Benjamin Newcomb

Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.

arising from N. C. Derecki et al. 484, 105–109 (2012); doi:10.1038/nature10907Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene MECP2 (ref. 1), and its treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome that can be reversed after Mecp2 re-expression. Recently, Derecki et al. reported that transplantation of wild-type bone marrow into lethally irradiated Mecp2-null (Mecp2tm1.1Jae/y) mice prevented neurological decline and early death by restoring microglial phagocytic activity against apoptotic targets, and clinical trials of bone marrow transplantation (BMT) for patients with Rett syndrome have thus been initiated. We aimed to replicate and extend the BMT experiments in three different Rett syndrome mouse models, but found that despite robust microglial engraftment, BMT from wild-type donors did not prevent early death or ameliorate neurological deficits. Furthermore, early and specific Mecp2 genetic expression in microglia did not rescue Mecp2-deficient mice.

Hst3 is regulated by Mec1-dependent proteolysis and controls the S phase checkpoint and sister chromatid cohesion by deacetylating histone H3 at lysine 56.

The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.

Distinct Signaling Roles of Ceramide Species in Yeast Revealed Through Systematic Perturbation and Systems Biology Analyses

Distinct transcriptional modules are regulated by specific ceramides. Unraveling Ceramide Signaling Specificity The extensive biochemical complexity of the ceramide class of lipids, with more than 30 species in yeast and hundreds in mammals, creates challenges for discerning the specific functions of individual species of ceramide or subgroups of these lipids. Montefusco et al. took a systems biology approach to tackle the functional complexity of ceramide-regulated events in yeast using a biosynthetic inhibitor to limit the number of species produced. The authors clustered changes in lipid abundance with functionally related transcriptional changes in yeast subjected to heat stress to identify changes in gene expression that correlated with specific classes of ceramides. Mapping of the lipid-correlated transcriptional profiles to transcription factors identified putative transcriptional modules regulated by changes in ceramide abundance, and two of these modules were experimentally verified in yeast. Thus, this approach and the data set will help unravel specificity in ceramide-mediated regulatory events. Ceramide, the central molecule of sphingolipid metabolism, is an important bioactive molecule that participates in various cellular regulatory events and that has been implicated in disease. Deciphering ceramide signaling is challenging because multiple ceramide species exist, and many of them may have distinct functions. We applied systems biology and molecular approaches to perturb ceramide metabolism in the yeast Saccharomyces cerevisiae and inferred causal relationships between ceramide species and their potential targets by combining lipidomic, genomic, and transcriptomic analyses. We found that during heat stress, distinct metabolic mechanisms controlled the abundance of different groups of ceramide species and provided experimental support for the importance of the dihydroceramidase Ydc1 in mediating the decrease in dihydroceramides during heat stress. Additionally, distinct groups of ceramide species, with different N-acyl chains and hydroxylations, regulated different sets of functionally related genes, indicating that the structural complexity of these lipids produces functional diversity. The transcriptional modules that we identified provide a resource to begin to dissect the specific functions of ceramides.

Sphingoid Bases and the Serine Catabolic Enzyme CHA1 Define a Novel Feedforward/Feedback Mechanism in the Response to Serine Availability

Background: The serine deaminase CHA1 responds to heat stress in a sphingolipid-dependent manner. Results: CHA1 requires de novo sphingoid base production for induction by serine, limiting growth-suppressing accumulation of sphingoid bases. Conclusion: Sphingoid bases are feedback sensors of serine availability, forming a feedforward/feedback loop through CHA1. Significance: This study defines a fundamental connection between sphingolipid and amino acid metabolic pathways with implications for disease. Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

The plant decapeptide OSIP108 prevents copper-induced apoptosis in yeast and human cells

We previously identified the Arabidopsis thaliana-derived decapeptide OSIP108, which increases tolerance of plants and yeast cells to oxidative stress. As excess copper (Cu) is known to induce oxidative stress and apoptosis, and is characteristic for the human pathology Wilson disease, we investigated the effect of OSIP108 on Cu-induced toxicity in yeast. We found that OSIP108 increased yeast viability in the presence of toxic Cu concentrations, and decreased the prevalence of Cu-induced apoptotic markers. Next, we translated these results to the human hepatoma HepG2 cell line, demonstrating anti-apoptotic activity of OSIP108 in this cell line. In addition, we found that OSIP108 did not affect intracellular Cu levels in HepG2 cells, but preserved HepG2 mitochondrial ultrastructure. As Cu is known to induce acid sphingomyelinase activity of HepG2 cells, we performed a sphingolipidomic analysis of OSIP108-treated HepG2 cells. We demonstrated that OSIP108 decreased the levels of several sphingoid bases and ceramide species. Moreover, exogenous addition of the sphingoid base dihydrosphingosine abolished the protective effect of OSIP108 against Cu-induced cell death in yeast. These findings indicate the potential of OSIP108 to prevent Cu-induced apoptosis, possibly via its effects on sphingolipid homeostasis.

Targeting (cellular) lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-B13 ester prodrugs.

Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.

Defining a Role for Acid Sphingomyelinase in the p38/Interleukin-6 Pathway

Background: Sphingolipids are important in multiple biological processes, but there is a lack of understanding of the pathways that mediate these effects. Results: Acid sphingomyelinase is involved in p38 activation, interleukin-6 production, and cancer cell invasion. Conclusion: Acid sphingomyelinase plays a role in inflammatory, proinvasive signaling in cancer cells. Significance: This work expands the understanding of acid sphingomyelinase signaling and downstream biology. Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid β-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4β-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid β-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.

A new twist to the emerging functions of ceramides in cancer: novel role for platelet acid sphingomyelinase in cancer metastasis

It is now appreciated that sphingolipids constitute a rich class of bioactive molecules that include ceramide, sphingosine, and sphingosine 1‐phosphate whose formation is controlled by a network of highly regulated enzymes (Hannun & Obeid, ). Notably, several stress stimuli induce the production of ceramide, which, as a single entity, has been traditionally associated with apoptotic and growth suppressive functions. However, recent data clearly suggest that this simplistic formulation is no longer tenable.

Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.

Identification of inhibitors of chromatin modifying enzymes using the yeast phenotypic screens.

A multitude of enzymes that modify histones and remodel nucleosomes are required for the formation, maintenance, and propagation of the transcriptionally repressed chromatin state in eukaryotes. Robust phenotypic screens in yeast S. cerevisiae have proved instrumental in identifying these activities and for providing mechanistic insights into epigenetic regulation. These phenotypic assays, amenable for high throughput small molecule screening, enable identification and characterization of inhibitors of chromatin modifying enzymes largely bypassing traditional biochemical approaches.