Alkaline sphingomyelinase deficiency impairs intestinal mucosal barrier integrity and reduces antioxidant capacity in dextran sulfate sodium-induced colitis.

BACKGROUND: Ulcerative colitis is a chronic inflammatory disease of the colon with an unknown etiology. Alkaline sphingomyelinase (alk-SMase) is specifically expressed by intestinal epithelial cells, and has been reported to play an anti-inflammatory role. However, the underlying mechanism is still unclear. AIM: To explore the mechanism of alk-SMase anti-inflammatory effects on intestinal barrier function and oxidative stress in dextran sulfate sodium (DSS)-induced colitis. METHODS: Mice were administered 3% DSS drinking water, and disease activity index was determined to evaluate the status of colitis. Intestinal permeability was evaluated by gavage administration of fluorescein isothiocyanate dextran, and bacterial translocation was evaluated by measuring serum lipopolysaccharide. Intestinal epithelial cell ultrastructure was observed by electron microscopy. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction were used to detect the expression of intestinal barrier proteins and mRNA, respectively. Serum oxidant and antioxidant marker levels were analyzed using commercial kits to assess oxidative stress levels. RESULTS: Compared to wild-type (WT) mice, inflammation and intestinal permeability in alk-SMase knockout (KO) mice were more severe beginning 4 d after DSS induction. The mRNA and protein levels of intestinal barrier proteins, including zonula occludens-1, occludin, claudin-3, claudin-5, claudin-8, mucin 2, and secretory immunoglobulin A, were significantly reduced on 4 d after DSS treatment. Ultrastructural observations revealed progressive damage to the tight junctions of intestinal epithelial cells. Furthermore, by day 4, mitochondria appeared swollen and degenerated. Additionally, compared to WT mice, serum malondialdehyde levels in KO mice were higher, and the antioxidant capacity was significantly lower. The expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in the colonic mucosal tissue of KO mice was significantly decreased after DSS treatment. mRNA levels of Nrf2-regulated downstream antioxidant enzymes were also decreased. Finally, colitis in KO mice could be effectively relieved by the injection of tertiary butylhydroquinone, which is an Nrf2 activator. CONCLUSION: Alk-SMase regulates the stability of the intestinal mucosal barrier and enhances antioxidant activity through the Nrf2 signaling pathway.

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.

Neurons regulate the esterification of bioactive lipid mediators in the brain of acid sphingomyelinase deficient mice.

Acid sphingomyelinase deficiency is a neurodegenerative lysosomal storage disorder caused by mutations in the sphingomyelin-degrading enzyme acid sphingomyelinase (ASM) gene. Upregulated neuroinflammation has been well-characterized in an ASM knockout mouse model of acid sphingomyelinase deficiency disease, but lipid mediator pathways involved in 'mediating' inflammation and inflammation-resolution have yet to be characterized. In this study, we 1) measured free (bioactive) and esterified (inactive) lipid mediators involved in inflammation and inflammation resolution in cerebellum and neuronal cultures of ASM knockout (ASMko) mice and wildtype (WT) controls, and 2) quantified the esterification of labeled pro-resolving free d11-14(15)-epoxyeicosatrienoic acid in cultured neurons from ASMko and WT mice. We found elevated concentrations of esterified pro-resolving lipid mediators and hydroxyeicosatrienoic acids typically destined for pro-resolving lipid mediator synthesis (e.g. lipoxins) in the cerebellum and neurons of ASMko mice compared to controls. Free d11-14(15)-epoxyeicosatrienoic acid esterification within neurons of ASMko mice was significantly elevated compared to WT. Our findings show evidence of increased inactivation of free pro-resolving lipid mediators through esterification in ASMko mice, suggesting impaired resolution as a new pathway underlying ASM deficiency pathogenesis.

Sphingomyelin 16:0 is a therapeutic target for neuronal death in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the SMPD1 gene encoding for the acid sphingomyelinase (ASM). While intravenous infusion of recombinant ASM is an effective treatment for the peripheral disease, the neurological complications of ASMD remain unaddressed. It has been shown that aberrantly high level of total brain sphingomyelin (SM) is a key pathological event leading to neurodegeneration. Using mice lacking ASM (ASMko), which mimic the disease, we here demonstrate that among the SM species, SM16:0 shows the highest accumulation and toxicity in ASMko neurons. By targeting lysosomes, SM16:0 causes permeabilization and exocytosis of these organelles and induces oxidative stress and cell death. We also show that genetic silencing of Ceramide Synthase 5, which is involved in SM16:0 synthesis and overexpressed in the ASMko brain, prevents disease phenotypes in ASMko cultured neurons and mice. The levels of SM16:0 in plasma also show a strong correlation with those in brain that is higher than in liver, even at early stages of the disease. These results identify SM16:0 both as a novel therapeutic target and potential biomarker of brain pathology in ASMD.

Similarities and differences between Gaucher disease and acid sphingomyelinase deficiency: An algorithm to support the diagnosis.

Lysosomal storage disorders are a group of inborn errors of metabolism due to defects in proteins crucial for lysosomal function. Gaucher disease is the most common autosomal recessive lysosomal storage disorder due to mutations in the GBA1 gene, resulting in the lysosomal deficiency of glucocerebrosidase activity. Gaucher disease is characterized by the toxic accumulation of glucosylceramide in the reticuloendothelial system. Acid sphingomyelinase deficiency (ASMD), previously known as Niemann Pick A/B disease, is also an autosomal recessive lysosomal storage disorder due to mutations in the SMPD1 gene, which result in acid sphingomyelinase deficiency and the accumulation of sphingomyelin in mononuclear phagocytic system and hepatocytes. The phenotypic expression of Gaucher disease type 1 (GD1), the most common type, and chronic visceral ASMD may overlap for several signs or symptoms. Splenomegaly is detectable in approximately 90% of the patients in both conditions; however, since GD1 is more frequent than ASMD, clinicians are more prone to suspect it, often neglecting the diagnosis of ASMD. Based on previous experience, a group of experts in the clinical and laboratory diagnosis, management, and treatment of lysosomal storage disorders developed an algorithm for both GD1 and ASMD to support physicians, including primary care providers, internists, and specialists (e.g., hepatologists, hematologists, and pulmonologists) to suspect and differentiate GD1 and ASMD and to provide the appropriate referral.

Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism.

Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.

Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease.

Lysosomal Storage Diseases (LSD) are genetic diseases causing systemic and nervous system dysfunction. The glia-derived lipid binding protein Apolipoprotein D (ApoD) is required for lysosomal functional integrity in glial and neuronal cells, ensuring cell survival upon oxidative stress or injury. Here we test whether ApoD counteracts the pathogenic consequences of a LSD, Niemann Pick-type-A disease (NPA), where mutations in the acid sphingomyelinase gene result in sphingomyelin accumulation, lysosomal permeabilization and early-onset neurodegeneration. We performed a multivariable analysis of behavioral, cellular and molecular outputs in 12 and 24 week-old male and female NPA model mice, combined with ApoD loss-of-function mutation. Lack of ApoD in NPA mice accelerates cerebellar-dependent motor deficits, enhancing loss of Purkinje neurons. We studied ApoD expression in brain sections from a NPA patient and age-matched control, and the functional consequences of ApoD supplementation in primary human fibroblasts from two independent NPA patients and two control subjects. Cell viability, lipid peroxidation, and lysosomal functional integrity (pH, Cathepsin B activity, Galectin-3 exclusion) were examined. ApoD is endogenously overexpressed in NPA patients and NPA mouse brains and targeted to lysosomes of NPA patient cells, including Purkinje neurons and cultured fibroblasts. The accelerated lysosomal targeting of ApoD by oxidative stress is hindered in NPA fibroblasts, contributing to NPA lysosomes vulnerability. Exogenously added ApoD reduces NPA-prompted lysosomal permeabilization and alkalinization, reverts lipid peroxides accumulation, and significantly increases NPA cell survival. ApoD administered simultaneously to sphingomyelin overload results in complete rescue of cell survival. Our results reveal that ApoD protection of lysosomal integrity counteracts NPA pathology. ApoD supplementation could significantly delay not only the progression of NPA disease, but also of other LSDs through its beneficial effects in lysosomal functional maintenance.

Finding pathogenic commonalities between Niemann-Pick type C and other lysosomal storage disorders: Opportunities for shared therapeutic interventions.

Lysosomal storage disorders (LSDs) are diseases characterized by the accumulation of macromolecules in the late endocytic system and are caused by inherited defects in genes that encode mainly lysosomal enzymes or transmembrane lysosomal proteins. Niemann-Pick type C disease (NPCD), a LSD characterized by liver damage and progressive neurodegeneration that leads to early death, is caused by mutations in the genes encoding the NPC1 or NPC2 proteins. Both proteins are involved in the transport of cholesterol from the late endosomal compartment to the rest of the cell. Loss of function of these proteins causes primary cholesterol accumulation, and secondary accumulation of other lipids, such as sphingolipids, in lysosomes. Despite years of studying the genetic and molecular bases of NPCD and related-lysosomal disorders, the pathogenic mechanisms involved in these diseases are not fully understood. In this review we will summarize the pathogenic mechanisms described for NPCD and we will discuss their relevance for other LSDs with neurological components such as Niemann- Pick type A and Gaucher diseases. We will particularly focus on the activation of signaling pathways that may be common to these three pathologies with emphasis on how the intra-lysosomal accumulation of lipids leads to pathology, specifically to neurological impairments. We will show that although the primary lipid storage defect is different in these three LSDs, there is a similar secondary accumulation of metabolites and activation of signaling pathways that can lead to common pathogenic mechanisms. This analysis might help to delineate common pathological mechanisms and therapeutic targets for lysosomal storage diseases.

Niemann-Pick Type A Disease: Behavior of Neutral Sphingomyelinase and Vitamin D Receptor.

Sphingomyelinase (SMase) is responsible for the breakdown of sphingomyelin (SM) with production of ceramide. The absence of acid sphingomyelinase (aSMase) causes abnormal synapse formation in Niemann-Pick type A (NPA) disease. Because high levels of ceramide in the NPA brain were demonstrated, the involvement of other SMases were supposed. In the present study we focused the attention on the neurogenic niches in the hippocampal gyrus dentatus (GD), a brain structure essential for forming cohesive memory. We demonstrated for the first time the increase of (Sex determining region Y)-box 2 (SOX2), and the down-regulation of glial fibrillary acidic protein (GFAP) NPA mice GD. Moreover, we found that the expression of Toll like receptors (TLRs), was increased in NPA mice, particularly TLR2, TLR7, TLR8 and TLR9 members. Although no significant change in neutral sphingomyelinase (nSMase) gene expression was detected in the NPA mice hippocampus of, protein levels were enhanced, probably because of the slower protein degradation rate in this area. Many studies demonstrated that vitamin D receptor (VDR) is expressed in the hippocampus GD. Unexpectedly, we showed that NPA mice exhibited VDR gene and protein expression up-regulation. In summary, our study suggests a relation between hippocampal cell differentiation defect, nSMase and VDR increase in NPA mice.

Niemann-Pick disease A or B in four pediatric patients and SMPD1 mutation carrier frequency in the Mexican population.

INTRODUCTION AND OBJECTIVES: Niemann-Pick disease type A (NPD-A) and B (NPD-B) are lysosomal storage diseases with a birth prevalence of 0.4-0.6/100,000. They are caused by a deficiency in acid sphingomyelinase, an enzyme encoded by SMPD1. We analyzed the phenotype and genotype of four unrelated Mexican patients, one with NPD-A and three with NPD-B. PATIENTS AND METHODS: Four female patients between 1 and 7 years of age were diagnosed with NPD-A or NPD-B by hepatosplenomegaly, among other clinical characteristics, and by determining the level of acid sphingomyelinase enzymatic activity and sequencing of the SMPD1 gene. Additionally, a 775bp amplicon of SMPD1 (from 11:6393835_6394609, including exons 5 and 6) was analyzed by capillary sequencing in a control group of 50 unrelated healthy Mexican Mestizos. RESULTS: An infrequent variant (c.1343A>G p.Tyr448Cys) was observed in two patients. One is the first NPD-A homozygous patient reported with this variant and the other a compound heterozygous NPD-B patient with the c.1829_1831delGCC p.Arg610del variant. Another compound heterozygous patient had the c.1547A>G p.His516Arg variant (not previously described in affected individuals) along with the c.1805G>A p.Arg602His variant. A new c.1263+8C>T pathogenic variant was encountered in a homozygous state in a NPD-B patient. Among the healthy control individuals there was a heterozygous carrier for the c.1550A>T (rs142787001) pathogenic variant, but none with the known pathogenic variants in the 11:6393835_6394609 region of SMPD1. CONCLUSIONS: The present study provides further NPD-A or B phenotype-genotype correlations. We detected a heterozygous carrier with a pathogenic variant in 1/50 healthy Mexican mestizos.

Chronic visceral acid sphingomyelinase deficiency (Niemann-Pick disease type B) in 16 Polish patients: long-term follow-up.

BACKGROUND: Acid sphingomyelinase deficiency (ASMD), due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, is divided into infantile neurovisceral ASMD (Niemann-Pick type A), chronic neurovisceral ASMD (intermediate form, Niemann-Pick type A/B) and chronic visceral ASMD (Niemann-Pick type B). We conducted a long-term observational, single-center study including 16 patients with chronic visceral ASMD. RESULTS: 12 patients were diagnosed in childhood and 4 others in adulthood, the oldest at the age of 50. The mean time of follow-up was approximately 10 years (range: 6 months - 36 years). Splenomegaly was noted in all patients at diagnosis. Hepatomegaly was observed in 88% of patients. Moderately elevated (several-fold above the upper limit of normal values) serum transaminases were noted in 38% of patients. Cherry-red spots were found in five Gypsy children from one family and also in one adult Polish patient, a heterozygote for p.delR610 mutation. Dyslipidemia was noted in 50% of patients. Interstitial lung disease was diagnosed in 44% of patients. Plasmatic lysosphingomyelin (SPC) was elevated in all the patients except one with p.V36A homozygosity and a very mild phenotype also presenting with elevated plasmatic SPC-509 but normal chitotriosidase activity. The most common variant of SMPD1 gene was p.G166R. We found a previously unreported variant in exon 2 (c.491G > T, p.G164 V) in one patient. CONCLUSIONS: Chronic visceral ASMD could constitute a slowly progressing disease with a relatively good outcome. The combined measurement of lysosphingomyelin (SPC) and lysospingomyelin-509 (SPC-509) is an essential method for the assessment of ASMD course.

Defects in sarcolemma repair and skeletal muscle function after injury in a mouse model of Niemann-Pick type A/B disease.

BACKGROUND: Niemann-Pick disease type A (NPDA), a disease caused by mutations in acid sphingomyelinase (ASM), involves severe neurodegeneration and early death. Intracellular lipid accumulation and plasma membrane alterations are implicated in the pathology. ASM is also linked to the mechanism of plasma membrane repair, so we investigated the impact of ASM deficiency in skeletal muscle, a tissue that undergoes frequent cycles of injury and repair in vivo. METHODS: Utilizing the NPDA/B mouse model ASM-/- and wild type (WT) littermates, we performed excitation-contraction coupling/Ca2+ mobilization and sarcolemma injury/repair assays with isolated flexor digitorum brevis fibers, proteomic analyses with quadriceps femoris, flexor digitorum brevis, and tibialis posterior muscle and in vivo tests of the contractile force (maximal isometric torque) of the quadriceps femoris muscle before and after eccentric contraction-induced muscle injury. RESULTS: ASM-/- flexor digitorum brevis fibers showed impaired excitation-contraction coupling compared to WT, a defect expressed as reduced tetanic [Ca2+]i in response to electrical stimulation and early failure in sustaining [Ca2+]i during repeated tetanic contractions. When injured mechanically by needle passage, ASM-/- flexor digitorum brevis fibers showed susceptibility to injury similar to WT, but a reduced ability to reseal the sarcolemma. Proteomic analyses revealed changes in a small group of skeletal muscle proteins as a consequence of ASM deficiency, with downregulation of calsequestrin occurring in the three different muscles analyzed. In vivo, the loss in maximal isometric torque of WT quadriceps femoris was similar immediately after and 2 min after injury. The loss in ASM-/- mice immediately after injury was similar to WT, but was markedly larger at 2 min after injury. CONCLUSIONS: Skeletal muscle fibers from ASM-/- mice have an impairment in intracellular Ca2+ handling that results in reduced Ca2+ mobilization and a more rapid decline in peak Ca2+ transients during repeated contraction-relaxation cycles. Isolated fibers show reduced ability to repair damage to the sarcolemma, and this is associated with an exaggerated deficit in force during recovery from an in vivo eccentric contraction-induced muscle injury. Our findings uncover the possibility that skeletal muscle functional defects may play a role in the pathology of NPDA/B disease.

Consensus recommendation for a diagnostic guideline for acid sphingomyelinase deficiency.

Disclaimer:This diagnostic guideline is intended as an educational resource and represents the opinions of the authors, and is not representative of recommendations or policy of the American College of Medical Genetics and Genomics (ACMG). The information should be considered a consensus based on expert opinion, as more comprehensive levels of evidence were not available in the literature in all cases. BACKGROUND: Acid sphingomyelinase deficiency (ASMD) is a rare, progressive, and often fatal lysosomal storage disease. The underlying metabolic defect is deficiency of the enzyme acid sphingomyelinase that results in progressive accumulation of sphingomyelin in target tissues. ASMD manifests as a spectrum of severity ranging from rapidly progressive severe neurovisceral disease that is uniformly fatal to more slowly progressive chronic neurovisceral and chronic visceral forms. Disease management is aimed at symptom control and regular assessments for multisystem involvement. PURPOSE AND METHODS: An international panel of experts in the clinical and laboratory evaluation, diagnosis, treatment/management, and genetic aspects of ASMD convened to review the evidence base and share personal experience in order to develop a guideline for diagnosis of the various ASMD phenotypes. CONCLUSIONS: Although care of ASMD patients is typically provided by metabolic disease specialists, the guideline is directed at a wide range of providers because it is important for primary care providers (e.g., pediatricians and internists) and specialists (e.g., pulmonologists, hepatologists, and hematologists) to be able to identify ASMD.Genet Med advance online publication 13 April 2017.

Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure.

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.

Types A and B Niemann-Pick disease.

Two distinct metabolic abnormalities are encompassed under the eponym Niemann-Pick disease (NPD). The first is due to the deficient activity of the enzyme acid sphingomyelinase (ASM). Patients with ASM deficiency are classified as having types A and B Niemann-Pick disease (NPD). Type A NPD patients exhibit hepatosplenomegaly in infancy and profound central nervous system involvement. They rarely survive beyond two years of age. Type B patients also have hepatosplenomegaly and pathologic alterations of their lungs, but there are usually no central nervous system signs. The age of onset and rate of disease progression varies greatly among type B patients, and they frequently live into adulthood. Recently, patients with phenotypes intermediate between types A and B NPD also have been identified. These individuals represent the expected continuum caused by inheriting different mutations in the ASM gene (SMPD1). Patients in the second NPD category are designated as having types C and D NPD. These patients may have mild hepatosplenomegaly, but the central nervous system is profoundly affected. Impaired intracellular trafficking of cholesterol causes types C and D NPD, and two distinct gene defects have been found. In this chapter only types A and B NPD will be discussed.

Altered Clathrin-Independent Endocytosis in Type A Niemann-Pick Disease Cells and Rescue by ICAM-1-Targeted Enzyme Delivery.

Pharmaceutical intervention often requires therapeutics and/or their carriers to enter cells via endocytosis. Therefore, endocytic aberrancies resulting from disease represent a key, yet often overlooked, parameter in designing therapeutic strategies. In the case of lysosomal storage diseases (LSDs), characterized by lysosomal accumulation of undegraded substances, common clinical interventions rely on endocytosis of recombinant enzymes. However, the lysosomal defect in these diseases can affect endocytosis, as we recently demonstrated for clathrin-mediated uptake in patient fibroblasts with type A Niemann-Pick disease (NPD), a disorder characterized by acid sphingomylinase (ASM) deficiency and subsequent sphingomyelin storage. Using similar cells, we have examined if this is also the case for clathrin-independent pathways, including caveolae-mediated endocytosis and macropinocytosis. We observed impaired caveolin-1 enrichment at ligand-binding sites in NPD relative to wild type fibroblasts, corresponding with altered uptake of ligands and fluid-phase markers by both pathways. Similarly, aberrant lysosomal storage of sphingomyelin induced by pharmacological means also diminished uptake. Partial degradation of the lysosomal storage by untargeted recombinant ASM led to partial uptake enhancement, whereas both parameters were restored to wild type levels by ASM delivery using model polymer nanocarriers specifically targeted to intercellular adhesion molecule-1. Carriers also restored caveolin-1 enrichment at ligand-binding sites and uptake through the caveolar and macropinocytic routes. These results demonstrate a link between lysosomal storage in NPD and alterations in clathrin-independent endocytosis, which could apply to other LSDs. Hence, this study shall guide the design of therapeutic approaches using viable endocytic pathways.

Improvement of biochemical and behavioral defects in the Niemann-Pick type A mouse by intraventricular infusion of MARCKS.

Niemann-Pick disease type A (NPDA) is a fatal disease due to mutations in the acid sphingomyelinase (ASM) gene, which triggers the abnormal accumulation of sphingomyelin (SM) in lysosomes and the plasma membrane of mutant cells. Although the disease affects multiple organs, the impact on the brain is the most invalidating feature. The mechanisms responsible for the cognitive deficit characteristic of this condition are only partially understood. Using mice lacking the ASM gene (ASMko), a model system in NPDA research, we report here that high sphingomyelin levels in mutant neurons lead to low synaptic levels of phosphoinositide PI(4,5)P2 and reduced activity of its hydrolyzing phosphatase PLCγ, which are key players in synaptic plasticity events. In addition, mutant neurons have reduced levels of membrane-bound MARCKS, a protein required for PI(4,5)P2 membrane clustering and hydrolysis. Intracerebroventricular infusion of a peptide that mimics the effector domain of MARCKS increases the content of PI(4,5)P2 in the synaptic membrane and ameliorates behavioral abnormalities in ASMko mice.

Nonclinical safety assessment of recombinant human acid sphingomyelinase (rhASM) for the treatment of acid sphingomyelinase deficiency:the utility of animal models of disease in the toxicological evaluation of potential therapeutics.

Recombinant human acid sphingomyelinase (rhASM) is being developed as an enzyme replacement therapy for patients with acid sphingomyelinase deficiency (Niemann-Pick disease types A and B), which causes sphingomyelin to accumulate in lysosomes. In the acid sphingomyelinase knock-out (ASMKO) mouse, intravenously administered rhASM reduced tissue sphingomyelin levels in a dose-dependent manner. When rhASM was administered to normal rats, mice, and dogs, no toxicity was observed up to a dose of 30mg/kg. However, high doses of rhASM≥10mg/kg administered to ASMKO mice resulted in unexpected toxicity characterized by cardiovascular shock, hepatic inflammation, adrenal hemorrhage, elevations in ceramide and cytokines (especially IL-6, G-CSF, and keratinocyte chemoattractant [KC]), and death. The toxicity could be completely prevented by the administration of several low doses (3mg/kg) of rhASM prior to single or repeated high doses (≥20mg/kg). These results suggest that the observed toxicity involves the rapid breakdown of large amounts of sphingomyelin into ceramide and/or other toxic downstream metabolites, which are known signaling molecules with cardiovascular and pro-inflammatory effects. Our results suggest that the nonclinical safety assessment of novel therapeutics should include the use of specific animal models of disease whenever feasible.

Pharmacological reversion of sphingomyelin-induced dendritic spine anomalies in a Niemann Pick disease type A mouse model.

Understanding the role of lipids in synapses and the aberrant molecular mechanisms causing the cognitive deficits that characterize most lipidosis is necessary to develop therapies for these diseases. Here we describe sphingomyelin (SM) as a key modulator of the dendritic spine actin cytoskeleton. We show that increased SM levels in neurons of acid sphingomyelinase knock out mice (ASMko), which mimic Niemann Pick disease type A (NPA), result in reduced spine number and size and low levels of filamentous actin. Mechanistically, SM accumulation decreases the levels of metabotropic glutamate receptors type I (mGluR1/5) at the synaptic membrane impairing membrane attachment and activity of RhoA and its effectors ROCK and ProfilinIIa. Pharmacological enhancement of the neutral sphingomyelinase rescues the aberrant molecular and morphological phenotypes in vitro and in vivo and improves motor and memory deficits in ASMko mice. Altogether, these data demonstrate the influence of SM and its catabolic enzymes in dendritic spine physiology and contribute to our understanding of the cognitive deficits of NPA patients, opening new perspectives for therapeutic interventions.