Sphingomyelin In Lysosomes Research Articles

A protective role of ABCA5 in response to elevated sphingomyelin levels in Parkinson’s disease

Parkinson’s disease (PD) is a chronic neurodegenerative disorder that affects the motor system. Increasing evidence indicates that lysosomal dysfunction is pivotal in the pathogenesis of PD, typically characterized by dysregulation of sphingolipids in lysosomes. ATP-binding cassette subfamily A member 5 (ABCA5) is a lysosomal transporter that mediates the removal of excess sphingomyelin from lysosomes. We therefore investigated whether the expression levels of ABCA5 are associated with sphingomyelin levels and α-synuclein pathology in PD. Firstly, we undertook a comprehensive assessment of the six sphingolipid classes that are part of the lysosomal salvage pathway in the disease-affected amygdala and disease-unaffected visual cortex using liquid chromatography-mass spectrometry. We found that sphingomyelin levels were significantly increased in PD compared to controls and correlated with disease duration only in the amygdala, whereas, the five other sphingolipid classes were slightly altered or unaltered. Concomitantly, the expression of ABCA5 was upregulated in the PD amygdala compared to controls and correlated strongly with sphingomyelin levels. Using neuronal cells, we further verified that the expression of ABCA5 was dependent on cellular levels of sphingomyelin. Interestingly, sphingomyelin levels were strongly associated with α-synuclein in the amygdala and were related to α-synuclein expression. Finally, we revealed that sphingomyelin levels were also increased in PD plasma compared to controls, and that five identical sphingomyelin species were increased in both the brain and the plasma. When put together, these results suggest that in regions accumulating α-synuclein in PD, ABCA5 is upregulated to reduce lysosomal sphingomyelin levels potentially as a protective measure. This process may provide new targets for therapeutic intervention and biomarker development for PD.

Niemann-Pick type A disease with new mutation: a case report

BackgroundNiemann-Pick type A (NP-A) is a congenital, hereditary disease caused by a deficiency in acid sphingomyelinase, a lysosomal enzyme. This deficiency results in an accumulation of sphingomyelin in lysosomes, leading to cellular apoptosis and ultimately to hepatosplenomegaly, neurodegenerative disorder and failure to thrive. Cherry-red spots in the macula and foamy cells in the bone marrow are other manifestations of the disease that help with diagnosis. Type A is a rare, untreatable disease with early manifestations and a poor prognosis, with newborns rarely surviving for 2–3 years.Case presentationA 1-year-old Persian boy was referred to our clinic due to abdominal distention and poor weight gain. He was the first male offspring of consanguineous parents. Other findings were neurodevelopmental delay, hepatosplenomegaly, severe hypotonia, difficulty in breathing, and a slightly coarse face with an open mouth and protruding tongue. The initial diagnosis was clinical mucopolysaccharidosis (MPS) based on the coarse facial features, but further workup ruled out this inherited disorder. Enzyme histochemistry revealed that the level of acid sphingomyelinase was lower than normal. In the genetic study, next-generation sequencing of all coding exons and flanking intronic regions of the patient’s DNA demonstrated a homozygous c.682T>G variant in the SMPD1 gene. This variant was classified as a variant of unknown significance. Further evaluation of DNA extract from his parents and examined using Sanger sequencing showed a heterozygous c.682T>G variant in the SMPD1 gene of both parents.ConclusionsWe describe a 1-year-old boy with neurodevelopmental delay, hepatosplenomegaly, and severe hypotonia. Further investigation demonstrated a new mutation for Niemann-Pick disease.

MEDB-32. Reducing treatment-related toxicity for children with WNT-activated medulloblastoma

WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care (SOC) surgical resection, radiotherapy, and chemotherapy. Unfortunately, while curative, this treatment is associated with major, long-term, debilitating motor, developmental, and neuroendocrine side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. Similarities have been demonstrated between cancer cell lysosomes and those of patients with Niemann-Pick, a lysosomal storage disease characterised by lysosomal fragility and sphingomyelin accumulation. A class of drugs known as Functional Inhibitors of Acid Sphingomyelinase (FIASMAs), increase lysosomal sphingomyelin and destabilise the cancer cell’s more fragile lysosomal membrane which leads to the induction of cell-death pathways via lysosomal membrane permeabilisation. Loratadine, an antihistamine with high FIASMA activity, consistently induced lysosomal membrane permeabilisation, leading to increased cell-death, in our panel of mouse and human WNT-medulloblastoma lines. Loratadine exhibited no detrimental effect on normal mouse embryonic stem cells from the lower rhombic lip – the putative cell of origin in WNT-medulloblastoma. Luciferase-expressing mouse WNT-medulloblastoma cells were orthotopically implanted into CD1-nude mice and monitored for tumour development via bioluminescent imaging. Upon tumour engraftment, mice were subjected to reduced SOC (radiotherapy and adjuvant vincristine) plus a clinically relevant dose of loratadine. Response and survival were compared to mice treated with full SOC (radiotherapy, vincristine, cisplatin, and etoposide). Mice treated with 2mg/kg/day of loratadine following reduced SOC demonstrated increased survival when compared to those treated with full SOC (p=0.02) along with a significant reduction in weight loss during treatment (p=<0.0001). This work suggests that loratadine, or other FIASMA compounds, may be good alternative adjuvant therapies for WNT-medulloblastoma. Using less toxic adjuvants could improve long-term outcomes through reducing therapeutic related toxicities for children with this devastating disease.

C-Abl Activation Linked to Autophagy-Lysosomal Dysfunction Contributes to Neurological Impairment in Niemann-Pick Type A Disease.

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Early secretory pathway-resident Zn transporter proteins contribute to cellular sphingolipid metabolism through activation of sphingomyelin phosphodiesterase 1.

Sphingomyelin phosphodiesterase 1 (SMPD1) converts sphingomyelin into ceramide and phosphocholine; hence, loss of SMPD1 function causes abnormal accumulation of sphingomyelin in lysosomes, which results in the lipid-storage disorder Niemann-Pick disease (types A and B). SMPD1 activity is dependent on zinc, which is coordinated at the active site of the enzyme, and although SMPD1 has been suggested to acquire zinc at the sites where the enzyme is localized, precisely how SMPD1 acquires zinc remains to be clarified. Here, we addressed this using a gene-disruption/reexpression strategy. Our results revealed that Zn transporter 5 (ZNT5)-ZNT6 heterodimers and ZNT7 homodimers, which localize in the compartments of the early secretory pathway, play essential roles in SMPD1 activation. Both ZNT complexes contribute to cellular sphingolipid metabolism by activating SMPD1 because cells lacking the functions of the two complexes exhibited a reduced ceramide to sphingomyelin content ratio in terms of their dominant molecular species and an increase in the sphingomyelin content in terms of three minor species. Moreover, mutant cells contained multilamellar body-like structures, indicative of membrane stacking and accumulation, in the cytoplasm. These findings provide novel insights into the molecular mechanism underlying the activation of SMPD1, a key enzyme in sphingolipid metabolism.

Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction.

Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.

Antidepressants act by inducing autophagy controlled by sphingomyelin\u2013ceramide

Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.

Enhanced Delivery and Effects of Acid Sphingomyelinase by ICAM-1-Targeted Nanocarriers in Type B Niemann-Pick Disease Mice

Acid sphingomyelinase deficiency in type B Niemann-Pick disease leads to lysosomal sphingomyelin storage, principally affecting lungs, liver, and spleen. Infused recombinant enzyme is beneficial, yet its delivery to the lungs is limited and requires higher dosing than liver and spleen, leading to potentially adverse reactions. Previous studies showed increased enzyme pulmonary uptake by nanocarriers targeted to ICAM-1, a protein overexpressed during inflammation. Here, using polystyrene and poly(lactic-co-glycolic acid) nanocarriers, we optimized lung delivery by varying enzyme dose and nanocarrier concentration, verified endocytosis and lysosomal trafficking invivo, and evaluated delivered activity and effects. Raising the enzyme load of nanocarriers progressively increased absolute enzyme delivery to all lung, liver, and spleen, over the naked enzyme. Varying nanocarrier concentration inversely impacted lung versus liver and spleen uptake. Mouse intravital and postmortem examination verified endocytosis, transcytosis, and lysosomal trafficking using nanocarriers. Compared to naked enzyme, nanocarriers increased enzyme activity in organs and reduced lung sphingomyelin storage and macrophage infiltration. Although old mice with advanced disease showed reactivity (pulmonary leukocyte infiltration) toinjections, including buffer without carriers, antibody, or enzyme, younger mice with mild disease did not. We conclude that anti-ICAM nanocarriers may result in effective lung enzyme therapy using low enzyme doses.

Induced Pluripotent Stem Cells for Disease Modeling and Evaluation of Therapeutics for Niemann-Pick Disease Type A.

Currently, there is no effective treatment for Niemann-Pick disease type A (NPA). To accelerate drug discovery for treatment of NPA, NPA-induced pluripotent stem cells were generated from patient dermal fibroblasts and differentiated into neural stem cells. By using the differentiated NPA neuronal cells as a cell-based disease model system, α-tocopherol, δ-tocopherol, and hydroxypropyl-β-cyclodextrin significantly reduced sphingomyelin accumulation in these NPA neuronal cells. Therefore, this cell-based NPA model can be used for further study of disease pathophysiology and for high-throughput screening of compound libraries to identify lead compounds for drug development.

Lyso-sphingomyelin is elevated in dried blood spots of Niemann–Pick B patients

Niemann–Pick disease type B (NPD-B) is caused by a partial deficiency of acid sphingomyelinase activity and results in the accumulation of lysosomal sphingomyelin (SPM) predominantly in macrophages. Notably, SPM is not significantly elevated in the plasma, whole blood, or urine of NPD-B patients. Here, we show that the de-acylated form of sphingomyelin, lyso-SPM, is elevated approximately 5-fold in dried blood spots (DBS) from NPD-B patients and has no overlap with normal controls, making it a potentially useful biomarker.

Elucidating Alzheimer’s disease neuronal death by the ‘calpain-cathepsin hypothesis'

Alzheimer's disease (AD) is characterized by slowly progressive neuronal death, but its molecular cascade remains elusive for over 100 years. Since accumulation of autophagic vacuoles (granulo-vacuolar degenerations) represents one of the histological hallmarks of degenerating neurons in AD, a causal link between autophagy failure and neuronal death is probable. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) as a molecular chaperone for damaged proteins and a guardian of lysosomal integrity. The aim of this study is at elucidating the underlying mechanism of AD by comparing ischemic neuronal death of monkeys and degenerative neuronal death of AD patients. Morphologic, immunoblotting, and proteomics analyses of both the ischemic monkey (Macaca fuscata, body weight of 5–10 Kg, n=20) and human AD brains were done. The ischemic monkey data in the author's laboratory were compared to the human AD patient data of the literature. The main topics are whether an age-related decrease in lysosomal and autophagic activities has a causal connection to programmed neuronal necrosis in sporadic AD, and how genetic factors such as apolipoprotein E (APOE) and presenilin 1 can facilitate lysosomal de stabilization. Both monkey and human brains exhibited evidence of transient or continual perturbed Ca 2+ homeostasis, calpain activation, and oxidative stresses upon Hsp70.1. Membrane lipids such as linoleic and arachidonic acids are vulnerable to the cumulative oxidative stresses, generating a toxic peroxidation product 'hydroxynonenal' that carbonylated Hsp70.1. In the monkey brain, both the carbonylation and calpain-mediated cleavage occurred at Arg469 (i.e., key site) of Hsp70.1. This caused decrease of acid sphingomyelinase activity and accumulation of lysosomal sphingomyelin. Electron microscopic data of the AD neurons showed evidence of lysosomal destabilization and multilamellar lysosomal lipidosis. Taken together, impairments of lysosomal autophagy and stabilization should be driven by the calpain-mediated cleavage of carbonylated Hsp70.1, and this causes lysosomal permeabilization and/or rupture with the resultant release of the cell degradation enzyme, cathepsins (calpain-cathepsin hypothesis: Yamashima T., J Neurochem. 120:477–494, 2012).

Hsp70 stabilizes lysosomes and reverts Niemann–Pick disease-associated lysosomal pathology

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70-BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

Pulmonary delivery of recombinant acid sphingomyelinase improves clearance of lysosomal sphingomyelin from the lungs of a murine model of Niemann–Pick disease

Systemic administration of recombinant acid sphingomyelinase (rhASM) into ASM deficient mice (ASMKO) results in hydrolysis of the abnormal storage of sphingomyelin in lysosomes of the liver, spleen and lung. However, the efficiency with which the substrate is cleared from the lung, particularly the alveolar macrophages, appears to be lower than from the other visceral tissues. To determine if delivery of rhASM into the air spaces of the lung could enhance clearance of pulmonary sphingomyelin, enzyme was administered to ASMKO mice by intranasal instillation. Treatment resulted in a significant and dose-dependent reduction in sphingomyelin levels in the lung. Concomitant with this reduction in substrate levels was a decrease in the amounts of the pro-inflammatory cytokine, MIP-1α, in the bronchoalveolar lavage fluids and an improvement in lung pathology. Maximal reduction of lung sphingomyelin levels was observed at 7 days post-treatment. However, reaccumulation of the substrate was noted starting at day 14 suggesting that repeated treatments will be necessary to effect a sustained reduction in sphingomyelin levels. In addition to reducing the storage abnormality in the lung, intranasal delivery of rhASM also resulted in clearance of the substrate from the liver and spleen. Hence, pulmonary administration of rhASM may represent an alternative route of delivery to address the visceral pathology associated with ASM deficiency.

Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins

Lysosomal breakdown of glycosphingolipids with short hydrophilic carbohydrate headgroups is achieved by the simultaneous action of specific hydrolases and sphingolipid activator proteins (SAPs). Activator proteins are considered to facilitate the enzyme/substrate interaction between water-soluble enzymes and membrane-bound substrates. Sphingomyelin, containing the small hydrophilic phosphorylcholine moiety, is hydrolysed by acid sphingomyelinase (acid SMase). Recent experimental data on the in vivo and in vitro role of activator proteins in sphingomyelin breakdown by acid SMase are reviewed. These data combined with the results using homogenous protein preparations as well as a liposomal assay system mimicking the physiological conditions suggest that lysosomal sphingomyelin degradation is not critically dependent on any of the known activator proteins. Moreover, evidence is provided that the assumed intramolecular activator domain of acid SMase and especially the presence of negatively charged lipids in the lysosomes are sufficient for sphingomyelin turnover.

Formation of bioactive sphingoid molecules from exogenous sphingomyelin in primary cultures of neurons and astrocytes

Exogenous sphingomyelin, radiolabelled at the sphingosine moiety, was administered to primary cultures of cerebellar granule cells and astrocytes for different pulse times (20 min–2 h) and the fate of the radioactivity was followed. Ceramide was the main metabolic product in both cells, whereas sphingosine, glucosyl-ceramide and gangliosides GM3 and GD3 were produced only in astrocytes. When endocytosis was prevented and the lysosomal apparatus inactivated, ceramide formation was reduced slightly in granule cells and almost completely blocked in astrocytes, with disappearance of sphingosine, glucosyl-ceramide, GM3 and GD3. These data indicate that (a) ceramide is rapidly produced in cerebellar granule cells and astrocytes, presumably at the level of the plasma membrane in the first cell type, and of the lysosomes in the second one; (b) sphingosine is produced in cerebellar astrocytes by lysosomal sphingomyelin degradation and is partly reused for glucosyl-ceramide and ganglioside biosynthesis.