Acid sphingomyelinase inhibition induces cerebral angiogenesis post-ischemia/reperfusion in an oxidative stress-dependent way and promotes endothelial survival by regulating mitochondrial metabolism.

Acid sphingomyelinase (ASM) inhibitors are widely used for the treatment of post-stroke depression. They promote neurological recovery in animal stroke models via neurorestorative effects. In a previous study, we found that antidepressants including amitriptyline, fluoxetine, and desipramine increase cerebral angiogenesis post-ischemia/reperfusion (I/R) in an ASM-dependent way. To elucidate the underlying mechanisms, we investigated the effects of the functional ASM inhibitor amitriptyline in two models of I/R injury, that is, in human cerebral microvascular endothelial hCMEC/D3 cells exposed to oxygen-glucose deprivation and in mice exposed to middle cerebral artery occlusion (MCAO). In addition to our earlier studies, we now show that amitriptyline increased mitochondrial reactive oxygen species (ROS) formation in hCMEC/D3 cells and increased ROS formation in the vascular compartment of MCAO mice. ROS formation was instrumental for amitriptyline's angiogenic effects. ROS formation did not result in excessive endothelial injury. Instead, amitriptyline induced a profound metabolic reprogramming of endothelial cells that comprised reduced endothelial proliferation, reduced mitochondrial energy metabolism, reduced endoplasmic reticulum stress, increased autophagy/mitophagy, stimulation of antioxidant responses and inhibition of apoptotic cell death. Specifically, the antioxidant heme oxygenase-1, which was upregulated by amitriptyline, mediated amitriptyline's angiogenic effects. Thus, heme oxygenase-1 knockdown severely compromised angiogenesis and abolished amitriptyline's angiogenic responses. Our data demonstrate that ASM inhibition reregulates a complex network of metabolic and mitochondrial responses post-I/R that contribute to cerebral angiogenesis without compromising endothelial survival.

ASMase is Essential for the Immune Response to Partial-Tumor Radiation Exposure.

BACKGROUND/AIMS: Tumor response to radiation is thought to depend on the direct killing of tumor cells. Our laboratory has called this into question. Firstly, we showed that the biology of the host, specifically the endothelial expression of acid sphingomyelinase (ASMase), was critical in determining tumor radiocurability. Secondly, we have shown that the immune system can enhance radiation response by allowing a complete tumor control in hemi-irradiated tumors. In this paper, we focus on the integration of these two findings. METHODS: We used Lewis Lung Carcinoma (LLC) cells, injected in the flank of either: (i) ASMase knockout or (ii) WT of matched background (sv129xBl/6) or (iii) C57Bl/6 mice. Radiation therapy (RT) was delivered to 50% or 100% of the LLC tumor volume. Tumor response, immune infiltration (CD8+ T cells), ICAM-1, and STING activation were measured. Radiotherapy was also combined with methyl-cyclodextrin, to inhibit the ASMase-mediated formation of ceramide-enriched lipid rafts. RESULTS: We recapitulated our previous finding, namely that tumor hemi-irradiation was sufficient for tumor control in the LLC/C57Bl/6 model. However, in ASMase KO mice hemi-irradiation was ineffective. Likewise, pharmacological inhibition of ASMase significantly reduced the tumor response to hemi-irradiation. Further, we demonstrated elevated ICAM-1 expression, increased levels of CD8+ T cells, ICAM-1, and STING activation in tumors growing in C57Bl/6 mice, as well as the ASMase WT strain. However, no such changes were seen in tumors growing in ASMase KO mice. CONCLUSION: ASMase and ceramide generation are necessary to mediate a radiation-induced anti-tumor immune response via STING activation.

Novel insights into the application of recombinant mutated phospholipases D as antigens for developing new strategies against Loxoscelism.

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Brain Region-Specific Expression Levels of Synuclein Genes in an Acid Sphingomyelinase Knockout Mouse Model: Correlation with Depression-/Anxiety-Like Behavior and Locomotor Activity in the Absence of Genotypic Variation.

Accumulating evidence suggests an involvement of sphingolipids, vital components of cell membranes and regulators of cellular processes, in the pathophysiology of both Parkinson's disease and major depressive disorder, indicating a potential common pathway in these neuropsychiatric conditions. Based on this interaction of sphingolipids and synuclein proteins, we explored the gene expression patterns of α-, ß-, and γ-synuclein in a knockout mouse model deficient for acid sphingomyelinase (ASM), an enzyme catalyzing the hydrolysis of sphingomyelin to ceramide, and studied associations with behavioral parameters. Normalized Snca, Sncb, and Sncg gene expression was determined by quantitative PCR in twelve brain regions of sex-mixed homozygous (ASM-/-, n = 7) and heterozygous (ASM+/-, n = 7) ASM-deficient mice, along with wild-type controls (ASM+/+, n = 5). The expression of all three synuclein genes was brain region-specific but independent of ASM genotype, with ß-synuclein showing overall higher levels and the least variation. Moreover, we discovered correlations of gene expression levels between brain regions and depression- and anxiety-like behavior and locomotor activity, such as a positive association between Snca mRNA levels and locomotion. Our results suggest that the analysis of synuclein genes could be valuable in identifying biomarkers and comprehending the common pathological mechanisms underlying various neuropsychiatric disorders.

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.

Acid sphingomyelinase deficiency in France: a retrospective survival study.

BACKGROUND: Acid sphingomyelinase deficiency (ASMD) or Niemann-Pick disease types A, A/B, and B is a progressive, life-limiting, autosomal recessive disorder caused by sphingomyelin phosphodiesterase 1 (SMPD1) gene mutations. There is a need to increase the understanding of morbidity and mortality across children to adults diagnosed with ASMD. METHODS: This observational retrospective survey analysed medical records of patients with ASMD with retrievable data from 27 hospitals in France, diagnosed/followed up between 1st January 1990 and 31st December 2020. Eligible records were abstracted to collect demographic, medical/developmental history, and mortality data. Survival outcomes were estimated from birth until death using Kaplan-Meier survival analyses; standardised mortality ratio (SMR) was also explored. RESULTS: A total of 118 medical records of patients with ASMD (type B [n = 94], type A [n = 15], and type A/B [n = 9]) were assessed. The majority of patients were males (63.6%); the median [range] age at diagnosis was 8.0 [1.0-18.0] months (type A), 1.0 [0-3] year (type A/B), and 5.5 [0-73] years (type B). Overall, 30 patients were deceased at the study completion date; the median [range] age at death for patients with ASMD type A (n = 14) was 1 [0-3.6] year, type A/B (n = 6) was 8.5 [3.0-30.9] years, and type B (n = 10) was 57.6 [3.4-74.1] years. The median [95% confidence interval (CI)] survival age from birth in patients with ASMD type A and type A/B was 2.0 [1.8-2.7] years and 11.4 [5.5-18.5] years, respectively. Survival analysis in ASMD type B was explored using SMR [95% CI] analysis (3.5 [1.6-5.9]), which showed that age-specific deaths in the ASMD type B population were 3.5 times more frequent than those in the general French population. The causes of death were mostly severe progressive neurodegeneration (type A: 16.7%), cancer (type B: 16.7%), or unspecified (across groups: 33.3%). CONCLUSIONS: This study illustrated a substantial burden of illness with high mortality rates in patients with ASMD, including adults with ASMD type B, in France.

Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy.

Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.

Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention.

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency.

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of a childhood neurodegenerative disorder caused by AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that mouse models of AMPD2 deficiency exhibit predominant degeneration of the hippocampal dentate gyrus, despite a general reduction of brain GTP levels. Neurodegeneration-resistant regions accumulate micron-sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis, while these filaments are barely detectable in the hippocampal dentate gyrus. Furthermore, we show that IMPDH2 filament disassembly reduces GTP levels and impairs growth of neural progenitor cells derived from individuals with human AMPD2 deficiency. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation, opening the possibility of exploring the induction of IMPDH2 assembly as a therapy for neurodegeneration.

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 (150 nm) 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.

ASM is a therapeutic target in dermatomyositis by regulating the differentiation of naive CD4 + T cells into Th17 and Treg subsets.

BACKGROUND: This study aims to investigate the involvement of acid sphingomyelinase (ASM) in the pathology of dermatomyositis (DM), making it a potential therapeutic target for DM. METHODS: Patients with DM and healthy controls (HCs) were included to assess the serum level and activity of ASM, and to explore the associations between ASM and clinical indicators. Subsequently, a myositis mouse model was established using ASM gene knockout and wild-type mice to study the significant role of ASM in the pathology and to assess the treatment effect of amitriptyline, an ASM inhibitor. Additionally, we investigated the potential treatment mechanism by targeting ASM both in vivo and in vitro. RESULTS: A total of 58 DM patients along with 30 HCs were included. The ASM levels were found to be significantly higher in DM patients compared to HCs, with median (quartile) values of 2.63 (1.80-4.94) ng/mL and 1.64 (1.47-1.96) ng/mL respectively. The activity of ASM in the serum of DM patients was significantly higher than that in HCs. Furthermore, the serum levels of ASM showed correlations with disease activity and muscle enzyme levels. Knockout of ASM or treatment with amitriptyline improved the severity of the disease, rebalanced the CD4 T cell subsets Th17 and Treg, and reduced the production of their secreted cytokines. Subsequent investigations revealed that targeting ASM could regulate the expression of relevant transcription factors and key regulatory proteins. CONCLUSION: ASM is involved in the pathology of DM by regulating the differentiation of naive CD4 + T cells and can be a potential treatment target.

ß-Sitosterol Inhibits Tumor Growth and Amplifies Rituximab Sensitivity through Acid Sphingomyelinase/Ceramide Signaling in Diffuse Large B-Cell Lymphoma.

Rituximab (RTX) resistance is a notable challenge in treating diffuse large B-cell lymphoma (DLBCL). ß-Sitosterol (ß-ST) is a plant sterol that has been found in a broad variety of fruits, spices, and medicinal plants. The antineoplastic properties of ß-ST are established in various solid malignancies; however, its effect on DLBCL is uncharted. This study investigates the role of ß-ST in DLBCL as well as the underlying mechanisms. Our findings indicated that ß-ST impeded DLBCL cell proliferation in a concentration- and time-dependent manner. ß-ST appeared to alter sphingolipid metabolism, facilitate acid sphingomyelinase (ASM) translocation to the plasma membrane, augment ceramide platforms through increased ceramide synthesis, and consequently induce apoptosis in DLBCL cells. Furthermore, we found that RTX initiated both apoptotic and survival pathways in vitro, with the former contingent on the transient activation of the ASM, and ß-ST could amplify the anti-DLBCL efficacy of RTX by modulating ASM/Ceramide (Cer) signaling. Collectively, our findings elucidate the mechanistic role of ß-ST in DLBCL and underscore its potential in amplifying the antineoplastic efficacy of RTX via ASM activation, proposing a potential avenue to improve the efficacy of RTX therapy.

Development and Evaluation of a Noninvasive Microfluidic-Based Paper Analytical Device for Leptospirosis Diagnosis.

Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-µPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-µPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-µPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-µPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

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 5 min 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.

Saikosaponin antidepressant mechanism: Improving the sphingolipid metabolism in the cortex via Apolipoprotein E and triggering neurovascular coupling.

BACKGROUND: Since the pathogenesis of depression is complex, antidepressant therapy remains unsatisfactory. Recent evidence suggests a link between depression and lipid metabolism. Saikosaponin (SS) exhibits antidepression and lipid-regulating effects in modern pharmacology. However, it is unknown whether lipid regulation is the key mechanism of the SS antidepressant effect and how it works. PURPOSE: In this study, we investigated the relationship between the antidepressant activity of SS and the regulation of lipid metabolism and explored potential mechanisms. METHODS: APOE-/- mice, in combination with the chronic unpredictable mild stress (CUMS) model, were used to study the relationship between SS antidepressant activity and lipid metabolism through behavioral, electrophysiological techniques, and non-targeted lipidomics. Western blot, primary cell culture technology, and laser speckle cerebral blood flow imaging were employed to elucidate potential mechanisms. GraphPad Prism was used for statistical analysis, and p < 0.05 was considered statistically significant. RESULTS: APOE-/- mice exhibit more severe depressive-like behavior and dysregulation of sphingolipid metabolism in CUMS. SS alleviates depressive behavior and cortical sphingolipid metabolism disorder caused by CUMS, but has no effect on APOE-/- mice. SS alleviates the imbalance between ceramide (Cer) and sphingomyelin (SM) through acidic sphingomyelinase (AMSase). In addition, SS regulates neuronal glutamate release via sphingolipid metabolism, thereby alleviating the CUMS-induced inhibition of neurovascular coupling (regulates metabotropic glutamate receptor and IP3 receptor), which ameliorates the reduction of cerebral blood flow in depressed mice. CONCLUSION: Our study highlights the role of lipid metabolism in the antidepressant activity of SS and explores its underlying mechanisms. This study provided new insights into the better understanding of the antidepressant mechanisms of phytomedicine while increasing the possibility of lipid metabolism as a therapeutic strategy for depression.

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

BACKGROUND: Traumatic 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. METHODS: Wildtype 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. RESULTS: In 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. CONCLUSIONS: Alterations 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.

Discovery of a Novel ASM Direct Inhibitor with a 1,5-Diphenyl-pyrazole Scaffold and Its Antidepressant Mechanism of Action.

Multiple studies have confirmed that acid sphingomyelinase (ASM) activity is associated with depression. The discovery of direct inhibitors against ASM is of great significance for exploring antidepressants and their mechanisms of action. Herein, a series of novel phenylpyrazole analogues were rationally designed and synthesized. Among them, compound 46 exhibited potent inhibitory activity (IC50 = 0.87 µM) and good drug-like properties. In vivo studies demonstrated that compound 46 was involved in multiple antidepressant mechanisms of action, which were associated with a decline of ceramide, including increasing the Bcl-2/Bax ratio and BDNF expression, down-regulating caspase-3 and caspase-9, ameliorating oxidative stress, reducing the levels of proinflammatory cytokines such as TNF-α, IL-1ß, and IL-6, and elevating 5-HT levels in the brains of mice, respectively. These meaningful results reveal for the first time that direct inhibitors exhibit remarkable antidepressant effects in the CUMS-induced mouse model through multiple mechanisms of antidepressant action.

Natural history of acid sphingomyelinase deficiency among European patients during childhood and adolescence: A retrospective observational study.

Acid sphingomyelinase deficiency (ASMD) is a rare, lysosomal storage disease with limited evidence on its natural history. This retrospective, medical record abstraction study aimed to characterize the natural history of ASMD (types B and A/B) during childhood and adolescence. Recruiting sites were European centers (i.e., France, Germany, Italy, and the United Kingdom) from the ASCEND-Peds trial (NCT02292654); these sites were targeted because of the rarity of ASMD and specialized care provided at these centers. The study population comprised ASMD trial patients (before exposure to treatment) and ASMD non-trial participants who were managed at the same trial sites. Overall, 18 patients were included (11 trials; 7 non-trials; median [Q1; Q3] age at ASMD diagnosis: 2.5 [1.0; 4.0] years). Median follow-up duration was 10.0 years. Frequently reported medical conditions were hepatobiliary (17 [94.4%]) and blood and lymphatic system disorders (16 [88.9%]). Adenoidectomy (3 [16.7%]) was the most commonly reported surgical procedure; gastroenteritis (5 [27.8%]) was the most frequently reported infection, and epistaxis (6 [33.3%]) was the most commonly reported bleeding event. Abnormal spleen (16 [88.9%]) and liver (15 [83.3%]) size and respiratory function (8 [44.4%]) were commonly reported during physical examination. Overall, 11 (61.1%) patients were hospitalized; 6 (33.3%) patients had emergency room visits. Findings were consistent with published literature and support the current understanding of natural history of ASMD.

ASM variants in the spotlight: A structure-based atlas for unraveling pathogenic mechanisms in lysosomal acid sphingomyelinase.

Lysosomal acid sphingomyelinase (ASM), a critical enzyme in lipid metabolism encoded by the SMPD1 gene, plays a crucial role in sphingomyelin hydrolysis in lysosomes. ASM deficiency leads to acid sphingomyelinase deficiency, a rare genetic disorder with diverse clinical manifestations, and the protein can be found mutated in other diseases. We employed a structure-based framework to comprehensively understand the functional implications of ASM variants, integrating pathogenicity predictions with molecular insights derived from a molecular dynamics simulation in a lysosomal membrane environment. Our analysis, encompassing over 400 variants, establishes a structural atlas of missense variants of lysosomal ASM, associating mechanistic indicators with pathogenic potential. Our study highlights variants that influence structural stability or exert local and long-range effects at functional sites. To validate our predictions, we compared them to available experimental data on residual catalytic activity in 135 ASM variants. Notably, our findings also suggest applications of the resulting data for identifying cases suited for enzyme replacement therapy. This comprehensive approach enhances the understanding of ASM variants and provides valuable insights for potential therapeutic interventions.

Whole-exome-based single nucleotide variants and copy number analysis for prenatal diagnosis of compound heterozygosity of SMPD4.

BACKGROUND: Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder. These variants have been identified in a group of children with neurodevelopmental disorders with microcephaly, arthrogryposis, and structural brain anomalies. SMPD4 encodes a sphingomyelinase that hydrolyzes sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes. MATERIALS AND METHODS: For the efficient prenatal diagnosis of rare and undiagnosed diseases, the parallel detection of copy number variants (CNVs) and single nucleotide variants using whole-exome analysis is required. A physical examination of the parents was performed. Karyotype and whole-exome analysis were performed for the fetus and the parents. RESULTS: A fetus with microcephaly and arthrogryposis; biallelic null variants (c.387-1G>A; Chr2[GRCh38]: g.130142742_130202459del) were detected by whole-exome sequencing (WES). We have reported for the first time the biallelic loss-of-function mutations in SMPD4 in patients born to unrelated parents in China. CONCLUSION: WES could replace chromosomal microarray analysis and copy number variation sequencing as a more cost-effective genetic test for detecting CNVs and diagnosing highly heterogeneous conditions.