A multi-omics dataset for the analysis of frontotemporal dementia genetic subtypes.

Understanding the molecular mechanisms underlying frontotemporal dementia (FTD) is essential for the development of successful therapies. Systematic studies on human post-mortem brain tissue of patients with genetic subtypes of FTD are currently lacking. The Risk and Modyfing Factors of Frontotemporal Dementia (RiMod-FTD) consortium therefore has generated a multi-omics dataset for genetic subtypes of FTD to identify common and distinct molecular mechanisms disturbed in disease. Here, we present multi-omics datasets generated from the frontal lobe of post-mortem human brain tissue from patients with mutations in MAPT, GRN and C9orf72 and healthy controls. This data resource consists of four datasets generated with different technologies to capture the transcriptome by RNA-seq, small RNA-seq, CAGE-seq, and methylation profiling. We show concrete examples on how to use the resulting data and confirm current knowledge about FTD and identify new processes for further investigation. This extensive multi-omics dataset holds great value to reveal new research avenues for this devastating disease.

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6‒8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

The antiangiogenic compound aeroplysinin-1 induces apoptosis in endothelial cells by activating the mitochondrial pathway.

Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo. In this work, we provide evidence of a selective induction of apoptosis by aeroplysinin-1 in endothelial cells. Studies on the nuclear morphology of treated cells revealed that aeroplysinin-1 induces chromatin condensation and nuclear fragmentation, and it increases the percentage of cells with sub-diploid DNA content in endothelial, but not in HCT-116, human colon carcinoma and HT-1080 human fibrosarcoma cells. Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism. Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound. The observation that aeroplysinin-1 prevents the phosphorylation of Bad relates to the mitochondria-mediated induction of apoptosis by this compound.

Massive parallel DNA pyrosequencing analysis of the tumor suppressor BRG1/SMARCA4 in lung primary tumors.

The tumor suppressor gene, SMARCA4 (or BRG1), which encodes the ATPase component of the chromatin remodeling complex SWI/SNF, is commonly inactivated by mutations and deletions in lung cancer cell lines. However, SMARCA4 alterations appear to be rare in lung primary tumors. Ultra-deep sequencing technologies provide a promising alternative to achieve a sensitivity superior to that of current sequencing strategies. Here we used ultra-deep pyrosequencing to screen for mutations over the entire SMARCA4 coding region in 12 lung tumors without detectable BRG1 protein. While automatic-fluorescence-based sequencing detected one somatic mutation (p.K586X), the pyrosequencing revealed additional variants, thus increasing the sensitivity. One of the variants, which affected a consensus splice site, was confirmed by individual cloning of PCR products, ruling out the possibility of PCR or pyrosequencing artifacts. This mutation, confirmed to be somatic, was present at a frequency of ten percent, suggesting normal cell contamination in the tumor. Our analysis also allowed us to determine the sensitivity and to identify some limitations of the technology. In conclusion, in addition to cell lines, SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, thereby constituting one of the most important genes contributing to the development of this type of cancer.

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

BRG1 and LKB1: tales of two tumor suppressor genes on chromosome 19p and lung cancer.

Losses of heterozygosity (LOH) of the short arm of chromosome 19 are frequent in lung cancer, suggesting that one or more tumor suppressor genes are present in this region. The LKB1 gene, also called STK11, is somatically inactivated through point mutations and large deletions in lung tumors, demonstrating that LKB1 is a target of the LOH of this chromosomal arm. Data from several independent groups have provided information about the profiles of lung tumors with LKB1 inactivation and it is generally agreed that this alteration strongly predominates in non-small cell lung cancer, in particular adenocarcinomas, in smokers. The LKB1 protein has serine-threonine kinase activity and is involved in the regulation of the cell energetic checkpoint through the phosphorylation and activation of adenosine monophosphate-dependent kinase (AMPK). LKB1 is also involved in other processes such as cell polarization, probably through substrates other than AMPK. Interestingly, another gene on chromosome 19p, BRG1, encoding a component of the SWI/SNF chromatin-remodeling complex, has emerged as a tumor suppressor gene that is altered in lung tumors. Similar to LKB1, BRG1 is somatically inactivated by point mutations or large deletions in lung tumors featuring LOH of chromosome 19p. These observations suggest an important role for BRG1 in lung cancer and highlight the need to further our understanding of the function of Brahma/SWI2-related gene 1 (BRG1) in cancer. Finally, simultaneous mutations at LKB1 and BRG1 are common in lung cancer cells, which exemplifies how a single event, LOH of chromosome 19p in this instance, targets two different tumor suppressors.

IB05204, a dichloropyridodithienotriazine, inhibits angiogenesis in vitro and in vivo.

In the course of a blind screening program for inhibitors of angiogenesis, IB05204 (4,8-dichloro-12-phenylpyrido[5',6':4'',5'';3',2':4,5]dithieno[3'',2''-d':3,2-d]-1,2,3-ditriazine) was selected for its ability to inhibit endothelial tubule-like network formation on Matrigel. IB05204 inhibits the in vivo angiogenesis in the chorioallantoic membrane (CAM) and the mouse Matrigel plug assays. Antiangiogenic activity seems to be highly dependent on the chloro substituents because their removal results in a complete loss of the in vitro inhibitory activity of endothelial differentiation and in vivo antiangiogenic activity in CAM assay. Although IB05204 inhibits the growth of endothelial and tumor cells in culture, its antiangiogenic activity seems to be mainly dependent on the prevention of endothelial capillary-like tube formation and inhibition of endothelial migration because these effects are recorded at lower concentrations. IB05204 treatment inhibits matrix metalloproteinase-2 (MMP-2) production in endothelial and tumor cells, down-regulates endothelial cyclooxygenase-2 expression, and represses phosphorylation of endothelial Akt in response to serum stimulation, suggesting that IB05204 interferes with molecular mechanisms of cell migration and survival. IB05204 induces apoptosis in endothelial cells through cytochrome c release and caspase activation. Data here shown altogether indicate that IB05204 is a compound that interferes with several key steps of angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.

Role of alterations in the apoptotic machinery in sensitivity of cancer cells to treatment.

Apoptosis is a genetically controlled and evolutionarily conserved form of cell death of critical importance for normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. The malfunction of the death machinery may play a primary or secondary role in various diseases, with essentially too little or too much apoptosis leading to proliferative or degenerative diseases, respectively. The machinery responsible for killing and degradation of the cell via apoptosis is expressed constitutively and become activated through various stimuli. Apoptotic mechanisms are operating during spontaneous regression of tumors as well as in response to treatment with anti-neoplastic drugs. The therapeutic goal in cancer treatment is to trigger tumor-selective cell death. However, resistance to treatment is a concern for many types of cancer. Since many anti-neoplastic agents induce an apoptotic type of death in susceptible cells, it is likely that defects or dysregulation of different steps of the apoptotic pathways might be an important determinant of resistance to anticancer drugs. These defects might appear at the initiation and/or execution stages of apoptosis and result in the insufficient elimination of tumor cells, which might lead either to acquired resistance to treatment, or to uncontrolled migration of cancer cells and metastasis. Hence, identification and targeting of the disabled pathway, which is most efficiently inactivated in a particular type of tumor might be the most successful approach in the future. Here we review current knowledge concerning function of apoptotic machinery in cancer cells, and how this information can be used to increase the efficiency of tumor treatment.

Inhibition of Mammalian thioredoxin reductase by some flavonoids: implications for myricetin and quercetin anticancer activity.

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, exerts a wide range of activities in cellular redox control, antioxidant function, cell viability, and proliferation. Recently, the selenocysteine (Sec)-containing mammalian TrxR has emerged as a new target for anticancer drug development because TrxR and Trx are overexpressed in many aggressive tumors and the tumor cells seem to be more dependent on Trx system than normal cells. Here we have investigated the inhibition of mammalian TrxR by flavonoids which have been presumed to be cancer chemoprevention agents because of their antioxidant activities. Myricetin and quercetin were found to have strong inhibitory effects on mammalian TrxRs with IC50 values of 0.62 and 0.97 micromol/L, respectively. The inhibition was shown to be concentration, NADPH, and time dependent and involved an attack on the reduced COOH-terminal -Cys-Sec-Gly active site of TrxR. Oxygen-derived superoxide anions enhanced the inhibitory effect whereas anaerobic conditions attenuated inhibition. Spectral analysis suggested that the flavonols might perform their inhibitory effects via semiquinone radicals. Additionally, the flavonols had the potential to inhibit the growth of A549 cells with the same potency as inhibition of TrxR. TrxR activity in the cell lysates was reduced on treatment with myricetin >50 micromol/L, which coincided with the oxidization of Trx. The cell cycle was arrested in S phase by quercetin and an accumulation of cells in sub-G1 was observed in response to myricetin. Thus, the anticancer activity of quercetin and myricetin may be due to inhibition of TrxR, consequently inducing cell death.

Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis.

Programmed cell death (PCD) is indispensable for eukaryotic development. In animals, PCD is executed by the caspase family of cysteine proteases. Plants do not have close homologues of caspases but possess a phylogenetically distant family of cysteine proteases named metacaspases. The cellular function of metacaspases in PCD is unknown. Here we show that during plant embryogenesis, metacaspase mcII-Pa translocates from the cytoplasm to nuclei in terminally differentiated cells that are destined for elimination, where it colocalizes with the nuclear pore complex and chromatin, causing nuclear envelope disassembly and DNA fragmentation. The cell-death function of mcII-Pa relies on its cysteine-dependent arginine-specific proteolytic activity. Accordingly, mutation of catalytic cysteine abrogates the proteolytic activity of mcII-Pa and blocks nuclear degradation. These results establish metacaspase as an executioner of PCD during embryo patterning and provide a functional link between PCD and embryogenesis in plants. Although mcII-Pa and metazoan caspases have different substrate specificity, they serve a common function during development, demonstrating the evolutionary parallelism of PCD pathways in plants and animals.

Homocysteine is a potent inhibitor of human tumor cell gelatinases.

Extracellular matrix-degrading gelatinases are mainly involved in tumor invasion and metastasis. Previous experimental data from our group and others suggested that homocysteine could have a potential modulatory role on the proteolytic balance at the extracellular matrix. Therefore, we studied the effects of homocysteine on extracellular matrix-degrading proteases using model human tumor cell lines and a combination of in vitro fluorogenic assay and zymographic techniques. Homocysteine is shown to be the thiol compound with the most potent inhibitory activity on matrix metalloproteinase 9. Zymographies reveal that matrix metalloproteinase 2 is, at least, as sensitive to inhibition by homocysteine as matrix metalloproteinase 9 is. This study opens new ways to the potential pharmacological use of thiol compounds.

Anti-angiogenic effects of homocysteine on cultured endothelial cells.

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.

Antiangiogenic activity of aeroplysinin-1, a brominated compound isolated from a marine sponge.

(+)-aeroplysinin-1, an antibacterial brominated compound produced by certain sponges, was selected during a blind high-throughput screening for new potential antiangiogenic compounds obtained from marine organisms. In a variety of experimental systems, representing the sequential events of the angiogenic process, aeroplysinin-1 treatment of endothelial cells resulted in strong inhibitory effects. Aeroplysinin-1 inhibited the growth of endothelial cells in culture and induced endothelial cell apoptosis. Capillary tube formation on Matrigel was completely abrogated by addition of aeroplysinin-1 at the low micromolar range. Aeroplysinin-1 also exhibited a clear inhibitory effect on the migration capabilities of endothelial cells. Zymographic assays showed that aeroplysinin-1 treatment produced a decrease in the concentration of matrix metalloproteinase-2 and urokinase in conditioned medium from endothelial cells. Finally, aeroplysinin-1 exhibited a dose-dependent inhibitory effect on the in vivo chorioallantoic membrane assay, showing potent apoptosis-inducing activity in the developing endothelium. The in vivo inhibition of angiogenesis by aeroplysinin-1 was confirmed by the Matrigel plug assay. Together, our data indicate that aeroplysinin-1 is a compound that interferes with key events in angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.