Expression Profile of Apoptosis-Related Genes during Differentiation in the Human Neuronal Progenitor Cell Line VM197

The development of the human central nervous system is accompanied by cell proliferation, differentiation and cell death. The massive loss of neural cells during neurogenesis suggests that apoptosis plays a critical role in human brain development. To characterize apoptotic processes during differentiation we analyzed the gene expression profiles of proliferating versus differentiating ReNCell VM197 at six different time points after induction of differentiation in vitro. The expression levels of about 1,117 cDNA/ESTs (expressed sequence tags) related to apoptosis pathways were found to be changed at least 2-fold higher or lower in differentiating cells than in proliferating cells. Consistent with our previous Western/Immunoblotting studies, we observed that pro-apoptotic proteins like Bax, Bid and cytochrome c showed increased gene expression during the first 6 h of differentiation and diminished expression levels at later stages of differentiation. In contrast the gene and protein expression levels of the anti-apoptotic protein Bcl-2 was found to be increased 6 h and 12 h respectively post-initiation of differentiation. We conclude that both, pro-apoptotic and anti-apoptotic processes occur during differentiation of VM197 cells and that Bcl-2 plays a protective role against apoptosis in fully differentiated neurons

Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening

UNLABELLED: Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication. IMPORTANCE: Q fever in humans is caused by the bacterium Coxiella burnetii. Infection with C. burnetii is marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets of C. burnetii infection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of the C. burnetii vacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol