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Etomoxir has been used for decades as a popular small molecule inhibitor of carnitine palmitoyltransferase I, Cpt1, to block mitochondrial fatty acid β-oxidation. To test the specificity of etomoxir, we generated click chemistry-enabled reagents to label etomoxir binding proteins in situ . Etomoxir bound to Cpt1, but also bound to a large array of diverse proteins that metabolize and transport fatty acids in the cytoplasm, peroxisome, and mitochondria. Many of the most abundant proteins identified in primary hepatocytes were peroxisomal proteins. The loss of Pex5, required for the import of peroxisomal matrix proteins, eliminated many of these etomoxir-labeled proteins. By utilizing the promiscuous, covalent, and fatty acid mimetic properties of etomoxir, etomoxir targets of fatty acid ω-oxidation were revealed following the loss of Pex5. These data demonstrate that etomoxir is not specific for Cpt1 and is not appropriate as a tool to distinguish the biological effects of fatty acid oxidation., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Lysophosphatidylcholine (LPC) is immunomodulatory in nonruminants; however, the actions of LPC on immunity in cattle are undefined. Our objective was to study the effects of LPC administration on measures of immunity, liver health, and growth in calves. Healthy Holstein heifer calves (n = 46; age 7 ± 3 d) were randomly assigned to 1 of 4 treatments (n = 10 to 11 calves/treatment): a milk replacer diet unsupplemented with lecithin in the absence (CON) or presence of subcutaneously (s.c.) administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other) or pure LPC (pLPC; 99% LPC-18:0), or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.-administered LPC (LYSO) for 5 wk. Calves received 5 s.c. injections of vehicle (10 mL of phosphate-buffered saline containing 20 mg of bovine serum albumin/mL; CON and LYSO) or vehicle containing mLPC or pLPC to provide 10 mg of total LPC per kilogram of BW per injection every 12 h during wk 2 of life. Calves were fed a milk replacer containing 27% crude protein and 24% fat at 1.75% of BW per day (dry matter basis) until wk 6 of life (start of weaning). Starter grain and water were provided ad libitum. Body measurements were recorded weekly, and clinical observations were recorded daily. Blood samples were collected weekly before morning feeding and at 0, 5, and 10 h, relative to the final s.c. injection of vehicle or LPC. Data were analyzed using a mixed model, with repeated measures including fixed effects of treatment, time, and their interaction. Dunnett's test was used to compare treatments to CON. Peak rectal temperatures were higher in mLPC or pLPC, relative to CON. Plasma LPC concentrations were greater in mLPC and LYSO calves 5 h and 10 h after the final injection, relative to CON. Calves receiving mLPC and pLPC also had higher circulating serum amyloid A concentrations, relative to CON. Calves receiving mLPC had greater serum aspartate aminotransferase, γ-glutamyltransferase, and glutamate dehydrogenase concentrations, relative to CON. Calves provided mLPC experienced lower average daily gain (ADG) after weaning, relative to CON. The LYSO treatment did not modify rectal temperatures, ADG, or measures of liver health, relative to CON. We conclude that LPC administered as s.c. injections induced an acute febrile response, modified measures of liver and immune function, and impaired growth in calves., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Cognitive decline in Alzheimer's disease (AD) is associated with hyperphosphorylated tau (pTau) propagation between neurons along synaptically connected networks, in part via extracellular vesicles (EVs). EV biogenesis is triggered by ceramide enrichment at the plasma membrane from neutral sphingomyelinase2 (nSMase2)-mediated cleavage of sphingomyelin. We report, for the first time, that human tau expression elevates brain ceramides and nSMase2 activity., Methods: To determine the therapeutic benefit of inhibiting this elevation, we evaluated PDDC, the first potent, selective, orally bioavailable, and brain-penetrable nSMase2 inhibitor in the transgenic PS19 AD mouse model. Additionally, we directly evaluated the effect of PDDC on tau propagation in a mouse model where an adeno-associated virus (AAV) encoding P301L/S320F double mutant human tau was stereotaxically-injected unilaterally into the hippocampus. The contralateral transfer of the double mutant human tau to the dentate gyrus was monitored. We examined ceramide levels, histopathological changes, and pTau content within EVs isolated from the mouse plasma., Results: Similar to human AD, the PS19 mice exhibited increased brain ceramide levels and nSMase2 activity; both were completely normalized by PDDC treatment. The PS19 mice also exhibited elevated tau immunostaining, thinning of hippocampal neuronal cell layers, increased mossy fiber synaptophysin immunostaining, and glial activation, all of which were pathologic features of human AD. PDDC treatment reduced these changes. The plasma of PDDC-treated PS19 mice had reduced levels of neuronal- and microglial-derived EVs, the former carrying lower pTau levels, compared to untreated mice. In the tau propagation model, PDDC normalized the tau-induced increase in brain ceramides and significantly reduced the amount of tau propagation to the contralateral side., Conclusions: PDDC is a first-in-class therapeutic candidate that normalizes elevated brain ceramides and nSMase2 activity, leading to the slowing of tau spread in AD mice., (© 2023. The Author(s).)

Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC., Methods: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples., Results: Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3β and MAPK signaling., Conclusions: Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target., (© 2023. The Author(s).)

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

Although HIV-1 Gag is known to drive viral assembly and budding, the precise mechanisms by which the lipid composition of the plasma membrane is remodeled during assembly are incompletely understood. Here, we provide evidence that the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) interacts with HIV-1 Gag and through the hydrolysis of sphingomyelin creates ceramide that is necessary for proper formation of the viral envelope and viral maturation. Inhibition or depletion of nSMase2 resulted in the production of noninfectious HIV-1 virions with incomplete Gag lattices lacking condensed conical cores. Inhibition of nSMase2 in HIV-1-infected humanized mouse models with a potent and selective inhibitor of nSMase2 termed PDDC [phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl) pyrrolidin-3-yl)-carbamate] produced a linear reduction in levels of HIV-1 in plasma. If undetectable plasma levels of HIV-1 were achieved with PDDC treatment, viral rebound did not occur for up to 4 wk when PDDC was discontinued. In vivo and tissue culture results suggest that PDDC selectively kills cells with actively replicating HIV-1. Collectively, this work demonstrates that nSMase2 is a critical regulator of HIV-1 replication and suggests that nSMase2 could be an important therapeutic target with the potential to kill HIV-1-infected cells.

Extracellular vesicles (EVs) have been proposed to regulate the deposition of Aβ. Multiple publications have shown that APP, amyloid processing enzymes and Aβ peptides are associated with EVs. However, very little Aβ is associated with EVs compared with the total amount Aβ present in human plasma, CSF, or supernatants from cultured neurons. The involvement of EVs has largely been inferred by pharmacological inhibition or whole body deletion of the sphingomyelin hydrolase neutral sphingomyelinase-2 (nSMase2) that is a key regulator for the biogenesis of at-least one population of EVs. Here we used a Cre-Lox system to selectively delete nSMase2 from pyramidal neurons in APP/PS1 mice (APP/PS1-SMPD3-Nex1) and found a ∼ 70% reduction in Aβ deposition at 6 months of age and ∼ 35% reduction at 12 months of age in both cortex and hippocampus. Brain ceramides were increased in APP/PS1 compared with Wt mice, but were similar to Wt in APP/PS1-SMPD3-Nex1 mice suggesting that elevated brain ceramides in this model involves neuronally expressed nSMase2. Reduced levels of PSD95 and deficits of long-term potentiation in APP/PS1 mice were normalized in APP/PS1-SMPD3-Nex1 mice. In contrast, elevated levels of IL-1β, IL-8 and TNFα in APP/PS1 mice were not normalized in APP/PS1-SMPD3-Nex1 mice compared with APP/PS1 mice. Mechanistic studies showed that the size of liquid ordered membrane microdomains was increased in APP/PS1 mice, as were the amounts of APP and BACE1 localized to these microdomains. Pharmacological inhibition of nSMase2 activity with PDDC reduced the size of the liquid ordered membrane microdomains, reduced the localization of APP with BACE1 and reduced the production of Aβ 1 - 40 and Aβ 1 - 42 . Although inhibition of nSMase2 reduced the release and increased the size of EVs, very little Aβ was associated with EVs in all conditions tested. We also found that nSMase2 directly protected neurons from the toxic effects of oligomerized Aβ and preserved neural network connectivity despite considerable Aβ deposition. These data demonstrate that nSMase2 plays a role in the production of Aβ by stabilizing the interaction of APP with BACE1 in liquid ordered membrane microdomains, and directly protects neurons from the toxic effects of Aβ. The effects of inhibiting nSMase2 on EV biogenesis may be independent from effects on Aβ production and neuronal protection., (Copyright © 2023. Published by Elsevier Inc.)

Wilson disease (WD) is a metabolic disorder caused by inactivation of the copper-transporting ATPase 2 (ATP7B) and copper (Cu) overload in tissues. Excess Cu causes oxidative stress and pathologic changes with poorly understood mechanistic connections. In Atp7b -/- mice with established liver disease, Cu overload activates the stress-sensitive transcription factor Nrf2 (nuclear factor erythroid-derived 2-like 2). Nrf2 targets, especially sulfotransferase 1e1 (Sult1e1), are strongly induced and cause elevation of sulfated sterols, whereas oxysterols are decreased. This sterol misbalance results in inhibition of the liver X receptor (LXR) and up-regulation of LXR targets associated with inflammatory responses. Pharmacological inhibition of Sult1e1 partially reverses oxysterol misbalance and LXR inhibition. Contribution of this pathway to advanced hepatic WD was demonstrated by treating mice with an LXR agonist. Treatment decreased inflammation by reducing expression of proinflammatory molecules, diminished fibrosis by down-regulating the noncanonical transforming growth factor-β signaling pathway, and improved liver morphology and function. Thus, the identified pathway is an important driver of WD.

Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid-β and hyperphosphorylated tau (pTau), which can spread throughout the brain via extracellular vesicles (EVs). Membrane ceramide enrichment regulated by the enzyme neutral sphingomyelinase 2 (nSMase2) is a critical component of at least one EV biogenesis pathway. Our group recently identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), the most potent (30 nM) and selective inhibitor of nSMase2 reported to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), modest brain penetration, and rapid clearance, limiting its clinical translation. To enhance its PK properties, we conjugated DPTIP to a hydroxyl-PAMAM dendrimer delivery system, creating dendrimer-DPTIP (D-DPTIP). In an acute brain injury model, orally administered D-DPTIP significantly reduced the intra-striatal IL-1β-induced increase in plasma EVs up to 72 h post-dose, while oral DPTIP had a limited effect. In a mouse tau propagation model, where a mutant hTau (P301L/S320F) containing adeno-associated virus was unilaterally seeded into the hippocampus, oral D-DPTIP (dosed 3× weekly) significantly inhibited brain nSMase2 activity and blocked the spread of pTau to the contralateral hippocampus. These data demonstrate that dendrimer conjugation of DPTIP improves its PK properties, resulting in significant inhibition of EV propagation of pTau in mice. Dendrimer-based delivery of DPTIP has the potential to be an exciting new therapeutic for AD.

People living with HIV (PLH) have significantly higher rates of cognitive impairment (CI) and major depressive disorder (MDD) versus the general population. The enzyme neutral sphingomyelinase 2 (nSMase2) is involved in the biogenesis of ceramide and extracellular vesicles (EVs), both of which are dysregulated in PLH, CI, and MDD. Here we evaluated EcoHIV-infected mice for behavioral abnormalities relevant to depression and cognition deficits, and assessed the behavioral and biochemical effects of nSMase2 inhibition. Mice were infected with EcoHIV and daily treatment with either vehicle or the nSMase2 inhibitor (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC) began 3 weeks post-infection. After 2 weeks of treatment, mice were subjected to behavior tests. EcoHIV-infected mice exhibited behavioral abnormalities relevant to MDD and CI that were reversed by PDDC treatment. EcoHIV infection significantly increased cortical brain nSMase2 activity, resulting in trend changes in sphingomyelin and ceramide levels that were normalized by PDDC treatment. EcoHIV-infected mice also exhibited increased levels of brain-derived EVs and altered microRNA cargo, including miR-183-5p, miR-200c-3p, miR-200b-3p, and miR-429-3p, known to be associated with MDD and CI; all were normalized by PDDC. In conclusion, inhibition of nSMase2 represents a possible new therapeutic strategy for the treatment of HIV-associated CI and MDD., (Copyright © 2021. Published by Elsevier Inc.)

Background: HIV infection results in immunometabolic reprogramming. While we are beginning to understand how this metabolic reprogramming regulates the immune response to HIV infection, we do not currently understand the impact of ART on immunometabolism in people with HIV (PWH)., Methods: Serum obtained from HIV-infected ( n = 278) and geographically matched HIV seronegative control subjects ( n = 300) from Rakai Uganda were used in this study. Serum was obtained before and ~2 years following the initiation of ART from HIV-infected individuals. We conducted metabolomics profiling of the serum and focused our analysis on metabolic substrates and pathways assocaited with immunometabolism., Results: HIV infection was associated with metabolic adaptations that implicated hyperactive glycolysis, enhanced formation of lactate, increased activity of the pentose phosphate pathway (PPP), decreased β-oxidation of long-chain fatty acids, increased utilization of medium-chain fatty acids, and enhanced amino acid catabolism. Following ART, serum levels of ketone bodies, carnitine, and amino acid metabolism were normalized, however glycolysis, PPP, lactate production, and β-oxidation of long-chain fatty acids remained abnormal., Conclusion: Our findings suggest that HIV infection is associated with an increased immunometabolic demand that is satisfied through the utilization of alternative energetic substrates, including fatty acids and amino acids. ART alone was insufficient to completely restore this metabolic reprogramming to HIV infection, suggesting that a sustained impairment of immunometabolism may contribute to chronic immune activation and comorbid conditions in virally suppressed PWH.

During metabolically demanding physiological states, ruminants and other mammals coordinate nutrient use among tissues by varying the set point of insulin action. This set point is regulated in part by metabolic hormones with some antagonizing (e.g., growth hormone and TNFα) and others potentiating (e.g., adiponectin) insulin action. Fibroblast growth factor-21 (FGF21) was recently identified as a sensitizing hormone in rodent and primate models of defective insulin action. FGF21 administration, however, failed to improve insulin action in dairy cows during the naturally occurring insulin resistance of lactation, raising the possibility that ruminants as a class of animals or lactation as a physiological state are unresponsive to FGF21. To start addressing this question, we asked whether FGF21 could improve insulin action in nonlactating ewes. Gene expression studies showed that the ovine FGF21 system resembles that of other species, with liver as the major site of FGF21 expression and adipose tissue as a target tissue based on high expression of the FGF21 receptor complex and activation of p44/42 extracellular signal-regulated kinase (ERK1/2) following exogenous FGF21 administration. FGF21 treatment for 13 days reduced plasma glucose and insulin over the entire treatment period and improved glucose disposal during a glucose tolerance test. FGF21 increased plasma adiponectin by day 3 of treatment but had no effect on the plasma concentrations of total, C16:0-, or C18:0-ceramide. Overall, these data confirm that the insulin-sensitizing effects of FGF21 are conserved in ruminants and raise the possibility that lactation is an FGF21-resistant state.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC 50 . Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1β or PDDC. IL-1β injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury., (Copyright © 2021. Published by Elsevier Inc.)