A protocol for measuring the activity of protein kinase C-delta in murine bone-marrow-derived dendritic cells.

Protein kinase C-δ (PKC-δ) is a key enzyme controlling growth, differentiation, and apoptosis in various cells, including immune cells. Here, we present a protocol to determine PKC-δ activation in response to increased membrane-bound diacylglycerol or phorbol-12-myristate-13-acetate treatment in murine bone-marrow-derived dendritic cells. We describe steps for dendritic cell differentiation, the isolation of plasma membrane lipids, and the quantification of diacylglycerol. We then detail procedures for measuring PKC-δ kinase activity by in vitro assay, indirect immunofluorescence, and western blotting experiments. For complete details on the use and execution of this protocol, please refer to Parsons et al.1.

Peroxisomal cholesterol metabolism regulates yap-signaling, which maintains intestinal epithelial barrier function and is altered in Crohn's disease.

Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn's Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn's Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis.

Towards early detection of neurodegenerative diseases: A gut feeling.

The gastrointestinal tract communicates with the nervous system through a bidirectional network of signaling pathways called the gut-brain axis, which consists of multiple connections, including the enteric nervous system, the vagus nerve, the immune system, endocrine signals, the microbiota, and its metabolites. Alteration of communications in the gut-brain axis is emerging as an overlooked cause of neuroinflammation. Neuroinflammation is a common feature of the pathogenic mechanisms involved in various neurodegenerative diseases (NDs) that are incurable and debilitating conditions resulting in progressive degeneration and death of neurons, such as in Alzheimer and Parkinson diseases. NDs are a leading cause of global death and disability, and the incidences are expected to increase in the following decades if prevention strategies and successful treatment remain elusive. To date, the etiology of NDs is unclear due to the complexity of the mechanisms of diseases involving genetic and environmental factors, including diet and microbiota. Emerging evidence suggests that changes in diet, alteration of the microbiota, and deregulation of metabolism in the intestinal epithelium influence the inflammatory status of the neurons linked to disease insurgence and progression. This review will describe the leading players of the so-called diet-microbiota-gut-brain (DMGB) axis in the context of NDs. We will report recent findings from studies in model organisms such as rodents and fruit flies that support the role of diets, commensals, and intestinal epithelial functions as an overlooked primary regulator of brain health. We will finish discussing the pivotal role of metabolisms of cellular organelles such as mitochondria and peroxisomes in maintaining the DMGB axis and how alteration of the latter can be used as early disease makers and novel therapeutic targets.

Modified protocol for culturing Drosophila S2 R+ cells and adult plasmatocytes to study actin cytoskeleton dynamics.

Here, we describe a protocol to culture Drosophila S2R+ cells and to extract plasmatocytes from adult flies. The modified seeding approach detailed here, in combination with coating of coverslips with concanvalin A, enables enhanced adhesion and spreading of cells. We describe the steps for confocal microscopy and a detailed quantification pipeline to evaluate changes in cortical actin cytoskeleton dynamics. The protocol can be applied to a variety of genetic or chemical perturbations. For complete details on the use and execution of this protocol, please refer to Nath et al. (2022).

Modulation of the cell membrane lipid milieu by peroxisomal ß-oxidation induces Rho1 signaling to trigger inflammatory responses.

Phagocytosis, signal transduction, and inflammatory responses require changes in lipid metabolism. Peroxisomes have key roles in fatty acid homeostasis and in regulating immune function. We find that Drosophila macrophages lacking peroxisomes have perturbed lipid profiles, which reduce host survival after infection. Using lipidomic, transcriptomic, and genetic screens, we determine that peroxisomes contribute to the cell membrane glycerophospholipid composition necessary to induce Rho1-dependent signals, which drive cytoskeletal remodeling during macrophage activation. Loss of peroxisome function increases membrane phosphatidic acid (PA) and recruits RhoGAPp190 during infection, inhibiting Rho1-mediated responses. Peroxisome-glycerophospholipid-Rho1 signaling also controls cytoskeleton remodeling in mouse immune cells. While high levels of PA in cells without peroxisomes inhibit inflammatory phenotypes, large numbers of peroxisomes and low amounts of cell membrane PA are features of immune cells from patients with inflammatory Kawasaki disease and juvenile idiopathic arthritis. Our findings reveal potential metabolic markers and therapeutic targets for immune diseases and metabolic disorders.