Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

Golgi-IP, a tool for multimodal analysis of Golgi molecular content.

The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signaling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid Golgi immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi-resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome, and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified proteins not previously associated with the Golgi. Metabolite profiling established the human Golgi metabolome and revealed the enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol, and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease.

Virtual reality mindfulness training for veterans in residential substance use treatment: Pilot study of feasibility and acceptability.

BACKGROUND: Mindfulness training is effective in recovery from substance use disorders; however, adoption can be difficult due to environmental and personal distractions. Virtual reality (VR) may help overcome these challenges by providing an immersive environment for practicing mindfulness, but there is currently limited knowledge regarding patient and provider perceptions of VR-based tools. OBJECTIVE: The present study investigated the feasibility and acceptability of VR mindfulness training for veterans in residential substance use treatment as well as potential benefits of VR mindfulness interventions in this population. We conducted a pilot feasibility/acceptability study as a first step toward conducting a larger randomized controlled trial (RCT). METHODS: The study recruited participants (N = 32) from a 30-day residential substance use program and collected both qualitative and quantitative feedback on the VR mindfulness intervention using a mixed-methods approach. Patients (n = 20) and providers (n = 12) rated the acceptability, usability, and satisfaction of the intervention. Using a within-subjects design, patients provided pre-post emotion ratings and reported on state mindfulness and VR presence after completing a single-session self-guided VR mindfulness intervention. Patients provided qualitative interview data on their overall impressions, while providers gave the same information via survey. RESULTS: Both patients and providers reported high satisfaction and confidence in the intervention. Moreover, within subjects t-tests showed that patients experienced significant reductions in negative affect and significant increases in positive affect from pre-post, along with high levels of state mindfulness and presence. Results of thematic analysis revealed that the intervention facilitated focused attention on the present moment, induced a state of calm and relaxation, and reduced negative thoughts and emotions. Participants requested improvements such as better integration of audiovisual elements, a more personalized and longer intervention, and more comfortable fitting headset. Finally, the intervention presented with several advantages compared to other mindfulness experiences including reduced distractions and a sense of safety and privacy. CONCLUSIONS: Self-guided VR mindfulness intervention is feasible and acceptable to patients and providers. VR mindfulness training provides an immersive experience that uplifts mood and reduces distractions. VR may provide a scaffolding tool to set the stage for deepening mindfulness skills. Results of the present study could inform further development and tailoring for future interventions.

An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's microglia.

Several genetic risk factors for Alzheimer's Disease (AD) implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and AD pathology remains poorly understood. Through single-nucleus RNA-sequencing of AD brain tissue, we have identified a microglial state defined by the expression of the lipid droplet (LD) associated enzyme ACSL1 with ACSL1-positive microglia most abundant in AD patients with the APOE4/4 genotype. In human iPSC-derived microglia (iMG) fibrillar Aß (fAß) induces ACSL1 expression, triglyceride synthesis, and LD accumulation in an APOE-dependent manner. Additionally, conditioned media from LD-containing microglia leads to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for AD with microglial LD accumulation and neurotoxic microglial-derived factors, potentially providing novel therapeutic strategies for AD.

Isotope tracing in health and disease.

Biochemical characterization of metabolism provides molecular insights for understanding biology in health and disease. Over the past decades, metabolic perturbations have been implicated in cancer, neurodegeneration, and diabetes, among others. Isotope tracing is a technique that allows tracking of labeled atoms within metabolites through biochemical reactions. This technique has become an integral component of the contemporary metabolic research. Isotope tracing measures substrate contribution to downstream metabolites and indicates its utilization in cellular metabolic networks. In addition, isotopic labeling data are necessary for quantitative metabolic flux analysis. Here, we review recent work utilizing metabolic tracing to study health and disease, and highlight its application to interrogate subcellular, intercellular, and in vivo metabolism. We further discuss the current challenges and opportunities to expand the utility of isotope tracing to new research areas.