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Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson’s Disease (PD), Amyotrophic Lateral Sclerosis (ALS), and Multiple Sclerosis (MS). One prominent feature of affected brain regions are iron-loaded microglia, but how iron overload influences microglia physiology and disease response is poorly understood. Here we show that microglia are highly susceptible to ferroptosis, an iron-dependent form of cell death. In a tri-culture of human iPSC-derived neurons, astrocytes, and microglia, under ferroptosis-inducing conditions, microglia undergo a drastic shift in cell state, with increased ferritin levels, disrupted glutathione homeostasis, and altered cytokine signaling. Similar ferroptosis-associated signature (FAS) microglia were uncovered in PD, and the signature was also found in a large cohort of PD patient blood samples, raising the possibility that ferroptosis can be identified clinically. We performed a genome-wide CRISPR screen which revealed a novel regulator of ferroptosis, the vesicle trafficking gene SEC24B. A small molecule screen also nominated several candidates which blocked ferroptosis, some of which are already in clinical use. These data suggest that ferroptosis sits at the interface of cell death and inflammation, and inhibition of this process in microglia and other brain cells may provide new ways for treating neurodegenerative disease.

In Gaucher disease (GD), genetic deficiency of acid β-glucosidase leads to accumulation of its substrate glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Lipid-laden cells, most prominently seen as macrophages engorged with GlcCer and GlcSph-laden lysosomes, trigger chronic metabolic inflammation and multisystemic phenotypes. Among the pleiotropic effects of inflammatory cascades, the induction of glucosylceramide synthase accentuates the primary metabolic defect. First-line therapies for adults with GD type 1 include Enzyme Replacement Therapy (ERT) and eliglustat Substrate Reduction Therapy (SRT). The ENCORE phase 3 clinical trial of eliglustat demonstrated non-inferiority compared to ERT. It is not known whether switching stable patients from long-term ERT to SRT results in the incremental reversal of the disease phenotype and its surrogate indicators. Herein, we report real-world experience from a single tertiary referral center of 38 adult GD type 1 patients, stable on long-term ERT (mean 13.3 years), who switched to eliglustat SRT (mean 3.1 years). After switch to SRT, there was significant reduction in spleen volume (P = 0.003) while liver volume, which was normal at baseline, remained unchanged. Platelet counts increased significantly (P = 0.026). Concomitantly, there was reduction of three validated biomarkers of Gaucher disease activity: plasma GlcSph decreased from 63.7 ng/ml (95% CI, 37.6-89.8) to 26.1 ng/ml (95% CI, 15.7-36.6) (P

Background: The recently developed single-cell RNA sequencing (scRNA-seq) has attracted a great amount of attention due to its capability to interrogate expression of individual cells, which is superior to traditional bulk cell sequencing that can only measure mean gene expression of a population of cells. scRNA-seq has been successfully applied in finding new cell subtypes. New computational challenges exist in the analysis of scRNA-seq data. Objective: We provide an overview of the features of different similarity calculation and clustering methods, in order to facilitate users to select methods that are suitable for their scRNA-seq. We would also like to show that feature selection methods are important to improve clustering performance. Results: We first described similarity measurement methods, followed by reviewing some new clustering methods, as well as their algorithmic details. This analysis revealed several new questions, including how to automatically estimate the number of clustering categories, how to discover novel subpopulation, and how to search for new marker genes by using feature selection methods. Conclusion: Without prior knowledge about the number of cell types, clustering or semisupervised learning methods are important tools for exploratory analysis of scRNA-seq data.

Receptor interacting protein kinase 1 (RIPK1) mediates cell death and inflammatory signaling and is increased in multiple sclerosis (MS) brain samples. Here, we investigate the role of glial RIPK1 kinase activity in mediating MS pathogenesis. We demonstrate RIPK1 levels correlate with MS disease progression. We find microglia are susceptible to RIPK1-mediated cell death and identify an inflammatory gene signature that may contribute to the neuroinflammatory milieu in MS patients. We uncover a distinct role for RIPK1 in astrocytes in regulating inflammatory signaling in the absence of cell death and confirm RIPK1-kinase-dependent regulation in human glia. Using a murine MS model, we show RIPK1 inhibition attenuates disease progression and suppresses deleterious signaling in astrocytes and microglia. Our results suggest RIPK1 kinase activation in microglia and astrocytes induces a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in progressive MS.

Single cell RNA sequencing (scRNA-seq) provides a more granular description of gene expression in a single cell. Many clustering methods for scRNA-seq data have been developed to understand cell development and cell differentiation. However, the high dimension and the strong noise make clustering scRNA-seq data challenging. To overcome this problem, we propose a method for clustering scRNA-seq data, called network enhancement-based similarity combined with Louvain (NES-Louvain). In NES-Louvain, the initial similarity matrix is denoised by using a network enhancement method. Then, a path-based similarity measurement is designed to introduce the nodes in high-order paths based on the assumption that including more relevant nodes would improve the similarity of node pairs. Finally, the Louvain community detection method is improved to clustering single cells. The experimental results show that NES and NES-Louvain achieve better performance than other methods. Furthermore, NES-Louvain shows robust to perturbation.

Engineering the gut microbiota to produce specific beneficial metabolites represents an important new potential strategy for treating chronic diseases. Our previous studies with bacteria engineered to produce N-acyl-phosphatidylethanolamines (NAPEs), the immediate precursors of the lipid satiety factors N-acyl-ethanolamides (NAEs), found that colonization of these bacteria inhibited development of obesity in C57BL/6J mice fed a high fat diet. Individual NAE species differ in their bioactivities. Intriguingly, colonization by our engineered bacteria resulted in increased hepatic N-stearoyl-ethanolamide (C18:0NAE) levels despite the apparent inability of these bacteria to biosynthesize its precursor N-stearoyl-phosphatidylethanolamine (C18:0NAPE) in vitro. We therefore sought to identify the factors that allowed C18:0NAPE biosynthesis by the engineered bacteria after colonization of the intestinal tract. We found that the species of NAPE biosynthesized by engineered bacteria depends on the species of dietary fatty acids available in the intestine, suggesting a simple method to fine-tune the therapeutic effects of modified microbiota.

Food intake induces synthesis of N-acylphosphatidylethanolamines (NAPEs) in the intestinal tract. While NAPEs exert leptin-like (leptogenic) effects, including reduced weight gain and food intake, the mechanisms by which NAPEs induce these leptogenic effects remain unclear. One key question is whether intestinal NAPEs act directly on cognate receptors or first require conversion to N-acylethanolamides (NAEs) by NAPE-hydrolyzing phospholipase D (NAPE-PLD). Previous studies using Nape-pld−/− mice were equivocal because intraperitoneal injection of NAPEs led to nonspecific aversive effects. To avoid the aversive effects of injection, we delivered NAPEs and NAEs intestinally using gut bacteria synthesizing these compounds. Unlike in wild-type mice, increasing intestinal levels of NAPE using NAPE-synthesizing bacteria in Nape-pld−/− mice failed to reduce food intake and weight gain or alter gene expression. In contrast, increasing intestinal NAE levels in Nape-pld−/− mice using NAE-synthesizing bacteria induced all of these effects. These NAE-synthesizing bacteria also markedly increased NAE levels and decreased inflammatory gene expression in omental adipose tissue. These results demonstrate that intestinal NAPEs require conversion to NAEs by the action of NAPE-PLD to exert their various leptogenic effects, so that the reduced intestinal NAPE-PLD activity found in obese subjects may directly contribute to excess food intake and obesity.

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a person’s microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

Lipid peroxidation generates a variety of lipid aldehydes, which have been recognized to modify protein and DNA, causing inflammation and cancer. However, recent studies demonstrate that phosphatidylethanolamine (PE) is a major target for these aldehydes, forming aldehyde-modified PEs (al-PEs) as a novel family of mediators for inflammation. This review summarizes our current understanding of these al-PEs, including formation, detection, structural characterization, physiological relevance and mechanism of action.

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.

The preparation of a series of monoquaternary pyridinium oximes bearing either a heterocyclic side chain or a functionalized aliphatic side chain and the corresponding in vitro evaluation for reactivation of paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE) are reported. Several newly synthesized compounds efficiently reactivated inhibited EeAChE, but were poor reactivators of inhibited rHuAChE. Compounds bearing a thiophene ring in the side chain (20, 23, 26 and 29) showed better reactivation (24-37% for EeAChE and 5-9% for rHuAChE) compared to compounds with furan and isoxazole heterocycles (0-8% for EeAChE and 2-3% for rHuAChE) at 10(-5)M. The N-pyridyl-CH(2)COOH analog 8 reactivated EeAChE (36%) and rHuAChE (15%) at 10(-4)M with a k(r) value better than 2-pyridine aldoxime methiodide (2-PAM) for rHuAChE.

Aims: Increased lipid peroxidation occurs in many conditions associated with inflammation. Because lipid peroxidation produces lipid aldehydes that can induce inflammatory responses through unknown mechanisms, elucidating these mechanisms may lead to development of better treatments for inflammatory diseases. We recently demonstrated that exposure of cultured cells to lipid aldehydes such as isolevuglandins (IsoLG) results in the modification of phosphatidylethanolamine (PE). We therefore sought to determine (i) whether PE modification by isolevuglandins (IsoLG-PE) occurred in vivo, (ii) whether IsoLG-PE stimulated the inflammatory responses of macrophages, and (iii) the identity of receptors mediating the inflammatory effects of IsoLG-PE. Results: IsoLG-PE levels were elevated in plasma of patients with familial hypercholesterolemia and in the livers of mice fed a high-fat diet to induce obesity and hepatosteatosis. IsoLG-PE potently stimulated nuclear factor kappa B (NFκB) activation and expression of inflammatory cytokines in macrophages. The effects of IsoLG-PE were blocked by the soluble form of the receptor for advanced glycation endproducts (sRAGE) and by RAGE antagonists. Furthermore, macrophages derived from the bone marrow of Ager null mice failed to express inflammatory cytokines in response to IsoLG-PE to the same extent as macrophages from wild-type mice. Innovation: These studies are the first to identify IsoLG-PE as a mediator of macrophage activation and a specific receptor, RAGE, which mediates its biological effects. Conclusion: PE modification by IsoLG forms RAGE ligands that activate macrophages, so that the increased IsoLG-PE generated by high circulating cholesterol levels or high-fat diet may play a role in the inflammation associated with these conditions. Antioxid. Redox Signal. 22, 1633–1645.

Lipid aldehydes including isolevuglandins (IsoLGs) and 4-hydroxynonenal modify phosphatidylethanolamine (PE) to form proinflammatory and cytotoxic adducts. Therefore, cells may have evolved mechanisms to degrade and prevent accumulation of these potentially harmful compounds. To test if cells could degrade isolevuglandin-modified phosphatidylethanolamine (IsoLG-PE), we generated IsoLG-PE in human embryonic kidney 293 (HEK293) cells and human umbilical cord endothelial cells and measured its stability over time. We found that IsoLG-PE levels decreased more than 75% after 6 h, suggesting that IsoLG-PE was indeed degraded. Because N-acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) has been described as a key enzyme in the hydrolysis of N-acyl phosphatidylethanoamine (NAPE) and both NAPE and IsoLG-PE have large aliphatic headgroups, we considered the possibility that this enzyme might also hydrolyze IsoLG-PE. We found that knockdown of NAPE-PLD expression using small interfering RNA (siRNA) significantly increased the persistence of IsoLG-PE in HEK293 cells. IsoLG-PE competed with NAPE for hydrolysis by recombinant mouse NAPE-PLD, with the catalytic efficiency (V(max)/K(m)) for hydrolysis of IsoLG-PE being 30% of that for hydrolysis of NAPE. LC-MS/MS analysis confirmed that recombinant NAPE-PLD hydrolyzed IsoLG-PE to IsoLG-ethanolamine. These results demonstrate that NAPE-PLD contributes to the degradation of IsoLG-PE and suggest that a major physiological role of NAPE-PLD may be to degrade aldehyde-modified PE, thereby preventing the accumulation of these harmful compounds.

Lipid aldehydes generated by lipid peroxidation induce cell damage and inflammation. Recent evidence indicates that γ-ketoaldehydes (isolevuglandins, IsoLGs) form inflammatory mediators by modifying the ethanolamine headgroup of phosphatidylethanolamines (PEs). To determine if other species of aldehyde-modified PEs (al-PEs) with inflammatory bioactivity were generated by lipid peroxidation, we oxidized liposomes containing arachidonic acid and characterized the resulting products. We detected PE modified by IsoLGs, malondialdehyde (MDA), and 4-hydroxynonenal (HNE), as well as a novel series of N-acyl-PEs and N-carboxyacyl-PEs in these oxidized liposomes. These al-PEs were also detected in high-density lipoproteins exposed to myeloperoxidase. When we tested the ability of al-PEs to induce THP-1 monocyte adhesion to cultured endothelial cells, we found that PEs modified by MDA, HNE, and 4-oxononenal induced adhesion with potencies similar to those of PEs modified by IsoLGs (∼2μM). A commercially available medium-chain N-carboxyacyl-PE (C11:0CAPE) also stimulated adhesion, whereas C4:0CAPE and N-acyl-PEs did not. PEs modified by acrolein or by glucose were only partial agonists for adhesion. These studies indicate that lipid peroxidation generates a large family of al-PEs, many of which have the potential to drive inflammation.