Regulation of Sphingomyelin Metabolism

Home , FIASMA, Lipid storage disorder

Pharmacological Reports 68 (2016) 570–581

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Pharmacological Reports

jou rnal homepage: www.elsevier.com/locate/pharep

Review article

Regulation of sphingomyelin metabolism

a a,b a a

Kamil Bienias , Anna Fiedorowicz , Anna Sadowska , Sławomir Prokopiuk , a,

Halina Car *

Department of Experimental Pharmacology, Medical University of Białystok, Białystok, Poland

Laboratory of Tumor Molecular Immunobiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław,

Poland

A R T I C L E I N F O A B S T R A C T

Article history: Sphingolipids (SFs) represent a large class of lipids playing diverse functions in a vast number of

Received 2 April 2015

physiological and pathological processes. Sphingomyelin (SM) is the most abundant SF in the cell, with

Received in revised form 24 November 2015

ubiquitous distribution within mammalian tissues, and particularly high levels in the Central Nervous

Accepted 28 December 2015

System (CNS). SM is an essential element of plasma membrane (PM) and its levels are crucial for the cell

Available online 11 January 2016

function. SM content in a cell is strictly regulated by the enzymes of SM metabolic pathways, which

activities create a balance between SM synthesis and degradation. The de novo synthesis via SM

Keywords:

synthases (SMSs) in the last step of the multi-stage process is the most important pathway of SM

Sphingomyelin

formation in a cell. The SM hydrolysis by sphingomyelinases (SMases) increases the concentration of

Sphingomyelin synthases

ceramide (Cer), a bioactive molecule, which is involved in cellular proliferation, growth and apoptosis. By

Acid sphingomyelinase

Neutral sphingomyelinase controlling the levels of SM and Cer, SMSs and SMases maintain cellular homeostasis. Enzymes of SM

cycle exhibit unique properties and diverse tissue distribution. Disturbances in their activities were

observed in many CNS pathologies. This review characterizes the physiological roles of SM and enzymes

controlling SM levels as well as their involvement in selected pathologies of the Central Nervous System,

such as ischemia/hypoxia, Alzheimer disease (AD), Parkinson disease (PD), depression, schizophrenia

and Niemann Pick disease (NPD).

Contents

Physiological roles of sphingomyelin (SM) and enzymes of SM cycle...... 571

SM – structure and function ...... 571

Sphingomyelin synthases (SMSs) ...... 572

SMS1 ...... 572

SMS2 ...... 572

SMSr...... 572

Sphingomyelinases (SMases) ...... 572

Acid Sphingomyelinase (a-SMase) ...... 572

Neutral sphingomyelinase (n-SMase) ...... 573

Alkaline SMase (alk-SMase) ...... 573

Abbreviations: a-syn, a-synuclein; Ab, amyloid b; AD, Alzheimer disease; APP, amyloid precursor protein; a-SMase, acid sphingomyelinase; alk-SMase, Alkaline SMase; Cer,

ceramide; CERT, ceramide transfer protein; CSF, cerebrospinal ﬂuid; D609, tricyclodecan-9-yl-xanthogenate; DAG, diacyglicerol; ER, endoplasmic reticulum; FasL, Fas ligand;

FIASMA, Functional Inhibitor of Acid SphingoMyelinAse; GSH, glutathione; IR, ischemia/reperfusion; l-SMaze, lysosome sphingomyelinase; MA-nSMase, mitochondrial

sphingomyelinase; MUFA, monounsaturated fatty acid; n-SMase, neutral sphingomyelinase; NPD, Niemann–Pick disease; PC-PLC, speciﬁc phospholipase C; PD, Parkinson

disease; PM, plasma membrane; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; SAFA, saturated fatty acids; SAM, sterile motif; SF, sphingolipid; SM,

sphingomyelin; SMase, sphingomyelinase; s-SMase, secretory sphingomyelinase; SMS, sphingomyelin synthase; SPT, serine palmitoyltransferase; START, protein-related

lipid transfer domain; TNF-a, tumor necrosis factor-a; TNFR1, tumor necrosis factor receptor 1.

* Corresponding author.

E-mail address: [email protected] (H. Car).

http://dx.doi.org/10.1016/j.pharep.2015.12.008

B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581 571

SM metabolism in selected pathologies of the CNS ...... 573

Ischemia/reperfusion ...... 573

Alzheimer disease (AD) ...... 574

Depression and schizophrenia ...... 576

Parkinson disease (PD) ...... 576

Niemann–Pick disease (NPD) ...... 576

Summary ...... 577

Conﬂict of interest ...... 577

Funding ...... 577

References ...... 577

Physiological roles of sphingomyelin (SM) and enzymes of SM stronger binding with cholesterol and form larger domains in

cycle comparison with racemic layers [15]. Importantly, SMs with

different fatty acid chains are not equally distributed in

SM – structure and function mammalian tissues [3], which results in unique compositions of

lipid rafts. It is worth noting that a myriad of signaling,

SM is the most abundant eukaryotic sphingolipid (SF) which cytoskeletal, mitochondrial [16] and GPI-anchored proteins have

constitutes one of the major components of the plasma membrane been discovered within lipid rafts [17].

(PM). The structural function of SM in a cell is determined by The de novo synthesis of SM is a multi-stage process which

chemical structure of a phospholipid. Speciﬁcally, the hydrophilic begins on the cytosolic side of the endoplasmic reticulum (ER) with

head and the hydrophobic tail of the phospholipid molecules the condensation of palmitoyl-CoA and serine through the action

enable them to form a double layer structure, known as a lipid of serine palmitoyltransferase (SPT). This reaction results in the

bilayer. SM (N-acyl-sphingosine-1-phosphorylcholine) is com- production of 3-ketosﬁnganine which is subsequently transformed

posed of ceramide which comprises a sphingoid backbone, an 18- into dihydrosphingosine (sphinganine) by 3-ketosphinganine

carbon aminoalcohol with one trans-double bond in position 4 [1] reductase. Next, the fatty acid chain is attached to sphinganine

with an attached hydrophobic fatty acid chain, and polar by dihydroceramide synthase to produce dihydroceramide. The

phosphocholine or, less frequently, phosphoethanolamine resi- reduction of a double bond in a molecule of dihydroceramide by

dues [2]. The naturally occurring SMs vary in their length of fatty dihydroceramide desaturase leads to the formation of ceramide

acid chain (usually from 16 to 24 carbons) and the level of their (Cer) [18]. For SM synthesis, Cer is transferred from ER to the Golgi

saturation and hydrogenation. Thus, SM can contain the saturated apparatus [19] by both vesicular and non-vesicular transport

fatty acids (SAFA), monounsaturated fatty acids (MUFA) and [20]. The latter is mediated by the ceramide transfer protein

polyunsaturated fatty acids (PUFA) [3,4]. The speciﬁc roles of the (CERT), which contains a steroidogenic acute regulatory protein-

diverse SM species in PM are poorly established, although it seems related lipid transfer domain (START), the site of Cer binding

that SM containing different fatty acids may regulate membrane [21]. Studies on the role of CERT in SM formation showed a

fluidity. There are some findings validating this statement, significant reduction in SM levels in CERT-deficient mouse

however, obtained mostly in the studies of artiﬁcial model embryos [22]. These ﬁndings point to a conclusion that the de

systems. For example, it was observed that MUFA-containing novo synthesis is the most important pathway of SM creation in the

species of SMs form more liquid domains than those with SAFA cell. Moreover, transport of Cer to the Golgi is considered as the

[5]. Furthermore, hydrogenation of the SM double bond results in rate limiting factor of the de novo SM synthesis [21]. In the Golgi

an increase in membrane raft melting temperature [6]. network, the phosphorylocholine group from phosphatidylo-

Being a major SF in PM, SM has the ability of binding cholesterol choline is transferred to Cer by sphingomyelin synthase 1

[7]. This feature of SM is particularly important in the formation of (SMS1), which eventually results in the formation of SM along

lipid rafts, which are cholesterol-enriched PM domains. The with the production of diacyglycerol (DAG) [23]. Thereafter,

speciﬁc composition of a lipid raft determines its gel ordered newly synthetized SM reaches PM in transport vesicles [24]. In

structure, which is separated from other parts of the membrane, the cell membrane, SM takes part in the lipid raft formation [5] or

thicker than the surroundings [8] and resistant to detergents undergoes hydrolysis. The common degradation process of SM

[9]. From 40% to 70% of cell SFs are gathered in lipid rafts [10], occurs in the lysosome, where SM, transported mainly from the

where SM represents approximately 2% of the total SF content. PM, is hydrolysed to Cer by the acidic SMase (a-SMase) [25]. The

Interestingly, SM accounts for 2%–15% of the total concentration of PM-localized SM can also be enzymatically degraded to Cer and

SFs in the cell [11]. Localization of a double bond in the SM fatty phosphocholine by the hydrolysis of phosphodiester bond by

acid chain inﬂuences the interactions of this lipid with cholesterol. two sphingomyelinases (SMases): secretory (s-SMase) or

13 15

Therefore, while double bonds at D or D positions do not affect neutral (n-SMase) SMase [26], which is often observed in

the SM binding with cholesterol, the double bond at D position pathological processes. Interestingly, Cers derived from SM

weakens this reaction. On the other hand, cholesterol does not hydrolysis can be reutilized for SM synthesis by sphingomyelin

seem to favor any SM species on the basis of their speciﬁc acyl synthase 2 (SMS2) [27].

chain length [12]. In addition, the interactions of cholesterol with Since SM is an essential modulator of PM properties which

the NH2 group of SM protect this lipid from oxidative degradation inﬂuences the membrane gathering of proteins involved in cellular

[13]. It has been reported that SM species with N-acyl branched proliferation, growth and apoptosis as well as being an important

chains form more ﬂuid and disordered lipid rafts compared to source of ceramide, its levels are critical for cell function. The SM

domains composed of unbranched SMs. Furthermore, the interac- content in a cell is strictly regulated by the enzymes of SM metabolic

tion of cholesterol with SM is impaired when SM N-acyl chains are pathways whose activities create a balance between SM synthesis

branched at D position [14]. Natural SM is optically active and and degradation. This manuscript will characterize the physiological

occurs in two isomers, d-erythro SM and l-threo SM. Monolayers roles of these enzymes as well as their involvement in selected

containing only one isomer are tighter, more ordered, exhibit pathologies of the Central Nervous System.

572 B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581

Sphingomyelin synthases (SMSs) [29]. The binding of SMS2 to PM is enabled by prior palmitoylation

of its COOH-terminal tail [47]. The highest content of SMS2 was

SMSs catalyze the reaction of SM synthesis from Cer. They found in the human brain, heart, kidney, liver, muscle and stomach

transfer the phosphorylocholine group from phosphatidylcholine [34]. The in vivo activity of this enzyme covers 20%–40% of all SMSs

on Cer in conjunction with the production of DAG. Recent reports activity in the cell [31,32], is cell speciﬁc and depends on the

have revealed that, apart from the production of SM, SMSs are availability of substrates [48]. Importantly, the major substrates

involved in the synthesis of the SM analog, ceramide phos- for SMS2 are Cers derived from the degradation of more complex

phoethanolamine (CPE) [28]. In addition, the synthesis of CPE can SFs, which is reﬂected by the predominantly membrane localiza-

be catalyzed by the sphingomyelin related protein (SMSr). To date, tion of SMS2 [34]. These characteristics of SMS2 strongly indicate

two SM synthases have been described, SMS1 and SMS2. its antiapoptotic activities. In other words, the overproduction of

Structurally, both SMSs are over 50% identical. Both enzymes cytotoxic Cers by SM hydrolysis in the PM is reduced by their

also share similarities in the C-terminus, responsible for their reutilization in SM synthesis which may constitute an important

localisations [29] as well as in the D3 and D4 motifs, essential for protective mechanism in the cell. Similarly to SMS1, SMS2

their catalytic activities. One of the most striking structural catalyses the reactions of both SM and CPE synthesis. The dual

differences between the two SMSs is the presence of SAM (sterile substrate speciﬁcity of SMS2 suggests a potential regulatory

motif) domain [30] in SMS1, which is absent in SMS2. The SAM mechanism of its activity. Thus, the multi-domain of SMS2 is

motif was considered essential for binding SMS1 to the Golgi oriented toward the exoplasmic leaﬂet of PM, where its activity can

apparatus [7]. However, recent ﬁndings have revealed that SAM is be negatively regulated by the PE. In physiological conditions, a

responsible neither for Golgi targeting nor for SMS1 activation low level of PE on the exoplasmic leaﬂet stimulates a SMS2-

[30]. catalised SM synthesis. In pathology, the structure of PM is

disordered and an increased concentration of PE moves SMS2

SMS1 activity toward the production of CPE instead of SM, which may

SMS1 activity covers from 60% to 80% of all SMS activity and is result in Cer accumulation [49]. Data on SMS2 physiological

limited by the substrate availability [31,32]. Among all human functions in the literature is rather scarce. Some important

tissues, the highest levels of SMS1 were reported in the kidney, observations have been made in the nervous system, in which

lung and liver [33]. SMS1 is encoded by the SGMS 1 gene [34], SMS2 is highly expressed. Consequently, the expression of SMS2

which is translated into 413-amino acid protein. The newly only (but not SMS1) has been registered in hippocampal neurites.

synthetized SMS1 is then transported to the Golgi apparatus, The localization of the enzyme in the proximity of SM clusters

where it catalyses the reaction of the de novo synthesis of SM suggests its involvement in creating the micro-domains anchoring

[7,31,34,35]. SMS1 is essential for maintaining optimal levels of signal transduction proteins [50]. Interestingly, Zhang et al.

SM in a cell since it is one of the major enzymes required for SM reported that SMS2 may be engaged in drug transport in the

synthesis. Hence, SMS1 is implicated in many key cellular brain. They found decreased levels of the drug transporter, Pgp

processes such as proliferation, cell growth, migration or apoptosis (Mdr1/P-glycoprotein), ezrin and b-actin in some cerebral areas in

[36–38]. It was demonstrated that SMS1 is engaged in astrocyte SMS2 knockout mice [51].

proliferation induced by basic ﬁbroblast growth factor (bFGF)

[39]. The exact mechanism of SMS1 involvement in proliferation Sphingomyelin synthase related protein (SMSr)

remains unclear. However, it is hypothesized that due to the SMSr is encoded by the SAMD8 gene and gene product is located

functional similarities of SMS1 and phosphatidylcholine speciﬁc in the endoplasmic reticulum [35]. SMSr catalyses the reaction of

phospholipase C (PC-PLC) [40], SMS1 may act by activating CPE synthesis from Cers, but its activity is very small in comparison

tyrosine kinases as it was observed for PC-PLC [41]. SMS1 has also with canonical SMSs [52]. Since SMSr is not directly involved in SM

been found to be required for maintaining cell morphology, production [28], its further description is beyond the scope of this

growth and migratory potential. This was reported in the Neuro- article.

2a cell line of murine neuroblasts, where SMS1 exerted its activity

through Akt kinase and cyclin D1 signaling [36]. The complex roles Sphingomyelinases (SMases)

which SMS1 can play in a cell have been vividly demonstrated in

studies on immune cells. Taguchi et al. showed that the IL-2- SMases (Sphingomyelin phosphodiesterases) catalyze the

mediated survival of natural killer cells depends on SMS1 reaction of SM hydrolysis. They hydrolyse the phosphodiester

activation which was suggested to occur through the action of bond of SM to generate Cer and phosphocholine. Three types of

phosphatidylinositol-3 kinase [42]. In addition, SMS1 controls the SMases can be distinguished on the basis of optimal pH values

fate of T cells. Dong et al. reported that SMS1, by involvement in required for their activation: acid (a-SMase), alkaline (alk-SMase)

lipid raft formation, provides sites for T cell activation through and neutral (n-SMase).

tyrosine phosphorylation [43]. SMS1 may also perform a

protective action toward the apoptosis of T cells evoked by Fas Acid Sphingomyelinase (a-SMase)

ligand (FasL) [38], presumably preventing Cer accumulation in the a-SMases encoded by the SMPD1 gene and translated into 629-

cells. Apart from controlling Cer levels, SMS1 regulates the aminoacid pro-protein [25], which gives rise to lysosomal (l-

concentration of the multifunctional lipid, DAG may play a SMase) and secretory (s-SMase) a-SMases [53]. Lysosomal locali-

structural function in the PM [44], but primarily it is an important zation of the enzyme requires the attachment of mannose residues

lipid second messenger, crucial in cellular proliferation and to the immature form of a-SMase, whereas lack of this modiﬁcation

differentiation processes [45]. Moreover, DAG is a precursor of in the a-SMase precursor protein leads to its secretion to the

more complex lipids such as phosphatidylethanolamine and extracellular space [54]. Both l-SMase and s-SMase are equipped

phosphatidylcholine [46]. with six potential glycosylation sites which are essential for the

activities of the enzymes and their anti-proteolytic protection

SMS2 [55]. a-SMases reach their full activity in an acidic environment,

SMS2 is a product of the SGMS2 gene [34]. It is an integral however, s-SMase can also act in neutral pH [56]. Furthermore, in

protein of PM, consisting of six trans-membrane domains and the contrast to s-SMase, [57] the activation of l-SMase do not depend

C-terminal catalytic region, localized in the outer leaﬂet of the PM on zinc ion availability in their environment [58].

B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581 573

The biological functions of a-SMases have been extensively metabolism remains controversial. According to Sawai et al., the

studied, although the vast majority of the reports have focused on overexpression of n-SMase1 in the MCF-7 cell line did not inﬂuence

the enzyme activities in pathological conditions and only few SM content, but rather contributed to the lyso-platelet-activating

examples of physiological roles of a-SMases exist in the literature factor (lyso-PAF) of metabolism [82]. Moreover, n-SMase1-

and refer mostly to s-SMase. Active forms of s-SMase were found in deﬁcient mice exhibited a lack of alterations in SM levels

synovial, salivary and cerebrospinal (CSF) ﬂuids, as well as in the [83]. On the other hand, n-SMase1 was reported to mediate T

sera, tears and urine of healthy people [59,60]. It has been cell receptor signaling through the production of Cers and

demonstrated that s-SMase plays an important role in the subsequent apoptosis [84]. Furthermore, zebraﬁsh studies

maintenance of platelet homeostasis as the enzyme is secreted revealed an engagement of n-SMase1 in apoptosis induced by

by thrombin-stimulated platelets [61,62]. Moreover, s-SMase was heat stress [85,86], which was stimulated by prior phosphorylation

reported to enhance glucose inﬂux into platelets [63], ATP release, of the enzyme by c-Jun N-terminal kinase (JNK) [86]. Interestingly,

and thrombus formation. s-SMase, by regulating the properties of novel ﬁndings by Guo et al. indicate that n-SMase1 is partially

platelet PM, inﬂuences phosphatidylserine translocation to PM, responsible for exosome biogenesis in the hypothalamic cell line

which is the central site of thrombin production regulation [64]. In [87].

addition, a-SMase mediates translocation of phospholipase A2 to The third n-SMase, n-SMase3, is a protein of 866 amino acids,

PM in platelet cells, which is most probably facilitated by PM localized to ER and trans-Golgi compartments. The strongest

rearrangement through the a-SMase- mediated ceramide produc- expression of n-SMase3 has been noted in striated and cardiac

tion [65]. muscles. Similarly to other n-SMases, n-SMase3 is a Mg -

dependent enzyme. Despite the fact that n-SMase3 shares no

Neutral sphingomyelinase (n-SMase) structural homology with other n-SMases, it exhibits activity

n-SMase are encoded by four genes, SMPD2–5 which are toward SM hydrolysis. To date, data concerning the functional

translated into four proteins, n-SMase1, n-SMase2, n-SMase3 roles of n-SMase3 have been limited, although it has been shown to

and mitochondria-associated SMase (MA-SMase) [66]. be upregulated in MCF-7 cells under TNF-a treatment [88]. In

The best characterized n-SMase is n-SMase2, which is a 655- addition, the n-SMase3 gene was overexpressed in colon cancer

amino acid protein containing the C-terminal catalytic domain and cells treated with genotoxic agents [89]. Novel ﬁndings revealed

two hydrophobic segments on its N-terminus [67]. The enzyme that n-SMase3 mediated the TNF-a-induced oxidant production in

also has two palmitoyl-binding cysteine clusters, the ﬁrst located skeletal muscle [90].

in the hydrophobic region of the protein and the second in its Identiﬁed as the last n-SMase, MA-SMase, is a 483-amino acid

active center. Palmitoylation of cysteine clusters is important for protein, localized in ER and mitochondria [91,92]. MA-SMase

the stability of n-SMase2 and its binding to PM [68]. Activation of expression has been found in the testis, brain, pancreas and

n-SMase2 occurs in neutral pH in the presence of Mg ions. n- epididymis. Lower levels of the enzyme have also been noted in the

SMase2 is ubiquitously expressed within mammalian tissues spleen, thymus and skin. The structure of MA-SMase is similar to

with the highest content in the brain [66]. Interestingly, Crivello that of n-SMase2, and similarly to the latter enzyme, requires Mg

et al. found that n-SMase2 activity in the brain underwent ions for its full activation. There are only few reports on the role of

changes during aging. They observed an increased activity of this MA-SMase in the cell. It was demonstrated that MA-SMase can

enzyme in the PM isolated from striatum, hippocampus and the mediate apoptosis by regulating Cer levels in the mitochondria

frontal cortex of aged brains [69]. n-SMase2 plays a multitude of [93]. On the other hand, the predominant expression of MA-SMase

roles in the CNS. Stoffel et al. demonstrated that the active form of in the testis [91] may indicate its essential role in fertilization

n-SMase2 was required for embryonic and postnatal develop- processes. Sperm contains considerable amounts of SM which

ment by inﬂuencing the hypothalamic function [70]. n-SMase2 decrease signiﬁcantly during fertilization [94]. MA-SMase, by SM

was also reported to mediate the trafﬁcking of NMDA receptors to hydrolysis, increases the level of Cers which are suspected to

PM, which may suggest its function in synaptic plasticity induce membrane fusion of spermatozoa with the ovum [95].

[71]. Furthermore, n-SMase2 can augment dopamine uptake

by the PC12 cells through Cer production and increase the Alkaline SMase (alk-SMase)

intracellular calcium level [72]. The n-SMase2 mediated regula- Alk-SMase is encoded by the ENPP7 gene and translated into

tion of intracellular calcium concentration goes beyond the CNS 458-aminoacid protein which is equipped with two hydrophobic

cells. In canine cerebrovascular smooth muscle cells, n-SMase2 domains on both ends of the protein, essential for signaling and

activation led to an enhancement of intracellular inﬂux of anchoring of the enzyme [96].

calcium required for proper contraction of the arteries [73]. In Alk-SMase differs structurally from the remaining SMases and

addition, Bautista-Pe´ rez et al. noted that the process of renal belongs to a different family of enzymes, nucleotide-pyropho-

vasoconstriction induced by angiotensin II involved Cers sphatase/phosphodiesterase [96]. It is not activated by divalent

originated from the n-SMase2 catalised SM hydrolysis [74]. On ions, but inhibited by Zn . In physiological terms, localization of

the other hand, n-SMase2 was reported to stimulate the alk-SMaseis limited to the intestine [97], where it digests

production of nitric oxide by endothelial cells, which induced exogenous SM [98].

vasorelaxation of the coronary arteries [75]. The important roles

of n-SMase2 were also found in T cell co-stimulation [76] as well SM metabolism in selected pathologies of the CNS

as skeletal tissue homeostasis reviewed by [77], where n-SMase2

contributed to proper bone mineralization [78] and cartilage Ischemia/reperfusion

development [79].

In contrast to n-SMase2, the physiological functions of other n- Cerebral ischemia is a condition of decreased tissue perfusion

SMases have been poorly researched. n-SMase1 is a 423-amino resulting in an inadequate supply of oxygen, glucose and other

acid protein which requires Mg ions to perform its enzymatic metabolites, and the subsequent reperfusion triggering multiple

activity [67]. n-SMase1 localizes predominantly to ER, but it has signaling cascades, which leads to brain injury. Various factors/

also been found in the Golgi apparatus [80] and the nucleus [81]. It pathways have been described in the literature as crucial for

is broadly expressed in human tissues such as the intestine, kidney, ischemia-induced neurodegeneration. Numerous changes, among

brain, liver, heart and lung [67]. Involvement of n-SMase1 in SM others, have been observed in SM metabolism, frequently noted as

574 B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581

essential for neuronal death or survival in ischemia/reperfusion accompanied by reduced ceramide utilization via glucosylcera-

(IR) processes. It was demonstrated that focal brain ischemia mide synthase [108]. Cer production, catalyzed by SMases, leads to

stimulated an early and irreversible SM hydrolysis and Cer alterations of SM proﬁles in a tissue. It was noted that the levels of

generation in the cerebral cortex [99]. Similarly, Nakane et al. almost all studied SM species were changed in NT-2 neuronal

described lethal forebrain ischemia in gerbil, an SM degradation precursor cells during hypoxia [112]. Likewise, the concentrations

leading to an increase in Cer levels in the hippocampus of the total SM and those SM species containing long chain fatty

[100]. Interestingly, it was shown that ischemia-induced Cers acids (C16–18) were elevated while the levels of very long chain

were localized to the astrocytic PMs in CA1 and DG regions of the (24C-) SMs were reduced in the white matter of postmortem

hippocampus, which suggested that the SMase pathway was frozen brain tissues of patients with subcortical ischemic vascular

involved in Cer production under ischemic conditions [101]. Soeda dementia D (SIVD) [113], the results being similar to those

et al. described in the pheochromocytoma cells (line PC-12) model obtained in the entorhinal cortex of AD human brains [114]. Im-

that the n-SMase activity peaked at 1 h and the increased activity portantly, the SM derangements observed in SIVD white matter

lasted thereafter for several hours. This observation was supported closely resembled the brain lipid proﬁles of ceramides synthase 2

by data that the diﬂuoromethylene analogs of sphingomyelin (CerS2)-null mice [115]. n-SMase appears to be activated by

(SMAs, especially SMA-7), which inhibit of n-SMase but not multiple stimuli but the molecular mechanisms for this regulation

ceramide synthase in the serum/glucose-deprived pheochromo- are not fully understood. A study by Gu et al. suggested that

cytoma cells line PC-12, could prevent rapid ceramide production ischemia-induced n-SMase2 activation might be partially depen-

and that they signiﬁcantly reduced the size of cerebral infarcts in dent on the TNF-aR signaling pathway [106]. It was described that

mice [102]. tumor necrosis factor (TNF) released during an ischemic stroke

Magnesium-dependent n-SMase2, mainly found in brain tissue, stimulated sphingomyelinase. n-SMase2 may be activated directly

is an important mediator of neuronal death induced by ischemia/ by binding to the TNF-a receptor/receptor for activated C kinase 1 –

hypoxia [102–106]. It was observed by Yoshimura et al., that the RACK1/embryonic ectoderm development – EED (TNF-aR/RACK1/

24-hour hypoxia of PC12 cells produced an increase in membrane- EED) in the early phase of ischemia [116,117]. However, the

bound n-SMase2 activities, but not in the activities of neutral inhibition of TNF-aR attenuated n-SMase2 activity to some extent,

magnesium-independent and acid SMases [104]. Comparative suggesting that the TNF-aR/RACK1/EED pathway plays a second-

analyses of n-SMase kinetics in PC12 cells cultured for 24 h under ary role in the upregulation of n-SMase2 activity in hippocampal

hypoxic conditions showed an elevated Vmax but not an altered astrocytes following ischemia.

Km of the enzyme [104]. In the in vivo model, Gu et al. observed It was observed that the activation of n-SMase was closely

early accumulation of Cers after 30-min cerebral ischemia due to linked to the initial depletion of glutathione (GSH) [118] and a high

the upregulation of n-SMase2 but not a-SMase activities [107]. The dose of vitamin E (50 or 100 mM) markedly reduced the activity of

downregulation of n-SMase2 by either a chemical inhibitor this enzyme in rat neurons during hypoxia and re-oxygenation

(GW4869) or siRNA, used prior to forebrain ischemia induction, [119]. Won and Singh stated that cellular redox potential and

reduced Cer production in rat hippocampal astrocytes and partially reactive oxygen species (ROS), which are regulated by GSH, were

reversed the neuronal damage as well as decreased mRNA levels of associated with SMase activation [120]. Similarly, Li et al. (2012)

pro-inﬂammatory cytokines [107]. In addition, based on the found ROS to be a factor activating a-SMase, which is in fact a

Transwell model, authors demonstrated that the effect of lysosomal enzyme important for cellular responses to ROS

activating the nSMase-2/ceramide pathway in the astrocytes can [121]. SMase activities in ischemia/hypoxia may also be regulated

be reversed by the inhibition of NF-kB signaling. Furthermore, the by the molecular pathways mediating cellular survival. In PC12

inhibition of a-SMAse with imipramine was not effective in cells over expressing Bcl-2, which exhibited strong resistance to

decreasing Cer levels. These ﬁndings suggest that at the early hypoxia, SM hydrolysis was decreased and n-SMase activation was

stages of cerebral ischemia, n-SMase2, but not a-SMase, is reduced [104]. Other studies have demonstrated that cerebral

responsible for SM hydrolysis in the rat hippocampus. In contrast, ischemia rapidly upregulated the p38 activity which in turn

other studies showed that the increase in Cer levels in ischemia is enhanced n-SMase2 activation by its phosphorylation [122]. More-

generated through the activation of both neutral and acid SMases over, p38 inhibition reversed ischemia-induced n-SMase2 activa-

[102,108]. The discrepancies among study ﬁndings presumably tion and reduced Cer levels [107]. In addition, A2B adenosine

resulted from different models of ischemia and experimental time receptor/p38 played an essential role in the activation of the n-

frames used by the authors. Thus, Ohtani et al. observed enhanced SMase2/ceramide pathway in rat hippocampus in response to

a-SMase activity 3 days after chronic cerebral ischemia induced by ischemia [107]. Under cerebral ischemia conditions, Cers are

clipping both common carotid arteries in rats [108]. The authors accumulated in the cells as a result of not only SMase over-activity

concluded that the hydrolysis of SM in the PM, catalyzed by n- but also a decreased expression of SMS1 mRNA in subcortex glia,

SMase2, was probably the ﬁrst cellular response to ischemia and which is probably associated with massive necrotic cell death in

the subsequent over-activation of the lysosomal/endosomal a- the infarct area [114,123]. The return of the SMS1 mRNA level to

SMase led to cell death. Additionally, it was suggested that the baseline 72 h after occlusion, may indicate active processes of cell

deﬁciency of a-SMase and the dysfunction of heat shock protein proliferation.

Hsp 70.1 occurred in cerebral ischemia and it was closely related to The understanding of whether SM hydrolysis is a cause or result

lysosomal rupture and CA1 neuronal death within a 7-day period of ischemic damage is still poor. However, based on scientiﬁc

[109]. literature, it appears that SMases could be promising new targets

It appears that the mechanism of ceramide accumulation for drugs designed to reduce ischemic brain lesions, particularly as

depends on the severity of an insult to the brain. Severe and lethal the pharmacological inhibition of SMase activities has been shown

cerebral ischemia/reperfusion resulted in ceramide accumulation to protect neurons, oligodendrocytes and astrocytes against Cer-

via the activation of a-SMase and SM hydrolysis [99,100]. More- induced cell death caused by ischemia [102].

over, the extent of brain tissue damage was lower in mice lacking

a-SMase [110]. On the other hand, in mild IR, Cer accumulation Alzheimer disease (AD)

resulted from the de novo Cer biosynthesis rather from SM

hydrolysis [111]. Furthermore, in chronic cerebral ischemia, Cers AD is one of the most common age-associated dementia.

were generated due to the activation of a-SMase which was Pathomorphological features of AD involve formation of

B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581 575

neuroﬁbrillary tangles, senile plaques with abnormal Ab symptomatic phases. In all experimental points, the levels of altered

(amyloid b) peptide accumulation and tau hyperphosphorylation SM species were reduced in the brains of APP/tau mice in

[124]. Although different aspects of AD are extensively studied, the comparison with controls, whereas in plasma of AD animals all

basic knowledge on the causes, diagnosis and treatment of this SM species were signiﬁcantly elevated [137].

disease, is still lacking. SM metabolism represents a promising area An event initiating cellular changes toward AD development is

of research on AD-associated processes. The alterations in SM cycle still unknown, nevertheless it seems that one of the ﬁrst steps in

were studied both in the context of seeking the potential diagnostic this process could be a structural reorganization of the PM. The

biomarkers as well as ﬁnding explanation of AD pathogenesis. alteration of PM properties could in turn lead to pathological Ab

Studying blood plasma/serum SM levels researchers obtained an processing, which occurs mainly in the lipid rafts. Ab, constitutes

incoherent results. Oresˇicˇ et al. reported that AD patients exhibited a the main component of amyloid plaques, is ﬁrstly cleaved from the

decreased content of serum SMs [125]. Mielke et al. analyzed the SM APP (amyloid precursor protein) by b-secretase. Further, Ab40 and

and Cer serum concentrations in one group of women being Ab42 peptides are produced by subsequent action of g-secretase

examined over 9 years of the study. They obtained ﬂuctuations in the [138,139]. The highly altered lipid compositions of lipid rafts were

levels of studied SFs corresponding with the time of memory found in frontal and entorhinal cortices in non-symptomatic and

impairment onset. This suggested that both Cers and SMs could mild dementia phases, in AD brains [140]. In addition, the levels of

serve as potential biomarkers of AD development [126]. Another 9- SMs associated with the lipid rafts were signiﬁcantly reduced in

year study, enrolling healthy women, showed a strong correlation frontal cortex at the early stages of AD. Authors hypothesized that

between increased Cer, but not SM, levels in serum of volunteers, such reorganization of lipid components at the beginning of the AD

and risk of AD development [127]. On the other hand, Kosicek et al. might provoke the Ab structural changes toward senile plaque

noted an augmentation of SM content in cerebrospinal ﬂuid (CSF) of creation. Dı´az et al. did some important observations on how the

individuals with probable AD [128]. Furthermore, studies on physicochemical features of the lipid rafts in AD brains change

patients with prodromal, mild and moderate stages of AD revealed with the development of the malady [141]. Using steady-state

the increments of SMs multiple species in CSF, but only in those ﬂuorescence polarization analyses, they reported more liquid-

participants with prodromal phase of the disease. Authors ordered and viscous structure of lipid rafts in frontal and

hypothesize that this early elevation of SMs in CSF of AD patients entorhinal cortices at the early stages of AD than in control brains.

could result from an increased level of Cers, which were Moreover, the authors stated that PM ﬂuidity did not correlate

subsequently used to SM synthesis [129]. Similarly, Mielke et al. with the SM content, but rather with the changes in saturation

have shown in the cohort study, that SMs from CSF may participate status of the phospholipids [141]. Importantly, altered lipid raft

in early events of AD. They analyzed levels of Cers and SMs in CSF of structure can greatly inﬂuence the functional properties of

healthy individuals with parental history of AD and found the proteins anchored in the PM, such as secretase/AbPP (amyloid-

correlation between concentrations of all studied SM species and b protein precursor) and impact APP processing [142] toward

levels of Ab, total tau, and some forms of p-tau [130]. A changed SM promotion of b-amyloidosis. This may lead to the conclusion that

metabolism was also reported in AD brains. Early studies revealed a changes in total levels of SM are probably less relevant for AD

general decline of SM content in AD brains [131,132]. Further development than the species composition of this sphingolipid. On

investigations showed and an elevation of Cer levels and reduction the other hand, it was reported that Ab peptides can induce the n-

of SM concentrations in human gray matter of frontotemporal area SMase activation. Thus, treatment of rat primary astrocytes with

in AD patients [133]. These changes were associated with increased Ab25–35 peptide increased n-SMase activity [143], whereas Ab42

activity and protein expression of a-SMase in membrane, but not activated n-SMase and decreased SM levels, which was modulated

cytosolic fraction of the tissue extracts. Taking into account that any by g-secretase activity [144].

apparent changes were reported for n-SMase, it is possible that in An interesting relation between n-SMase2 activity and produc-

AD, lysosomal a-SMase translocates to the PM and becomes a tion of exosomes was found in APP/PS1 transgenic mice [145]. An

primary enzyme responsible for the Cer over-production. Cers inhibition of n-SMase-2 led to lowering the amount of exosomes in

generated from the SM hydrolysis can participate in formation of the brain and serum as well as reduction of Ab1–42 concentration

ceramide-enriched platforms, which are required for clustering of in the plaques and levels of almost all Cer species analyzed in

some speciﬁc receptors, to trigger their molecular pathway serum. Authors suggested that n-SMase2 might inﬂuence the

[134]. Interesting data was obtained from AD plaque analysis. The Ab1–42 aggregation through the exosomal signaling. In addition,

senile plaques isolated post mortem from the brains of AD patients in vitro experiments revealed that exosomes stimulated the

contained an increased concentration of predominantly Cer-C20:0 changes in Ab conformation toward reduction of toxicity of

in comparison with surrounding neuropil. On the other hand, the amyloid aggregates and enabled their engulﬁng by the microglia

level of SM-C20:0 was only slightly reduced. Authors suggested that cells. Moreover, exosome secretion from neurons was regulated by

elevated Cers could partially originated from the SM hydrolysis since n-SMase2 and SMS2 [146]. A new mechanism of a-SMase action in

the expressions of both a-SMase and n-SMase were found in corona, the AD was proposed by Lee et al. [147]. They noted an increment

and in some cases core, of senile plaques [135]. Changes in SM in a-SMAse activities in brains, plasma and ﬁbroblasts obtained

concentrations in the brain were also studied in AD animal models. from patients with familiar AD or APP/PS1 (amyloid precursor

Gonza´lez-Domı´nguez et al. noted that the levels of SMs containing protein/presenilin 1) transgenic mice. The enhanced activation of

saturated acyl groups were augmented in the hippocampus, the enzyme disturbed autophagy-lysosome pathway and dimin-

whereas those with unsaturated fatty acid chains were reduced ished autophagic degradation. Suppression or even restoration of

in the cerebellum and cortex of APP/PS1 (amyloid precursor protein/ a-SMase activity in mice to the normal range reversed this

presenilin 1) mice [136]. Interestingly, decrement of SM species with processes. Furthermore, reduction of a-SMase expression lowered

very long chain fatty acid chains was detected in all studied brain also Ab content as well as improved the memory dysfunctions in

areas: hippocampus, cortex, cerebellum, striatum and olfactory mice [147]. Bandaru et al. presented an interesting observation

bulbs [136]. The lipid analysis in the brains and blood plasma of mice concerning ApoE4 gene variant, which highly predisposes its

with expression of mutated human amyloid precursor protein (APP) carriers to AD development [148]. Authors have found that ApoE4

and tau protein (Tg2576 Â JNPL3) (APP/tau mice) revealed the is not sufﬁcient by itself to induce the SM and Cer alterations in the

changes in several SM species concentrations at different stages of brain since healthy people with ApoE4 genotype did not present

symptom development, namely pre-symptomatic and early or late any changes in the levels of these lipids. In opposite, ApoE4 AD

576 B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581

patients demonstrated multiple variations in SF concentrations in PD is caused by extensive damage to the dopaminergic neurons in

several brain regions [148]. the substantia nigra. The principal pathomorphological feature of

PD lesions is the formation of Lewy bodies and neurites, which are

Depression and schizophrenia intracellular deposits of protein aggregates containing predomi-

nantly a-synuclein (a-syn) [164]. The etiology of PD is still

Major depression is one of the most widespread mood unknown. Xilouri et al. [165] hypothesized that the disease may

disorders. Although it affects mental functions of the patients, commence with lysosomal malfunctions in a-syn degradation.

several systemic alterations have been found in the course of the This process may involve the lysosomal enzyme, a-SMAse

disease [149–151]. Interestingly, it was shown that depression [165]. Indeed, mutations in its coding sequence were found to

inﬂuences a SM metabolism, which is reﬂected in the changes of correlate with PD incidence [166,167]. Moreover, Gan-Or et al.

some SM-related blood parameters. A Dutch cohort study revealed suggested that mutations in the SMPD1 gene led to abnormalities

that depressed patients presented an altered pattern of plasma in the lysosome functions, which resulted in disturbed aS

SMs, wherein the SM-23:1/SM-16:0 ratio negatively correlated clearance [168]. The dysregulated SM metabolism can also

with the symptoms of the disease [152]. In addition, Kornhuber inﬂuence the process of neurotransmission in PD. It is well

et al. found over-activity of a-SMase in peripheral blood known that a-syn, by aggregation in PM of neuronal cells, causes a

mononuclear cells of patients suffering from major depression deterioration of the synaptic functions [169]. Studies by Leftin

[153]. et al. [170] on model membranes indicate that a-syn aggregate is

The pathophysiology of depression involves several systems of likely in the SM-enriched domains, which perturbs PM organiza-

neurotransmission, however, the majority of the clinical treat- tion. Disorders in the structure of PM may result in alterations of

ments are designed to regulate the activities of serotoninergic membrane fusion [170], which may have important consequences

synapses [154]. Serotonin generates the excitatory/inhibitory for the exocytosis of neurotransmitters.

signals at the synapse through binding to its receptors, 5-HT (1– In order to establish whether SM levels underwent changes

7) [155]. Sjogren et al. have shown that the inhibition of SM with PD progression, several studies on post mortem brain tissue

biosynthesis in HeLa cell line stably transfected with 5-HT(7) of PD patients have been conducted. Sidransky et al. [171] found

receptor, reduced the serotonin binding to the receptor [156]. In- that in subjects displaying mutations in the gene encoding

terestingly, a decrease in SM level changed neither the afﬁnity of lysosomal enzyme, glucocerebrosidase, which is an important

serotonin to 5-HT(7) nor the concentration of the latter. This risk factor for PD development [171] as well as in patients with

suggests that the strength of serotonin-5-HT(7) binding may be sporadic PD no alterations in SM content were observed

regulated by the SM content in the lipid rafts of the PM [156]. On [172]. On the other hand, Fabelo et al. noted that SM levels

the other hand, serotonin can inﬂuence SM levels, which was noted were slightly decreased in patients with early and incidental PD

in mice with chemically reduced neurotransmitter [157]. The well- [173]. However, novel ﬁndings have revealed that PD-related

established medication of mood disorders is based, in a large changes in SM levels may differ depending on the brain region.

extent, on antidepressants targeting the serotoninergic system. Thus, in the anterior cingulate cortex, the brain area affected by

Surprisingly, many of these drugs, particularly tricyclic antide- PD, SM content was lower in comparison with healthy controls

pressants (TCAs), turned out to exert additional activities of while in the occipital cortex, which did not display signs of

indirect a-SMase inhibitors, so-called ‘‘FIASMA’’ (Functional neurodegeneration, no alterations in SM levels were found

Inhibitor of Acid SphingoMyelinAse) [158]. A role of a-SMase [174]. In addition, the authors noted that in the cingulate cortex

regulation by the TCAs in major depression was shown in mouse of PD brains the several SM molecular species, such as SM

model by Gulbins et al. [159]. Thus, the amitriptyline and containing C23:0, C24:1, and C26:1 fatty acids, were reduced

ﬂuoxetine treatments of stressed mice led to a reduction in a- versus controls, whereas the concentrations of C18:1- and C20:0-

SMase activity in the hippocampus and improved depressive-like containing SMs were augmented.

behavior of the animals, which was not observed in mice with a- Another interesting hypothesis concerning the involvement of

SMase deﬁciency. Interestingly, mice with a-SMase overexpression SM metabolism in PD pathogenesis connects the SM cycle and

showed stressed behavior, even without its prior induction by classical inﬂammatory pathways [175]. Thus, NF-kB translocation

stressors. The treatment with TCAs ameliorated a performance of to the nuclei of dopaminergic neurons in PD [176] may trigger the

the animals [159]. formation of free radicals, which in turn results in promoting SM

The disturbed SM metabolism has also been reported in other hydrolysis leading to Cer generation and apoptosis.

serious psychiatric condition, schizophrenia. In 1993, Keshavan

et al. showed an increased level of SM in the erythrocyte PM of Niemann–Pick disease (NPD)

psychotic patients [160]. Several years later, Ponizovsky et al.

found a correlation between erythrocyte SM content and negative NPD was one of the ﬁrst discovered syndromes caused by the

schizophrenic symptoms [161]. On the other hand, SM levels in the altered metabolism of SM. This genetic lipid storage disorder [177]

thalamus of schizophrenic patients were demonstrated to be shed new light on a-SMase function in SM hydrolysis. There are

signiﬁcantly decreased [162]. Interestingly, the altered neuronal three main types of NPD. NPD type A and B, differing in clinical

content of SM may regulate the activity of the a7 nicotinic outcomes [178], are both a result of a-SMase deﬁciency due to

acetylcholine receptor, a potential target for the treatment of autosomal mutation of the SMPD1 gene. A large number of

schizophrenia. Thus, Colo´ n-Sa´ez et al. observed that the enzymatic mutations found in this gene [179–184] determine the differences

hydrolysis of SM by SMase in rat primary hippocampal neurons in a-SMase levels (from an almost complete absence for type A to

changed the characteristics of the a7 nicotinic acetylcholine approximately 10% of the normal level for type B), which results in

receptor desensitization, presumably inﬂuencing its anchoring in diverse phenotypes of NPD. a-SMase deﬁciency leads to disturbed

the lipid rafts [163]. SM degradation and accumulation of this lipid in the cells. NPD type

C (C1 or C2) is caused by mutation in the NPC1 and 2 genes, the

Parkinson disease (PD) coding proteins taking part in cholesterol trafﬁcking [185,186],

which has consequences in cholesterol accumulation in virtually all

PD is one of the most common neurodegenerative disorders, body organs. An increase in SM levels as well as the reduced activity

which affects predominantly the motor functions of the patients. of a-SMase are also observed in NPD type C, however, as a multi-lipid

B. Kamil et al. / Pharmacological Reports 68 (2016) 570–581 577

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