Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy.

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

A Role of Sphingosine in the Intracellular Survival of Neisseria gonorrhoeae.

Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells.

Prenatal and postnatal experiences associated with epigenetic changes in the adult mouse brain.

To analyze the influences of early-life history on the brain epigenome, the offspring of mouse dams kept in an enriched or standard environment were exposed postnatally to enriched, standard, or adverse conditions. The methylation patterns of 7 candidate genes (9 loci) involved in developmental programming of stress vulnerability/resilience and psychiatric disease were analyzed in 6 brain regions of adult male and female mice. Exposure to an enriched prenatal environment was associated with widespread epigenetic changes (all of small effect size), affecting 29 of 324 (9%) gene/region-specific methylation patterns. The effects of either adverse or enriched postnatal conditions were tested separately in the two prenatal cohorts. Significant changes were observed in 2 of 324 (0.6%) loci in offspring of dams in a standard environment and 6 of 324 (1.9%) loci in animals that were exposed prenatally to an enriched environment. Prenatal life experiences appear to have a bigger effect on the adult brain epigenome than postnatal experiences. Positive prenatal life experiences may increase epigenetic plasticity of the brain later in life. All observed between-group differences were sex-specific, consistent with largely different developmental trajectories of the male and female brain. Multiple changes of small effect size are consistent with a multifactorial model of developmental programming of adult behavior and disease susceptibility.

Romidepsin induces caspase-dependent cell death in human neuroblastoma cells.

Neuroblastoma is the most common extracranial pediatric solid tumor, arising from the embryonic sympathoadrenal lineage of the neural crest, and is responsible for 15% of childhood cancer deaths. Although survival rates are good for some patients, those children diagnosed with high-risk neuroblastoma have survival rates as low as 35%. Thus, neuroblastoma remains a significant clinical challenge and the development of novel therapeutic strategies is essential. Given that there is widespread epigenetic dysregulation in neuroblastoma, epigenetic pharmacotherapy holds promise as a therapeutic approach. In recent years, histone deacetylase (HDAC) inhibitors, which cause selective activation of gene expression, have been shown to be potent chemotherapeutics for the treatment of a wide range of cancers. Here we examined the ability of the FDA-approved drug Romidepsin, a selective HDAC1/2 inhibitor, to act as a cytotoxic agent in neuroblastoma cells. Treatment with Romidepsin at concentrations in the low nanomolar range induced neuroblastoma cell death through caspase-dependent apoptosis. Romidepsin significantly increased histone acetylation, and significantly enhanced the cytotoxic effects of the cytotoxic agent 6-hydroxydopamine, which has been shown to induce cell death in neuroblastoma cells through increasing reactive oxygen species. Romidepsin was also more potent in MYCN-amplified neuroblastoma cells, which is an important prognostic marker of poor survival. This study has thus demonstrated that the FDA-approved chemotherapeutic drug Romidepsin has a potent caspase-dependent cytotoxic effect on neuroblastoma cells, whose effects enhance cell death induced by other cytotoxins, and suggests that Romidepsin may be a promising chemotherapeutic candidate for the treatment of neuroblastoma.

A Small Molecule Activator of p300/CBP Histone Acetyltransferase Promotes Survival and Neurite Growth in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.