Secretory Sphingomyelinase in Health and Disease

Home , Acid sphingomyelinase, FIASMA, Glycolipid, Sphingolipid

Biol. Chem. 2015; 396(6-7): 707–736

Review Open Access

Johannes Kornhuber*, Cosima Rhein, Christian P. Müller and Christiane Mühle Secretory sphingomyelinase in health and disease

Abstract: Acid sphingomyelinase (ASM), a key enzyme may be both a promising clinical chemistry marker and a in sphingolipid metabolism, hydrolyzes sphingomy- therapeutic target. elin to ceramide and phosphorylcholine. In mammals, the expression of a single gene, SMPD1, results in two Keywords: ceramide; inflammation; lipids; secretory forms of the enzyme that differ in several characteristics. sphingomyelinase; sphingomyelin; sphingomyelinase. Lysosomal ASM (L-ASM) is located within the lysosome, 2+ requires no additional Zn ions for activation and is gly- DOI 10.1515/hsz-2015-0109 cosylated mainly with high-mannose oligosaccharides. Received January 20, 2015; accepted February 16, 2015; previously By contrast, the secretory ASM (S-ASM) is located extra- published online March 24, 2015 cellularly, requires Zn2+ ions for activation, has a complex glycosylation pattern and has a longer in vivo half-life. In this review, we summarize current knowledge regarding the physiology and pathophysiology of S-ASM, includ- Introduction ing its sources and distribution, molecular and cellular Acid sphingomyelinase (ASM, EC 3.1.4.12) plays a major mechanisms of generation and regulation and relevant role in sphingolipid metabolism because it catalyzes the in vitro and in vivo studies. Polymorphisms or mutations hydrolysis of sphingomyelin (SM) to ceramide and phos- of SMPD1 lead to decreased S-ASM activity, as detected in phorylcholine. Ceramide and related products, such as patients with Niemann-Pick disease B. Thus, lower serum/ spingosine-1-phosphate, are important lipid signaling plasma activities of S-ASM are trait markers. No genetic molecules. These molecules are involved in a variety of causes of increased S-ASM activity have been identified. molecular and cellular processes and play a central role Instead, elevated activity is the result of enhanced release in a growing number of human diseases. The multiple (e.g., induced by lipopolysaccharide and cytokine stimu- physiological sources of ceramide in mammalian cells lation) or increased enzyme activation (e.g., induced by (de novo synthesis from palmitoyl-CoA and serine, syn- oxidative stress). Increased S-ASM activity in serum or thesis from sphingosine and fatty acid, SM catabolism, plasma is a state marker of a wide range of diseases. In hydrolysis of glycosylceramide and galactosylceramide, particular, high S-ASM activity occurs in inflammation of dephosphorylation of ceramide-1-phosphate) do not con- the endothelium and liver. Several studies have demon- tribute equally to the pool, but the degradation of SM by strated a correlation between S-ASM activity and mortality the ASM is an important source of ceramide. The promi- induced by severe inflammatory diseases. Serial measure- nent position of sphingomyelinases is mainly attributed ments of S-ASM reveal prolonged activation and, there- to the abundance of their substrate SM in cell membranes fore, the measurement of this enzyme may also provide (van Meer et al., 2008). Because of its spatially bulky head information on past inflammatory processes. Thus, S-ASM group, SM is restricted to the membrane leaflet where it is generated, i.e., the luminal Golgi leaflet or – after vesicular transport – the outer leaflet of the plasma membrane *Corresponding author: Johannes Kornhuber, Department of (Gault et al., 2010), unless flipping is aided by a specific Psychiatry and Psychotherapy, Friedrich Alexander University of flippase (Sharom, 2011). However, of the known flip- Erlangen-Nürnberg (FAU), Schwabachanlage 6, D-91054 Erlangen, pases, none is active toward SM (Takatsu et al., 2014). Germany, e-mail: [email protected] Mammalian cells contain a single gene for ASM (SMPD1), Cosima Rhein, Christian P. Müller and Christiane Mühle: Department of Psychiatry and Psychotherapy, Friedrich Alexander which is responsible for the generation of both the secre- University of Erlangen-Nürnberg (FAU), Schwabachanlage 6, tory (S-ASM) and lysosomal (L-ASM) forms (Schissel D-91054 Erlangen, Germany et al., 1996a). S- and L-ASM originate from the same gene

©2015, Johannes Kornhuber et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. 708 J. Kornhuber et al.: Secretory sphingomyelinase without the involvement of alternative RNA splicing as lability at 55°C (Spence et al., 1979) and exceptionally they can be produced from the same full-length cDNA longer half-life (Jenkins et al., 2010), different molecular (Schissel et al., 1998b). The common mRNA is also trans- weight of the protein core due to an intact C-terminus lated in the same reading frame because the resulting (Jenkins et al., 2011b) and different N-terminal proteolytic L- and S-ASM proteins are similar in size and are both rec- processing. In addition, S-ASM has a complex N-glyco- ognized by antibodies prepared against L-ASM (Schissel sylation pattern (Ferlinz et al., 1994; Hurwitz et al., 1994b) et al., 1996a, 1998b). By contrast, in the nematode Cae- that enables resistance to endoglycosidase H and results norhabditis elegans, three genes have been identified (Lin in a different localization (Schissel et al., 1998b) com- et al., 1998; Kim and Sun, 2012) on different chromosomes pared to L-ASM with high-mannose oligosaccharides for (Yook et al., 2012): the product of asm-1 is almost entirely lysosomal targeting (Kornfeld, 1987). Whether L-ASM and secreted but is Zn2+-independent, whereas only 20% of S-ASM act on different SM pools in the cell, which would the asm-2 product is secreted and requires Zn2+. The asm-3 result in distinct effects via differential ceramide genera- protein with the strongest homology to human ASM and tion, remains unclear (Jenkins et al., 2010). sensitivity to desipramine and clomipramine (Lin et al., 1998; Kim and Sun, 2012) may correspond to L-ASM. The presence of separate genes for the secreted sphingomyeli- Genetic impact on S-ASM activity nase in C. elegans with developmentally regulated expression emphasizes the critical importance of the secreted A number of genetic sequence variations and mutations enzyme. However, in mammalian organisms the role, influence S-ASM activity and the process of generating L- function and regulation of S-ASM are not as well under- or S-ASM. The repeat number variation of a hexanucleo- stood as those of L-ASM. Several previous reviews cover tide sequence – c.108GCTGGC(3_8)/p.37LA(3)_8) – that various aspects of S-ASM physiology and pathophysiology leads to the modification of the signal peptide of the pre- (Tabas, 1999; Goni and Alonso, 2002; Smith and Schuch- pro-form of ASM (Wan and Schuchman, 1995), was associ- man, 2008; Jenkins et al., 2009; Pavoine and Pecker, 2009; ated with S-ASM activity but not L-ASM activity in a cell He and Schuchman, 2012). In this review, we focus on the culture model. In addition, analysis of a human sample progress made in recent years specifically on mamma- resulted in a significant association of S-ASM activity lian S-ASM and some aspects of secreted L-ASM, and we with the number of hexanucleotide repeats; levels were examine novel studies investigating S-ASM as a mediator highest in subjects homozygous for six repeats, inter- of pathogenic processes, a potential therapeutic target and mediate in subjects homozygous for five repeats, and a biomarker in human diseases. The sequence numbering lowest in subjects homozygous for four repeats. One of at the DNA and protein level in this review is based on the the most frequent SMPD1 sequence variations, rs1050239/ standard database reference sequence NM_000543.4 with c.1522G > A/p.G508R (Rhein et al., 2014), has been associ- 631 amino acids, which leads to a shift of +2 amino acids ated with S-ASM activity but not L-ASM activity. S-ASM when referring to some published positions because of activity was measured in the blood plasma of healthy an additional leucine-alanine dipeptide within the poly young adults and was highest in subjects homozygous morphic signal peptide. for the major G allele, intermediate in heterozygous subjects and lowest in subjects homozygous for the A allele (Reichel et al., 2014). Importantly, the influences of these two polymorphic sites on S-ASM activity are independ- Molecular and cellular regulatory ent (Rhein et al., 2014). The nonsynonymous variation mechanisms of S-ASM activity rs141641266/c.1460C > T/p.A487V, previously assumed to be a missense mutation causing Niemann-Pick disease Two distinct enzymes arise from the single SMPD1 gene (NPD) type B (Simonaro et al., 2002), leads to normal because of differential modification and trafficking pro- L-ASM and only slightly lower S-ASM activity levels (Rhein cesses. The lysosomal form (L-ASM) is located in the et al., 2013). endolysosomal compartment, whereas the secretory form More than 100 clinically relevant missense mutations (S-ASM) is released by the secretory pathway (Schissel in the SMPD1 gene leading to enzymes with decreased cat- et al., 1996a; Jenkins et al., 2010). S-ASM differs from the alytic activity, the cause of autosomal recessive NPD types lysosomal form by its dependence on exogenously added A and B (Brady et al., 1966; Schuchman, 2010), have been Zn2+ ions (Schissel et al., 1998b) associated with its inhi- deposited in the Human Gene Mutation Database (www. bition by ethylenediaminetetraacetic acid (EDTA), heat hgmd.cf.ac.uk). While genotype/phenotype correlations J. Kornhuber et al.: Secretory sphingomyelinase 709 are typically complex, the DR608 deletion mutation how S-ASM activity is influenced by alternative splicing appears to protect patients against the development of has not been investigated. The unexpectedly mild NPD-B the more severe neuropathic NPD form type A (Schuch- phenotype and 20–25% residual L-ASM activity observed man and Miranda, 1997). L-ASM activity in cultured skin in two patients homozygous for either p.M1_W32del or fibroblasts of patients correlates with the respective phe- p.W32X and, consequently, complete absence of full- notype, with residual L-ASM activity of < 5% correspond- length ASM, suggests that in the absence of a functional ing to the more severe NPD type A (Desnick et al., 2010), first initiation codon, the second initiation codon (ATG for which – similar to other neurodegenerative sphin- coding for p.33M) may still produce a relatively functional golipidoses – effective therapeutic options are missing enzyme (Pittis et al., 2004). (Eckhardt, 2010). The determination of S-ASM activity in blood plasma can be used diagnostically, particularly to discriminate between NPD patients and NPD carriers (He Trafficking and post-translational processing et al., 2003). However, clinical data on S-ASM activity in relevant for S-ASM and L-ASM activity NPD patients remain limited because in most studies only cellular L-ASM activity is determined (Vanier et al., 1980; The generation of L-ASM versus S-ASM is determined by Pavlu-Pereira et al., 2005). Because lysosomal hypertro- the differential processing and trafficking of the common phy is a hallmark of lysosomal storage disorders, a parallel precursor ASM protein (Figure 1). Upon entry of the assessment of the acidic compartment volume combined nascent ASM polypeptide (631 amino acids correspond- with L- and S-ASM activities could provide a basis for gen- ing to 70 kDa) into the rough endoplasmic reticulum (ER) otype-phenotype correlations, particularly to explain the (Hasilik, 1992), cleavage of the putative N-terminal signal large heterogeneity of symptom severity observed in NPD peptide (Wan and Schuchman, 1995), which is postulated patients with an intermediate phenotype (Pavlu-Pereira to extend to p.A48 (Schuchman et al., 1991) yields the pre- et al., 2005; Wasserstein et al., 2006). Interestingly, there pro-form with a molecular weight of 75 kDa after glycosyla- is growing support for a genetic convergence of lysosomal tion (65 kDa protein core). The signal peptide is essential storage disorders and Parkinson’s disease (Deng et al., for the production of enzymatically active mature L-ASM. 2014). Mutations such as p.L304P (Gan-Or et al., 2013), ASM constructs with N-terminal truncations of the signal which may be limited to Ashkenazi Jews (Wu et al., 2014), sequence lack catalytic L-ASM activity (Ferlinz et al., or variants in SMPD1, e.g., p.R591C, p.P533L (Foo et al., 1994). Despite the predicted absence of the signal peptide 2013), have not only been associated with altered ASM in the mature enzyme, the length of the hydrophobic core activities but also appear to be linked to an up to 9-fold of the polymorphic ASM signal peptide affects the secre- increased risk for Parkinson’s disease (Spatola and Wider, tion and activity of S-ASM, whereas L-ASM activity is unaf- 2014). This effect is likely mediated via ceramide because fected (Rhein et al., 2014). plasma ceramide and monohexosylceramide levels are L- and S-ASM undergo differential glycosylation increased and associated with cognitive impairment in processing inside the Golgi giving rise to the ASM pro- Parkinson’s disease patients lacking a risk-conveying form of 72–75 kDa (63–64 kDa protein core). L-ASM has glucocerebrosidase mutation (Mielke et al., 2013). a high mannose N-glycan composition, which provides stabilization and protection against proteolysis within the lysosome, whereas S-ASM possesses a complex Transcriptional and translational effects N-glycosylation pattern (Hurwitz et al., 1994b; Schissel on ASM activity et al., 1998b). This differential glycosylation is evident in the susceptibility of L-ASM to endoglycosidase H, which ASM is up-regulated at the transcriptional level in differ- is specific for high mannose-type N-linked oligosac- ent cell culture models, but only effects on L-ASM activ- charides (Yamamoto, 1994). In contrast, S-ASM is com- ity have been investigated (Langmann et al., 1999; Murate pletely resistant to endoglycosidase H, and both enzymes et al., 2002). A variety of alternatively spliced ASM tran- are susceptible to peptide-N-glycosidase F (Schissel scripts have been identified, of which only one, ASM-1, et al., 1998b). In the cis-Golgi, L-ASM acquires mannose- encodes an enzyme with complete protein domains and phosphate residues via the typical sequential action of L-ASM activity. The isoforms ASM-2 to -7 lack protein N-acetylglucosamine-1-phosphotransferase (Reitman and domains that are essential for ASM activity. Some ASM iso- Kornfeld, 1981) and N-acetylglucosamine phosphodies- forms have a dominant-negative effect on cellular L-ASM terase on the mannose residues of the precursor (Hurwitz activity levels (Rhein et al., 2012). However, whether and et al., 1994b; Schissel et al., 1998b), whereas S-ASM is 710 J. Kornhuber et al.: Secretory sphingomyelinase

Figure 1: L- and S-ASM arise from a common ASM precursor protein. The ASM-encoding gene SMPD1 gives rise to a common mannosylated precursor protein, pre-pro-ASM, which is cleaved to yield pro-ASM. Differential glycosylation and N-terminal as well as C-terminal processing inside the Golgi then lead to the generation of two distinct ASM forms. Whereas S-ASM is released into the extracellular space via the constitutive secretory pathway and requires Zn2+ ions for activation, L-ASM is shuttled into the lysosomal trafficking pathway via its mannose 6-phosphate groups and encounters Zn2+ ions on its way to the endolysosomal compartment. The details of the exocytotic and endocytotic steps have not been fully elucidated (dotted lines). Data regarding the molecular weight of the mature enzyme with the deglycosylated protein core in brackets are based on the most recently reported data (Edelmann et al., 2011; Jenkins et al., 2011b) and protein molecular weight calculations (protcalc.sourceforge.net) after modifications cited in the text; they may vary from other reports. spared of this modification. Bovine DNase I is an example protein misfolding (Newrzella and Stoffel, 1996; Ferlinz of a suboptimal substrate for the phosphotransferase that et al., 1997). is required to confer the mannose 6-phosphate moieties The S-ASM pro-form is constitutively secreted via the for the lysosomal pathway, and this feature is assumed to default Golgi secretory pathway resulting in a 75–80 kDa give rise to an intralysosomal as well as a secretory form extracellular enzyme with a protein core of 64 kDa (Jenkins of bovine DNaseI. A similar phenomenon may occur for et al., 2010, 2011b). The L-ASM pro-form is targeted mostly ASM (Nishikawa et al., 1997; Schissel et al., 1998b). The via cation-dependent and cation-independent mannose ASM pro-form contains six potential N-glycosylation sites 6-phosphate receptors and to a lesser extent via sortilin- (N88, N177, N337, N397, N505, N522), five of which appear mediated transport to the endolysosomal compartment to be occupied in purified ASM (Ferlinz et al., 1997). The (Ni and Morales, 2006). The importance of the mannose two C-terminal N-glycosylation sites (N505 and N522) are 6-phosphate recognition system is evident in the 8-fold essential for proper trafficking and affect S-ASM secre- increase in the ratio of secreted to intracellular ASM activ- tion and the enzymatic activity of L-ASM (Ferlinz et al., ity in cells deficient in mannose phosphorylation. Alanine 1997). Although presumably not glycosylated, removal of scanning mutagenesis of the 13 lysine residues revealed the fifth site reduces enzymatic activity to < 20% due to that p.K95 is critical for the specific three-dimensional J. Kornhuber et al.: Secretory sphingomyelinase 711 arrangement required for mannose phosphorylation, and acids, resulting in a protein core of 62 kDa (Jenkins et al., p.K95A replacement results in an overall 2-fold increase 2011b). However, another recent study indicates that TNF- in the ratio of secreted to intracellular ASM activity via mediated stimulation leads to the direct interaction of a reduction of intracellular activity and an increase in caspase-7 with the 72-kDa pro-ASM and proteolytic cleav- Zn2+-dependent secretory activity (Takahashi et al., 2005). age of the zymogen at the non-canonical cleavage site after Similarly, deglycosylation treatment or expression of a p.D253 within TNF receptosomes, generating an active dominant-negative sortilin variant results in trapping of 57-kDa L-ASM (Edelmann et al., 2011). This active L-ASM the enzyme within the Golgi in its degradation-vulner- consists of a protein core of 43 kDa and corresponds to the able form (Bartelsen et al., 1998; He et al., 1999; Ni and previously proposed active form after N-terminal cleav- Morales, 2006). age between the second and third glycosylation sites with Sequencing of purified enzymes has revealed that minimal trimming at the C-terminus (Ferlinz et al., 1994). the N-terminus of S-ASM begins at the amino acid p.H62 Unlike its lysosomal counterpart, the C-terminus of S-ASM (numbered according to the standard database reference remains intact (Jenkins et al., 2011b), enabling regulation sequence NM_000543.4 throughout the review, p.H60 in via its unbound p.C631 (Qiu et al., 2003). the publication), whereas that of L-ASM begins at p.G68 Functional inhibitors of ASM (FIASMAs), e.g., anti- (Schissel et al., 1998b) after typical N-terminal processing depressant drugs such as desipramine and fluoxetine of lysosomal proteins (Hasilik, 1992). The positions of the (Kornhuber et al., 2008, 2010, 2011), lead indirectly to the N-termini correspond to molecular weights of the remain- inactivation of L-ASM (Hurwitz et al., 1994a; Kölzer et al., ing deglycosylated full C-terminal portions of 63.6 kDa for 2004b) by leupeptin-sensitive proteolytic cleavage to a S-ASM and 63.0 kDa for L-ASM. The removal of only a few 52-kDa form (Jenkins et al., 2011b). The half-life of L-ASM, N-terminal amino acids – six in ASM – is reminiscent of as determined by the addition of the protein synthesis the maturation of another lysosomal enzyme, the β-chain inhibitor cycloheximide and pulse chase experiments, of hexosaminidase (Quon et al., 1989). By contrast, p.G85 of approximately 5–6 h (Hurwitz et al., 1994b; Schissel (61.0 kDa) has been identified as the first amino acid of et al., 1998b; Jenkins et al., 2010) is exceptionally short purified human placental ASM (Lansmann et al., 1996). compared to most lysosomal enzymes, which have half- While the exact molecular weight values differ slightly lives of days to weeks. By contrast, S-ASM is not further between publications, the presence of markedly different processed after the Golgi and is detected as a 75–80-kDa subunits across human tissues (Jobb and Callahan, 1987) form following its secretion into the extracellular space. has not been confirmed. ASM may also be delivered from In contrast to L-ASM, after secretion, S-ASM is unusually the Golgi directly to the phagosome, bypassing fusion stable and remains at the same activity level over at least with lysosomal compartments. Sortilin participates in this 25 h, with profound implications for chronic conditions process via its short cytoplasmic tail (Wähe et al., 2010), in which small changes in continued secretion over time which is similar to the cytoplasmic segment of the cation- compound to high total S-ASM levels (Jenkins et al., 2010). independent mannose 6-phosphate receptor (Petersen ASM activity is influenced by further post-transla- et al., 1997; Nielsen et al., 2001). tional modifications. An activating effect on L-ASM has In the endolysosomal system, the 72-kDa enzyme is been described for the phosphorylation of serine residue processed at the C-terminus to the 65-kDa L-ASM (55 kDa p.S510 in the C-terminal domain by the protein kinase Cδ. for the deglycosylated protein). This processing step within It has been postulated that this phosphorylation results the acidic compartment is similar to that for other lysoso- in the translocation of L-ASM to the plasma membrane mal hydrolases (Erickson and Blobel, 1983; Arunachalam (Zeidan and Hannun, 2007a; Zeidan et al., 2008b). The et al., 2000) and is essential to generate the catalytically ASM mutant p.S510A, in which this phosphorylation site highly active enzyme, most likely by promoting the coor- was altered by site-specific mutagenesis, retains L-ASM dination of lysosomal Zn2+. By contrast, the low activity activity but is not secreted (Jenkins et al., 2010). This phos- of the non-lysosomal precursor may prevent enzymatic phorylation site seems to be essential for constitutive as activity in the Golgi (Jenkins et al., 2011b). The role of the well as regulated secretion and thus the activity of S-ASM. C-terminal region in the proteolytic maturation of L-ASM Furthermore, stimulated exocytosis of ASM is dependent is indicated by the altered localization and virtual absence on the t-SNARE protein syntaxin 4 (Perrotta et al., 2010). of Zn2+-independent activity in the recombinant NPD L- and S-ASM are activated in vitro via interactions mutants p.R602H, p.R602P, and p.ΔR610 (Jenkins et al., with Zn2+ ions (Schissel et al., 1998b). Zn2+ acquisition 2011b). The preliminary detection of p.Q622 as the C-ter- itself may represent a form of L-ASM regulation because minus of L-ASM would suggest a loss of only nine amino the partial Zn2+-dependence of L-ASM isolated from 712 J. Kornhuber et al.: Secretory sphingomyelinase lysosomal-rich fractions but not standard cell homogen- 2008a; Milhas et al., 2010). However, stimulating agents ates suggests an incomplete saturation of this form with can have differential effects on L- and S-ASM activity. The Zn2+ (Schissel et al., 1998b), although the isolate might co-regulation of S- and L-ASM activity has been addressed have contained Zn2+-dependent pre-lysosomal forms in several cell culture systems. Cultured human platelets in addition to the fully mature lysosomal 65-kDa ASM exhibit increased S-ASM release but decreased L-ASM (Jenkins et al., 2011b). Massive overexpression of ASM, activity upon stimulation with thrombin (Romiti et al., which typically results in the saturation of the mannose 2000). S-ASM activity is increased and L-ASM activity is 6-phosphate receptor shuttling mechanism and secretion decreased in MCF7 breast carcinoma cells treated with of an otherwise lysosomally targeted enzyme (Ioannou phorbol myristate acetate or ammonium chloride (Jenkins et al., 1992), yielded a Zn2+-dependent mannose 6-phos- et al., 2010). S-ASM activity is increased upon stimulation phate receptor-binding species in the supernatant. This with interleukin (IL) 1β or tumor necrosis factor-alpha finding demonstrates that Zn2+ acquisition is not glyco- (TNFα), with no effect on L-ASM activity (Jenkins et al., sylation-dependent but occurs during lysosomal traffick- 2010). The same pattern was observed following the treating (Schissel et al., 1998b). ment of human umbilical vein endothelial cells with IL-1β Eight disulfide bonds are formed among 17 cysteine or interferon γ; S-ASM activity was increased by 2–3.5-fold, residues within mature ASM, which leaves the C-terminal whereas L-ASM activity was slightly decreased (Marathe p.C631 unbound (Lansmann et al., 2003). Oxidation, dele- et al., 1998). Oxidized low-density lipoprotein (LDL) and tion or replacement of this residue activates recombinant oxidized LDL-containing immune complexes differen- human S-ASM by favoring the hydration of Zn2+. Activa- tially regulate L-ASM activity and S-ASM release in mac- tion is also achieved by copper-promoted dimerization rophages (Truman et al., 2012). A special role of S-ASM in via cysteine residues and by C-terminal truncation by chemokine elaboration was detected following the stimu- carboxypeptidase Y (Qiu et al., 2003) and likely resembles lation of MCF7 cells with TNFα because the chemokine the cysteine switch mechanism of matrix metalloprotein- CCL5 appears to be an initial target of the pathway that ases (Van Wart and Birkedal-Hansen, 1990) in post-trans- is selectively induced by S-ASM activity (Jenkins et al., lational regulation. However, this activation is assumed 2011a). Cytokines also affect the ratio of L- and S-ASM to be limited to S-ASM in which the entire C-terminus is activity in a genetic manner (Rhein et al., 2014). Thus, retained; this cysteine is lost in fully mature L-ASM during cytokine stimulation of cultured cells may have a short- the final processing step (Jenkins et al., 2011b). term effect on S-ASM and appear to increase S-ASM activity and secretion while decreasing L-ASM activity. This effect could evolve by redirecting the ASM precursor Regulation of S-ASM and L-ASM secretion protein away from the lysosomal pathway into the secretory pathway. The involvement in this redirection of the The ASM enzyme consists of distinct domains, a proline- cis-Golgi enzyme N-acteylglucosaminyl-1-phosphotrans- rich domain, a metallophosphoesterase domain and the ferase, which phosphorylates the mannose residues of the C-terminal domain. Moreover, strong sequence homol- precursor protein for the lysosomal trafficking pathway, ogy (Ponting, 1994) and an analogous arrangement of has been proposed (Tabas, 1999). disulfide bonds (Lansmann et al., 2003) suggest that Although originally thought to localize only to the ASM contains its own intramolecular sphingolipid acti- lysosome, under stress conditions, L-ASM may translo- vator protein (SAP)-type domain as the fourth functional cate from intracellular compartments to the extracellu- domain and is thus, unlike other lysosomal hydrolases, lar leaflet of the cell membrane, where it hydrolyzes SM independent of the presence of SAPs as a cofactor. SAPs to ceramide to form signaling platforms (Cifone et al., are small, enzymatically inactive lysosomal glycoproteins 1994); however, L-ASM may also contribute to extracellu- that facilitate sphingolipid degradation at the inner mem- lar ASM activity. The designation of the ASM that is trans- brane of acidic compartments by promoting substrate located to the outer leaflet of the plasma membrane as binding and lipid extraction (Kolter and Sandhoff, 2005; a response to specific stimuli is not consistent and may, Remmel et al., 2007). The addition of SAPs can partially in fact, represent S- or L-ASM or a combination of both rescue inactive ASM with mutations in conserved amino species. While some reports refer to the enzyme as ‘secre- acids within its SAP domain (Kölzer et al., 2004a). tory’ ASM, other authors suggest it is L-ASM by assuming The selective activation of L-ASM by a variety of a lysosomal origin. Although the vesicles that appear to stress stimuli has been comprehensively investigated fuse and empty their contents at the extracellular surface (Charruyer et al., 2005; Rotolo et al., 2005; Zeidan et al., may contain the secretory form of ASM, direct data on J. Kornhuber et al.: Secretory sphingomyelinase 713 specific properties or Zn2+-dependence are lacking in most a negative curvature that leads to inward vesiculation studies. For example, extracellular ASM activity increased (Trajkovic et al., 2008), ceramide generation caused by following the repair of myoblasts from mechanical injury the activation of the neutral sphingomyelinase species as a result of vesicle docking and exocytosis (Defour localized to the inner leaflet of the plasma membrane pro- et al., 2014). Cationic cell-penetrating peptides as poten- motes an outward curvature and thus allows the cell to tial molecular transporters of membrane-impermeable shed membrane-containing toxin pores into the extracel- molecules induce the translocation of ASM to the plasma lular space (Draeger and Babiychuk, 2013). membrane, resulting in an increase in ceramide formation and, in turn, enhancing its own uptake (Verdurmen et al., 2010). Similarly, treatment of Jurkat T cells with ultra- Sources of S-ASM violet light type C also induces the rapid translocation of ASM to the membrane, hydrolysis of extracellular SM Similar to the ubiquitous expression of L-ASM, which and raft clustering (Rotolo et al., 2005). In addition to the presumably reflects an important housekeeping role, a ASM-independent functions of CD95, transmission elec- wide variety of cells have been demonstrated to secrete tron microscopy and fluorescence-activated cell sorting substantial amounts of Zn2+-dependent S-ASM. In addi- analysis have demonstrated that CD95 stimulation also tion to the production of L-ASM, murine brain microglial causes ASM to translocate from intracellular, presumably cells, mouse peritoneal macrophages, J774 macrophages, vesicular, compartments to the extracellular leaflet. This human skin fibroblasts and several other (untransfected) process hydrolyzes extracellularly oriented SM, which, in cultured cells, including Chinese hamster ovary and COS-7 turn, mediates CD95 clustering as an essential step toward cells, have also been reported to secrete ASM that is acti- CD95-triggered cell death (Grassmé et al., 2001). Together vated by physiological levels of Zn2+. This activity is mark- with CD95, TNF-related apoptosis-inducing ligand is edly up-regulated during the differentiation of human involved in the activation of ASM via a redox mechanism monocytes to macrophages (Schissel et al., 1996a). that results in the generation of ceramide and the forma- Cultured human coronary artery and umbilical vein tion of ceramide-enriched membrane platforms (Dumitru endothelial cells secrete massive amounts of ASM both and Gulbins, 2006), potentially via the release of ASM of apically and basolaterally (Marathe et al., 1998). However, lysosomal origin, as indicated by cell lysate activity in the in contrast to the almost complete Zn2+-dependence of absence of Zn2+. The application of hydrogen peroxide as S-ASM originating from macrophages and fibroblasts, the primary form of reactive oxygen species in mammals endothelium-derived S-ASM exhibits partial activity in the also induces very rapid calcium-dependent ASM release absence of exogenous Zn2+. The up to 20-fold higher levels by lysosomal exocytosis, as detected by the exposure of of basal secretion compared to macrophages are further lysosome-associated protein LAMP1 (Li et al., 2012). The increased 2- to 3-fold by incubation with inflammatory lysosomal origin of released ASM is also supported by the cytokines such as IL-1β or interferon-gamma (Marathe concomitant appearance of LAMP1 on the sarcolemma et al., 1998), demonstrating that human vascular endothe- surface and the release of the lysosomal β-hexosaminidase lial cells are a rich and regulatable source of S-ASM. The in a wound repair study (Corrotte et al., 2013). ability of ependymal cells to secrete ASM has not yet been In fact, ASM has recently emerged as a major regu- analyzed, although these cells are responsible for most lator facilitating wound repair in eukaryotic cells, such daily cerebrospinal fluid (CSF) production and could be as from pore-forming toxins (Tam et al., 2010; Corrotte the source of S-ASM detected therein (Mühle et al., 2013). et al., 2013). Plasmalemmal injury-triggered Ca2+ influx In addition to various endothelial cells, human platelets induces the fusion of lysosomes with the plasma mem- not only exhibit ASM activity but also release the enzyme brane and exocytosis of ASM. Through ceramide genera- in response to stimulation by thrombin (Simon et al., tion, ASM in turn triggers lesion removal via endocytosis 1998; Romiti et al., 2000), without a significant concomi- and intracellular degradation (Tam et al., 2013; Andrews tant change in ceramide levels (Simon and Gear, 1999). et al., 2014). Transcriptional silencing of ASM abolishes As a likely source of ASM in human tear fluid, human this process, while the addition of exogenous enzyme corneal cells constitutively secrete ASM and respond with restores it (Tam et al., 2010). Trypanosoma cruzi sub- increased secretion to agents that induce stress, includ- verts the ASM-dependent ceramide-enriched endosomes ing ultraviolet B radiation and hyperosmolarity (Robciuc in the plasma membrane repair pathway to invade host et al., 2014). cells (Fernandes et al., 2011). While ceramide production Furthermore, extracellular ASM activity has been by ASM in the outer leaflet promotes tighter packing and detected in muscle cell supernatants after mechanical 714 J. Kornhuber et al.: Secretory sphingomyelinase injury and repair but may represent the lysosomal enzyme Distribution of S-ASM that is exocytosed after docking of lysosomes to the plasma membrane (Defour et al., 2014). The release of In contrast to the nearly ubiquitous distribution of the ASM into the culture medium of human fibroblasts and L-ASM enzyme in mammalian tissues (Weinreb et al., 1968) mouse L-cells has been detected in the presence of the and almost uniform expression across human brain regions lysosomotropic agent ammonium chloride (Weitz et al., (Spence et al., 1979), there is only some basic evidence for 1983). Similarly, a number of studies have reported that the presence of S-ASM in various body fluids other than upon stimulation with various agents, vesicles of unspeci- serum and plasma. Early studies indicated ASM activity in fied identity containing ASM translocate to the plasma amniotic fluid and urine and confirmed this activity based membrane and release the enzyme to the outer leaflet or on phosphorylcholine liberation as well as lower activity to the extracellular space. Some authors refer to this ASM levels in patients with NPD (Harzer and Benz, 1973). The species as L-ASM (Li et al., 2012; Defour et al., 2014), some enzyme has been purified from human urine and exten- as S-ASM (Rotolo et al., 2005) and some do not directly sively characterized (Weitz et al., 1985; Quintern et al., 1987; specify the affiliation (Grassmé et al., 2001; Verdurmen Quintern and Sandhoff, 1991). Compared to ASM activity et al., 2010), which thus remains to be elucidated. Regard- in leukocytes, greater variations in activity were observed less, the exocytosed ASM localizes to the extracellular in human urine and were best correlated with 24-h creati- space or the outer leaflet of the plasma membrane, in con- nine excretion in long-term tests (Seidel et al., 1978). Taka- trast to the orientation of the neutral sphingomyelinase, hashi et al. (2000) detected slightly weaker S-ASM activity in which the catalytic site is thought to face the cytosolic in human urine compared to sera and nearly 2-fold higher inner leaflet (Tani and Hannun, 2007). activity in synovial fluid aspirated from patients with either While S-ASM is secreted by various cell types, the rheumatoid arthritis or osteoarthritis. Significant stimula- enzyme can also be internalized and trafficked to the lyso- tion of SM degradation by Zn2+ ions indicated the presence some, where its contact with Zn2+ sufficiently activates the of S-ASM rather than L-ASM in these samples. Very high enzyme such that subsequent cell homogenates exhibit Zn2+-dependent S-ASM activity has been observed in sali- sphingomyelinase activity in the absence of added Zn2+, vary fluid and may participate in the digestion of the abun- as demonstrated in ASM-negative cells treated with S-ASM dant SM in the normal diet. Similarly, ASM activity detected (Schissel et al., 1998b). Similar to other lysosomal enzyme in human milk efficiently degraded SM as the dominant defects, this feature renders NPD amenable to enzyme phospholipid in milk (Nyberg et al., 1998). replacement therapy. After intravenous administration of Unexpectedly, ASM activity is more than 10-fold recombinant ASM in ASM knock-out (KO) mice, more than higher in tear fluid than in serum. Although the lack of a 90% of the injected enzyme was taken up by the liver, of response to the addition of Zn2+ ions in that study (Taka- which the majority was delivered to the lysosome. The hashi et al., 2000) may indicate that this activity is the mannose 6-phosphate receptor system was responsible result of L-ASM released from cells, a more recent investi- for 30–50% of the internalization of the enzyme (He et al., gation identified both L-ASM and S-ASM in all five tested 1999), although in ASM KO cells, the mannose receptor tear fluid samples (Robciuc et al., 2014). By optimizing the plays the major role (Dhami and Schuchman, 2004). As reaction conditions, particularly with respect to the type little as four injections resulted in the reversal of symp- and concentration of detergent, Zn2+-dependent S-ASM toms in reticuloendothelial organs, even in older KO mice, activity has been detected in human CSF and character- but not in neurological improvement because the enzyme ized (Mühle et al., 2013). The identity of this enzymatic cannot traverse the blood-brain-barrier (Miranda et al., SM-hydrolyzing activity as S-ASM encoded by SMPD1 2000). was confirmed by its absence in Smpd1 KO mice and Estimations of ASM activity in human blood indicate overexpression in Smpd1 transgenic mice, which exhibit that approximately 17% of the total activity in healthy extraordinarily high levels of S-ASM compared to L-ASM adults is present as L-ASM in the mixture of cells repre- overexpression. ASM activity in human or boar seminal senting peripheral blood mononuclear cells, while the plasma is not influenced by divalent metal ions or chelat- remaining 83% is detectable as Zn2+-dependent S-ASM in ing agents. Thus, these enzymes most likely originate from the corresponding plasma (C. Mühle, unpublished data). the lysosome (Vanha-Perttula, 1988). A SM-degrading This proportion, or even absolute ASM levels, may vary enzyme with a slightly acidic pH optimum and a distinct between species, as suggested by the observation of ele- pattern of activation by Co2+ and Mn2+ has been purified vated S-ASM activity in fetal calf serum, mouse and par- from bovine seminal plasma but is assumed to differ from ticularly rat serum (C. Mühle, unpublished data). S-ASM (Vanha-Perttula, 1988; Vanha-Perttula et al., 1990). J. Kornhuber et al.: Secretory sphingomyelinase 715

In a mouse model of NPD type C, lysosomal enlarge- dose-dependent up-regulation of S-ASM secretion with ment in the brain – a feature of these lysosomal storage selective production of C16-ceramide at the expense of disorders – correlated with that in circulating B cells (te C16-SM, elevations in L-ASM activity are associated with Vruchte et al., 2014). Thus, measuring the relative acidic increases in selective, very-long-chain ceramide species, compartment volume of these peripheral cells using a e.g., C26:1-ceramide (Jenkins et al., 2010). Michaelis- fluorescent probe may represent a surrogate marker for Menten kinetic analysis revealed roughly similar values of the progression of lipid storage in the brain. While these the Michaelis constant Km for SM affinity despite the use data suggest an association between brain and peripheral of differently labeled substrates: 77 μm for soluble ASM in L-ASM activities, the higher levels of S-ASM activity in tear the human epidermis (Bowser and Gray, 1978), 5–25 μm fluid compared to serum (Takahashi et al., 2000) and the for ASM purified from placenta (Jones et al., 1981, 1983; lack of a correlation between activities in serum and CSF Sakuragawa, 1982), 65 μm for ASM from Bacillus cereus (Mühle et al., 2013) indicate tissue-specific regulation of (Fujii et al., 2004), 47 μm for ASM from human fibroblasts S-ASM secretion. (Sato et al., 1988), 25 μm for recombinant ASM from the conditioned medium of infected insect cells (Bartelsen et al., 1998), 20 μm for ASM from human CSF (Mühle et al., 2013), 0.2–0.6 μm for ASM released from human fibroblasts In vitro characterization and and mouse L-cells, respectively (Weitz et al., 1983), 11 μm function of S-ASM with a VMAX of 21 nmol/h/mg protein for HEK 293 whole

cell lysates and 2 μm and a VMAX of 4.3 μmol/h/mg protein Coordinated breakdown of SM, the most abundant for ASM immunoprecipitated from Jurkat cells (Gulbins complex sphingolipid in mammalian cells, is essential and Kolesnick, 2000). Specific activities ranged from 600 for membrane homeostasis. Different structures such to 2500 μmol/h/mg (Sakuragawa, 1982; Yamanaka and as the plasma membrane, axonal myelin sheaths, plas- Suzuki, 1982; Jones et al., 1983; Weitz et al., 1985; Lans- matic lipoproteins, various particles or vesicles as well as mann et al., 1996). membranes of exogenous origin such as particles could Defects in N-glycosylation reduce enzyme stabil- serve as physiological substrates for S-ASM. As a bilayer- ity and activity but do not affect KM values (Newrzella destabilizing lipid with a considerably smaller polar head and Stoffel, 1996). Moreover, pH plays an important role group and thus reduced area within the leaflet compared in substrate binding but not catalytic velocity (Callahan to SM (López-Montero et al., 2007), ceramide imposes a et al., 1983). The stimulation of cell surface receptors can negative curvature at the bilayer interface (Veiga et al., lead to an increase in VMAX′ of 2–3-fold with little or no

1999). Thereby, ceramide generation by ASM in the outer change in KM values (Schwandner et al., 1998). In a thera- leaflet of the plasma membrane facilitates the formation peutically aimed approach, the dipolar aprotic solvent of endocytic vesicles (Zha et al., 1998). Moreover, the dimethyl sulfoxide caused a slow but marked up to 2-fold altered fluidity and exclusion of cholesterol promote the increase in sphingomyelinase activity (Vmax) at pH 5.0 and formation of large ceramide-enriched platforms, which, in 7.5 over a period of days. This activity was dependent on turn, lead to clustering of receptors and signaling mole- protein synthesis and did not substantially change the Km cules (Sot et al., 2008; Staneva et al., 2008, 2009). In addi- value (Sakuragawa et al., 1985; Sato et al., 1988). tion to SM, ASM may also cleave phosphatidylglycerol and phosphatidylcholine (Quintern et al., 1987; Oninla et al., 2014), although contradictory evidence indicates that Reaction conditions neither phosphatidylcholine nor lysophosphatidylcholine or glycerophosphocholine undergo hydrolysis (Spence The influence of detergents on ASM activity was exam- et al., 1989). ined by Spence et al. (1989) using fetal bovine serum, with an 8–15-fold increase in response to the addition of Triton X-100 to the reaction. It was also noticeable by the Enzyme kinetics discrepancy in an order of magnitude between optimal Nonidet P-40 concentrations in assays for human serum- Experiments using a secretion-incompetent p.S510A vs. CSF-derived S-ASM (Mühle et al., 2013). Detergent also mutant enzyme suggest distinct metabolic roles of S-ASM influences the immunoprecipitation of soluble ASM from and L-ASM in cellular ceramide formation with respect human urine and placenta (Driessen et al., 1985), but to specific substrates: while Il-1β induces a time- and details regarding the different detergent optima remain 716 J. Kornhuber et al.: Secretory sphingomyelinase limited. Storage temperature appears to be an impor- zinc-dependent enzymes in a process that is dynamically tant parameter for comparing absolute S-ASM activities controlled by its interactions with the glutathione redox because the activity of endogenous ASM from plasma, couple (Jiang et al., 1998). Consequently, exogenous Zn2+ CSF and cell lysates (Mühle et al., 2013) as well as that of ions are not essential for endothelial cell secreted S-ASM human recombinant ASM in cell supernatants (Qiu et al., activity and cause only 2-fold stimulation (Marathe et al., 2003) is higher following long-term storage at -20°C com- 1998). pared to -80°C. This activation is attributed to the loss of the single free cysteine residue p.C631 by chemical modification during the freezing process (Qiu et al., 2003). Activity at neutral pH However, the enzymatic activity remains relatively stable during the storage of CSF (Mühle et al., 2013) as well as Given the optimal reaction rate of the enzyme in an acidic undialyzed urine (Seidel et al., 1978) at room temperature range of pH 5.0–5.5 (Spence et al., 1989; Mühle et al., 2013), for several days. activity in the neutral pH of blood or CSF of 7.4 appears The dependence on the addition of Zn2+ ions is sup- counterintuitive. Indeed, the ability of S-ASM to hydrolyze ported by the stimulation of S-ASM activity by Zn2+, which SM in a neutral milieu remains controversial and has led is increased in case of prior chelation of endogenous Zn2+ some authors to refer to S-ASM as ‘secretory sphingomyeli- by EDTA, and its inhibition by EDTA or the more zinc- nase’, omitting the specification ‘acidic’ despite its proven specific chelator 1,10-phenanthroline (He et al., 1999). By origin from the ASM-encoding gene SMPD1. However, this contrast, typical short-term chelation of L-ASM with EDTA name would only be unambiguous in the absence of any is insufficient to strip the enzyme of its metal ion, consist- other reported sphingomyelinase species, e.g., neutral or ent with other zinc metalloenzymes (Little and Otnäss, alkaline enzymes that could potentially be detectable as 1975). In contrast to neutral sphingomyelinases, Mg2+ secreted or otherwise released proteins in the extracellu- and Mn2+ were ineffective for ASM activity (Spence et al., lar space, which is currently the case (Milhas et al., 2010). 1989). The role of other metal cations has not been well Data concerning the extent of S-ASM activity at investigated, with the exception of a potentially differ- neutral pH are inconsistent. Both S-ASM activities in ent ASM species that was partially purified from seminal saliva and tear fluid peak at pH 4–5 and are markedly plasma (Vanha-Perttula, 1988). The optimal Zn2+ concen- decreased at pH 6, with negligible residual activities at trations are similar to those for matrix metalloproteinases pH 7 (Takahashi et al., 2000). This behavior corresponds (Sorbi et al., 1993) and lie well within the range present well to the pH profiles described for tissue L-ASM (Sch- in the extracellular space, e.g., approximately 100 μm neider and Kennedy, 1967; Bowser and Gray, 1978), ASM in human whole blood or serum (Buxaderas and Farré- released from human fibroblasts (optimal pH 4.4) and Rovira, 1985), although the actual concentration depends mouse L-cells (pH 4.8) (Weitz et al., 1983) and our data for on factors such as dietary intake or pregnancy (Donan- S-ASM in human CSF (Mühle et al., 2013), serum, plasma, gelo and Chang, 1981). Zn2+ ions move freely through the saliva and urine, which indicate < 5% of maximal Zn2+- cerebrospinal and extracellular fluid compartments and dependent activity at pH 6.5 and nearly background levels are differentially distributed in various regions of the at pH 7.0 (C. Mühle, unpublished data). Purified recombi- brain (Takeda et al., 1994), exhibiting variations in half- nant human ASM from CHO cells binds tightly to SM at pH life (Takeda et al., 1995) and age-dependence (Sawash- 4.0, while binding is not detectable at pH 8.0 (He et al., ita et al., 1997) that may influence local S-ASM activity. 1999). By contrast, S-ASM from fetal bovine serum (Spence Moreover, Zn2+ levels are elevated in both atherosclerotic et al., 1989) or recombinant ASM species (Mintzer et al., and inflammatory lesions (Mendis, 1989; Milanino et al., 2005) exhibit clearly detectable hydrolysis at a neutral pH 1993), and Zn2+ is released during intense neuronal activa- of 7.4 of up to 20% of that at the optimal acidic pH. This tion (Assaf and Chung, 1984; Frederickson and Moncrieff, discrepancy may be attributable to the different charac- 1994). In addition, the regulation and cell-type-dependent teristics of human endogenous vs. purified (Spence et al., variation of the subcellular concentration and localization 1989) bacterial or recombinant, sometimes tagged, S-ASM of Zn2+ ions (Csermely et al., 1987; Brand and Kleineke, (Mintzer et al., 2005) species utilized in these experiments, 1996) may render S-ASM partially or fully Zn2+-independ- different reaction conditions, substrates and purity or gly- ent under specific conditions. For example, the cysteine- cosylation patterns (Jenkins et al., 2009) of the enzymes. rich protein metallothionein acts as a temporary cellular Interestingly, neutral sphingomyelinase C from Staphylo- reservoir of free zinc. Metallothionein may keep cellular coccus aureus selectively catalyzes the hydrolysis of SM in concentrations very low or release zinc to intracellular the plasma membrane (Slotte et al., 1990). Its application J. Kornhuber et al.: Secretory sphingomyelinase 717 to hippocampal slices results in the generation of long- lipoproteins of these mice enhances their susceptibility to chain ceramides and in a positive reversible regulation of ASM (Jeong et al., 1998). The most prominent difference neuron excitability via a sphingosine-1-phosphate-medi- was a 10-fold increase in S-ASM activity at pH 7.4 in LDL ated mechanism (Norman et al., 2010). Because extracel- extracted from atherosclerotic lesions compared to that in lular ASM catalyzes comparable reactions at the plasma plasma LDL from the same human donor (Schissel et al., membrane, similar activity can be expected. 1998a). Although the biological role of S-ASM is not precisely Moreover, extracellular SM degradation by S-ASM clear, the enzyme has been implicated in the metabo- could occur in pockets of acidity that are present in lism of lipoprotein-bound SM to ceramide, the subse- inflammatory lesions of the arterial wall (Menkin, 1934) quent aggregation and retention of LDL particles and the and in artherosclerotic plaques (Naghavi et al., 2002; acceleration of lesion progression as key initial steps in Sneck et al., 2012), in proximity to glycosaminoglycans atherogenesis (Marathe et al., 2000a; Tabas et al., 2007; (Maroudas et al., 1988), activated macrophages (Tapper Devlin et al., 2008). After observing that SM-degrading and Sundler, 1992) and osteoclasts (Silver et al., 1988) activity of several arterial wall cell types is responsible for or close to the cell surface, e.g., in acidic microdomains subendothelial retention and aggregation of atherogenic located predominantly in the distal dendrites of oligo- lipoproteins in rabbit and human atherosclerotic lesions dendrocytes (Ro and Carson, 2004). Such acidic micro- (Schissel et al., 1996b), Schissel et al. (1998a) identified this environments have been linked to lipid microdomains activity as a Zn2+-dependent S-ASM capable of hydrolyzing (Steinert et al., 2008) as the site of optimal ASM action. SM associated with atherogenic lipoproteins at a neutral The translocation of lysosomal V1 H+-ATPase to the cell pH. Signs of SM hydrolysis in lesional LDL (Öörni et al., membrane is a critical contribution to the formation of a 1998) as well as ASM-induced LDL aggregation (Walters local acid microenvironment to facilitate membrane ASM and Wrenn, 2011), depending on the sphingomyelinase- activation and, consequently, redox signalosome forma- to-LDL molar ratio (Guarino et al., 2006), confirmed the tion in coronary arterial endothelial cells (Xu et al., 2012). role of ASM and extended its promotion of aggregation In addition, substrate binding but not catalytic velocity to small very-low-density and intermediate-density lipo- are predominantly mediated by pH (Callahan et al., 1983). protein particles (Öörni et al., 2005). However, another S-ASM may also be active in acidic endosomes following group detected sphingomyelinase activity as an intrinsic endocytosis of the enzyme by cell surface receptors (Korn- property of the multifunctional LDL constituent apoli- feld, 1987). poprotein B-100 based on the sequence homology of the Human plasma itself exerts an inhibitory effect on the apoliprotein B-100 α2 domain with bacterial sphingomy- reaction if sample volumes > 2% of the total assay volume elinase or at least as very tightly associated with LDL and are used. This inhibition is only partially explained by the absent in oxidized LDL or other lipoproteins (Holopainen presence of low amounts of EDTA originating from anti- et al., 2000; Kinnunen and Holopainen, 2002). A number coagulation vials. Inhibition has also been observed to a of atherogenesis-associated modifications of lipoprotein- lesser degree for lithium-heparin-plasma as well as serum bound SM appear to increase its susceptibility to hydroly- (C. Mühle, unpublished data). The effect is not observed sis: while native LDL is only hydrolyzed and aggregated for CSF but is expected to limit the SM-hydrolyzing activity at acidic pH, oxidation, treatment with phospholipase A2, of S-ASM in the blood in vivo. or enrichment with apolipoprotein CIII promotes prompt hydrolysis at pH 7.4 (Schissel et al., 1998a). LDL particles undergo lipolysis by ASM more readily after prior proteoly- Endogenous inhibition of ASM activity sis by the extracellular hydrolases chymase and cathepsin S, and this pre-treatment was even essential for the action The activity of S-ASM is inhibited by a number of endog- of the secretory phospholipases A2 (sPLA2-V). This sensi- enous agents, including adenosine monophosphate tization of LDL by proteolysis may enhance extracellular and high concentrations of ZnCl2 (6 mm) (Schissel et al., accumulation of LDL-derived lipids during atherogenesis 1996a; Jenkins et al., 2010), as well as in a time- and dose- (Plihtari et al., 2010). Moreover, a high SM-to-phosphati- dependent manner by dithiothreitol and beta-mercapto dylcholine ratio appears to promote hydrolysis and aggre- ethanol (He et al., 1999). Interestingly, S-ASM appears to gation by ASM at neutral pH, as observed for plasmatic be less stable than L-ASM at elevated temperatures (Schis- lipoproteins from apolipoprotein E KO mice, which are sel et al., 1996a). Inorganic phosphate, other nucleotides, prone to atherosclerosis (Schissel et al., 1998a). Simi- dolichol phosphate and phosphoinositides are potent larly, the high SM content in the slowly cleared remnant noncompetitive inhibitors of ASM (Callahan et al., 1983; 718 J. Kornhuber et al.: Secretory sphingomyelinase

Watanabe et al., 1983). Interestingly, while phosphati- of cells to the pathogen (Gupta et al., 2008). Surface-local- dylinositol 3,5-bisphosphate (Kölzer et al., 2003) and ized ASM activity and the presence of SM are required for phosphatidylinositol 3,4,5-trisphosphate (Testai et al., the efficient infection of cells by Ebola virus (Miller et al., 2004) both inhibit ASM in the low mm range, the enzyme 2012). Moreover, there is evidence that pathogens utilize is potently activated by other phosphate-containing lipids their own sphingolipid repertoire as virulence factors, such as endocytosed bismonoacylgycerophosphate (Linke such as the sphingomyelinase of Streptococcus aureus et al., 2001) or by plasma membrane-derived phosphati- (Cohen and Barenholz, 1978), or continue to modulate dylglycerol and phosphatidic acid (Oninla et al., 2014). the sphingolipid metabolism of the host after internaliza- Cholesterol, another endogenous lipid, potently inhibits tion to replicate, survive, and finally infect neighboring ASM (Reagan et al., 2000). Removal of lipoproteins from cells (reviewed in Zeidan and Hannun, 2007b). An under- the culture medium of fibroblasts obtained from NPD-C standing of these interaction processes is a prerequisite patients who suffer from an additional secondary ASM for future antimicrobial therapies. In addition, ASM is deficiency restored the activity of ASM (Thomas et al., required for the proper fusion of late phagosomes with 1989). The presence of sterols is likely responsible for the lysosomes to efficiently transfer lysosomal antibacterial inhibitory effect on ASM activity, which may be relevant hydrolases into phagosomes (Schramm et al., 2008). to inhibitor development because various members of the sterol class, such as 7-ketocholesterol, exhibit inhibitory potential in vitro (Maor et al., 1995). Involvement of ASM in other cellular processes

Role of ASM in microorganism infection In erythrocytes, the activity of sphingomyelinase induces a transition from a discoid to spherical shape, followed Viral, bacterial and parasitic pathogens are assumed by phosphatidylserine exposure and finally a loss of cyto- to induce changes in the composition and structure of plasmic content (Dinkla et al., 2012). In the context of membranes and microdomains therein to permit the elevated S-ASM levels in sepsis (Claus et al., 2005; Kott invasion of epithelial cells. The triggered translocation of et al., 2014), the resulting enhanced erythrocyte clearance ASM from endolysosomal compartments to the host cell is likely to contribute to anemia. The sensitivity of eryth- surface followed by SM hydrolysis to form ceramide-rich rocytes increases further during blood bank storage and domains seems to play a key role in microbial internaliza- physiological aging (Dinkla et al., 2012). tion (Riethmüller et al., 2006). ASM has been associated In contrast to the vast number of experimental con- with infection and host defense against Pseudomonas ditions that result in increased ASM activity, only one aeruginosa (Grassmé et al., 2003), Listeria monocytogenes study has reported a condition leading to a reduction of (Utermöhlen et al., 2003) and Cryptosporidium parvum ASM activity. Mechanical injury-triggered ASM secretion (Nelson et al., 2006). ASM mediates the entry of Neisseria is reduced by 70% in C2C12 myoblasts upon knockdown of gonorrhoeae into nonphagocytic epithelial (Grassmé dysferlin expression, thus mimicking the cells of patients et al., 1997) and phagocytic (Hauck et al., 2000) cells with the progressive muscle wasting disease dysferlinopa- via the enrichment of CEACAM receptors for the bacte- thy. The defect in cell membrane repair in the immortal- rium in ceramide domains, thus functioning as a portal ized patient myoblasts was fully reversed by treating the of microbial entry. Infection of macrophages with Salmo- cells with extracellular ASM (Defour et al., 2014). nella typhimurium induces a dynamic change in the cel- While the ASM/ceramide pathway has been well lular distribution of ASM, with a reduction in L-ASM and established as a bona fide system in the chemothera- a surge in S-ASM activity (McCollister et al., 2007). L-ASM peutic treatment of cancers and many commonly used is rapidly activated during the entry of sindbis virus in chemotherapeutic agents mediate cell death via ASM- neuroblastoma cells (Jan et al., 2000). Viruses such as induced ceramide formation [reviewed in Henry et al., human immunodeficiency virus (Popik et al., 2002) and (2013)], recent evidence (Don et al., 2014) suggests that rhinoviruses (Grassmé et al., 2005) appear to employ reducing ASM activity with functional inhibitors such as sphingomyelin-enriched microdomains to invade epithe- desipramine may induce lysosomal rupture in cancer cells lial cells. SM functions as a novel receptor for the entry (Petersen et al., 2013). Although unexpected with respect of Helicobacter pylori vacuolating cytotoxin into epithelial to reduced SM levels in cancerous compared to normal cells, and treatment with sphingomyelinase to deplete tissue (Hendrich and Michalak, 2003; Barceló-Coblijn plasma membrane SM significantly reduces the sensitivity et al., 2011), ASM activity is down-regulated in cancer J. Kornhuber et al.: Secretory sphingomyelinase 719

(Petersen et al., 2013) and may consequently sensitize 2014). In patients with Alzheimer’s disease, the activity of these cells to further decreases in enzyme activity levels. S-ASM is increased and the concentration of SM is reduced Lysosomal stability and integrity requires L-ASM activity in the plasma, while ceramide levels are unchanged (Lee (Kirkegaard et al., 2010), while the cytoprotective proper- et al., 2014). S-ASM activity is elevated in the plasma of ties of ASM down-regulation in cancer cells are assumed APP/PS1 mice, while the concentrations of SM and cera- to relate to the secreted form required for vesicle and mide are unchanged (Lee et al., 2014). Feeding rats grape membrane turnover (Magenau et al., 2011). In the context seed oil vs. butter leads to increased S-ASM activity and, of reduced SM levels in cancer, the potent antitumor agent simultaneously, decreased ceramide concentrations 2-hydroxyoleic acid selectively kills cancer cells by stim- (Drachmann et al., 2007). The induction of S-ASM activ- ulating SM synthases, leading to strong augmentation ity by adeno-associated virus in ApoE-/- mice resulted in and restoration of the SM concentration (Barceló-Coblijn only minor changes in SM and ceramide: a small reduc- et al., 2011). tion of SM and ceramide in plasma at several time points after injection and a reduction of SM and unaltered ceramide concentrations in the liver (Leger et al., 2011). Cor- relations between S-ASM in plasma SM and ceramide In vivo effects of S-ASM activity on were previously reported only sporadically (Sathishkumar sphingolipid levels et al., 2005). In the literature, the ratio between SM and ceramide has been used as an indirect measure of S-ASM S-ASM activity in plasma or serum is increased under activity (Drobnik et al., 2003); such an approach is ques- various pathological conditions in humans (Figure 2) and tionable in light of the data described above. animal models. This increase should result in a reduced It is unclear why the available in vivo data on the concentration of SM and an increased concentration of relationship between S-ASM activity and sphingolipid ceramide. In addition, a negative correlation between concentrations are so heterogeneous and predominantly S-ASM and SM and a positive correlation between S-ASM negative. Various explanations may be considered. (1) and ceramide should be observed. However, such phe- The measurement of total sphingolipids easily overlooks nomena have been observed in only a few studies. Jenkins significant and relevant changes in subspecies. This is et al. (2013) observed increased S-ASM activity in serum exemplified in the work of Jenkins et al. (2013). While the with a parallel increase in different ceramide species total ceramide concentration is moderately increased in in patients with lymophohistiocytosis. A particularly patients with lymphohistiocytosis, the analysis of single large increase in C16-ceramide was observed, and this ceramide species reveals a more differentiated picture, result was compatible with cell-based studies in which with highly elevated concentrations of C16-ceramide a specific increase in C16-ceramide via S-ASM was noted but decreased concentrations of C24-ceramide. (2) If rel- (Jenkins et al., 2010). Increased S-ASM activity and par- evant changes in sphingolipid concentrations occur only allel increases in ceramide levels were observed by Pan in small subcompartments, they may be missed in the et al. (2014) in patients with heart disease and by Hammad overall analysis. (3) Ceramides may rapidly enter different et al. (2012) in patients with post-traumatic stress disor- metabolic pathways after formation (the enzymes involved der. Irradiation induces increases in S-ASM activity in include sphingomyelin synthase, ceramidase, glucosyl- some but not all tumor patients, and a positive correla- ceramide synthase, and ceramide kinase). The measure- tion between S-ASM activity and serum ceramide concen- ment of static concentrations provides little information tration has been observed (Sathishkumar et al., 2005). about turnover. Therefore, future studies should analyze In rodents, increasing age is associated with increased not only total sphingolipids but sphingolipid species at S-ASM activity and a simultaneous increase in the concen- greater spatial and temporal resolution and assess the tration of individual ceramide species (Kobayashi et al., activities of the enzymes involved. 2013). Other in vivo studies did not report the expected relationship between S-ASM activity and sphingolipid concentrations. As expected, S-ASM activity is elevated in Pharmacology patients with non-alcoholic fatty liver disease, with a parallel increase in the concentration of ceramide. However, The enzymes involved in sphingolipid metabolism are S-ASM activity is increased even further in patients with considered important therapeutic targets (Billich and hepatitis C, although no concomitant change in the con- Baumruker, 2008; Arenz, 2010; Kornhuber et al., 2010). A centration of ceramide was observed (Grammatikos et al., pronounced increase in the activity of S-ASM would have 720 J. Kornhuber et al.: Secretory sphingomyelinase

Figure 2: Relative S-ASM activities in plasma or serum across different studies in humans. In clinical studies, the activity values in the control population were set as 100%; in genetic studies, activities of homocygotes for the major allele were set as 100%. The activities range from approximately 0.2% in NPD-B to nearly 2000% in lymphohistiocytosis, thus represent- ing four orders of magnitude. Severe reductions as well as elevations of peripheral S-ASM activities appear to be associated with human diseases and occur via different mechanisms. Decreased S-ASM activities are caused by genetic mechanisms: NPD-B involves mutations in the SMPD1 gene. However, heterozygous gene carriers also exhibit reduced S-ASM activity. Common or rare polymorphisms in the SMPD1 gene (rs1050239, rs141641266) and a shorter repeat length in the signal peptide also lead to reduced peripheral S-ASM activity. Because of the association of the rs1050239 polymorphism with allergy, slightly decreased values of peripheral S-ASM are found in allergy suffer- ers. Elevated levels of S-ASM are found in a variety of diseases, such as Alzheimer’s disease, heart disease, diabetes mellitus II, alcohol dependence and severe inflammatory diseases such as hepatitis C, sepsis, systemic inflammatory response syndrome, inflammatory renal disease, systemic vasculitis and lymphohistiocytosis. The mechanisms underlying these elevations of S-ASM include enhanced release of the enzyme and activation of the enzyme by oxidative stress and cytokine activation. Reduced S-ASM activity is mediated by genetic mechanisms and must therefore be regarded as a trait marker, while increased activity is mediated by cytokines, LPS or oxidative stress and must therefore be regarded as a state marker of a condition. Please note that the values obtained in different studies are not directly comparable because S-ASM activities were determined using different methods and different reference values. SIRS, systemic inflammatory response syndrome; NPD, Niemann-Pick disease. Literature (in alphabetical order of the description in the Figure): acute myocardial infarction (Pan et al., 2014), alcohol detoxification (Reichel et al., 2011), allergy (Reichel et al., 2014), Alzheimer’s disease (Lee et al., 2014), chronic heart failure (Pan et al., 2014), diabetes mellitus II (Górska et al., 2003), hepatitis C (Grammatikos et al., 2014), inflammatory renal disease (Kip- rianos et al., 2012), lymphohistiocytosis (Takahashi et al., 2002; Jenkins et al., 2013), non-alcoholic fatty liver (Grammatikos et al., 2014), NPD-B (Abe et al., 1999; He et al., 2003), NPD-B carrier (He et al., 2003), post-operative (Kott et al., 2014), post-traumatic stress disorder (Hammad et al., 2012), rs1050239 (Reichel et al., 2014), rs141641266 (Rhein et al., 2013), sepsis (Claus et al., 2005), signal peptide repeat 4/4 (Rhein et al., 2014), SIRS (Kott et al., 2014), stable angina pectoris (Pan et al., 2014), systemic vasculitis (Kiprianos et al., 2012), unstable angina pectoris (Pan et al., 2014), and Wilson disease (Lang et al., 2007).

obvious negative clinical consequences, spurring inter- FIASMAs such as desipramine or fluoxetine (Kornhuber est in the development of inhibitors of S-ASM. In addition et al., 2008, 2010, 2011) accumulate in acidic intracel- to classifications based on endogenous and exogenous lular environments (Trapp et al., 2008) and induce a inhibitors (Canals et al., 2011), we can also distinguish detachment of L-ASM from the inner lysosomal mem- direct, competitive inhibitors from FIASMAs. An indirect brane by shielding the abundant negatively charged mechanism of irreversible inactivation or degradation of bis(monoacylglycero)phosphate, resulting in subsequent cellular ASM and lipidosis by cationic amphiphilic drugs proteolytic degradation of L-ASM (Hurwitz et al., 1994a; was assumed in the early 1980s (Albouz et al., 1981, 1983; Kölzer et al., 2004b). Interestingly, treatment with the Yoshida et al., 1985) but was not further pursued directly. conserved chaperone heat shock protein 70, which binds J. Kornhuber et al.: Secretory sphingomyelinase 721 to endolysosomal bis(monoacylglycero)phosphate to tumor cells or sensitize them to chemotherapy (Smith and provide lysosomal stability, effectively corrects the NPD Schuchman, 2008; He and Schuchman, 2012; Savic and phenotype in vitro (Kirkegaard et al., 2010). In agreement Schuchman, 2013). with this model, the prototypical FIASMA desipramine does not affect purified ASM in in vitro micellar assays (Yoshida et al., 1985) or S-ASM in a cell culture system (Jenkins et al., 2011b). However, another report suggested Animal models an inhibition of endogenous as well as TNFα-stimulated secretion of recombinant S-ASM by desipramine in stably S-ASM in brain function and behavior transfected MCF7 cells (Jenkins et al., 2011a). Thus, it is conceivable that FIASMAs not only act within acidic intra- The development of ASM KO mice has permitted the inves- cellular compartments but also extracellularly by another tigation of the role of ASM in behavior. Homozygous ASM mechanism. Although they possess some degree of selec- KO mice develop normally and do not exhibit impairments tivity (Kornhuber et al., 2010), these FIASMA drugs are not until they are 8 weeks of age. Mild tremor and locomotor completely specific for ASM and exhibit inhibitory effects ataxia then emerge. Tottering with zigzag movements against other lysosomal enzymes, including acid cerami- becomes evident and is characteristic of cerebellar dys- dase (Zeidan et al., 2006). function. Between 12 and 16 weeks, ASM KO mice become Potent and selective competitive inhibitors of ASM lethargic and unresponsive to stimuli. They increasingly are rare. In a cell-free drug screen of a wide range of com- experience problems feeding and much reduced weight pounds, no drug-like competitive inhibitors of S-ASM were compared to WT mice. Beyond 4 months of age, these identified (Mintzer et al., 2005). More recently, new com- animals exhibit severe ataxia and die between 6 and petitive inhibitors of ASM have been identified and/or 8 months of age. ASM KO animals exhibit a significant developed (Roth et al., 2009a,b, 2010; Arenz, 2010) that reduction in brain volume. A striking finding was the should function against both L-ASM and S-ASM. Available significant loss of Purkinje cells in the cerebellum and compounds include substrate analogues such as L-car- atrophy of the mesencephalon beginning at 10 weeks of nitine, phosphate analogues such as phosphatidylino- age (Horinouchi et al., 1995; Otterbach and Stoffel, 1995). sitol-3,5-bisphosphate and natural compounds such as Both regions appear to be involved in movement control α-mangostin (Arenz, 2010). The systemic availability of and motivated behavior. Homozygous ASM KO mice exhib- these molecules remains unclear, and most of these sub- ited reduced ceramide levels in the hippocampus and dis- stances are not yet available for clinical use. Competitive played some signs of reduced anxiety and a reduction of inhibitors of ASM that cannot pass through the cell mem- depression-related behaviors. Young ASM KO mice did not brane and therefore have no intracellular effects may help exhibit altered synaptic structure or functioning at the clarify the physiological effects of S-ASM relative to L-ASM. level of the hippocampus (Gulbins et al., 2013). Interest- However, there are applications for increasing ASM ingly, heterozygous ASM KO mice, which possess approxi- activity. In addition to obvious therapeutic applications mately 50% of normal ASM activity, did not exhibit any for patients with NPD-A and -B, including enzyme replace- motor problems, reduced feeding, or enhanced mortality. ment and gene therapy, NPD-C patients may benefit from This finding may suggest that 50% enzyme activity is suf- ASM treatment despite their intact SMPD1 gene. Mutations ficient for normal brain function and a normal behavio- in NPC1 or NPC2 result in impaired cholesterol traffick- ral phenotype (Horinouchi et al., 1995), consistent with ing in the endolysosome (Madra and Sturley, 2010) and the rare phenotype observed in NPD carriers (Lee et al., in a secondary deficiency in ASM activity (Tamura et al., 2003; Schuchman, 2007). While these findings suggest 2006). Restoration of ASM to normal levels in NPC1-defi- an important role of ASM in brain function and locomo- cient cells improves several defects, including a reduction tor behavior, it was unclear if these effects were mediated in lysosomal cholesterol (Devlin et al., 2010), underlin- by L-ASM or S-ASM because the KO affects both subtypes. ing the important role of ASM in this disease. Another In a subsequent study, this question was addressed by field of application of ASM is cancer therapy, with the fusing the Smpd1 gene with the lysosomal Lamp1 gene and aim of pharmacologically modifying the deregulated thus targeting all ASM to the lysosome. The mutant mice sphingolipid metabolism that occurs in many cancers, as still exhibited low levels of L-ASM activity (11.5–18.2% of evident, for example, in the reduced levels of ASM mRNA. WT) but a complete absence of S-ASM activity. These mice A number of studies have revealed the potential of ASM exhibited neither brain pathology nor signs of locomotor overexpression or supplementation to either directly kill impairment. This finding suggests that severe deficits in 722 J. Kornhuber et al.: Secretory sphingomyelinase locomotor function in ASM KO mice are mediated pre- S-ASM and atherosclerosis dominantly by a lack of L-ASM, while S-ASM may not be required (Marathe et al., 2000b). Inflammation plays an important role in the develop- The role of ASM in depression/anxiety has been ment of atherosclerosis. In mice, inflammation induced investigated in animal studies utilizing a transgene for by lipopolysaccharide (LPS) increases S-ASM activity in ASM (tgASM). These mice exhibited higher ASM activ- the serum 3 h after injection. This increase was depend- ity and ceramide production in the hippocampus than ent on IL-1 activity. Serum S-ASM was also increased in WT mice (Gulbins et al., 2013). The enhanced ceramide mice that were treated with IL-1β or TNFα. These find- levels in the hippocampus were paralleled by a decline ings raise the possibility that an increase in S-ASM in neurogenesis, neuronal maturation, and neuronal sur- activity contributes to inflammatory cytokine activity vival (Gulbins et al., 2013), which are depression-related in atherosclerosis (Wong et al., 2000). The ASM activ- neuronal markers (Santarelli et al., 2003; Krishnan and ity present in atherosclerotic lesions (Schissel et al., Nestler, 2008). S-ASM activity was increased more than 1996b) is dependent on S-ASM and is associated with 10-fold in the CSF of tgASM mice compared to the WT con- the extracellular arterial wall. In this bound state, trols (Mühle et al., 2013). At the behavioral level, tgASM S-ASM retains enzymatic activity that stimulates sub- mice exhibited a depression/anxiety-like phenotype in endothelial retention and aggregation of atherogenic several tests (Gulbins et al., 2013); for a review see Korn- lipoproteins (Marathe et al., 1999). When mice are fed huber et al. (2014). This ASM effect is most likely mediated an atherogenic diet containing saturated fats and cho- in the brain because the chronic application of C16-cera- lesterol, serum S-ASM activity is up-regulated, which mide in the dorsal hippocampus of mice induced the same facilitates the conversion of SM to ceramide (Deevska depression-like behavioral phenotype observed in tgASM et al., 2012). However, another study found increased mice (Gulbins et al., 2013). This finding is consistent with activity of S-ASM in rats fed for 14 weeks with a diet rich the effects of chronic unpredictable stress, which not in n-3 polyunsaturated fatty acids (PUFA) vs. butter, and only double hippocampal ceramide levels and reduced a strong negative correlation was observed between the neurogenesis and neuronal maturation but also induce serum concentration of n-3-PUFA and S-ASM activity depression-like behavior in mice (Gulbins et al., 2013). (Drachmann et al., 2007). The association of atheroscle- Further support for the role of ceramide is provided by the rosis with increased age is mediated, at least in part, by elevated plasma ceramide levels detected in patients with S-ASM. S-ASM is increased in mice at 65 weeks of age. major depression (Gracia-Garcia et al., 2011). Whether Apolipoprotein E-/- (ApoE-/-) mice develop atheroscle- these effects are mediated by S-ASM or L-ASM remains to rosis at 15 weeks of age. Plasma ceramide levels are be determined. generally higher in ApoE-/- than WT mice but decrease Many antidepressant drugs are functional inhibitors with age in parallel with an increase in S-ASM activ- of ASM (Albouz et al., 1983; Kornhuber et al., 2008, 2010, ity. When ApoE-/- mice develop atherosclerosis, several 2011). These drugs can normalize the effects of chronic ceramide species increase in the aorta compared to WT unpredictable stress in WT and tgASM but not ASM KO mice: C18:0, C22:0 and C24:0. At 65 weeks of age, C16:0 mice. This observation suggests that ASM functional inhi- and C24:1 species are increased compared to WT mice. bition is a major pathway for the pharmacological effects These findings indicate that an age-related increase in of antidepressants (Gulbins et al., 2013; Kornhuber et al., S-ASM activity, which results in an elevation of certain 2014). This effect, however, predominantly affects L-ASM. ceramide species, may contribute to age-related ath- Interestingly, evidence suggests that serum zinc levels are erosclerosis (Kobayashi et al., 2013). When ApoE-/- mice significantly lower in patients with major depression (Lev- are treated with a recombinant adeno-associated virus enson, 2006; Swardfager et al., 2013a). Low dietary zinc (AAV) that constitutively expresses high levels of human intake has been associated with a greater incidence of ASM in the liver and plasma, S-ASM levels are persis- depression (Vashum et al., 2014), and an antidepressant tently elevated. However, this phenomenon leads to effect of zinc supplementation has been observed in labo- only a short reduction in plasma SM and no changes in ratory rodents (reviewed in Levenson, 2006, and Sward- serum lipoprotein levels. Plaque formation in the aortic fager et al., 2013b) and in human trials (Lai et al., 2012; sinus after 17 weeks did not differ in mice treated with Swardfager et al., 2013b). While the underlying mecha- a control AAV. These findings suggest that S-ASM has nism remains a subject of speculation, a possible connec- no accelerating or exacerbating role in atherosclerotic tion to the stimulatory effects of zinc on S-ASM activity has lesion formation in the ApoE-/- model of atherosclerosis not been explored. (Leger et al., 2011). J. Kornhuber et al.: Secretory sphingomyelinase 723

S-ASM in diabetes lungs, which results in a 28% increase in strictly Zn2+- dependent serum ASM activity without altering the activi- Diabetes mellitus, a metabolic disorder characterized by ties of neutral sphingomyelinase or ceramide synthase. hyperglycemia, is associated with changes in ceramide The application of ceramide-specific antisera, pharma- metabolism. The induction of a diabetic state with strep- cological disruption of ceramide formation or functional tozotocin causes a significant increase in blood glucose inhibition of ASM all specifically prevent PAF-triggered in rats. Eight weeks after induction, plasma and renal pulmonary edema. These data corroborate the hypothesis ceramide levels appear to increase. While neutral sphin- that PAF-induced pulmonary edema is mediated by ASM gomyelinase levels are unchanged in the liver and kidney, and ceramide as the second major pathway in addition plasma S-ASM activity increases. These findings suggest to the activation of EP-3-receptors by PAF (Göggel et al., that the induction of diabetes increases plasma ceramide 2004). levels by enhancing S-ASM activity (Kobayashi et al., A slow, modest but significant increase in S-ASM but 2013). not L-ASM or neutral sphingomyelinase was also noted in a pulmonary emphysema mouse model after VEGF receptor blockade in which the prompt and up to 3-fold S-ASM in sepsis activation of ceramide synthase was the main contributor to increased lung ceramide levels associated with alveo- Early studies show that ASM KO mice are protected against lar septal destruction (Petrache et al., 2005). The authors effects of LPS (Haimovitz-Friedman et al., 1997). However, speculate that, due to the high abundance of S-ASM from the differential role of L- and S-ASM cannot be derived endothelial cells, injured lung capillary endothelial cells from these studies. Claus et al. (2005) find increased aggravate cellular damage in a vicious cycle, resulting in S-ASM activity 24 h after endotoxin application in mice; the sustained lung damage observed in patients despite this increase did not occur after pre-treatment of mice with smoking cessation. In a neonatal pigled model of pulmo- the FIASMA NB6. In a subsequent study, the group dem- nary inflammation, S-ASM activity and ceramide concen- onstrates an increase of the S-ASM activity after induction tration are reduced in serum after treatment of lungs with of sepsis in WT mice, but not in ASM KO mice (Jbeily et al., inhaled surfactant factor combined with the FIASMA imi- 2013). ASM KO mice exhibit enhanced basal ceramide pramine (von Bismarck et al., 2008; Preuß et al., 2012). levels in the blood but a significantly reduced ceramide Wilson disease, an autosomal recessive disorder, is response when sepsis is experimentally induced by peri- characterized by an accumulation of Cu2+, which leads toneal contamination and infection. There is an increase to severe consequences including progressive liver cir- in bacterial burden, increased phagocytic activity and an rhosis, neurological and psychiatric symptoms and, occa- enhanced cytokine storm in the blood after sepsis induc- sionally, anemia. Patients with Wilson disease exhibit a tion in ASM KO mice. Thus, in murine models of sepsis, constitutive increase in ASM activity in the blood plasma S-ASM activity is increased, which may contribute to the even during treatment with D-penicillamine or trientine, effective defense of septic pathogens. S-ASM triggered which decrease total but not free Cu2+ concentrations hydrolysis of membrane-bound SM may play an important (Lang et al., 2007). In isolated murine hepatocytes or cell role in the primary defense against invading microorgan- lines, Cu2+ activates ASM but not neutral sphingomyeli- isms (Jbeily et al., 2013). nase and triggeres ceramide release within 10–20 min after application. Both effects were absent or reduced in ASM KO cells and cells preincubated with the antioxidants S-ASM in pulmonary edema N-acetylcysteine and Tiron, suggesting that Cu2+ may directly or indirectly stimulate ASM via oxygen radicals. In In the context of non-cardiogenic pulmonary edema, whole blood and in purified mouse leukocytes, Cu2+ treat- treatment of mice with platelet-activating factor (PAF) ment results in the stimulation and secretion of ASM into leads to a 50% increase in lung tissue concentrations of the supernatant or plasma, followed by apoptosis-like ceramides, and this increase is thought to contribute to changes including shrinkage of erythrocytes and phos- the development of severe edema (Göggel et al., 2004). phatidylserine exposure. In patients, increased plasma However, in contrast to the PAF-stimulated transient accu- ASM activity also correlates with a constitutively increased mulation of ceramide in P388D1 macrophages by de novo number of erythrocytes exposing ceramide or phosphati- synthesis (Balsinde et al., 1997), this increase in ceramide dylserine on the cell surface, which may mediate the rapid is attributed to the PAF-induced release of ASM from the clearance of erythrocytes and thus result in anemia. The 724 J. Kornhuber et al.: Secretory sphingomyelinase causal connection between Wilson disease symptoms and and cytokines (Marathe et al., 1998; Wong et al., 2000; ASM is supported by the observation of a delayed onset of Jenkins et al., 2010). Increased activity of S-ASM is induced disease and protection against the development of fibrosis by LPS, cytokines or oxidative stress and must therefore be and liver failure in a rat model of Wilson disease treated regarded as a state marker of these conditions. The data in with amitriptyline as a pharmacological inhibitor of ASM Figure 2 show no clear boundaries, and thus up to 200% (Lang et al., 2007). activation of S-ASM is considered a mild increase. Mildly elevated activities are observed in Alzheimer’s disease (Lee et al., 2014), type II diabetes mellitus (Górska et al., 2003), stable angina pectoris, acute myocardial infarction Clinical studies (Pan et al., 2014), chronic heart failure (Doehner et al., 2007), post-traumatic stress disorder (Hammad et al., 2012) Cross-sectional clinical studies and in response to ionizing radiation treatment in cancer patients (Sathishkumar et al., 2005). Marked increases in Several studies have investigated peripheral S-ASM activi- activity ( > 200%) are observed in unstable angina pectoris ties in humans under various conditions. Most of these (Pan et al., 2014), Wilson disease (Lang et al., 2007), sepsis studies analyzed serum or plasma. Some studies have (Claus et al., 2005), systemic inflammatory response syn- also examined urine, salivary fluid, tear fluid, synovial drome (Kott et al., 2014), lymphohistiocytosis (Takahashi fluid (Takahashi et al., 2000) and CSF (Mühle et al., 2013). et al., 2002; Jenkins et al., 2013), non-alcoholic fatty liver, Figure 2 summarizes all available studies using plasma hepatitis C (Grammatikos et al., 2014), systemic vasculitis, or serum that also included a control group. The consid- inflammatory renal disease (Kiprianos et al., 2012) and in eration of relative changes allows comparisons between patients with alcohol dependency undergoing withdrawal different types of disorders. However, the studies used dif- (Reichel et al., 2011). In sepsis, lymphohistiocytosis, sys- ferent methods, and S-ASM activity was related to a certain temic inflammatory response syndrome and chronic heart volume in most studies but to the amount of protein in failure, high S-ASM activity is associated with higher others. Many of these studies suffer from the limitation mortality (Claus et al., 2005; Doehner et al., 2007; Jenkins of a small number of investigated cases. Furthermore, the et al., 2013; Kott et al., 2014). In accordance with this, the study designs are heterogeneous; some studies used a ret- plasma ceramide content is an independent risk factor for rospective design, while other studies used a prospective mortality in patients with chronic heart failure (Yu et al., design. However, Figure 2 illustrates the variation of S-ASM 2015). High S-ASM activity has been related to reduced in different clinical situations. Reduced activity values skeletal muscle strength (Doehner et al., 2007). In several arise because of genetic variations in the SMPD1 gene. Very studies, a negative correlation between S-ASM activity low activity values ( < 5%) are measured in NPD-B patients and body mass index (BMI) has been observed (Doehner (Abe et al., 1999; He et al., 2003). Heterozygous NPD-B gene et al., 2007; Reichel et al., 2011; Mühle et al., 2014), while carriers exhibit intermediate S-ASM activity levels (≈20%) this has not been detected in other studies (Grammatikos (He et al., 2003) (the NPD patients reported in the publica- et al., 2014). In patients with chronic heart failure, S-ASM tions by Takahashi et al. (2000, 2002) are likely identical activity increases with age (Doehner et al., 2007), in to those reported in Abe et al. (1999) and were therefore agreement with studies conducted in mice (Kobayashi omitted in Figure 2). Only slightly reduced activity values et al., 2013). By contrast, Grammatikos et al. (2014) did (40–90%) are observed for the polymorphisms rs1050239 not observe a correlation between age and S-ASM activ- (Reichel et al., 2014) and rs141641266 (Rhein et al., 2013) ity. A diet rich in n-3 PUFAs reduces S-ASM activity in rats and short repeat lengths in the signal peptide (Rhein (Drachmann et al., 2007) but not in humans (measured et al., 2014). Because the minor A allele of polymorphism in samples from the study by Rees et al. (2006); Lars I. rs1050239 is associated with allergy, slightly reduced Hellgren, personal communication). Indirect evidence for values of peripheral S-ASM are also observed in allergy increased S-ASM activity based on an elevated ceramide patients (Reichel et al., 2014). Based on current informa- to SM ratio in septic patients has been reported (Drobnik tion, decreased S-ASM activity is genetically determined et al., 2003). In the plasma or CSF of patients with Alz- and must therefore be regarded as a trait marker. heimer’s disease, increases in this ratio have been asso- Until now, increased S-ASM activity has not been associated with greater cognitive progression (Mielke et al., ciated with genetic changes in the SMPD1 gene but has 2011). Although increased ceramide levels could originate been related to the enhanced release of the enzyme and from an inhibition of ceramide-degrading enzymes (Satoi enhanced activation via oxidative stress, inflammation et al., 2005), aberrantly high concentrations of total and J. Kornhuber et al.: Secretory sphingomyelinase 725 individual ceramide species correlate with an up-regula- 2014). Serial measurements of S-ASM during the days after tion of ASM transcription in patients with neurodegenera- abdominal surgery revealed increased S-ASM values that tion (Filippov et al., 2012). remained high until the end of the observation period at L-ASM activity is reduced in the lysosomes of cells day 5 (Kott et al., 2014). Serial measurements indicated from patients with I-cell disease (mucolipidosis II) who are increased ceramide/SM ratios as an indirect measure of deficient in N-acetylglucosaminyl-1-phosphotransferase S-ASM activity in septic patients at day 11 in non-survivors activity and thus unable to add mannose 6-phosphate compared to survivors (Drobnik et al., 2003). These studies residues to enzymes targeted to the lysosome, including all suggest a relatively slow decrease in S-ASM activity in L-ASM (Wenger et al., 1976; Weitz et al., 1983). Although response to treatment for diseases in which S-ASM activity either increased S-ASM activity in the plasma of these is initially increased. This effect may be related to the slow patients or intracellular retention would be expected, the turnover of S-ASM. In cell culture experiments, S-ASM fate and localization of the assumed excessive enzymes activity remains high over 24 h despite the inhibition of remain unknown. protein synthesis, whereas L-ASM activity rapidly declines (Jenkins et al., 2010). The slow turnover of S-ASM indicates that small persistent changes in the release of S-ASM Longitudinal clinical studies result in profound and long-lasting changes in peripheral S-ASM activity. If the pathological condition is removed, Several clinical studies have longitudinally investigated such as during alcohol withdrawal treatment (Reichel the activity of S-ASM, yielding interesting conclusions et al., 2011; Mühle et al., 2014), S-ASM activity slowly regarding the value of S-ASM as a biomarker. In contrast to decreases to control levels. This underscores the notion of a pronounced decrease in C-reactive protein (CRP), S-ASM increased ASM activity as a state marker of disease. activity only normalizes to a minor extent within 2 weeks in patients with systemic vasculitis undergoing immuno- suppressive therapy, resulting in a dissociation of S-ASM S-ASM in clinical routine activity and CRP-levels during the treatment of these patients (Kiprianos et al., 2012). Increased S-ASM activ- Should S-ASM activity be used as a chemistry marker in ity was measured at baseline in male alcohol-dependent the clinical routine? A moderate reduction of S-ASM activ- patients entering an alcohol withdrawal program. S-ASM ities per se is not clearly related to the pathophysiology of activity steadily declined from day 0 (baseline) to days disease. The association of slightly reduced S-ASM activity 1 and 2 and nearly reached control levels on days 7–10 with allergy appears to be indirectly mediated via genetic (Reichel et al., 2011). Similar results were reported in an mechanisms; however, it is unlikely that reduced S-ASM independent sample of alcohol-dependent subjects, with activity results in an increased risk of allergy. A severe males exhibiting higher initial S-ASM activities compared reduction of S-ASM activity is observed in patients with to females (Mühle et al., 2014). In a single patient with NPD-B. However, all diseases that are associated with lymphohistiocytosis, S-ASM activities slowly declined increased S-ASM activity are also associated with mecha- over several weeks in response to active treatment with nisms that lead to activation of S-ASM, namely oxidative dexamethasone (Takahashi et al., 2002). In patients stress, increased cytokine production and/or endothelial with unstable angina pectoris, high S-ASM activity was damage. While ASM and its reaction product ceramide detected on the day of symptom onset; only slightly nor- have been implicated in all of these diseases, the specific malized values were observed on day 1 after onset, and role of S-ASM in relation to L-ASM remains to be clari- slightly increased S-ASM values were observed on the day fied. Diseases with hepatic and endothelial involvement of percutaneous coronary intervention. In patients with preferentially appear to present a marked elevation of acute myocardial infarction who underwent conventional peripheral S-ASM activity. This elevation may be related to treatment, high S-ASM activity was observed up to at least the characteristics of the endothelium as a rich source of day 7 after onset (Pan et al., 2014). In a group of septic S-ASM (Marathe et al., 1998). The mechanisms leading to patients, S-ASM activity was increased compared to con- marked elevations of S-ASM activity must be investigated trols at baseline. S-ASM activity decreased in survivors, in future studies. Because of the slow turnover of S-ASM, while S-ASM activity further increased in non-survivors its measured activity may represent not only the current (Claus et al., 2005). Similarly, in patients with severe status but also past events. systemic inflammation, S-ASM activity predicted mortal- A determination of peripheral S-ASM activity appears ity in patients with low procalcitonin values (Kott et al., to be useful in suspected NPD patients and carriers and 726 J. Kornhuber et al.: Secretory sphingomyelinase in severe acute inflammatory diseases. Increased S-ASM –– A detailed understanding of the mechanisms of phar- activity is observed in many different disease states and macological inhibition of both ASM enzymes, or spe- thus can be used as a general marker that reflects inflam- cifically L- or S-ASM, would facilitate the development mation, cytokine release, oxidative stress and endothelial of common and selective inhibitors for in vitro experi- damage as a pathological state. Although it is not appli- mental work as well as for clinical applications. cable as a biomarker of a single disease, S-ASM levels –– To date, only a few in vitro and in vivo models are may be helpful for monitoring disease progression and able to separate the effects of L-and S-ASM (Marathe severity and therapeutic efficiency or for evaluating the et al., 2000b; Jenkins et al., 2010). The development prognosis as well as the differentiation between subtypes of further models that permit distinction between S- of disorders with variable involvement of inflammatory and L-ASM is urgently needed to study the differential processes. In severely diseased patients, S-ASM activity is effects and responses of these enzyme species in a associated with mortality and low BMI. variety of normal and pathological conditions. Induc- ible and tissue-specific knockdown or overexpression of S- or L-ASM will aid in dissecting the biological Open questions function of these enzyme species. –– The methods used to determine S-ASM activities S-ASM is a potential biomarker and therapeutic target, but should be standardized to enable comparisons of important questions remain to be resolved before its inclu- values across studies and to establish reference val- sion in the clinical routine: ues; in the recent literature, different radioactive and –– The precise origin of S-ASM activity in the blood fluorescent methods with various conditions, sub- should be identified to further elucidate the relevance steps and detection techniques have been applied. of altered (mainly increased) S-ASM activity under Other biological materials including saliva, urine, various clinical conditions. Indeed, there seems to be tear fluid as well as synovial or peritoneal fluid could no clear consensus on the subcellular origin of ASM be studied for the correlation of S-ASM activities that is translocated to the outer leaflet of the plasma with that in blood. The potential of these materials membrane in response to different stimulatory sig- as sources of S-ASM as a biomarker remains largely nals: the lysosome, other vesicles or compartments at unexplored. or near the cell surface or even from cytosolic pools. –– The relationships between S-ASM activities and spe- Similarly, the fate of endocytosed S-ASM has not yet cific clinical features remain to be elucidated for both been determined. very low as well as high S-ASM activities. Cut-off val- –– Many details of regulation and processing, including ues and sensitivity and specificity data for diagnos- modifications in the Golgi complex, trafficking, dif- tic (such as NPD) and prognostic purposes (such as ferentiation between L- and S-ASM and activation, mortality) should be established when using S-ASM remain elusive. Moreover, we have observed higher activity either alone or in combination with other S-ASM activity in bovine and rodent serum compared to biomarkers. human serum. However, the activity of ASM and related –– In future clinical studies, known confounders such enzymes of sphingolipid metabolism from different as alcohol consumption, BMI, and genetic variants species in relation to their evolution and regulation has should be controlled for. To obtain a complete under- not been systematically investigated, despite the use of standing of sphingolipid turnover, the activities of various animal species in models of human diseases. other enzymes such as L-ASM, neutral sphingomy- –– With respect to enzymatic analysis, there are insuffi- elinase, ceramidases, sphingosine kinase, ceramide cient data regarding reaction requirements for S- (and synthases and lipids such as ceramide and sphingo- L-) ASM originating from different sources, including sine-1-phosphate should be quantified in parallel to cofactors, inhibitors and pH profiles as well as the the Zn2+-dependent and Zn2+-independent fractions substrate specificity such as fatty acid chain length of S-ASM activity. The levels of inhibitors and acti- and explanations for so far observed differences are vators of S-ASM, such as Zn2+ ions, TNFα, oxidative lacking. The reported activation of S-ASM from the stress, and inflammatory markers should be deter- cell supernatant, serum and CSF following storage at mined concomitantly. Further confounders such as -20°C is relevant for patient material and clinical stud- fasting, chronobiology or epigenetic influences, as ies, but specific mechanisms and temperature profiles well as the influence of sex and developmental stage, are not yet available. should be investigated. J. Kornhuber et al.: Secretory sphingomyelinase 727

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