Sphingomyelinase Research Articles

SMPD4-mediated sphingolipid metabolism regulates brain and primary cilia development.

Genetic variants in multiple sphingolipid biosynthesis genes cause human brain disorders. A recent study looked at people from 12 unrelated families with variants in the gene SMPD4, a neutral sphingomyelinase that metabolizes sphingomyelin into ceramide at an early stage of the biosynthesis pathway. These individuals have severe developmental brain malformations, including microcephaly and cerebellar hypoplasia. The disease mechanism of SMPD4 was not known and so we pursued a new mouse model. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia, a crucial organelle mediating cellular signaling. We found that the mouse model has cerebellar hypoplasia due to failure of Purkinje cell development. Human induced pluripotent stem cells lacking SMPD4 exhibit neural progenitor cell death and have shortened primary cilia, which is rescued by adding exogenous ceramide. SMPD4 production of ceramide is crucial for human brain development.

Dual-Synergistic Nanomodulator Alleviates Exosomal PD-L1 Expression Enabling Exhausted Cytotoxic T Lymphocytes Rejuvenation for Potentiated iRFA-Treated Hepatocellular Carcinoma Immunotherapy.

The tumor immunosuppressive microenvironment (TME) induced by incomplete radiofrequency ablation (iRFA) in hepatocellular carcinoma (HCC) is a critical driver of tumor progression and metastasis. Herein, we proposed a therapeutic strategy aimed at remodeling the post-iRFA TME by targeting exosome biogenesis, secretion, and PD-L1 expression, thereby rejuvenating cytotoxic T lymphocyte function to mitigate the progression and metastasis of HCC. Leveraging the versatile properties of polydopamine nanomodulators, we have engineered a tailored delivery platform for GW4869 and amlodipine (AM), enabling precise and tumor-specific release of these therapeutic agents. Initially, GW4869, a neutral sphingomyelinase inhibitor, synergized with AM, an intracellular calcium modulator, to suppress exosome biogenesis and secretion. Subsequently, AM triggered the autophagic degradation of PD-L1. In vitro and in vivo experiments demonstrated that this synergistic approach significantly enhanced the robust activation and proliferation of various functional T-cell subsets following iRFA, particularly CD8+T cells, IFN-γ+ CD8+ cytotoxic T cells, natural killer cells, and innate lymphoid cells. Concurrently, it effectively reduced the infiltration of immunosuppressive cell types, including regulatory T cells and myeloid-derived suppressor cells. This favorable remodeling of the TME substantially inhibited the progression and metastasis of HCC post-iRFA. Collectively, our study presented a promising paradigm for enhancing HCC treatment efficacy by integrating radiofrequency ablation with advanced immune modulation strategies.

Challenging the conventional wisdom: Re-evaluating Smpd3's role in extracellular vesicle biogenesis.

Extracellular vesicles (EVs) are pivotal in intercellular communication, impacting diverse physiological and pathological processes. Current in vitro EV biogenesis studies often utilize pharmacological inhibitors, inducing off-target effects and overlooking cell-specific production nuances. Addressing these limitations, we utilized CRISPR/Cas9 to generate heterozygous full-body and conditional sphingomyelin phosphodiesterase 3 (Smpd3) knockout (KO) transgenic mice. Smpd3, also known as neutral sphingomyelinase 2 (nSMase2), triggers membrane curvature through sphingomyelin hydrolysis to ceramide, thereby influencing exosome release. Intriguingly, Smpd3 deficiency demonstrated no impact on EV release both in vitro and in vivo, underscoring its potential cell-type-specific role in EV biogenesis. Notably, bone marrow derived macrophages (BMDMs) did exhibit reduced EV release upon Alix deletion. Our findings open avenues for subsequent inquiries, enriching our knowledge of EV biogenesis and illuminating intercellular communication in health and disease.

The Effect of Competence, Discipline, and Work Motivation on Job Satisfaction and Affective Commitment of School Administration Staff (TAS) at SMA Se Sunggal District, Deli Serdang Regency

The research aims to analyze the effects of: (1) competence on job satisfaction; (2) discipline towards job satisfaction; (3) work motivation on job satisfaction; (4) competence towards affective commitment; (5) discipline towards affective commitment; (6) work motivation towards affective commitment; (7) job satisfaction with affective commitment; (8) job satisfaction in moderating competence towards affective commitment; (9) job satisfaction in moderating discipline towards affective commitment; and (10) job satisfaction in moderating work motivation towards affective commitment to School Administration Personnel in High Schools in Sunggal District. The research population is School Administration Personnel in high schools in Sunggal District with a sample of 136 people. The research method is a quantitative method with Smart-PLS analysis. The results of the study concluded: (1) the influence of competency variables on job satisfaction was 27.1%; (2) the influence of the discipline variable on job satisfaction was 26.5%; (3) the magnitude of the influence of the work motivation variable on job satisfaction was 34.8%; (4) the influence of competency variables on affective commitment is 12.5%; (5) the influence of the discipline variable on affective commitment was 71.4%; (6) the influence of work motivation variables on affective commitment is 26.5%; (7) the magnitude of the influence of the job satisfaction variable on affective commitment was 16.6%; (8) the influence of the competency variable on affective commitment with job satisfaction moderation was 11.8%; (9) the influence of the discipline variable on affective commitment with job satisfaction moderation was 11.7%; and (10) the influence of the work motivation variable on affective commitment with job satisfaction moderation was 12.3%. Overall, the results of the study showed that affective commitment was influenced by competence, discipline, and work motivation on job satisfaction by 74.0%; While the remaining 26.0% is determined by other factors.

Transcriptomics analysis reveals potential regulatory role of nSMase2 (Smpd3) in nervous system development and function of middle-aged mouse brains.

Neutral sphingomyelinase-2 (nSMase2), gene name sphingomyelin phosphodiesterase-3 (Smpd3), is a key regulatory enzyme responsible for generating the sphingolipid ceramide. The function of nSMase2 in the brain is still controversial. To better understand the functional roles of nSMase2 in the aging mouse brain, we applied RNA-seq analysis, which identified a total of 1462 differentially abundant mRNAs between +/fro and fro/fro, of which 891 were increased and 571 were decreased in nSMase2-deficient mouse brains. The most strongly enriched GO and KEGG annotation terms among transcripts increased in fro/fro mice included synaptogenesis, synapse development, synaptic signaling, axon development, and axonogenesis. Among decreased transcripts, enriched annotations included ribosome assembly and mitochondrial protein complex functions. KEGG analysis of decreased transcripts also revealed overrepresentation of annotations for Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington disease (HD). Ingenuity Pathway Analysis (IPA) tools predicted lower susceptibility to these neurodegenerative disorders, as well as predictions agreeing with stronger synaptic function, learning, and memory in fro/fro mice. The IPA tools identified signaling proteins, epigenetic regulators, and microRNAs as likely upstream regulators of the broader set of genes encoding the affected transcripts. It also revealed 16 gene networks, each linked to biological processes identified as overrepresented annotations among the affected transcripts by multiple analysis methods. Therefore, the analysis of these RNA-seq data indicates that nSMase2 impacts synaptic function and neural development, and may contribute to the onset and development of neurodegenerative diseases in middle-aged mice.

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.

Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

Ceramide-mediated orchestration of oxidative stress response through filopodia-derived small extracellular vesicles.

Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H2O2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia-derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopodia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress-induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S-palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD-ceramide. NBD-ceramide-labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclusion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide-rich sEVs that target mitochondria and propagate cell death.

Sphingolipid changes in mouse brain and plasma after mild traumatic brain injury at the acute phases

BackgroundTraumatic brain injury (TBI) causes neuroinflammation and can lead to long-term neurological dysfunction, even in cases of mild TBI (mTBI). Despite the substantial burden of this disease, the management of TBI is precluded by an incomplete understanding of its cellular mechanisms. Sphingolipids (SPL) and their metabolites have emerged as key orchestrators of biological processes related to tissue injury, neuroinflammation, and inflammation resolution. No study so far has investigated comprehensive sphingolipid profile changes immediately following TBI in animal models or human cases. In this study, sphingolipid metabolite composition was examined during the acute phases in brain tissue and plasma of mice following mTBI.MethodsWildtype mice were exposed to air-blast-mediated mTBI, with blast exposure set at 50-psi on the left cranium and 0-psi designated as Sham. Sphingolipid profile was analyzed in brain tissue and plasma during the acute phases of 1, 3, and 7 days post-TBI via liquid-chromatography-mass spectrometry. Simultaneously, gene expression of sphingolipid metabolic markers within brain tissue was analyzed using quantitative reverse transcription-polymerase chain reaction. Significance (P-values) was determined by non-parametric t-test (Mann–Whitney test) and by Tukey’s correction for multiple comparisons.ResultsIn post-TBI brain tissue, there was a significant elevation of 1) acid sphingomyelinase (aSMase) at 1- and 3-days, 2) neutral sphingomyelinase (nSMase) at 7-days, 3) ceramide-1-phosphate levels at 1 day, and 4) monohexosylceramide (MHC) and sphingosine at 7-days. Among individual species, the study found an increase in C18:0 and a decrease in C24:1 ceramides (Cer) at 1 day; an increase in C20:0 MHC at 3 days; decrease in MHC C18:0 and increase in MHC C24:1, sphingomyelins (SM) C18:0, and C24:0 at 7 days. Moreover, many sphingolipid metabolic genes were elevated at 1 day, followed by a reduction at 3 days and an absence at 7-days post-TBI. In post-TBI plasma, there was 1) a significant reduction in Cer and MHC C22:0, and an increase in MHC C16:0 at 1 day; 2) a very significant increase in long-chain Cer C24:1 accompanied by significant decreases in Cer C24:0 and C22:0 in MHC and SM at 3 days; and 3) a significant increase of C22:0 in all classes of SPL (Cer, MHC and SM) as well as a decrease in Cer C24:1, MHC C24:1 and MHC C24:0 at 7 days.ConclusionsAlterations in sphingolipid metabolite composition, particularly sphingomyelinases and short-chain ceramides, may contribute to the induction and regulation of neuroinflammatory events in the early stages of TBI, suggesting potential targets for novel diagnostic, prognostic, and therapeutic strategies in the future.

Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure.

Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

Activation of Piezo-1 Prevents Human Microvascular Endothelial Dysfunction During Acute Inhibition of Ceramide Formation

Despite the well-known damaging effects of elevated intracellular ceramides on human microvascular function, our recent work has shown that inhibition of ceramide formation through neutral sphingomyelinase (NSmase) promotes endothelial dysfunction in arterioles from healthy adults, manifested as endothelial production of hydrogen peroxide (H2O2), rather than nitric oxide (NO), in response to flow (flow-induced dilation; FID). Recent evidence suggests that ceramide prevents inactivation of Piezo-1, a shear-sensitive Ca2+ channel and inhibition of Piezo-1 reduces endothelial nitric oxide synthase (eNOS) activation in response to flow. Together, we hypothesized that ceramide-induced Piezo-1 activation is required to maintain NO-mediated FID in human microvessels and thus agonism of Piezo-1 during NSmase inhibition will prevent endothelial dysfunction. Arterioles (100-250μm) from healthy adults were dissected from discarded surgical adipose tissue. Vessels were cannulated in organ chambers, pre-constricted with endothelin-1, and changes in internal diameter in response to increases in flow were measured with videomicroscopy. Peroxy yellow 1 (PY1) fluorescent probe was used to assess endothelial H2O2 production in human umbilical vein endothelial cells (HUVECs). Mean% maximal dilation or fluorescent intensity±SE with two-way repeated measures ANOVA is reported. FID was preserved with Piezo-1 agonism (Yoda-1, 1μM, 30min) during NSmase inhibition (GW4869 4μM, 30min; 73±16.8, n=4 vs control 73±8.2, n=9; p>0.05), however FID was diminished in the presence of the eNOS inhibitor L-NAME (100μM; -2.6±11.3, n=5; p<0.05). A similar switch to NO-mediated FID was seen with C2-ceramide (5μM)+GW4869 (70.1±9.7 vs +L-NAME -3±9.7, n=4 both; p<0.05). NSmase inhibition increased H2O2 production in HUVECs (5.2±7.4 vs 83.0±31.6, n=6; p<0.05), an effect that was diminished with Yoda-1 (5.1±7.5, n=4; p<0.05) and C2-ceramide (10.7±9.2, n=5; p<0.05). These data suggest a role for Piezo-1 in NSmase-induced NO-signaling and provide mechanistic insight into how ceramide may exert beneficial effects within the human microvasculature. This research is supported by the National Institute of Health (NHLBI) R01HL160752 (JKF) and the American Heart Association (AHA) Predoctoral Fellowship 909315 (GSK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Astragaloside IV inhibits colorectal cancer metastasis by reducing extracellular vesicles release and suppressing M2-type TAMs activation

Ethnopharmacological relevanceTumour-derived extracellular vesicles (TEVs) have been confirmed to facilitate colorectal cancer (CRC) metastasis by remodelling the tumour microenvironment (TME). Drugs targeted TEVs is considered as a promising therapeutic strategy for cancer treatment. Traditional Chinese medicine (TCM) plays a vital role in improving the prognosis of CRC patients and eventually CRC patients with distant metastasis. Although the anti-tumour effects of active compounds from TCM prescriptions are observed widely, the molecular mechanisms remain unknown. Aim of the studyThis study aims to investigate the effects of active compounds in our library of TCM on preventing CRC metastasis, and also explore the potential mechanisms from the perspective of TEVs. Materials and methods: The effects of active compounds on the proliferation of CRC cells were determined by CCK-8 assay. TEVs were extracted from MC38 cells by ultracentrifugation and characterized by electron microscopy, Nanosight NS300 and western blotting. The TEV particles were quantified by Nanosight NS300. The potential mechanism by which astragaloside IV (ASIV) reduced TEV secretion was determined by western blotting. RAW264.7 cells were cocultured with the conditioned medium (CM) of MC38 cells treated with or without ASIV, and the activation of tumour-associated macrophages (TAMs) was assessed by immunofluorescence and quantitative polymerase chain reaction (qPCR). The migration of CRC cells was measured by wound healing and Transwell assay. A spleen-to-liver metastasis model of colorectal cancer was used to confirm the efficiency of ASIV in vivo. Liver metastatic tumours of the mice were used for liver weight measures and H&E staining. Immunofluorescence was applied to observe the infiltration of TAMs, the expression of neutral sphingomyelinase 2 (nSMase2) and Rab27a. ResultsBy screening our TCM monomer library, we found that ASIV, which is mainly extracted from Radix Astragali, reduced the release of TEVs from CRC cells in a time- and concentration-dependent manner. Mechanistically, ASIV inhibited the production and secretion of TEVs by downregulating nSMase2 and Rab27a expression in CRC cells. CM from ASIV-treated CRC cells reshaped the polarization of TAMs by decreasing M2-type polarization, increasing M1-type polarization. Consequently, the repolarization of M2-type to M1-type macrophages led to reduced invasion and migration of CRC cells. Moreover, we confirmed that ASIV inhibited the liver metastasis of CRC, reduced M2-type macrophage infiltration and decreased the expression of nSMase2 and Rab27a in liver metastases. ConclusionsASIV inhibited CRC metastasis by reducing EVs release and suppressing M2-type TAMs activation. All these findings reveal a new insight into the mechanisms of ASIV in preventing CRC progression and provide a promising approach for anti-tumour therapy.

Chronic Estrogen Treatment Promotes Human Microvascular Endothelial Dysfunction Through Sex-Specific Regulation of Ceramide Formation

Risk for cardiovascular disease (CVD) is elevated in individuals assigned female at birth and women of trans experience (assigned male at birth) undergoing long-term estrogen therapy (e.g. contraception, gender-affrmation). We have previously shown that chronic estrogen treatment induces microvascular endothelial dysfunction in human arterioles collected from those assigned male and female at birth. The mechanism via which estrogen causes microvascular damage remains unknown. Estrogen can increase expression of the ceramide-forming enzyme neutral sphingomyelinase (NSmase), and elevated ceramides promote cellular oxidative stress. Together, we hypothesize that estrogen induces microvascular endothelial dysfunction via NSmase-mediated ceramide formation. Human arterioles from otherwise healthy adults were treated with 17β-estradiol (E2; 100nM) +/- NSmase inhibitor (GW4869; 4μM, 24hrs). Vascular response to increasing flow rates was assessed using live videomicroscopy. Sex-specific human umbilical vein endothelial cells were treated with E2+/-GW4869 (48hrs), and H2O2 levels were measured using the fluorescent indicator peroxy yellow 1 (PY1). Endothelial ceramide levels were measured using liquid chromatography, tandem mass spectrometry. Exposure to estrogen impaired dilation to flow in arterioles from those assigned male and female at birth, an effect that was prevented by inhibition of NSmase (%Mean Dilation±SE; E2 vs E2+GW4869; male: 11.20±4.99% vs 48.43±7.15%, n=3 both, p=0.037; female: 1.78±1.99% vs 40.35±8.46%, n=5 both, p=0.006; two-way ANOVA). Estrogen treatment increased endothelial H2O2 production in cells from both sexes which was diminished by GW4869 treatment (%Change from control±SE; E2 vs E2+GW4869; male: 20.90±5.86% vs 2.85±1.99%, n=11 both, p=0.009*; female: 63.33±13.65%, n=10 vs 25.25±7.64%, n=9, p=0.031*, unpaired t-test*). Endothelial long-chain ceramide levels were higher in cells from biological males compared to females (pmol/nmol phosphate; male 5.90±2.19, n=4 vs female 2.18±0.86, n=5; p=0.009*). While estrogen treatment increased cellular ceramides in both sexes, ceramide accumulation was greater in cells from biological males compared to females (%change from baseline: 65.97±53.64% vs 6.08±7.86%, p=0.041*). Together, these results show that estrogen-induced microvascular endothelial dysfunction can be prevented through inhibition of NSmase-mediated ceramide formation. There is also notable sexual dimorphism in endothelial ceramide levels both at baseline and in response to estrogen. This study offers mechanistic insight into how chronic estrogen therapy may contribute to microvascular dysfunction and elevate the risk of CVD in patients. Furthermore, it presents the inhibition of NSmase as a plausible therapeutic approach to safeguard microvascular integrity during estrogen therapy. NHLBI R01HL160752 (J.K.F.), NHLBI K08HL141562 (J.K.F.), and AHA predoctoral fellowship grant 909315 (G.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neutral sphingomyelinase controls acute and chronic alcohol effects on brain activity

Alcohol consumption is a widespread phenomenon throughout the world. However, how recreational alcohol use evolves into alcohol use disorder (AUD) remains poorly understood. The Smpd3 gene and its coded protein neutral sphingomyelinase (NSM) are associated with alcohol consumption in humans and alcohol-related behaviors in mice, suggesting a potential role in this transition.Using multiparametric magnetic resonance imaging, we characterized the role of NSM in acute and chronic effects of alcohol on brain anatomy and function in female mice.Chronic voluntary alcohol consumption (16 vol% for at least 6 days) affected brain anatomy in WT mice, reducing regional structure volume predominantly in cortical regions. Attenuated NSM activity prevented these anatomical changes. Functional MRI linked these anatomical adaptations to functional changes: Chronic alcohol consumption in mice significantly modulated resting state functional connectivity (RS FC) in response to an acute ethanol challenge (i.p. bolus of 2 g kg−1) in heterozygous NSM knockout (Fro), but not in WT mice. Acute ethanol administration in alcohol-naïve WT mice significantly decreased RS FC in cortical and brainstem regions, a key finding that was amplified in Fro mice. Regarding direct pharmacological effects, acute ethanol administration increased the regional cerebral blood volume (rCBV) in many brain areas. Here, chronic alcohol consumption otherwise attenuated the acute rCBV response in WT mice but enhanced it in Fro mice. Altogether, these findings suggest a differential role for NSM in acute and chronic functional brain responses to alcohol. Therefore, targeting NSM may be useful in the prevention or treatment of AUD.

Soluble CD27 is an intrathecal biomarker of T-cell-mediated lesion activity in multiple sclerosis

ObjectiveSoluble CD27 is a promising cerebrospinal fluid inflammatory biomarker in multiple sclerosis. In this study, we investigate relevant immune and neuro-pathological features of soluble CD27 in multiple sclerosis.MethodsProtein levels of soluble CD27 were correlated to inflammatory cell subpopulations and inflammatory cytokines and chemokines detected in cerebrospinal fluid of 137 patients with multiple sclerosis and 47 patients with inflammatory and non-inflammatory neurological disease from three independent cohorts. Production of soluble CD27 was investigated in cell cultures of activated T and B cells and CD27-knockout T cells. In a study including matched cerebrospinal fluid and post-mortem brain tissues of patients with multiple sclerosis and control cases, levels of soluble CD27 were correlated with perivascular and meningeal infiltrates and with neuropathological features.ResultsWe demonstrate that soluble CD27 favours the differentiation of interferon-γ-producing T cells and is released through a secretory mechanism activated by TCR engagement and regulated by neutral sphingomyelinase. We also show that the levels of soluble CD27 correlate with the representation of inflammatory T cell subsets in the CSF of patients with relapsing-remitting multiple sclerosis and with the magnitude of perivascular and meningeal CD27 + CD4 + and CD8 + T cell infiltrates in post-mortem central nervous system tissue, defining a subgroup of patients with extensive active inflammatory lesions.InterpretationOur results demonstrate that soluble CD27 is a biomarker of disease activity, potentially informative for personalized treatment and monitoring of treatment outcomes.

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells.

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation.

Non-alcoholic fatty liver disease (NAFLD) is manifested by hepatic steatosis, insulin resistance, hepatocyte death, and systemic inflammation. Obesity induces steatosis and chronic inflammation in the liver. However, the precise mechanism underlying hepatic steatosis in the setting of obesity remains unclear. Here, we report studies that address this question. After 14 weeks on a high-fat diet (HFD) with high sucrose, C57BL/6 mice revealed a phenotype of liver steatosis. Transcriptional profiling analysis of the liver tissues was performed using RNA sequencing (RNA-seq). Our RNA-seq data revealed 692 differentially expressed genes involved in processes of lipid metabolism, oxidative stress, immune responses, and cell proliferation. Notably, the gene encoding neutral sphingomyelinase, SMPD3, was predominantly upregulated in the liver tissues of the mice displaying a phenotype of steatosis. Moreover, nSMase2 activity was elevated in these tissues of the liver. Pharmacological and genetic inhibition of nSMase2 prevented intracellular lipid accumulation and TNFα-induced inflammation in in-vitro HepG2-steatosis cellular model. Furthermore, nSMase2 inhibition ameliorates oxidative damage by rescuing PPARα and preventing cell death associated with high glucose/oleic acid-induced fat accumulation in HepG2 cells. Collectively, our findings highlight the prominent role of nSMase2 in hepatic steatosis, which could serve as a potential therapeutic target for NAFLD and other hepatic steatosis-linked disorders.

Exosome inhibition improves response to first-line therapy in small cell lung cancer.

Exosomes are recognized as important mediators of cell-to-cell communication, facilitating carcinogenesis. Although there have been significant advancements in exosome research in recent decades, no drugs that target the inhibition of sEV secretion have been approved for human use. For this study, we employed GW4869 and Nexinhib20 as inhibitors of exosome synthesis and trafficking combined. First, we found that Nexinhib20 and GW4869 effectively inhibited RAB27A and neutral sphingomyelinase 2 (nSMase2) nsMase2. Interestingly, the inhibition of nsMase2 and RAB27A decreased expression of CD9, CD63 and Tsg101, both at RNA and protein levels. We used a combination treatment strategy of cisplatin/etoposide plus GW4869 or Nexinhib20 on small cell lung cancer (SCLC) cell lines. The combination treatment of GW4869 or Nexinhib20 effectively enhanced the inhibitory effects of first-line chemotherapy on the SCLC cells. Furthermore, we demonstrated that reducing exosome release through GW4869 and Nexinhib20 treatment effectively reduced cellular proliferation and significantly induced apoptosis in SCLC cells. Also, we showed that combining exosome inhibition with chemotherapy has a significant synergistic effect on cellular proliferation. We also found increased p53 and p21 expressions with western blot and significantly changing Bax, BCL2, caspase-3 and caspase-9 expressions. Inhibiting the exosome pathway offers opportunities for developing novel, effective treatment strategies for SCLC.

Sex-specific pleiotropic changes in emotional behavior and alcohol consumption in human α-synuclein A53T transgenic mice during early adulthood.

Point mutations in the α-synuclein coding gene may lead to the development of Parkinson's disease (PD). PD is often accompanied by other psychiatric conditions, such as anxiety, depression, and drug use disorders, which typically emerge in adulthood. Some of these point mutations, such as SNCA and A30T, have been linked to behavioral effects that are not commonly associated with PD, especially regarding alcohol consumption patterns. In this study, we investigated whether the familial PD point mutation A53T is associated with changes in alcohol consumption behavior and emotional states at ages not yet characterized by α-synuclein accumulation. The affective and alcohol-drinking phenotypes remained unaltered in female PDGF-hA53T-synuclein-transgenic (A53T) mice during both early and late adulthood. Brain region-specific activation of ceramide-producing enzymes, acid sphingomyelinase (ASM), and neutral sphingomyelinase (NSM), known for their neuroprotective properties, was observed during early adulthood but not in late adulthood. In males, the A53T mutation was linked to a reduction in alcohol consumption in both early and late adulthood. However, male A53T mice displayed increased anxiety- and depression-like behaviors during both early and late adulthood. Enhanced ASM activity in the dorsal mesencephalon and ventral hippocampus may potentially contribute to these adverse behavioral effects of the mutation in males during late adulthood. In summary, the A53T gene mutation was associated with diverse changes in emotional states and alcohol consumption behavior long before the onset of PD, and these effects varied by sex. These alterations in behavior may be linked to changes in brain ceramide metabolism.

Sphingomyelinases as modulators of synaptic transmission

The brain has a high content of sphingomyelin, which is involved in the formation of plasma membranes and myelin, and is also an important for the organization of membrane microdomains (lipid rafts). Lipid rafts, as well as derivatives of sphingomyelin hydrolysis (ceramide, sphingosine, sphingosine-1-phosphate), are vital for synaptic transmission and its regulation. One of the main pathways to control the level of sphingomyelin and its derivatives is cleavage of membrane sphingomyelin by sphingomyelinases. Sphingomyelinases are localized inside the cell (in association with the plasma membrane, in lysosomes, endosomes, Golgi complex and endoplasmic reticulum) as well as can be secreted into the extracellular space. The levels and activity of sphingomyelinases significantly increase under the action of various stressful stimuli (including inflammation). At the same time, sphingomyelinase activity deficiency causes diseases with severe neurological manifestations. In the present review, we summarized the data on the currently known effects of acidic and neutral sphingomyelinases on pre- and postsynaptic processes, as well as about the synaptic localization of sphingomyelinases. In addition, a brief analysis of possible synaptic dysfunction due to hypo- or hyperfunction of sphingomyelinases in a number of neurological diseases is given. Thus, sphingomyelinases are considered as important modulators of synaptic transmission at the pre- and postsynaptic levels in normal and pathological conditions.