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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.

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.

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

Background: Sphingolipids are myelin components and inflammatory signaling intermediates. Sphingolipid metabolism may be altered in people with multiple sclerosis (PwMS), but existing studies are limited by small sample sizes. Objectives: To compare the levels of serum ceramides between PwMS and healthy controls (HCs) and to determine whether ceramide levels correlate with disability status, as well as optical coherence tomography (OCT)-derived rates of retinal layer atrophy. Methods: We performed targeted lipidomics analyses for 45 ceramides in PwMS ( n = 251) and HCs ( n = 68). For a subset of PwMS, baseline and 5-year Expanded Disability Status Scale (EDSS) assessments ( n = 185), or baseline and serial spectral-domain OCT ( n = 180) were assessed. Results: Several ceramides, including hexosylceramides, lactosylceramides, and dihydroceramides, were altered in PwMS compared with HCs. Higher levels of Cer16:0 were associated with higher odds of EDSS worsening at 5 years in univariable (odds ratio (OR) = 3.84, 95% confidence interval (CI) = 1.41–10.43) and multivariable analyses accounting for age, sex, and race (OR = 2.97, 95% CI = 1.03–8.59). Each 1 ng/mL higher concentration of Hex-Cer22:0 and DH-HexCer22:0 was associated with accelerated rates (μm/year) of ganglion cell + inner plexiform layer (–0.138 ± 0.053, p = 0.01; –0.158 ± 0.053, p = 0.003, respectively) and peripapillary retinal nerve fiber layer thinning (–0.305 ± 0.107, p = 0.004; –0.358 ± 0.106, p = 0.001, respectively). Conclusion: Ceramide levels are altered in PwMS and may be associated with retinal neurodegeneration and physical disability.

Introduction: We have demonstrated that asymptomatic cerebral small vessel disease (cSVD) measured by white matter hyperintensity volume is associated with reduced manipulative manual dexterity on the Grooved Peg Board Test (GPBT) in middle-aged healthy individuals with a family history of early coronary artery disease. In this current study, we aim to identify the association of subcortical white matter microstructural impairment measured by diffusion tensor imaging, manual dexterity measured by GPBT and circulating serums ceramide, another marker for white matter injury. We hypothesize that lower regional fractional anisotropy (rFA) is associated with worse performance on GPBT and elevated serum ceramides in the same study population. Methods: rFA of 48 regions representing the subcortical white matters were analyzed in GeneSTAR participants in addition to serum ceramides and GPBT scores. Unadjusted univariable analyses with Bonferroni correction for multiple comparisons were completed using Spearman correlation for testing the associations between ceramides, rFA of subcortical white matter, and GPBT performance. Subsequently, sensitivity analyses were performed after excluding the participants that had any physical limitation that may influence their performance on GPBT. Finally, in the adjusted analysis using generalized estimating equation, linear regression models were performed for the areas that met significance threshold in the unadjusted analyses. Results: 112 subjects (age [49 ± 11], 51% female, 39.3% African American) were included. Adjusted analyses for the significant correlations that met the Bonferroni correction threshold in the unadjusted univariable analyses identified significant negative associations between rFA of the right fornix (RF) and log-GPBT score (β = −0.497, p = 0.037). In addition, rFA of RF negatively correlated with log serum ceramide levels (C18: β = −0.03, p = 0.003, C20: β = −0.0002, p = 0.004) and rFA of left genu of corpus callosum negatively correlated with log C18 level (β = −0.0103, p = 0.027). Conclusions: These results demonstrate that subcortical microstructural white matter disruption is associated with elevated serum ceramides and reduced manual dexterity in a population with cSVD. These findings suggest that injury to white matter tracts undermines neural networks, with functional consequences in a middle-aged population with cardiovascular risk factors.

Cognitive complications persist in antiretroviral therapy(ART)-treated people with HIV. However, the pattern and severity of domain-specific cognitive performance is variable and may be exacerbated by ART-mediated neurotoxicity. 929 women with HIV(WWH) from the Women's Interagency HIV Study who were classified into subgroups based on sociodemographic and longitudinal behavioral and clinical data using semi-parametric latent class trajectory modelling. Five subgroups were comprised of: 1) well-controlled HIV with vascular comorbidities(n = 116); 2) profound HIV legacy effects(CD4 nadir250 cells/μL; n = 275); 3) primarily45 year olds with hepatitis C(n = 165); 4) primarily 35-55 year olds(n = 244), and 5) poorly-controlled HIV/substance use(n = 129). Within each subgroup, we fitted a constrained continuation ratio model via penalized maximum likelihood to examine adjusted associations between recent ART agents and cognition. Most drugs were not associated with cognition. However, among the few drugs, non-nucleoside reverse transcriptase inhibitor (NNRTIs) and protease inhibitors(PIs) were most commonly associated with cognition, followed by nucleoside reverse transcriptase inhibitors(NRTIs) and integrase inhibitors(IIs). Directionality of ART-cognition associations varied by subgroup. Better psychomotor speed and fluency were associated with ART for women with well-controlled HIV with vascular comorbidities. This pattern contrasts women with profound HIV legacy effects for whom poorer executive function and fluency were associated with ART. Motor function was associated with ART for younger WWH and primarily 35-55 year olds. Memory was associated with ART only for women with poorly-controlled HIV/substance abuse. Findings demonstrate interindividual variability in ART-cognition associations among WWH and highlight the importance of considering sociodemographic, clinical, and behavioral factors as an underlying contributors to cognition. Are antiretroviral agents a risk factor for cognitive complications in women with HIV? We examind associations between ART-agents and cognitive function among similar subgroups of women with HIV from the Women's Interagency HIV study. The patterns of associations depended on sociodemographic, clinical, and behavioral characteristics of women.

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor in adults. Despite the multimodal standard treatments for GBM, the median survival is still about one year. Analysis of brain tissues from GBM patients shows that lipid droplets are highly enriched in tumor tissues while undetectable in normal brain tissues, yet the identity and functions of lipid species in GBM are not well understood. The aims of the present work are to determine how GBM utilizes fatty acids, and assess their roles in GBM proliferation. Treatment of U138 GBM cells with a monounsaturated fatty acid, oleic acid, induces accumulation of perilipin 2-coated lipid droplets containing triglycerides enriched in C18:1 fatty acid, and increases fatty acid oxidation. Interestingly, oleic acid also increases glucose utilization and proliferation of GBM cells. In contrast, pharmacologic inhibition of monoacylglycerol lipase attenuates GBM proliferation. Our findings demonstrate that monounsaturated fatty acids promote GBM proliferation via triglyceride metabolism, suggesting a novel lipid droplet-mediated pathway which may be targeted for GBM treatment.

The fungal isolate myriocin inhibits serine palmitoyltransferase and de novo ceramide synthesis in rodents; however, the effects of myriocin on ceramide concentrations and metabolism have not been previously investigated in ruminants. In our study, 12 non-lactating crossbred ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. Ewes consumed a high-energy diet from day 1 to 14 and were nutrient-restricted (straw only) from day 15 to 17. Blood was collected preprandial and at 1, 6, and 12 h relative to bolus and nutrient restriction. Tissues were collected following euthanasia on day 17. Plasma was analyzed for free fatty acids (FFAs), glucose, and insulin. Plasma and tissue ceramides were quantified using mass spectrometry. HIGH selectively decreased metabolizable energy intake, BW, and plasma insulin, and increased plasma FFA (Dose, P < 0.05). Myriocin linearly decreased plasma very-long-chain (VLC) ceramide and dihydroceramide (DHCer) by day 13 (Linear, P < 0.05). During nutrient restriction, fold-change in FFA was lower with increasing dose (P < 0.05). Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH (Dose × Time, P < 0.05). Myriocin linearly decreased most ceramide and DHCer species in the liver and omental and mesenteric adipose, VLC ceramide and DHCer in the pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue (Linear, P ≤ 0.05). We conclude that the intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h decreases circulating and tissue ceramide without modifying energy intake in ruminants.

The ability to generate human‐induced pluripotent stem cell (hiPSC)‐derived neural cells displaying region‐specific phenotypes is of particular interest for modeling central nervous system biology in vitro. We describe a unique method by which spinal cord hiPSC‐derived astrocytes (hiPSC‐A) are cultured with spinal cord hiPSC‐derived motor neurons (hiPSC‐MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC‐A enhance hiPSC‐MN electrophysiological maturation in a time‐dependent fashion. The sequence of plating, density, and age in which hiPSC‐A are cocultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to coculture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron crosstalk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity, which confirm hiPSC‐MN maturation. These findings are supported by immunocytochemistry and real‐time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and coculture with neurons influences astrocyte maturation as well. Taken together, these data indicate that in a human in vitro spinal cord culture system, astrocytes support hiPSC‐MN maturation in a time‐dependent and species‐specific manner and suggest a closer approximation of in vivo conditions. stem cells translational medicine 2019;8:1272&1285, We describe a fully human, spinal cord‐specific, coculture platform with human‐induced pluripotent stem cell‐derived motor neurons and astrocytes for multielectrode array recording. We show that human‐induced pluripotent stem cell‐derived motor neurons/human‐induced pluripotent stem cell‐derived astrocytes bidirectional morphological and molecular maturation is reflected by electrophysiological recordings with multielectrode array recording.

Extracellular vesicles have now emerged as key players in cell-to-cell communication. This is particularly important in the central nervous system, where glia-neuron cross-talk helps maintain normal neuronal function. Astrocyte-derived extracellular vesicles (ADEVs) secreted constitutively promote neurite outgrowth and neuronal survival. However, extracellular stimuli can alter the cargo and downstream functions of ADEVs. For example, ADEVs secreted in response to inflammation contain cargo microRNAs and proteins that reduce neurite outgrowth, neuronal firing, and promote neuronal apoptosis. We performed a comprehensive quantitative proteomic analysis to enumerate the proteomic cargo of ADEVs secreted in response to multiple stimuli. Rat primary astrocytes were stimulated with a trophic stimulus (adenosine triphosphate, ATP), an inflammatory stimulus (IL-1β) or an anti-inflammatory stimulus (IL10) and extracellular vesicles secreted within a 2 hr time frame were collected using sequential ultracentrifugation method. ADEVs secreted constitutively without exposure to any stimulus were used a control. A tandem mass tag-based proteomic platform was used to identify and quantify proteins in the ADEVs. Ingenuity pathway analysis was performed to predict the downstream signaling events regulated by ADEVs. We found that in response to ATP or IL10, ADEVs contain a set of proteins that are involved in increasing neurite outgrowth, dendritic branching, regulation of synaptic transmission, and promoting neuronal survival. In contrast, ADEVs secreted in response to IL-1β contain proteins that regulate peripheral immune response and immune cell trafficking to the central nervous system.

Supplemental Digital Content is available in the text, Objective: Almost half of HIV-positive people on antiretroviral therapy have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal insulin therapy improves cognition in Alzheimer's disease and diabetes. Here we tested intranasal insulin therapy in a model of HIV-NCI in EcoHIV-infected conventional mice. Design and methods: Insulin pharmacokinetics following intranasal administration to mice was determined by ELISA. Mice were inoculated with EcoHIV to cause NCI; 23 days or 3 months after infection they were treated daily for 9 days with intranasal insulin (2.4 IU/mouse) and examined for NCI in behavioral tests and HIV burdens by quantitative PCR. Some animals were tested for hippocampal neuronal integrity by immunostaining and expression of neuronal function-related genes by real time-quantitative PCR. The effect of insulin treatment discontinuation on cognition and neuropathology was also examined. Results: Intranasal insulin administration to mice resulted in μIU/ml levels of insulin in cerebrospinal fluid with a half-life of about 2 h, resembling pharmacokinetic parameters of patients receiving 40 IU. Intranasal insulin treatment starting 23 days or 3 months after infection completely reversed NCI in mice. Murine NCI correlated with reductions in hippocampal dendritic arbors and downregulation of neuronal function genes; intranasal insulin reversed these changes coincident with restoration of cognitive acuity, but they returned within 24 h of treatment cessation. Intranasal insulin treatment reduced brain HIV DNA when started 23 but not 90 days after infection. Conclusion: Our preclinical studies support the use of intranasal insulin administration for treatment of HIV-NCI and suggest that some dendritic injury in this condition is reversible.

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.

ObjectiveTo determine the association of insulin sensitivity and metabolic status with declining cognition in HIV-infected individuals.MethodsWe conducted targeted clinical and metabolic measures in longitudinal plasma samples obtained from HIV-infected patients enrolled in the Central Nervous System HIV Anti-Retroviral Therapy Effects Research Study (CHARTER). Findings were validated with plasma samples from the Multicenter AIDS Cohort Study (MACS). Patients were grouped according to longitudinally and serially assessed cognitive performance as having stably normal or declining cognition.ResultsPatients with declining cognition exhibited baseline hyperinsulinemia and elevated plasma c-peptide levels with normal c-peptide/insulin ratios, suggesting that insulin production was increased, but insulin clearance was normal. The association of hyperinsulinemia with worsening cognition was further supported by low high-density lipoprotein (HDL), high low-density lipoprotein/HDL ratio, and elevated cholesterol/HDL ratio compared to patients with stably normal cognition.ConclusionsThese findings suggest that hyperinsulinemia and impaired insulin sensitivity are associated with cognitive decline in antiretroviral therapy–treated HIV-infected patients.

Brain injury and inflammation induces a local release of extracellular vesicles (EVs) from astrocytes carrying proteins, RNAs, and microRNAs into the circulation. When these vesicles reach the liver, they stimulate the secretion of cytokines that mobilize peripheral immune cell infiltration into the brain, which can cause secondary tissue damage and impair recovery. Recent studies suggest that suppression of EV biosynthesis through neutral sphingomyelinase 2 (nSMase2) inhibition may represent a new therapeutic strategy. Unfortunately, currently available nSMase2 inhibitors exhibit low potency (IC50 ≥ 1 μM), poor solubility and/or limited brain penetration. Through a high throughput screening campaign of >365,000 compounds against human nSMase2 we identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), a potent (IC50 30 nM), selective, metabolically stable, and brain penetrable (AUCbrain/AUCplasma = 0.26) nSMase2 inhibitor. DPTIP dose-dependently inhibited EV release in primary astrocyte cultures. In a mouse model of brain injury conducted in GFAP-GFP mice, DPTIP potently (10 mg/kg IP) inhibited IL-1β-induced astrocyte-derived EV release (51 ± 13%; p in vitro or in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The discovery of novel biomarkers for peripartal diseases in dairy cows can improve our understanding of normal and dysfunctional metabolism, and lead to nutritional interventions that improve health and milk production. Our objectives were to characterize the plasma lipidome and identify metabolites associated with common markers of metabolic disease in peripartal dairy cattle. Multiparous Holstein cows (n = 27) were enrolled 30 d prior to expected parturition. Blood and liver samples were routinely collected through to d 14 postpartum. Untargeted lipidomics was performed using quadrupole time-of-flight mass spectrometry. Based on postpartum measures, cows were categorized into low or high total fatty acid area under the curve (total FAAUC, d 1–14 postpartum, 4915 ± 1369 vs. 12,501 ± 2761 (μmol/L × 14 d), n = 18), β-hydroxybutyrate AUC (BHBAAUC, 4583 ± 459 vs. 7901 ± 1206 (μmol/L × 14 d), n = 18), or liver lipid content (d 5 and 14 postpartum, 5 ± 1 vs. 12 ± 2% of wet weight, n = 18). Cows displayed decreases in plasma triacylglycerols and monoalkyl-diacylglycerols, and the majority of phospholipids reached a nadir at parturition. Phosphatidylcholines (PC) 32:3, 35:5, and 37:5 were specific for high total FAAUC, PC 31:3, 32:3, 35:5, and 37:5 were specific for high BHBAAUC, and PC 31:2, 31:3, and 32:3 were specific for high liver lipid content. PC 32:3 was specific for elevated total FA, BHBA, and liver lipid content. Lipidomics revealed a dynamic peripartal lipidome remodeling, and lipid markers associated with elevated total FA, BHBA, and liver lipid content. The effectiveness of nutrition to impact these lipid biomarkers for preventing excess lipolysis and fatty liver warrants evaluation.

Extracellular vesicles (EVs) are indispensable mediators of intercellular communication, but they can also assume a nefarious role by ferrying pathological cargo that contributes to neurological, oncological, inflammatory, and infectious diseases. The canonical pathway for generating EVs involves the endosomal sorting complexes required for transport (ESCRT) machinery, but an alternative pathway is induced by the enrichment of lipid membrane ceramides generated by neutral sphingomyelinase 2 (nSMase2). Inhibition of nSMase2 has become an attractive therapeutic strategy for inhibiting EV biogenesis, and a growing number of small-molecule nSMase2 inhibitors have shown promising therapeutic activity in preclinical disease models. This review outlines the function of EVs, their potential role in disease, the discovery and efficacy of nSMase2 inhibitors, and the path to translate these findings into therapeutics.

Objective: To test the hypothesis that increased ketone body production with a ketogenic diet (KD) will correlate with reductions in pro-inflammatory cytokines, lipid subspecies, and improved clinical outcomes in adults treated with an adjunctive ketogenic diet for super-refractory status epilepticus (SRSE). Methods: Adults (18 years or older) were treated with a 4:1 (fat:carbohydrate and protein) ratio enteral KD as adjunctive therapy to pharmacologic seizure suppression for SRSE. Blood and urine samples and clinical measurements were collected at baseline (n=10), after 1 week (n=8), and after 2 weeks of KD (n=5). Urine acetoacetate, serum β-hydroxybutyrate, lipidomics, pro-inflammatory cytokines (IL-1β, IL-6), chemokines (CCL3, CCL4, CXCL13), and clinical measurements were obtained at these 3 time points. Univariate and multivariate data analyses were performed to determine the correlation between ketone body production and circulating lipids, inflammatory biomarkers, and clinical outcome. Results: Changes in lipids included an increase in ceramides, mono-hexosyl ceramide, sphingomyelin, phosphocholine, and phosphoserines, and there was a significant reduction in IL-6 and CXCL13 seen at 1 and 2 weeks of KD. Higher blood β-hydroxybutyrate levels at baseline correlated with better clinical outcome however, ketone body production did not correlate with other variables during treatment. Higher pro-inflammatory chemokine CCL3 levels following treatment correlated with greater length of intensive care unit stay, higher modified Rankin Scale score (worse neurologic disability) at discharge and 6-month follow up. Conclusions: Adults receiving an adjunctive enteral ketogenic diet for super-refractory status epilepticus have alterations in select pro-inflammatory cytokines and lipid species that may predict response to treatment.

Chronic inflammation is thought to contribute to the early pathogenesis of Alzheimer's disease (AD). However, the precise mechanism by which inflammatory cytokines promote the formation and deposition of Aβ remains unclear. Available data suggest that applications of inflammatory cytokines onto isolated neurons do not promote the formation of Aβ, suggesting an indirect mechanism of action. Based on evidence astrocyte derived extracellular vesicles (astrocyte derived EVs) regulate neuronal functions, and data that inflammatory cytokines can modify the molecular cargo of astrocyte derived EVs, we sought to determine if IL‐1β promotes the formation of Aβ indirectly through actions of astrocyte derived EVs on neurons. The production of Aβ was increased when neurons were exposed to astrocyte derived EVs shed in response to IL‐1β (astrocyte derived EV‐IL‐1β). The mechanism for this effect involved an enrichment of Casein kinase 1 (CK1) in astrocyte derived EV‐IL‐1β. This astrocyte derived CK1 was delivered to neurons where it formed a complex with neuronal APC and GSK3 to inhibit the β‐catenin degradation. Stabilized β‐catenin translocated to the nucleus and bound to Hnrnpc gene at promoter regions. An increased cellular concentration of hnRNP C promoted the translation of APP by outcompeting the translational repressor fragile X mental retardation protein (FMRP) bound to APP mRNA. An increased amount of APP protein became co‐localized with BACE1 in enlarged membrane microdomains concurrent with increased production of Aβ. These findings identify a mechanism whereby inflammation promotes the formation of Aβ through the actions of astrocyte derived EV‐IL‐1β on neurons.

Choline and methionine may serve unique functions to alter hepatic energy metabolism. Our objective was to trace carbon flux through pathways of oxidation and glucose metabolism in bovine hepatocytes exposed to increasing concentrations of choline chloride (CC) and d,l-methionine (DLM). Primary hepatocytes were isolated from 4 Holstein calves and maintained for 24 h before treatment with CC (0, 10, 100, 1000 μmol/L) and DLM (0, 100, 300 μmol/L) in a factorial design. After 21 h, [1-14C]C16:0 or [2-14C]pyruvate was added to measure complete and incomplete oxidation, and cellular glycogen. Reactive oxygen species (ROS), cellular triglyceride (TG), and glucose and ß-hydroxybutyrate (BHB) export were quantified. Exported very-low density lipoprotein particles were isolated for untargeted lipidomics and to quantify TG. Interactions between CC and DLM, and contrasts for CC (0 vs. [10, 100, 1000 μmol/L] and linear and quadratic contrast 10, 100, 1000 μmol/L) and DLM (0 vs. [100, 300 μmol/L] and 100 vs. 300 μmol/L) were evaluated. Presence of CC increased complete oxidation of [1-14C]C16:0 and decreased BHB export. Glucose export was decreased, but cellular glycogen was increased by the presence of CC and increasing CC. Presence of CC decreased ROS and marginally decreased cellular TG. No interactions between CC and DLM were detected for these outcomes. These data suggest a hepato-protective role for CC to limit ROS and cellular TG accumulation, and to alter hepatic energy metabolism to support complete oxidation of FA and glycogen storage regardless of Met supply.

Cognitive impairment remains frequent and heterogeneous in presentation and severity among virally suppressed (VS) women with HIV (WWH). We identified cognitive profiles among 929 VS-WWH and 717 HIV-uninfected women from 11 Women's Interagency HIV Study sites at their first neuropsychological (NP) test battery completion comprised of: Hopkins Verbal Learning Test-Revised, Trail Making, Symbol Digit Modalities, Grooved Pegboard, Stroop, Letter/Animal Fluency, and Letter-Number Sequencing. Using 17 NP performance metrics (T-scores), we used Kohonen self-organizing maps to identify patterns of high-dimensional data by mapping participants to similar nodes based on T-scores and clustering those nodes. Among VS-WWH, nine clusters were identified (entropy = 0.990) with four having average T-scores ≥45 for all metrics and thus combined into an “unimpaired” profile (n = 311). Impaired profiles consisted of weaknesses in: (1) sequencing (Profile-1; n = 129), (2) speed (Profile-2; n = 144), (3) learning + recognition (Profile-3; n = 137), (4) learning + memory (Profile-4; n = 86), and (5) learning + processing speed + attention + executive function (Profile-5; n = 122). Sociodemographic, behavioral, and clinical variables differentiated profile membership using Random Forest models. The top 10 variables distinguishing the combined impaired vs. unimpaired profiles were: clinic site, age, education, race, illicit substance use, current and nadir CD4 count, duration of effective antiretrovirals, and protease inhibitor use. Additional variables differentiating each impaired from unimpaired profile included: depression, stress-symptoms, income (Profile-1); depression, employment (Profile 2); depression, integrase inhibitor (INSTI) use (Profile-3); employment, INSTI use, income, atazanavir use, non-ART medications with anticholinergic properties (Profile-4); and marijuana use (Profile-5). Findings highlight consideration of NP profile heterogeneity and potential modifiable factors contributing to impaired profiles.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). Results were compared with standard EV characterization techniques such as transmission electron microscopy (TEM) and Western blot (WB). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized per MISEV2018 guidelines. Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode and NFCM were able to detect at least two markers on the same particle. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and may contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In human ALS tissues and biofluids, we observe that higher levels of Cx43 correlate with accelerated disease progression. Using human iPSC-derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we show that Cx43 is upregulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We then demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes, during a specific temporal window, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Our data identify Cx43 hemichannels as novel conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

Background Increased plasma free fatty acids (FFAs) impair insulin sensitivity in dairy cows via unknown mechanisms. In nonruminants, saturated FFAs upregulate the hepatic synthesis and secretion of ceramide, which inhibits insulin action. Objective We aimed to determine whether an increase in plasma FFAs promotes hepatic and plasma ceramide accumulation in dairy cows. Methods Six nonpregnant, nonlactating Holstein cows were used in a study with a crossover design and treatments consisting of intravenous infusion of either saline (control) or triacylglycerol emulsion (TG; 20 g/h) for 16 h. The feeding level was set at 120% of energy requirements. Blood was collected at regular intervals and liver was biopsied at 16 h. Ceramides, monohexosylceramides (Glc/Gal-Cer), lactosylceramides (LacCer), and sphingomyelins (SMs) in plasma and liver were profiled. Hepatic expression of ceramide synthases was determined. Data were analyzed with the use of mixed models, regressions, and Spearman rank correlations. Results After 16 h of infusion, plasma FFA concentrations were >5-fold and liver triacylglycerol concentrations were 4-fold greater in TG cows, relative to control. Plasma total and very long-chain ceramide (e.g., C24:0-ceramide) concentrations increased ∼4-fold in TG over control by hour 16 of infusion, while C16:0-ceramide were not modified by TG. Infusion of TG increased plasma Glc/Gal-Cer (e.g., C16:0-Glc/Gal-Cer, 4-fold by hour 16) relative to control, but did not alter LacCer or SM concentrations. Hepatic ceramide concentrations increased with TG relative to control (e.g., C24:0-ceramide by 1.7-fold). Hepatic expression of ceramide synthase 2 was 60% greater after TG infusion compared with the control. Circulating ceramides were related to circulating FFA and hepatic triacylglycerol concentrations (e.g., C24:0-ceramide, ρ = 0.73 and 0.80, respectively; P Conclusion Hepatic ceramide synthesis is associated with elevations in circulating FFAs and hepatic triacylglycerol during the induction of hyperlipidemia in dairy cows. This work supports the emerging evidence for the role of ceramide during hepatic steatosis and insulin antagonism in cows.

Objective Identify metabolic changes produced by dimethyl fumarate (DMF) treatment and link them to immunological effects. Methods We enrolled 18 MS patients and obtained blood prior to DMF and 6 months postinitiation. We also enrolled 18 healthy controls for comparison. We performed global metabolomics on plasma and used weighted correlation network analysis (WGCNA) to identify modules of correlated metabolites. We identified modules that changed with treatment, followed by targeted metabolomics to corroborate changes identified in global analyses. We correlated changes in metabolite modules and individual metabolites with changes in immunological parameters. Results We identified alterations in lipid metabolism after DMF treatment – increases in two modules (phospholipids, lysophospholipids and plasmalogens) and reduction in one module (saturated and poly‐unsaturated fatty acids) eigen‐metabolite values (all P

Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants inPCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase α (CCTα), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences ofPCYT1Avariants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-editedPCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCTα steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). WhilePCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers or sizes as compared to controls. Lipid droplet phenotypes inPCYT1A-null HEK293 cells were rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His humanPCYT1AcDNAs. While both edited cellular models had normal morphology and proliferation rates compared to unedited controls,Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-200 lipid levels inPCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as compared to wild-type controls. The specific lipids altered and extent of change varied by cell type. Importantly, bothPCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting compensatory PC production through increased LPC remodeling by LPCAT or decreased conversion of PC to LPC by phospholipase A2. Our results show that all testedPCYT1Aalleles associated with SMD-CRD are hypomorphic and suggest involvement ofPCYT1Ain chondrocyte differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation.Author SummaryRare genetic disorders can reveal the function of genes on an organismal scale. When normal gene activity is lost, patients can experience a range of symptoms, often dependent on the residual activity of the encoded protein. Rare variants in the genePCYT1Acan cause multiple inherited disorders, including a disorder of the skeleton and the retina characterized by short stature, bone abnormalities, and blindness.PCYT1Ais required for normal cellular function, particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. To determine consequences of genetic variants in patients with this disorder, we made and studied a series of cellular models, including cells cultured from patients and CRISPR-edited cell lines lacking normal copies ofPCYT1A. Here we show that patient variants lead to reducedPCYT1Aexpression and/or function and have adverse consequences on cell biology and lipid metabolism that are often cell-type specific. This work advances understanding of the role of lipid metabolism in skeletal and eye development.

Astrocytes are known to be critical regulators of neuronal function. However, relatively few mediators of astrocyte to neuron communication have been identified. Recent advancements in the biology of extracellular vesicles have begun to implicate astrocyte derived extracellular vesicles (ADEV) as mediators of astrocyte to neuron communication, suggesting that alterations in the release and/or composition of ADEVs could influence gliotransmission. TNFα and IL-1β are key mediators of glial activation and neuronal damage, but the effects of these cytokines on the release or molecular composition of ADEVs is unknown. We found that ADEVs released in response to IL-1β (ADEV-IL-1β) and TNFα (ADEV-TNFα) were enriched with miRNAs that target proteins involved in neurotrophin signaling. We confirmed that miR-125a-5p and miR-16-5p (both enriched in ADEV-IL-1β and ADEV-TNFα) targeted NTKR3 and its downstream effector Bcl2. Downregulation of these targets in neurons was associated with reductions in dendritic growth, dendritic complexity, reduced spike rates, and burst activity. Molecular interference of miR-125a-5p and miR-16-5p prevented ADEV-IL-1β from reducing dendritic complexity, spike, and burst rates. These findings suggest that astrocytes respond to inflammatory challenge by modifying the miRNA cargo of ADEVs to diminish the activity of target neurons by regulating the translational expression of proteins controlling programs essential for synaptic stability and neuronal excitability.

At present, accurate and reliable biomarkers to ascertain the presence, severity, or prognosis of blast traumatic brain injury (bTBI) are lacking. There is an urgent need to establish accurate and reliable biomarkers capable of mbTBI detection. Currently, there are no studies that identify changes in miRNA and lipids at varied severities of bTBI. Various biological components such as lipids, circulating mRNA, and miRNA, could potentially be detected using advanced techniques such as next-generation sequencing and mass spectroscopy. Therefore, plasma analysis is an attractive approach with which to diagnose and treat brain injuries. Subacute changes in plasma microRNA (miRNA) and lipid composition for sphingolipids were evaluated in a murine model of mild-to-moderate bTBI using next-generation sequencing and mass spectroscopy respectively. Animals were exposed at 17, 17 × 3, and 20 psi blast intensities using a calibrated blast simulator. Plasma lipid profiling demonstrated decreased C18 fatty acid chains of sphingomyelins and increased ceramide levels when compared with controls. Plasma levels of brain-enriched miRNA, miR-127 were increased in all groups while let-7a, b, and g were reduced in the 17 × 3 and 20 psi groups, but let 7d was increased in the 17 psi group. The majority of the miRs and lipids are highly conserved across different species, making them attractive to explore and potentially employ as diagnostic markers. It is tempting to speculate that sphingolipids, miR-128, and the let-7 family could predict mTBI, while a combination of miR-484, miR-122, miR-148a, miR-130a, and miR-223 could be used to predict the overall status of injury following blast injury.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteins in 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 IC50. 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.