Summary The majority of research on reactive oxygen species (ROS) has focused on their cellular toxicities. Stem cells generally have been thought to maintain low levels of ROS as a protection against these processes. However, recent studies suggest that ROS can also play roles as second messengers, activating normal cellular processes. Here, we investigated ROS function in primary brain-derived neural progenitors. Somewhat surprisingly, we found that proliferative, self-renewing multipotent neural progenitors with the phenotypic characteristics of neural stem cells (NSC) maintained a high ROS status and were highly responsive to ROS stimulation. ROS-mediated enhancements in self-renewal and neurogenesis were dependent on PI3K/Akt signaling. Pharmacological or genetic manipulations that diminished cellular ROS levels also interfered with normal NSC and/or multipotent progenitor function both in vitro and in vivo. This study has identified a redox-mediated regulatory mechanism of NSC function which may have significant implications for brain injury, disease, and repair.

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1 0 U-containing, 2 0 O-methylated 31-37 nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3 0 -5 0 processing of 31-37 nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.

SUMMARY Piwi proteins play essential roles in germline development, stem cell self-renewal, epigenetic regulation, and transposon silencing, and are implicated in oncogenesis [1-7]. Piwi proteins bind to a complex class of small non-coding RNAs called Piwi-Interacting RNAs (piRNAs) [8]. Mammalian Piwi proteins such as Mili are localized in the cytoplasm of spermatogenic cells, where they are associated with a germline-specific organelle called the nuage or its derivative, the chromatoid body, as well as with polysomes [9]. To investigate the molecular mechanisms mediated by Mili, we searched for Mili interacting proteins using co-immunoprecipitation and mass spectrometry. Here we report that Mili specifically interacts with Tudor Domain Containing Protein 1 (Tdrd1; a.k.a. Mouse Tudor Repeat 1, Mtr-1), a germline protein that contains multiple Tudor domains [10, 11]. This RNA-independent interaction is mediated through the N-terminal domain of Mili and the N-terminal region of Tdrd1 containing the Myeloid Nervy DEAF-1 (MYND) domain and first two Tudor domains. In addition, Mili positively regulates Tdrd1 expression at the mRNA level. Furthermore, Mili and Tdrd1 mutants share similar spermatogenic defects. However, Tdrd1, unlike Mili, is not required for piRNA biogenesis. Our results suggest that Mili interacts with Tdrd1 in the nuage and chromatoid body. This interaction does not contribute to piRNA biogenesis, but represents a regulatory mechanism critical for spermatogenesis.

Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

We report a novel connection between the phosphatidylinositol (PI) metabolic pathway and the DNA replication and damage checkpoint pathway discovered from an unbiased chemical genomics screen. Substrates and products of PI kinases are important signaling molecules that affect a wide range of biological processes. The full collection of yeast deletion strains was screened to identify genes that confer altered sensitivity to the natural product wortmannin, a PI kinase inhibitor. These experiments have allowed us to explore metabolomic and proteomic implications of PI synthesis and turnover. This study also uncovers other biological processes affected by wortmannin treatment, including proteasome-mediated degradation and chromatin remodeling. Bioinformatic analyses were used to reveal the relative distances among cellular processes affected by wortmannin and protein-protein interactions in the wortmannin-sensitive proteomic subnetwork. These results illustrate the great utility of using a whole-genome approach in annotating the biological effects of small molecules and have clear implications for pharmacogenomics. Furthermore, our discovery points to a route to overcoming genome instability, a result of defective DNA damage signaling͞ repair and a hallmark of cancer.

Glioblastoma multiforme (GBM) is amongst the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells amongst which a tumor initiating and treatment-resistant subpopulation, here termed GBM stem cells (GSC), have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GSC. The paradigm uses a tissue culture model to enrich for GSC derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from seven chemical libraries that we characterized and ranked based on their effect on a panel of GSC-enriched cultures as well as their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds demonstrated selectivity by inhibiting multiple GSC-enriched cultures compared to non-enriched cultures: Emetine, N-Arachidonoyldopamine (NADA), N-Oleoyldopamine (OLDA), and N-Palmitoyldopamine (PALDA). ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, Emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GSC in self-renewal assays in vitro and in a xenograft model in vivo. These studies demonstrate that our screening strategy provides potential candidates as well as a blueprint for lead compound identification in larger scale screens or screens involving other cancer types.

High-throughput identification of small molecules that selectively modulate molecular, cellular, or systems-level properties of the mammalian brain is a significant challenge. Here we report the chemical genetic identification of the orphan ligand phosphoserine (P-Ser) as an enhancer of neurogenesis. P-Ser inhibits neural stem cell/progenitor proliferation and self-renewal, enhances neurogenic fate commitment, and improves neuronal survival. We further demonstrate that the effects of P-Ser are mediated by the group III metabotropic glutamate receptor 4 (mGluR4). siRNA-mediated knockdown of mGluR4 abolished the effects of P-Ser and increased neurosphere proliferation, at least in part through upregulation of mTOR pathway activity. We also found that P-Ser increases neurogenesis in human embryonic stem cell-derived neural progenitors. This work highlights the tremendous potential of developing effective small-molecule drugs for use in regenerative medicine or transplantation therapy.

Anthrax lethal toxin is one of the fundamental components believed to be responsible for the virulence of Bacillus anthracis. In order to find novel compounds with anti-lethal toxin properties, we used a cell-based assay to screen a collection of approximately 500 small molecules. Nineteen compounds that blocked lethal toxinmediated killing of RAW 264.7 macrophages were identified, and we report here on the characterization of the two most potent antitoxic compounds, amiodarone and bepridil. These drugs are used to treat cardiac arrhythmia or angina in humans at doses similar to those that provide protection against lethal toxin in vitro. Our results support a model whereby the antitoxic properties of both drugs result from their ability to block endosomal acidification, thereby blocking toxin entry. Amiodarone was tested in vivo and found to significantly increase survival of lethal toxin-challenged Fischer rats.Bacillus anthracis is a gram-positive bacterium that is the causative agent of anthrax. Two major virulence factors that contribute to disease are lethal toxin (LT) and edema toxin (ET), and these genes are located on an extrachromosomal plasmid, pXO1 (10). Both LT and ET are bipartite toxins that rely on the same binding moiety, protective antigen (PA), to mediate the delivery of their respective enzymatic subunits into the host cytosol. LT consists of PA and lethal factor (LF), a metalloprotease that cleaves the N terminus of mitogen-activated protein kinase kinases 1 to 4, 6, and 7 (15, 49). ET, on the other hand, consists of PA and edema factor (EF), a calciumand calmodulin-dependent adenylate cyclase that raises cyclic AMP (cAMP) levels once inside the host cell (30).Cellular entry of either EF or LF begins with PA binding to a cell surface receptor. Receptor-bound PA undergoes furin cleavage, a step that allows it to heptamerize and form a structure known as the prepore (26, 34). The prepore then associates with up to three catalytic subunits and undergoes receptor-mediated endocytosis (38). Acidic pH of the endocytic compartment triggers conformational changes in the PA heptamer, causing it to insert into the membrane of the endosome (34). Insertion results in formation of a pore through which LF and EF translocate, thus gaining access to the cytosol of the host cell (28, 29, 52).Importantly, both LT and ET are lethal when administered independently to laboratory animals, suggesting a role for both toxins in the pathogenic process (14,17,35). At the cellular level, however, the response to each toxin depends on cell type. LT treatment of macrophages derived from certain inbred mouse strains results in rapid cell lysis (6, 18), whereas a less dramatic cytotoxic effect has been seen in endothelial cells and dendritic cells, among others, where prolonged treatment over several days results in reduced viability (reviewed in reference 3). Yet, most cell types tested so far do not die in response to LT. Rather, more subtle effects have been characterized mostly in immune cells, such as neutrophils and ...