Aim: Homocysteine (Hcy) can elicit neuronal cell death, and hyperhomocysteinemia is a strong independent risk factor for Alzheimer's disease. The aim of this study was to examine the effects of hydrogen sulfide (H 2 S) on Hcy-induced endoplasmic reticulum (ER) stress and neuronal apoptosis in rat hippocampus. Methods: Adult male SD rats were intracerebroventricularly (icv) injected with Hcy (0.6 μmol/d) for 7 d. Before Hcy injection, the rats were treated with NaHS (30 or 100 μmol·kg -1 ·d -1 , ip) and/or k252a (1 μg/d, icv) for 2 d. The apoptotic neurons were detected in hippocampal coronal slices with TUNEL staining. The expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and BDNF in the hippocampus were examined using Western blotting assays. The generation of H 2 S in the hippocampus was measured with the NNDPD method. Results: Hcy markedly inhibited the production of endogenous H 2 S and increased apoptotic neurons in the hippocampus. Furthermore, Hcy induced ER stress responses in the hippocampus, as indicated by the upregulation of GRP78, CHOP, and cleaved caspase-12. Treatment with the H 2 S donor NaHS increased the endogenous H 2 S production and BDNF expression in a dosedependent manner, and significantly reduced Hcy-induced neuronal apoptosis and ER stress responses in the hippocampus. Treatment with k252a, a specific inhibitor of TrkB (the receptor of BDNF), abolished the protective effects of NaHS against Hcy-induced ER stress in the hippocampus. Conclusion: H 2 S attenuates ER stress and neuronal apoptosis in the hippocampus of Hcy-treated rats via upregulating the BDNF-TrkB pathway.

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 MELK is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail-and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein.

Snail-like transcription factors affect stem cell function through mechanisms that are incompletely understood. In the Caenorhabditis elegans neurosecretory motor neuron (NSM) neuroblast lineage, CES-1 Snail coordinates cell cycle progression and cell polarity to ensure the asymmetric division of the NSM neuroblast and the generation of two daughter cells of different sizes and fates. We have previously shown that CES-1 Snail controls cell cycle progression by repressing the expression of cdc-25.2 CDC25. However, the mechanism through which CES-1 Snail affects cell polarity has been elusive. Here, we systematically searched for direct targets of CES-1 Snail by genome-wide profiling of CES-1 Snail binding sites and identified >3000 potential CES-1 Snail target genes, including pig-1, the ortholog of the oncogene maternal embryonic leucine zipper kinase (MELK). Furthermore, we show that CES-1 Snail represses pig-1 MELK transcription in the NSM neuroblast lineage and that pig-1 MELK acts downstream of ces-1 Snail to cause the NSM neuroblast to divide asymmetrically by size and along the correct cell division axis. Based on our results we propose that by regulating the expression of the MELK gene, Snail-like transcription factors affect the ability of stem cells to divide asymmetrically and, hence, to self-renew. Furthermore, we speculate that the deregulation of MELK contributes to tumorigenesis by causing cells that normally divide asymmetrically to divide symmetrically instead.

Caspases have functions other than in apoptosis. Here, we report that Caenorhabditis elegans CED-3 caspase regulates asymmetric cell division. Many of the 131 cells that are “programmed” to die during C. elegans development are the smaller daughter of a neuroblast that divides asymmetrically by size and fate. We have previously shown that CED-3 caspase is activated in such neuroblasts, and that before neuroblast division, a gradient of CED-3 caspase activity is formed in a ced-1 MEGF10 (multiple EGF-like domains 10)-dependent manner. This results in the nonrandom segregation of active CED-3 caspase or “apoptotic potential” into the smaller daughter. We now show that CED-3 caspase is necessary for the ability of neuroblasts to divide asymmetrically by size. In addition, we provide evidence that a pig-1 MELK (maternal embryonic leucine zipper kinase)-dependent reciprocal gradient of “mitotic potential” is formed in the QL.p neuroblast, and that CED-3 caspase antagonizes this mitotic potential. Based on these findings, we propose that CED-3 caspase plays a critical role in the asymmetric division by size and fate of neuroblasts, and that this contributes to the reproducibility and robustness with which the smaller daughter cell is produced and adopts the apoptotic fate. Finally, the function of CED-3 caspase in this context is dependent on its activation through the conserved egl-1 BH3-only, ced-9 Bcl-2, and ced-4 Apaf-1 pathway. In mammals, caspases affect various aspects of stem cell lineages. We speculate that the new nonapoptotic function of C. elegans CED-3 caspase in asymmetric neuroblast division is relevant to the function(s) of mammalian caspases in stem cells.

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.