Alzheimer’s disease model. Interestingly, a very marked increase in NMDAR–NCS1 complexes was identified in neurons and a marked increase of both NMDAR–NCS1 and NMDAR–CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor–calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APPSw,Ind mouse embryos/pups

Functional Analysis of the Kinome of the Wheat Scab Fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study

The role of interactions between Prorocentrum minimum and Heterosigma akashiwo in bloom formation

We examined the growth and interactions between the bloom-forming flagellates Prorocentrum minimum and Heterosigma akashiwo using bi-algal culture experiments. When both species were inoculated at high cell densities, growth of H. akashiwo was inhibited by P. minimum. In other combinations of inoculation densities, the species first reaching the stationary phase substantially suppressed maximum cell densities of the other species, but the growth inhibition effect of P. minimum was stronger than that of H. akashiwo. We used a mathematical model to simulate growth and interactions of P. minimum and H. akashiwo in bi-algal cultures. The model indicated that P. minimum always out-competed H. akashiwo over time. Additional experiments showed that crude extracts from P. minimum and H. akashiwo cultures did not affect the growth of either species, but both strongly inhibited the growth of the bloom-forming diatom Skeletonema costatum. Further experiments showed that it was unlikely that reactive oxygen species produced by H. akashiwo were responsible for the inhibition of P. minimum growth