Acid Sphingomyelinase Activity Research Articles

Adult niemann-pick disease: A case report

Adult niemann-pick disease: A case report

Mitochondrial ALDH2 improves ß-cell survival and function against doxorubicin-induced apoptosis by targeting CK2 signaling.

The aim of this study was to better understand how the chemotherapy drug doxorubicin contributes to the development of β-cell dysfunction and to explore its relationship with mitochondrial aldehyde dehydrogenase-2 (ALDH2). In order to investigate this hypothesis, doxorubicin was administered to INS-1 cells, a rat insulinoma cell line, either with or without several target protein activators and inhibitors. ALDH2 activity was detected with a commercial kit and protein levels were determined with western blot. Mitochondrial ROS, membrane potential, and lipid ROS were determined by commercial fluorescent probes. The cell viability was measured by CCK-assay. Exposure of INS-1 cells to doxorubicin decreased active insulin signaling resulting in elevated ALDH2 degradation, compared with control cells by the induction of acid sphingomyelinase mediated ceramide induction. Further, ceramide induction potentiated doxorubicin induced mitochondrial dysfunction. Treatment with the ALDH2 agonist, ALDA1, blocked doxorubicin-induced acid sphingomyelinase activation which significantly blocked ceramide induction and mitochondrial dysfunction mediated cell death. Treatment with the ALDH2 agonist, ALDA1, stimulated casein kinase-2 (CK2) mediated insulin signaling activation. CK2 silencing neutralized the function of ALDH2 in the doxorubicin treated INS-1 cells. Mitochondrial ALDH2 activation could inhibit the progression of doxorubicin induced pancreatic β-cell dysfunction by inhibiting the acid sphingomyelinase induction of ceramide, by regulating the activation of CK2 signaling. Our research lays the foundation of ALDH2 activation as a therapeutic target for the precise treatment of chemotherapy drug induced β-cell dysfunction.

Contribution of Gasdermin D Pore Formation to NLRP3 Inflammasome Product Secretion and Pyroptosis in Podocytes during Obesity-Induced Glomerulopathy

NLRP3 inflammasome activation in podocytes has been reported to play an important role in the development of glomerular inflammation and sclerosis during obesity. Although, the molecular mechanism mediating the secretion of NLRP3 inflammasome products to induce local inflammatory response remains poorly understood. Given that gasdermin D (GSDMD) has been found to form oligomeric pores on plasma membrane for release of inflammasome product and execution of pyroptosis, the present study tested whether GSDMD pore contributes to NLRP3 inflammasome product secretion and pyroptosis in podocytes and thereby initiates glomerular inflammation and sclerosis in obesity-induced glomerulopathy (ORG). By Western blot analysis, we demonstrated that palmitic acid (PA) as an obesity-related danger factor dose-dependently stimulated the reduction of total GSDMD and elevation of cleaved GSDMD (GSDMD-NT) in podocytes. In vivo, glomerular decrease in total GSDMD and increase in GSDMD-NT were remarkable in wild type (WT/WT) mice on high-fat diet (HFD) compared to control mice. Urinary excretion of NLRP3 inflammasome products was also significantly increased by HFD treatment. Moreover, we found that pre-treatment with amitriptyline or MCC950 blocked NLRP3 inflammasome activation as well as GSDMD pore formation on plasma membrane of podocytes, while disulfiram prevented GSDMD pore formation without affecting NLRP3 inflammasome activation. Since acid sphingomyelinase (ASM)-ceramide signaling pathway plays a vital role in the development of ORG, we examined whether ASM activity determines GSDMD pore formation in podocytes. By atom force microscopy, a 3D topography map of a single podocyte was generated, and pore-like structures were observed in the membrane of PA-treated WT/WT podocytes. Podocytes lacking Smpd1 (gene code of ASM) gene did not show pore formation, but Smpd1 gene overexpression amplified PA-induced pore formation in podocytes. Moreover, it was found that HFD-induced GSDMD cleavage and pyroptosis in glomeruli were blocked by Smpd1 gene deletion in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression amplified GSDMD-NT production and pyroptosis in glomeruli. Correspondingly, HFD-induced podocyte foot process effacement and glomerular sclerosis in mice were inhibited by Smpd1 gene knockout but exaggerated by podocyte-specific Smpd1 gene overexpression. Taken together, our findings suggest that ASM-ceramide signaling pathway may control GSDMD pore formation in podocytes to determine glomerular inflammation and sclerosis in ORG. This study was supported by NIH grants DK054927 and DK120491. 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.

Gaucher Disease or Acid Sphingomyelinase Deficiency? The Importance of Differential Diagnosis.

Background: Gaucher disease is a lysosomal storage disorder caused by functional glucocerebrosidase enzyme deficiency. Hepatosplenomegaly and hematological complications are found in both Gaucher disease and Acid Sphingomyelinase Deficiency, which is caused by acid sphingomyelinase dysfunction. The possible overlap in clinical presentation can cause diagnostic errors in differential diagnosis. For this reason, in patients with an initial clinical suspicion of Gaucher disease, we aimed to carry out a parallel screening of acid sphingomyelinase and glucocerebrosidase. Methods: Peripheral blood samples of 627 patients were collected, and enzymatic activity analysis was performed on both glucocerebrosidase and acid sphingomyelinase. The specific gene was studied in samples with null or reduced enzymatic activity. Specific molecular biomarkers helped to achieve the correct diagnosis. Results: In 98.7% of patients, normal values of glucocerebrosidase activity excluded Gaucher disease. In 8 of 627 patients (1.3%), the glucocerebrosidase enzymatic activity assay was below the normal range, so genetic GBA1 analysis confirmed the enzymatic defect. Three patients (0.5%) had normal glucocerebrosidase activity, so they were not affected by Gaucher disease, and showed decreased acid sphingomyelinase activity. SMPD1 gene mutations responsible for Acid Sphingomyelinase Deficiency were found. The levels of specific biomarkers found in these patients further strengthened the genetic data. Conclusions: Our results suggest that in the presence of typical signs and symptoms of Gaucher disease, Acid Sphingomyelinase Deficiency should be considered. For this reason, the presence of hepatosplenomegaly, thrombocytopenia, leukocytopenia, and anemia should alert clinicians to analyze both enzymes by a combined screening. Today, enzyme replacement therapy is available for the treatment of both pathologies; therefore, prompt diagnosis is essential for patients to start accurate treatment and to avoid diagnostic delay.

The Genetic Basis, Lung Involvement, and Therapeutic Options in Niemann-Pick Disease: A Comprehensive Review.

Niemann-Pick Disease (NPD) is a rare autosomal recessive disease belonging to lysosomal storage disorders. Three types of NPD have been described: NPD type A, B, and C. NPD type A and B are caused by mutations in the gene SMPD1 coding for sphingomyelin phosphodiesterase 1, with a consequent lack of acid sphingomyelinase activity. These diseases have been thus classified as acid sphingomyelinase deficiencies (ASMDs). NPD type C is a neurologic disorder due to mutations in the genes NPC1 or NPC2, causing a defect of cholesterol trafficking and esterification. Although all three types of NPD can manifest with pulmonary involvement, lung disease occurs more frequently in NPD type B, typically with interstitial lung disease, recurrent pulmonary infections, and respiratory failure. In this sense, bronchoscopy with broncho-alveolar lavage or biopsy together with high-resolution computed tomography are fundamental diagnostic tools. Although several efforts have been made to find an effective therapy for NPD, to date, only limited therapeutic options are available. Enzyme replacement therapy with Olipudase α is the first and only approved disease-modifying therapy for patients with ASMD. A lung transplant and hematopoietic stem cell transplantation are also described for ASMD in the literature. The only approved disease-modifying therapy in NPD type C is miglustat, a substrate-reduction treatment. The aim of this review was to delineate a state of the art on the genetic basis and lung involvement in NPD, focusing on clinical manifestations, radiologic and histopathologic characteristics of the disease, and available therapeutic options, with a gaze on future therapeutic strategies.

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.

Mouse Model of Nitrogen Mustard Ocular Surface Injury Characterization and Sphingolipid Signaling.

Vesicating chemicals like sulfur mustard (SM) or nitrogen mustard (NM) can cause devastating damage to the eyes, skin, and lungs. Eyes, being the most sensitive, have complicated pathologies that can manifest immediately after exposure (acute) and last for years (chronic). No FDA-approved drug is available to be used as medical counter measures (MCMs) against such injuries. Understanding the pathological mechanisms in acute and chronic response of the eye is essential for developing effective MCMs. Here, we report the clinical and histopathological characterization of a mouse model of NM-induced ocular surface injury (entire surface) developed by treating the eye with 2% (w/v) NM solution for 5 min. Unlike the existing models of specific injury, our model showed severe ocular inflammation, including the eyelids, structural deformity of the corneal epithelium and stroma, and diminished visual and retinal functions. We also observed alterations of the inflammatory markers and their expression at different phases of the injury, along with an activation of acidic sphingomyelinase (aSMase), causing an increase in bioactive sphingolipid ceramide and a reduction in sphingomyelin levels. This novel ocular surface mouse model recapitulated the injuries reported in human, rabbit, and murine SM or NM injury models. NM exposure of the entire ocular surface in mice, which is similar to accidental or deliberate exposure in humans, showed severe ocular inflammation and caused irreversible alterations to the corneal structure and significant vision loss. It also showed an intricate interplay between inflammatory markers over the injury period and alteration in sphingolipid homeostasis in the early acute phase.

Desipramine ameliorates fine particulate matter-induced hepatic insulin resistance by modulating the ceramide metabolism in mice

Recent research has highlighted a correlation between exposure to ambient fine particulate matter (PM2.5) and the development of systemic insulin resistance (IR) along with an elevated risk of diabetes. Ceramide has emerged as one of the pathogenic mechanisms contributing to IR. The inhibition of acid sphingomyelinase (ASMase) activity by desipramine (DES) has been shown to effectively reduce ceramide levels. In the present study, 24 female C57BL/6 N mice were randomized into one of the four groups: the filtered air exposure (FA) group, the concentrated PM2.5 exposure (PM) group, the concentrated PM2.5 treated with low-dose DES (DL) group, and the concentrated PM2.5 treated with high-dose DES (DH) group. The PM, DL and DH groups were exposed to PM2.5 for an 8-week period within a whole-body exposure system. The study encompassed extensive examinations of glucose homeostasis, liver lipid profile, ceramide pathway, and insulin signaling pathway. Our results demonstrated that PM2.5 exposure caused impaired glucose tolerance, elevated ceramide levels, increased phosphorylation PP2A, reduced Akt phosphorylation, and hindered GLUT2 expression. Remarkably, DES administration mitigated PM2.5-induced IR by effectively lowering ceramide levels. In conclusion, the reduction of ceramide levels by DES may be a promising therapeutic strategy for coping PM2.5-induced IR.

Effects of Nutraceuticals on Cisplatin-Induced Cytotoxicity in HEI-OC1 Cells.

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo®) or hydrogen sulfide (H2S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H2S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Identification of a Novel Acid Sphingomyelinase Activity Associated with Recombinant Human Acid Ceramidase.

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the accumulation of ceramides and other sphingolipids in various cells and tissues. In addition to ceramide hydrolysis, several other activities have been attributed to AC, including a reverse reaction that synthesizes ceramide from free fatty acids and sphingosine, and a deacylase activity that removes fatty acids from complex lipids such as sphingomyelin and glycosphingolipids. A close association of AC with another important enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM), has also been observed. Herein, we used a highly purified recombinant human AC (rhAC) and novel UPLC-based assay methods to investigate the recently described deacylase activity of rhAC against three sphingolipid substrates, sphingomyelin, galactosyl- and glucosylceramide. No deacylase activities were detected using this method, although we did unexpectedly identify a significant ASM activity using natural (C-18) and artificial (Bodipy-C12) sphingomyelin substrates as well as the ASM-specific fluorogenic substrate, hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (HMU-PC). We showed that this ASM activity was not due to contaminating, hamster-derived ASM in the rhAC preparation, and that the treatment of ASM-knockout mice with rhAC significantly reduced sphingomyelin storage in the liver. However, unlike the treatment with rhASM, this did not lead to elevated ceramide or sphingosine levels.

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Niemann-Pick Disease With Bilateral Adrenal Mass

Abstract Niemann–Pick disease (NPD) is a heterogeneous group of lysosomal storage disorders with autosomal recessive inheritance pattern. There are 4 types of NPD. Patients with NPD type B generally have better prognosis, allowing them to survive into adulthood. They have a widespread clinical presentation, affecting multiple organs but rarely neurological involvement. Here, we describe the case of a 38-year-old woman with unexplained hepatosplenomegaly and young hypertensive intracranial bleed. Evaluation for young hypertension further revealed a nonfunctional bilateral adrenal mass and gross hepatosplenomegaly. NPD was confirmed through dried blood spot investigation. It showed low activity of acid sphingomyelinase and genetic testing also detected 2 pathogenic mutations. She is being managed by a multidisciplinary team for supportive treatment that includes regular symptoms monitoring and genetic counseling.

Factor VIIa releases phosphatidylserine-enriched extracellular vesicles from endothelial cells by activating acid sphingomyelinase

BackgroundOur recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched with phosphatidylserine (PS) and contribute to the hemostatic effect of FVIIa in thrombocytopenia and hemophilia. ObjectiveTo investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS. MethodsFVIIa activation of acid sphingomyelinase (aSMase) was evaluated by its translocation to the cell surface. The role of aSMase in the biogenesis of FVIIa-induced EVs and their enrichment with PS was investigated using specific siRNAs and inhibitors of aSMase and its downstream metabolites. Wild-type and aSMase-/- mice were injected with a control vehicle or FVIIa. EVs released into circulation were quantified by nanoparticle tracking analysis. EVs hemostatic potential was assessed in a murine thrombocytopenia model. ResultsFVIIa activation of aSMase is responsible for both the externalization of PS and the release of EVs in endothelial cells. FVIIa-induced aSMase activation led to ceramide generation and de novo expression of transmembrane protein 16F. Inhibitors of ceramidases, sphingosine kinase, or sphingosine-1-phosphate receptor modulator blocked FVIIa-induced expression of transmembrane protein 16F and PS externalization without interfering with FVIIa release of EVs. In vivo, FVIIa release of EVs was markedly impaired in aSMase-/- mice compared with wild-type mice. Administration of a low dose of FVIIa, sufficient to induce EVs release, corrected bleeding associated with thrombocytopenia in wild-type mice but not in aSMase-/- mice. ConclusionOur study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.

Acid sphingomyelinase mediates ferroptosis induced by high glucose via autophagic degradation of GPX4 in type 2 diabetic osteoporosis

BackgroundFerroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), although the specific underlying mechanisms remain largely unknown. This study aimed to clarify the role and possible mechanism of acid sphingomyelinase (ASM)-mediated osteoblast ferroptosis in T2DOP.MethodsWe treated hFob1.19 cells with normal glucose (NG) and different concentrations of high glucose (HG, 26.25 mM, 35 mM, or 43.75 mM) for 48 h. We then measured cell viability and osteogenic function, quantified ferroptosis and autophagy levels, and measured the levels of ASM and ceramide in the cells. To further investigate the specific mechanism, we examined these indicators by knocking down ASM expression, hydroxychloroquine (HCQ) treatment, or N-acetylcysteine (NAC) treatment. Moreover, a T2DOP rat model was induced and microcomputed tomography was used to observe the bone microstructure. We also evaluated the serum levels of iron metabolism-associated factors, ceramide and lipid peroxidation (LPO) and measured the expression of ASM, LC3 and GPX4 in bone tissues.ResultsHG inhibited the viability and osteogenic function of osteoblasts by inducing ferroptosis in a concentration-dependent manner. Furthermore, the expression of ASM and ceramide and autophagy levels were increased by HG treatment, and these factors were required for the HG-induced reactive oxygen species (ROS) generation and LPO. Similarly, inhibiting intracellular ROS also reduced HG-induced ASM activation and autophagy. ASM-mediated activation of autophagy was crucial for HG-induced degradation of GPX4, and inhibiting ASM improved osteogenic function by decreasing HG-induced autophagy, GPX4 degradation, LPO and subsequent ferroptosis. We also found that inhibiting ASM could alleviated ferroptosis and autophagy and improved osteogenic function in a T2DOP rat model.ConclusionASM-mediated autophagy activation induces osteoblast ferroptosis under HG conditions through the degradation of GPX4, providing a novel mechanistic insight into the treatment and prevention of T2DOP.

Ultrasound-stimulated microbubbles enhancement of fractionated radiation for tumor treatment.

Radiation therapy (XRT) causes numerous biological changes in tumor microenvironment. Radiation vascular response, due to endothelial disruption, can influence treatment outcomes in a dose-dependent manner. Ultrasound-stimulated microbubbles (USMB) have also been demonstrated to create a vascular response in the tumor microenvironment and enhance tumor response when used in combination with XRT. Single doses of 8-10Gy are known to induce activation of acid sphingomyelinase (ASMase)-induced ceramide production, causing vascular damage. Destruction of vasculature results in endothelial apoptosis followed by tumor cell death. The effect of tumor response is known to be synergistic by 10-fold higher cell kill observed when USMB is combined with radiation. In this study, we used an USMB approach in combination with conventional low dose fractionated radiation to enhance endothelial cell responses to XRT in human PC3 prostate cancer xenograft model. Mice were divided into untreated, USMB therapy, fractionated XRT, and combined USMB therapy followed by XRT (USMB + XRT) groups. USMB therapy was delivered twice per week in the USMB-alone and combined USMB + XRT treatment groups over four weeks. Radiation treatments were delivered in fractions of 2Gy/day (total 40Gy in 20 fractions, BED10 = 48Gy) in the XRT-alone and combined USMB + XRT groups. The treatment outcome was evaluated using histopathology, power Doppler, and immunohistochemistry assays. Tumor growth assessment showed that sizes of tumors increased in the control and the single treatment groups over a treatment period of four weeks, but significantly decreased with the combined treatments of USMB + XRT. Immunohistochemical analysis indicated a statistically significant vascular disruption in mice that received treatment involving a full 4-week schedule of combined (USMB + XRT) treatments. A statistically significant increase in vascular disruption was demonstrated through CD68 and trichrome fibrosis staining. Changes in local perfusion assessed using high-frequency power Doppler imaging demonstrated attenuated blood flow in the combined group. This work demonstrates the efficacy of using USMB as a radiation sensitizer in a mouse model of human PC3 tumor xenograft. This radiation treatment enhancement modality has the advantage of targeting tumor vasculature with ultrasound stimulation that can be implemented prior to radiation treatment.

LRRK2 exonic variants are associated with lysosomal hydrolase activities and lysosphingolipid alterations in Parkinson's disease

Last data demonstrated that exonic variants of LRRK2 (p.G2019S, p.M1646T) may affect the catalytic activity of lysosomal enzyme glucocerebrosidase (GCase) probably through the phosphorylation of Rab10 protein. We aimed to evaluate an association of LRRK2 exonic variants previously associated with alteration of phosphorylation levels for Rab10Thr73 with PD risk in Russian population and analyze an impact of p.G2019S mutation and selected LRRK2 variants on lysosomal hydrolase activities. LRRK2 variants were determined by full sequencing of LRRK2 in 508 PD patients and 470 controls from Russian population. Activity of lysosomal enzymes (glucocerebrosidase (GCase), alpha-galactosidase A (GLA), acid sphingomyelinase (ASMase) and concentrations of their corresponded substrates (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM), respectively) were estimated in 211 PD patients and 179 controls by liquid chromatography with tandem mass spectrometry (LC-MS-MS) in dry blood spots. p.M1646T and p.N2081D were associated with PD (OR = 2.33, CI 95%: 1.1215 to 4.8253, p = 0.023; OR = 1.89, 95%CI: 1.0727 to 3.3313, p = 0.028, respectively) in Russian population. An increased LysoGb3 concentration was found in p.G2019S and p.N2081D LRRK2 carriers among PD patients compared to both PD patients and controls (p.G2019S: p = 0.00086, p = 0.0004, respectively; p.N2081D: p = 0.012, p = 0.0076, respectively). A decreased ASMase activity in p.G2019S LRRK2 carriers among PD patients (p = 0.014) was demonstrated as well. Our study supported possible involvement of LRRK2 dysfunction in an alteration of sphingolipid metabolism in PD.

A zebrafish model of combined saposin deficiency identifies acid sphingomyelinase as a potential therapeutic target.

Sphingolipidoses are a subcategory of lysosomal storage diseases (LSDs) caused by mutations in enzymes of the sphingolipid catabolic pathway. Like many LSDs, neurological involvement in sphingolipidoses leads to early mortality with limited treatment options. Given the role of myelin loss as a major contributor toward LSD-associated neurodegeneration, we investigated the pathways contributing to demyelination in a CRISPR-Cas9-generated zebrafish model of combined saposin (psap) deficiency. psap knockout (KO) zebrafish recapitulated major LSD pathologies, including reduced lifespan, reduced lipid storage, impaired locomotion and severe myelin loss; loss of myelin basic protein a (mbpa) mRNA was progressive, with no changes in additional markers of oligodendrocyte differentiation. Brain transcriptomics revealed dysregulated mTORC1 signaling and elevated neuroinflammation, where increased proinflammatory cytokine expression preceded and mTORC1 signaling changes followed mbpa loss. We examined pharmacological and genetic rescue strategies via water tank administration of the multiple sclerosis drug monomethylfumarate (MMF), and crossing the psap KO line into an acid sphingomyelinase (smpd1) deficiency model. smpd1 mutagenesis, but not MMF treatment, prolonged lifespan in psap KO zebrafish, highlighting the modulation of acid sphingomyelinase activity as a potential path toward sphingolipidosis treatment.

Modulation of Dietary Choline Uptake in a Mouse Model of Acid Sphingomyelinase Deficiency.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the gene-encoding acid sphingomyelinase (ASM). ASMD impacts peripheral organs in all patients, including the liver and spleen. The infantile and chronic neurovisceral forms of the disease also lead to neuroinflammation and neurodegeneration for which there is no effective treatment. Cellular accumulation of sphingomyelin (SM) is a pathological hallmark in all tissues. SM is the only sphingolipid comprised of a phosphocholine group linked to ceramide. Choline is an essential nutrient that must be obtained from the diet and its deficiency promotes fatty liver disease in a process dependent on ASM activity. We thus hypothesized that choline deprivation could reduce SM production and have beneficial effects in ASMD. Using acid sphingomyelinase knock-out (ASMko) mice, which mimic neurovisceral ASMD, we have assessed the safety of a choline-free diet and its effects on liver and brain pathological features such as altered sphingolipid and glycerophospholipid composition, inflammation and neurodegeneration. We found that the choline-free diet was safe in our experimental conditions and reduced activation of macrophages and microglia in the liver and brain, respectively. However, there was no significant impact on sphingolipid levels and neurodegeneration was not prevented, arguing against the potential of this nutritional strategy to assist in the management of neurovisceral ASMD patients.

Polyphyllin D punctures hypertrophic lysosomes to reverse drug resistance of hepatocellular carcinoma by targeting acid sphingomyelinase

Hypertrophic lysosomes are critical for tumor progression and drug resistance; however, effective and specific lysosome-targeting compounds for cancer therapy are lacking. Here we conducted a lysosomotropic pharmacophore-based in silico screen in a natural product library (2,212 compounds), and identified polyphyllin D (PD) as a novel lysosome-targeted compound. PD treatment was found to cause lysosomal damage, as evidenced by the blockade of autophagic flux, loss of lysophagy, and the release of lysosomal contents, thus exhibiting anticancer effects on hepatocellular carcinoma (HCC) cell both invitro and invivo. Closer mechanistic examination revealed that PD suppressed the activity of acid sphingomyelinase (SMPD1), a lysosomal phosphodieserase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine, by directly occupying its surface groove, with Trp148 in SMPD1 acting as a major binding residue; this suppression of SMPD1 activity irreversibly triggers lysosomal injury and initiates lysosome-dependent cell death. Furthermore, PD-enhanced lysosomal membrane permeabilization to release sorafenib, augmenting the anticancer effect of sorafenib both invivo and invitro. Overall, our study suggests that PD can potentially be further developed as a novel autophagy inhibitor, and a combination of PD with classical chemotherapeutic anticancer drugs could represent a novel therapeutic strategy for HCC intervention.

The Air-Liquid Interface Reorganizes Membrane Lipids and Enhances the Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colonoid Monolayers.

Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl-/HCO3- antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air-liquid interface (ALI) was created by removing apical media for a specified number of days; from 12-14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.