GangliosidesandGangliosidoses:PrinciplesofMolecular … · 2013. 7. 16. · 2000). Gangliosides...

Disease Focus

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Gangliosides and Gangliosidoses: Principles of Molecularand Metabolic Pathogenesis

Konrad Sandhoff1 and Klaus Harzer21LIMES, Kekulé-Institut, 53121 Bonn, Germany and 2Neurometabolic Laboratory, Klinik für Kinder- und Jugendmedizin, University of Tübingen, 72076Tübingen, Germany

Gangliosides are the main glycolipids of neuronal plasma membranes. Their surface patterns are generated by coordinated processes,involving biosynthetic pathways of the secretory compartments, catabolic steps of the endolysosomal system, and intracellular traffick-ing. Inherited defects in ganglioside biosynthesis causing fatal neurodegenerative diseases have been described so far almost exclusivelyin mouse models, whereas inherited defects in ganglioside catabolism causing various clinical forms of GM1- and GM2-gangliosidoseshave long been known. For digestion, gangliosides are endocytosed and reach intra-endosomal vesicles. At the level of late endosomes,they are depleted of membrane-stabilizing lipids like cholesterol and enriched with bis(monoacylglycero)phosphate (BMP). Lysosomalcatabolism is catalyzed at acidic pH values by cationic sphingolipid activator proteins (SAPs), presenting lipids to their respectivehydrolases, electrostatically attracted to the negatively charged surface of the luminal BMP-rich vesicles. Various inherited defects ofganglioside hydrolases, e.g., of �-galactosidase and �-hexosaminidases, and of GM2-activator protein, cause infantile (with tetraparesis,dementia, blindness) and different protracted clinical forms of GM1- and GM2-gangliosidoses. Mutations yielding proteins with smallresidual catabolic activities in the lysosome give rise to juvenile and adult clinical forms with a wide range of clinical symptomatology.Apart from patients’ differences in their genetic background, clinical heterogeneity may be caused by rather diverse substrate specificitiesand functions of lysosomal hydrolases, multifunctional properties of SAPs, and the strong regulation of ganglioside catabolism bymembrane lipids. Currently, there is no treatment available for neuronal ganglioside storage diseases. Therapeutic approaches in mousemodels and patients with juvenile forms of gangliosidoses are discussed.

Introduction1Gangliosides had been discovered byErnst Klenk in the 1930s (Klenk, 1937,1939) when he analyzed postmortembrain tissues of patients with Tay–Sachsdisease, a fatal infantile form of amauroticidiocy. At that time the clinical diagnosis“amaurotic idiocy” comprised an inher-ited disease characterized by high num-

bers of lipid-laden cells in the nervoussystem and the visceral organs, mental re-tardation, and impaired vision or blind-ness. With the help of biochemical,enzymatic, molecular genetic, morpho-logical, and animal studies, the clinicalpicture of amaurotic idiocy has been re-solved in the last five decades into two ma-jor groups of diseases at the molecularlevel: lysosomal ganglioside storage dis-eases, comprising GM1-gangliosidosesand 4 forms of GM2-gangliosidoses, and10 forms of neuronal ceroid lipofuscino-ses poorly characterized so far (Jalankoand Braulke, 2009).

Gangliosides are typical components ofneuronal plasma membranes. The first gan-glioside structure, that of ganglioside GM1(Fig. 1), was elucidated by Kuhn and Wie-

gand (1963). Six major and many minorganglioside species have been identified inmammalian brains (Yu et al., 2011). Inher-ited defects in ganglioside metabolism cancause fatal neurodegenerative diseases. Bestknown so far are inherited defects in lyso-somal ganglioside catabolism, the GM1-and GM2-gangliosidoses.

Up to now, no defects in gangliosidebiosynthesis have been described in hu-mans with the exception of two reports onthree patients with an infantile onset re-fractory epilepsy (Simpson et al., 2004;Fragaki et al., 2013), caused by a defi-ciency of ganglioside GM3-synthase. Itis associated with psychomotor delay,blindness, deafness, and respiratory chaindysfunction. In addition, one patient witha genetically undisclosed basis of infantile

Received Feb. 19, 2013; revised April 30, 2013; accepted May 9, 2013.This work was supported by the Deutsche Forschungsgemeinschaft.

Dedicated on the occasion of her 60th birthday to Ingeborg Krägeloh-Mann, professor of Neuropediatrics, Tübingen.

Correspondence should be addressed to either of the following: KonradSandhoff, LIMES, Kekulé-Institut, Gerhard-Domagk-Str. 1, 53121 Bonn,Germany, E-mail: [email protected]; or Klaus Harzer, NeurometabolicLaboratory, Klinik für Kinder- und Jugendmedizin, University of Tübingen,72076 Tübingen, Germany, E-mail: [email protected].

DOI:10.1523/JNEUROSCI.0822-13.2013Copyright © 2013 the authors 0270-6474/13/3310195-14$15.00/0

The Journal of Neuroscience, June 19, 2013 • 33(25):10195–10208 • 10195

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Figure 1. Pathway of lysosomal sphingolipid degradation. The eponyms of known metabolic diseases and those of SAPs (red) necessary for in vivo degradation are indicated. Hydrolases are givenin green. Heterogeneity in the lipid part of the sphingolipids is not indicated. Variant AB, Variant AB of GM2-gangliosidosis �deficiency of GM2-activator protein (GM2-AP)�; Sap, sphingolipidactivator protein (modified from Sandhoff and Kolter, 1995).

10196 • J. Neurosci., June 19, 2013 • 33(25):10195–10208 Sandhoff and Harzer • Gangliosides and Gangliosidoses

GM2-synthase deficiency, a gangliosideGM3 storage and loss of higher ganglio-sides has been described, passed away atage 3.5 months (Fishman et al., 1975).

However, blocks in ganglioside bio-synthesis generated in gene-manipulatedmice cause a broad spectrum of patholog-ical phenotypes (Proia, 2003).

Ganglioside biosynthesis and its defectsin genetically manipulated miceGlycolipids are expressed in a cell-type-and stage-specific manner, gangliosidesbeing the main glycolipids of neuronalsurfaces. Their surface patterns are gener-ated by coordinated processes, involvingbiosynthetic and catabolic steps and intra-cellular trafficking.

Their hydrophobic membrane an-chors, the ceramide residues, are formedin the endoplasmic reticulum (ER), glyco-sylation of ceramide takes place at theGolgi and trans-Golgi network (TGN),and their constitutive degradation mainlyin the endolysosomal compartment (Kolterand Sandhoff, 1999). After formation ofglucosylceramide (GlcCer) from cer-amide (Cer) at the cytosolic surface ofGolgi membranes and its flip to the lumi-nal surface of Golgi membranes, GlcCergets converted to lactosylceramide (Lac-Cer). At the luminal surfaces of Golgi andTGN membranes, LacCer is sialylated toform small precursor gangliosides (GM3,GD3, and GT3). They are then convertedby membrane-bound promiscuous glyco-syltransferases to complex gangliosidestructures, which reach cellular surfacesby vesicular transport of the secretorypathway. This concept of “combinatorialganglioside biosynthesis” (Kolter et al.,2002) allows the generation of cell-specific patterns on neuronal surfaces de-pending on the availability of precursormolecules, expression of biosynthetic en-zymes and their regulation. Generationand analysis of mutant mice with defectsin the pathways of ganglioside biosynthe-sis supported this model (Kolter et al.,2002; Sandhoff and Kolter, 2003). Mutantmice defective in b-series gangliosideswere viable and without apparent neuro-logical abnormalities (Kawai et al., 2001;Okada et al., 2002) and reach an almostnormal life span. Mice defective inGalNAc-transferase, missing the majorbrain gangliosides, were viable but suf-fered from instability of brain structureswith detachment of myelin sheets fromaxonal membranes (Schnaar, 2010) andmale infertility. A combined defect ofGM3-synthase and GM2-synthase causesan early fatal disease. Here, the absence of

all a-series and b-series gangliosides triggersthe formation of 0-series gangliosides,which are not observed in the wild-typebrain. Their additional synthesis, however,cannot compensate the loss of all the majorneuronal ganglioside structures. Surprisingwas that the total ganglioside content in thebrains of the various mutant mice stayed al-most unchanged at the wild-type level(Sandhoff, 2012).

The ubiquitous block of GlcCer syn-thesis, the precursor of the main glyco-sphingolipids (GSLs), is lethal in mice atembryonic day 6 (Yamashita et al., 1999).However, its cell-type-specific knock outin neurons (Jennemann et al., 2005),hepatocytes (Jennemann et al., 2010), orkeratinocytes (Jennemann et al., 2007)still allowed complete embryogenesis butwith the birth of fatally sick animals.Apparently, some complex glycolipids de-rived from GlcCer such as the stage-specific embryonic antigens (Kannagi etal., 1983; Eggens et al., 1989) involved incellular adhesion processes are needed atearly embryonic stages for developmentof a multicellular organism (Kolter et al.,2000).

Gangliosides have been discovered asGSLs of neuronal membranes by lipidanalysis of Tay–Sachs disease (Klenk,1937, 1939), but their complex chemicalstructure was elucidated only later in the1960s (Kuhn and Wiegandt, 1963). Theirstorage in neuronal lysosomes paved theway for the analysis of their cellularmetabolism and lysosomal catabolism(Sandhoff et al., 1969; Sandhoff, 1977)

Clinical phenotypes, correlations topathological and biochemicalphenotypes

All types of gangliosidoses are caused byinherited defects in ganglioside catabo-lism (Fig. 1) and can manifest with severe,extremely variable clinical symptoms (Ta-ble 1) in almost any age group, althoughmost patients have an early infantile onsetand a late infantile or juvenile, fatal end oftheir disease, which is dominated by thecentral nervous involvement (Fig. 2A)(Lyon et al., 1996a). The cerebral degen-eration is marked by the lipid storagepresent in neurons with the ultrastruc-turally characteristic “membranous cy-toplasmic bodies,” which are secondary,“storage” lysosomes (Fig. 2B). These stor-age bodies distend the neurons (Fig. 2C),which eventually are lost, but the exactreason is unclear (for example, cell inac-tivity by ganglioside-induced impairmentof synaptic transmission is discussed).The peripheral and autonomous nerve

systems are also involved, but with minorclinical relevance, though lysosomal stor-age bodies are demonstrable at places.

In addition to the neuronal involve-ment, an extraneural involvement (Table1) is striking in infantile and partly presentin juvenile patients with GM1-gangli-osidosis; even an intrauterine manifesta-tion with hydrops fetalis is possible, and adysmorphic (gargoyle-like) face is regularin early infantile patients. Juvenile andadult GM1-gangliosidosis patients have apredominant or almost pure neurologicpicture (Suzuki et al., 2001). Extraneuralmanifestations are absent in GM2-gangliosidosis with the exception of vari-ant 0, also called Sandhoff disease, inwhich slight visceromegaly is frequent(Gravel et al., 2001) and some involve-ment of bones and even the heart can oc-cur (Venugopalan and Joshi, 2002).

The clinical phenotypes in the gangli-osidoses can, though with some caveats,be correlated to the different types andnumbers of substrates, which are accumu-lated in these diseases. Examples of thesubstrates of the acid hydrolases whosedeficiencies define the diseases are ganglio-sides, asialogangliosides, other glycolipids,specific oligosaccharides and, in case of acid�-galactosidase, mucopolysaccharides. Inthe deficiencies, the patterns of accumulatedsubstrates can be explained in a simplifiedformat as follows. The main substratesof the acid �-galactosidase, which aredeficient in GM1-gangliosidosis, are GM1-ganglioside (Fig. 1), specific oligosaccha-rides, and keratan sulfate, so that this diseaseis marked not only by ganglioside storage(whose main pathologic correlate is neuro-nal degeneration) but also by oligosacchari-dosis and mucopolysaccharidosis, with theextraneuronal clinical involvement dueto the latter. The main substrate of the�-hexosaminidase A deficient in GM2-gangliosidosis variant B (also calledTay–Sachs disease) or suppressed in theGM2-activator-deficient variant AB, isGM2-ganglioside, so the disease is “only” aganglioside storing disorder. The main sub-strates of the �-hexosaminidases A and Bdeficient in GM2-gangliosidosis variant 0are GM2-ganglioside and oligosaccharides,so the disease includes gangliosidosis andoligosaccharidosis, with some visceral in-volvement due to the latter.

Another correlation can, though withsome reasonable doubts, be assumed be-tween the time (as patients’ age) of onsetor manifestation, or the duration (chro-nicity) of the disease, and the rudimentarycatabolic functions of the defective (if notcompletely absent) acid hydrolases in the

Sandhoff and Harzer • Gangliosides and Gangliosidoses J. Neurosci., June 19, 2013 • 33(25):10195–10208 • 10197

patients. This “residual activity hypothe-sis” postulates higher residual functionsfor late starting/manifesting or chronicforms of gangliosidoses than for early (in-fantile) disease forms (see Protracted clin-ical forms of gangliosidoses and thethreshold theory, below). Although thelow residual, probably pathophysiologi-cally relevant activities of the defective hy-drolases cannot always reliably be assessedunder laboratory conditions in vitro, thathypothesis seemed to be acceptable formany patients who were studied enzymat-ically. As to those residual functions, it isinteresting that in late manifesting GM1-gangliosidosis the almost only substraterelevant for the biochemical phenotype isthe ganglioside accumulation, particu-larly in neurons, while for the breakdownof other substrates the residual functionsseem to be nearly sufficient.

However, in a certain type of geneticacid �-galactosidase deficiency, also a suf-ficient catabolism of GM1-ganglioside ispossible, whereas the breakdown of othersubstrates is severely impaired, and the re-sulting disease is not gangliosidosis but mu-copolysaccharidosis IV (Morquio) type B(Suzuki et al., 2001). This indicates different

substrate (e.g., the ganglioside/activatorprotein complex) binding sites, protein-folding-relevant sites, or subdomains in theenzyme, which are required for the catabo-lism of different substrates, and suggeststhat these sites are differentially or alterna-tively defective in different patients whohave then very variable diseases caused bytheir �-galactosidase defects. On the otherhand, a special GM1-ganglioside-storingdisease (associated with oligosaccharidestorage) results from an indirect deficiencyof acid �-galactosidase (and sialidase, and aspecific sulfatase) in galactosialidosis, whichis caused by the genetic deficiency of the so-called protective protein (identical to car-boxypeptidase A) (d’Azzo et al., 2001).When this protein is defective, the func-tional complex of the normal protein withacid �-galactosidase and sialidase (and thesulfatase) cannot be formed, and these en-zymes, though having a normal structure,are subject to rapid decay. The resultingclinical phenotype is very similar to that inclassic GM1-gangliosidosis (and to that inthe genetic sialidase defect, sialidosis). So anenzymatic diagnosis of GM1-gangliosidosishas always to be confirmed by demonstrat-

ing the presence of normal sialidase activity,or by molecular gene analysis.

Summary outline ofclinical symptomatologyThe main symptomatology of the gangli-osidoses is given as a partially simplifiedsummary in Table 1. Clinical detailsshould be found in the special literature(Lyon et al., 1996a, b; Gravel et al., 2001;Suzuki et al., 2001). Pathologists havefound that in gangliosidosis the brain issometimes slightly infiltrated with inflam-matory cells considered to be a responseto the dysfunction of the lipid-laden neu-rons and to the abundance of antigeni-cally acting gangliosides. Here followsome supplementary clinical remarks.Neuroimaging has no essential role in thecharacterization of gangliosidoses, but inlate disease stages cerebral and cerebellaratrophy can often be documented andslight T2-weighted hyperintensities mayappear earlier in the brainstem and whitematter (Wilken et al., 2008). Magneticresonance spectroscopy (N-acetyl aspar-tate, myoinositol, and other signals) maybe helpful in documenting neuronal lossand inflammation (Assadi et al., 2008).

Table 1. The main clinical hallmarks and symptoms in gangliosidoses

Disease Manifestation Regular symptoms/signs Facultative symptoms/signs Remarks

GM1-gangliosidosis Infantilea,b Dysmorphic face, severe dysostosis, hepato-megaly, unable to sit, muscle hypotonia,later on spasticity, visual failure, foam cellsin bone marrow, oligosacchariduria,mucopolysacchariduria

Cherry-red macular spotc, nystagmus,seizures, splenomegaly, joint contrac-tures, hematologic signs, vacuolatedblood lymphocytesd

Rapid or 1–2 year downhill course withpsychomotor deterioration, generaldystrophy, feeding problems

Juvenilea By 4 years or later: dysarthria, extrapyramidalsigns including dystonia

Slight intellectual impairment, mild bonechanges, foam cells in bone marrow

Some patients have also parts or attenu-ated forms of the symptoms listed forthe infantile disease, other patientsbelong to the chronic group

Adulta As in juvenile disease, but with more insidiousonset

Psychosis Disease is chronic rather than with onlyadult manifestation

Chronic Combines late infantile, juvenile, andsometimes adult manifestations inwhich the above listed symptoms arepartly or gradually developed

GM2-gangliosidosis, variants B,B1 and 0

Infantilee Loss of head/trunk control, smiling, by about 6months, cherry-red macular spot c, highstartle response to noise, muscle hypotoniaby 1 year, epilepsy, tetraparesis with somespasticity, dementia, blindness

Megalencephaly, visceral involvement andoligosacchariduria only in variant 0

Downhill course mostly less rapid than inearly GM1-gangliosidosis

Juvenile/subacute Onset by 3 to 6 years, dysarthria, loss ofspeech, spastic paraparesis, later onparaplegyf, pyramidal signsf, cerebellarataxia, mental deterioration

Seizures, irritability, psychiatric signs,denervation muscle atrophy (EMG),areflexia, cherry red macular spotc

Clinical variability even in siblingsg, aboveremark to juvenile GM1-gangliosidosishere also apt

Adult Clumsiness, lower motor neuron disease,denervation muscle atrophy, cerebellarataxia

Psychosis High clinical variability, oldest patient was76 years

Chronic See remark to chronic GM1-gangliosidosisGM2-gangliosidosis, variant AB Most as in variants B, B1, 0 Cherry red macular spotc No adult patients knownaGrouping of GM1-gangliosidosis patients as type I, II, and III of the disease according to the age at clinical onset is questionable by the often ambiguous onset or insidious or chronic manifestation through all, except the early infantile, agegroups (Lyon et al, 1996b). bEarlier named “generalized gangliosidosis.” cSee Fig. 2D. dSee Fig. 2E. eEarlier named “infantile amaurotic idiocy”; relatively high prevalence of variant B (Tay–Sachs disease) in Ashkenazim. fSee Fig. 2A. gManypatients are Ashkenazim, but a genetic isolate of variant B1 is in Spain.


The prenatal and postnatal laboratory di-agnosis of gangliosidoses is usually madeenzyme biochemically (e.g., in blood sam-ples), and/or molecularly. Many aspectsof genotype–phenotype correlation inthese diseases were described in the specialliterature. Therapeutic strategies such asbone marrow transplantation (BMT),use of drugs with substrate reducing,chaperon-like and anti-inflammatory ef-fects, and intracerebral or intrathecal en-zyme replacement are under investigation(see Therapeutic approaches, below).

Pathway of ganglioside degradationTopology of endocytosis and lysosomaldigestion of gangliosidesLysosomes are intracellular stomachs. Theydegrade macromolecules and release theircomponents as nutrients into the cytosol forsalvage pathways and energy metabolism.

Macromolecules and membrane compo-nents reach the lysosomal compartment fordigestion by autophagy (Florey and Over-holtzer, 2012), phagocytosis (Delves et al.,2006; Florey and Overholtzer, 2012), and byendocytotic pathways (Kolter and Sandhoff,2005). Whereas water-soluble lysosomalhydrolases can attack water-soluble macro-molecules directly, the digestion of ganglio-sides, GSLs, and membranes needs a morecomplex cooperation between soluble hydro-lases, lipid binding and transfer proteins, andluminal intra-endolysosomal vesicles (Kolterand Sandhoff, 2005; Kolter and Sandhoff,2010). Due to the lipid phase problem, water-soluble glycosidases hardly attack amphiphilicGSLs as components of vesicular membranes,but need the help of membrane perturbingand lipid binding proteins, the sphingolipidactivator proteins (SAPs).

Our view of the topology of lysosomalganglioside digestionFor their digestion, gangliosides of cellularsurfaces reach luminal intra-endolysosomalvesicles or intra-endosomal membranes(IMs) (Fig. 3; Burkhardt et al., 1997; Möbiuset al., 1999), which are generated during en-docytosis. Luminal vesicles are formed bysuccessive steps of vesicle budding and fis-sion controlled by the endosomal-sortingcomplex proteins (Wollert and Hurley,2010). The vesicles are prepared for lyso-somal digestion by a lipid-sorting processbeginning at the level of endosomes (Kolterand Sandhoff, 2005; Gallala et al., 2011).Cholesterol is sorted out by two sterol bind-ing proteins, NPC-2 and NPC-1 (Abdul-Hammed et al., 2010), and sphingomyelinis degraded by acid sphingomyelinase(ASM; V. Oninla, B. Breiden, K. Sandhoff,

Figure 2. Clinical and morphological observations in gangliosidoses. A, Near end stage in a Spanish 13-year-old patient with GM2-gangliosidosis variant B1 (photograph courtesy of H.H. Goebel,Mainz). B, GM2-gangliosidosis variant 0 in a 23-week-old fetus; and part of a cortical neuron; early stage of membranous cytoplasmic bodies (two clusters marked by crosses), i.e., lipid storingsecondary lysosomes, �36,000 (courtesy W. Schlote, Tübingen). C, The large cells are neurons distended by lysosomal storage in the medulla oblongata of a 1.5-year-old patient with GM1-gangliosidosis. The small dark bodies are nuclei from increased numbers of glial cells (“reactive gliosis”). Cresyl violet stain �300 (courtesy of H.U. Benz, Tübingen). D, Cherry red macular spot in aninfantile patient with GM2-gangliosidosis variant B (Tay–Sachs disease). The dark red spot in the middle of the light central area is secondary to lipid storage in neuronal cells in this area; the storingcells have lost their processes that normally cover the fovea centralis. The fovea normally appears as yellow, but is changed to the red macular spot showing the color of the choroidea behind theretina. E, Lymphocytes with clusters of vacuoles (storage lysosomes) in blood smear of an early infantile patient with GM1-gangliosidosis, indicating the “generalized storage disorder.” May-Grünwald stain; �1500 (courtesy of H.U. Benz, Tübingen).


unpublished observations). Anionic bis-(monoacylglycero)phosphate (BMP) thatstimulates ganglioside catabolic steps sub-stantially is formed from phosphatidyl glyc-erol in the IMs (Gallala and Sandhoff, 2011;Gallala et al., 2011). Whereas the lysosomalperimeter membrane seems to be quite re-sistant against lysosomal degradation, IMsare digested with the help of lipid bindingproteins (SAPs) and hydrolytic enzymes.Perimeter membranes are protected bya thick glycocalyx enriched in polylac-tosamine structures, and by a high choles-terol level (Hay, 1989; Appelqvist et al.,2012), the chaperon HSP70 (Kirkegaard etal., 2010), and presumably also by a highlateral pressure attenuating the interactionwith GM2AP, a lipid binding protein essen-tial for ganglioside catabolism (Giehl et al.,1999).

Biochemistry of ganglioside catabolismand its diseasesGanglioside catabolism proceeds in a stepwiseprocedure (Fig. 1) at the surface of luminal in-

tralysosomal vesicle and membrane struc-tures. These are enriched with anionic BMP,which attracts polycationic proteins, glycosi-dases,andSAPs, totheganglioside-containingmembrane surfaces in the acidic intralyso-somal environment. Whereas GM1 hydrolyz-ing �-galactosidase and GM2 splitting HexAbind to these anionic surfaces, they do not at-tack the membrane bound ganglioside sub-strates intheabsenceofmembraneperturbinglipid binding and transfer proteins. The firstcaseofGM1storagewasdescribedin1963asaspecial form of infantile amaurotic idiocy(JatzkewitzandSandhoff,1963).Hydrolysisofvesicle-bound GM1 by lysosomal ganglioside�-galactosidase needs the presence ofGM2AP or saposin B (Sap-B; Wilkening etal., 2000). Anionic lipids like BMP in theGM1 carrying vesicles stimulate GM1 hy-drolysis efficiently. Inherited defects of theGM1 degrading �-galactosidase causeGM1-gangliosidoses (Okada and O’Brien,1968).

The nonspecific and versatile GM1 de-grading �-galactosidase occurs as part of alysosomal multi-enzyme complex, contain-ing sialidase, cathepsin A (the so-called pro-tective protein) (d’Azzo et al., 2001), andN-acetylaminogalacto-6-sulfate sulfa-tase (Pshezhetsky and Ashmarina, 2001).Due to changes of the substrate specificityof variant patient enzymes, an inheriteddefect of GM1-�-galactosidase may alsolead to a major accumulation of galac-tose containing keratan sulfate and oligo-saccharides, a phenotype called Morquiosyndrome, type B (mucopolysaccharido-sis IV B) (Neufeld and Muenzer, 2001;Okumiya et al., 2003).

Hydrolysis of GM2 is facilitated at an-ionic vesicular surfaces by the cooperationof two proteins, HexA (�,�) and themembrane perturbing and lipid bindingprotein GM2AP (Fig. 4) (Kolter andSandhoff, 2006). At acidic pH values, thelatter one is a protonated cationic amphi-philic protein and can bind to the anionic,

Figure 3. Proposed topology of endocytosis and lysosomal lipid and membrane digestion (Gallala et al., 2011). “A section of the plasma membrane is internalized by way of coated pits orcaveolae. These membrane patches include GSLs (red) and receptors such as epidermal growth factor receptor (EGFR; blue). These vesicles fuse with the early endosomes, which mature to lateendosomes. Endosomal perimeter membranes form invaginations, controlled by ESCRT proteins (Wollert and Hurley, 2010), which bud off, forming intra-endosomal vesicles. Lipid sorting occurs atthis stage. The pH of the lumen is �5. At this pH, ASM is active and degrades sphingomyelin of the intra-endosomal vesicles to ceramide, whereas the perimeter membrane is protected against theaction of ASM by the glycocalyx facing the lumen. This decrease in sphingomyelin, coupled with the increase in ceramide, facilitates the binding of cholesterol to NPC2 and its transport to theperimeter membrane of the late endosome where it is transferred to NPC1 (Kwon et al., 2009). This protein mediates the export of cholesterol through the glycocalyx, eventually reaching cholesterolbinding proteins in the cytosol. Ultimately, late endosomes fuse with lysosomes. The GSLs are harbored in intralysosomal vesicles that face the lumen of the lysosome, and are degraded by hydrolaseswith the assistance of SAPs. The products of this degradation are exported to the cytosol or loaded on CD1b immunoreceptors and exported to the plasma membrane for antigen presentation.Gradients of pH in the lysosol, and intra-endolysosomal vesicle content of cholesterol (Chol), BMP, sphingomyelin (SM, hypothetical), and ceramide (Cer, hypothetical) are shown” (modified fromKolter and Sandhoff, 2010).


BMP-rich membrane surfaces, and lift aganglioside molecule (Fig. 4). A HexAbinding helix of the GM2AP facilitatesformation of the Michaelis–Menten com-plex for the degradation of GM2, the Tay–Sachs ganglioside, and glycolipid GA2.Inherited defects of either of the proteinsresult in fatal neurodegenerative diseases(Kolter and Sandhoff, 2006). Tay–Sachsdisease is caused by mutations in theHexA gene with loss of HexA (�,�) and S(�,�) activity. HexA mutations that stillallow the formation of the heterodimericHexA (�,�) and affect only its activityagainst anionic substrates result in B1variant of GM2-gangliosidosis (Kytzia etal., 1983; Kytzia and Sandhoff, 1985). Onthe other hand, inherited defects in HexBgene cause the loss of HexA (�,�) andHexB (�,�) activities in Sandhoff disease.Here, the combined deficiency of bothmajor hexosaminidase isoenzymes causes

the additional storage of neutral glycolip-ids, especially of globoside in the visceralorgans, and of oligosaccharides. The raredefect of GM2AP results in AB-variant ofGM2-gangliosidoses (Sandhoff et al.,1989; Sandhoff and Kolter, 2003). Genet-ically manipulated knock-out mice areavailable as models for these diseases tostudy their course and pathogenesis, andto investigate therapeutic approaches.

Protracted clinical forms ofgangliosidoses and the threshold theoryComplete or nearly complete loss of GM1or GM2 catabolic activities results in fatalinfantile forms of storage diseases. How-ever, allelic mutations, which produceproteins with some residual catabolic ac-tivities, give rise to protracted clinicalforms often described as late infantile, ju-venile, or chronic diseases (Sandhoff et al.,1989) with a wide range of clinical symp-

tomatology. At the biochemical level, theclinical heterogeneity is paralleled by avariation of the extent and the pattern ofglycolipid accumulation and by differentlevels of residual catabolic activities de-tected in cultures of patients’ fibroblasts(Fig. 5). Catabolic activities were assayedin cell cultures using the physiological, ra-diolabeled lipid substrates instead of thewater-soluble synthetic substrates, whichserve widely for diagnostic purposes. Thewater-soluble substrates may well lead toerroneous data because of the lipid phaseproblem and the absent control byGM2AP (and other activators) of the ca-tabolism of physiological lipid substratessuch as GM2 ganglioside. According toFigure 5, GM2 cleavage by HexA was al-most absent in fibroblasts of infantile pa-tients, and significantly higher in those ofjuvenile and adult patients (Kytzia et al.,1984; Leinekugel et al., 1992). This obser-

Figure 4. Mechanism of GM2-AP-Liftase. A, Model for the interaction of GM2-activator protein with luminal lysosomal membranes (IM) in the degradation of ganglioside GM2 (modified fromWendeler et al., 2004a). “GM2-AP interacts with the membrane by dipping the two exposed hydrophobic loops, V90 –W94 and V153–L163, into the apolar part of the membrane. Ganglioside GM2is recognized by specific sites at the rim of the cavity. In the open protein conformation, the large hydrophobic area reaching from the apolar phase of the membrane to the activator’s cavity lowersthe energy barrier for lipids leaving the membrane in an upward direction. After the ceramide tail has arrived inside the activator’s cavity, the inward-bending of the hydrophobic loop V153–L163is favored, and the conformation changes to the closed form (Wright et al., 2003). This folding in of the hydrophobic loop leaves a more polar patch close to the membrane. The activator is thenanchored only by the loop V90 –W94. It may now rotate slightly upward to expose all polar patches more fully to the solvent, and it may also leave the membrane and interact with the degradingenzyme. The photoaffinity label analogs C(n � 5,1)-TPD-GM2 were photo-incorporated specifically into the region V153–L163” (Sandhoff, 2012). B, BMP and GM2-AP stimulate the hydrolysis ofLUV-bound ganglioside GM2. The degradation of ganglioside GM2 inserted into LUVs doped with 0 mol % BMP (E), 5 mol % BMP(�), 10 mol % BMP (·), 20 mol % BMP (Œ), or 25 mol % BMP (f)was measured in the presence of increasing concentrations of GM2-AP (0 –2.2 �M) (Werth et al., 2001).


vation can well be modeled on the basis ofa greatly simplified kinetic model (Con-zelmann and Sandhoff, 1983, 1991;Leinekugel et al., 1992) presented in Fig-ure 5. A nice correlation was observed be-tween the predicted and the measuredlipid substrate turnover, and also betweenthe decrease of the ganglioside GM2 turn-over and the clinical course of the disease.Ten to 20% of normal GM2 cleaving ac-tivity appear already compatible withnormal life. Similar observations werereported for metachromatic leukodystro-phy (MLD), Gaucher, Sandhoff, andASM-deficient Niemann–Pick disease(Kudoh et al., 1983; Kytzia et al., 1984;Leinekugel et al., 1992; Graber et al.,1994).

Of course, additional factors inducedby the mutation and the storage process in

the disease will also contribute to the clin-ical course and disease pathogenesis, e.g.,misregulation of proteins and intracellu-lar trafficking, formation of lytic and toxiccompounds (Nilsson et al., 1982; Igisuand Suzuki, 1984), depletion of precursorpools, alterations of composition andfunction of membranes, mislocalizedstorage compounds like GM1, which acti-vate ER-chaperons and trigger neuronalapoptosis (Tessitore et al., 2004), and al-tered regulation of endolysosomal pro-teins and enzymes by an increased load oflipids (see below). Also the formation ofmeganeurites and an increase in synapticspines may well disturb the connectivityin the nervous system (Purpura and Su-zuki, 1976).

A genetic mutation and loss of a cata-bolic activity may not only result in accu-

mulation of nondegradable substrates butalso affect the transcription rate of manyother genes. Mutations in the HexB geneand the subsequent loss of HexA andHexB activity in genetically manipulatedmice trigger a significant downregulationof �300 transcripts and a significant up-regulation of another �250 transcripts in-cluding genes of the immune system(Myerowitz et al., 2002).

Genetically manipulated mice are notalways faithful copies of the human dis-ease. In contrast to Tay–Sachs patients,HexA knock-out mice reach an almostnormal life span and hardly get sick. Thereason seems to be a small difference inthe substrate specificity of murine and hu-man sialidase. Whereas human sialidase isalmost inactive, murine sialidase has a mi-nor but significant cleaving activity on the

Figure 5. Residual catabolic activity correlates with clinical forms of GM2 gangliosidoses (modified from Sandhoff, 2012). A, Steady-state substrate concentration as a function of enzymeconcentration and activity (Conzelmann and Sandhoff, 1983). The model underlying this theoretical calculation assumes influx of the substrate into the lysosomal compartment at a constant rate(vi) and its subsequent utilization by the catabolic enzyme. Blue line� [S]eq steady-state substrate concentration;

………… � theoretical threshold of enzyme activity; ——-� critical thresholdvalue, taking limited solubility of substrate into account; red line � turnover rate of substrate (flux rate). B, To experimentally verify the basic assumptions for this model, studies were performedin cell culture (Leinekugel et al., 1992). The radiolabeled substrate ganglioside GM2 was added to cultures of skin fibroblasts with different activities of �-hexosaminidase A and its uptake andturnover measured. The correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation rate of ganglioside GM2 increased steeply withresidual activity, to reach the control level at a residual activity of �10 –15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of thesefeeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.


storage compound ganglioside GM2 andthereby opens a catabolic bypass (Sand-hoff and Jatzkewitz, 1967; Sango et al.,1995). Mutant mice develop only a minorstorage process in some neurons, e.g., inthe pyramidal cells of the cortex (Taniikeet al., 1995). Apparently, the catabolic ac-tivity of the bypass (sequential action ofmurine sialidase to convert GM2 to GA2,and hex B to convert GA2 to LacCer) issufficient in almost all neurons of the ce-rebral cortex except in the murine pyra-midal cells and in a few others. These cellsprobably have a rather high biosyntheticrate for gangliosides, thereby slightly ex-ceeding their maximal catabolic capacity(Vmax of the catabolic system; Fig. 5) incase of hex A knocked out. Further patho-genetic cascades activated in these dis-eases, altered lipid trafficking, alteredautophagy, and induction of inflamma-tion, have been reviewed (Vitner et al.,2010; Xu et al., 2010).

Many proteins involved in gangliosidemetabolism are nonspecificThe inherited defect of an endolysosomalprotein usually affects only one step inganglioside and sphingolipid catabolism.However, a more careful investigation ofthe proteins revealed that many of themhave rather promiscuous functions and,therefore, have a broader effect on metab-olism than originally expected. For glyco-sidases this has been known for a longtime, and it was no surprise to find outthat GM1-�-galactosidase hydrolyzesmany other �-galactosides in addition toganglioside GM1 and GA1 (Kolter andSandhoff, 2006). Mutations in the�-galactosidase gene can even change theenzyme’s substrate specificity and affectthe catabolism of GSLs and glycosamino-glycans differently.

It was also no surprise to realize thathuman �-hexosaminidases have broadand partially overlapping substrate speci-ficities. Again some mutations in theHexA gene may also change substratespecificity of the patients’ HexA. Thosethat disrupt enzyme activity only againstanionic substrates (like GM2) and notagainst neutral substrates (like globoside)cause B1 variant of GM2-gangliosidosis(Kytzia et al., 1983), whereas those thataffect HexA activity against all substratesincluding neutral substrates cause Tay–Sachs disease (variant B of GM2-gangliosidosis). Active �-hexosaminidasesare the homodimers HexB (�,�) and HexS(�,�), and the heterodimer HexA (�,�).Each of them carries two active sites at thedomain interfaces (Maier et al., 2003; Le-

mieux et al., 2006). Therefore, mutationsaffecting the dimer formation produce inac-tive monomeric subunits causing infantilediseases. The broad and overlapping speci-ficity of murine hexosaminidases A, B, and Sis highlighted by mice lacking both subunitsof �-hexosaminidase. They store ganglio-sides and glycosaminoglycans and displayphenotypic, pathologic, and biochemicalfeatures of mucopolysaccharidoses, notseen in Tay–Sachs and Sandhoff disease(Sango et al., 1996).

The first activator protein described,the sulfatide activator protein, is nowcalled Sap-B. It was discovered as an es-sential cofactor for the lysosomal catabo-lism of sulfatides (Mehl and Jatzkewitz,1964). Its deficiency causes a late infantileform of MLD with a pronounced sulfatidestorage (Mehl and Jatzkewitz, 1964; Ste-vens et al., 1981). Sap-B combines withsulfatides, and the formed stoichiometricsoluble complex then forms a Michaelis–Menten complex with arylsulfatase A(Mehl and Jatzkewitz, 1964; Mraz et al.,1976). Though this suggested a high lipidbinding specificity of Sap-B, we nowknow that the human Sap-B is not specificfor sulfatide binding, but rather a promis-cuous lipid binding protein (Gärtner etal., 1983; Sun et al., 2008). It even stimu-lates the hydrolysis of bacterial lipids bybacterial hydrolases (Li et al., 1988). Alsothe other saposins have broad functions(Doering et al., 1999) and are rather un-specific lipid binding proteins, like theGM2AP (Wendeler et al., 2004b). Even theglycosyltransferases involved in the biosyn-thesis of complex gangliosides at Golgimembranes are rather versatile. There areonly one GalNAc-, one Gal-, and two sialyl-transferases available in Golgi membranesto convert the precursors LacCer, GM3,GD3, and GT3 into the different complexgangliosides of the 0-, a-, b-, and c-series(Kolter et al., 2002).

Membrane lipids are regulators ofganglioside catabolismPromiscuous functions of endolysosomalproteins and enzymes make it difficult tounderstand the precise molecular pathol-ogy of the diseases caused by a defect ofany of these proteins. The complexity isfurther increased by the strong lipid de-pendency of many endolysosomal pro-teins, given that the lipid composition ofmany organelle membranes is mostly un-known, especially in patients’ tissues. Invitro experiments have shown that cata-bolic steps at surfaces of substrate carryingliposomes—simulating luminal endoly-sosomal vesicles—not only need low pH

values, enzymes, and SAPs for significantdegradation rates, but also a sufficientconcentration of anionic lipids such asBMP in the liposomal membranes.

Both, GM2AP and Sap-B, allow that�-galactosidase hydrolyzes GM1. There-fore, neither a defect of GM2AP nor ofSap-B causes a GM1 accumulation, sincethe other one left efficiently facilitates thereaction. However, the reaction is ratherslow in neutral liposomes and is stimu-lated 2- to 9-fold in the presence of ananionic phospholipid such as BMP, phos-phatidylinositol, dolicholphosphate, andphosphatidylserine (Wilkening et al.,2000). Even at acidic, lysosomal pH val-ues, these phospholipids convey negativecharges to the vesicular surfaces, which at-tract and bind the protonated and posi-tively charged hydrolases and SAPs tocatalyze the hydrolytic reaction. This viewalso applies to the degradation of lipo-somal ganglioside GM2 by HexA in thepresence of GM2AP. GM2AP is essentialfor the enzymatic hydrolysis of ganglio-side GM2, even in the presence of highBMP concentrations. Its absence causesthe fatal neurodegenerative AB variant ofGM2-gangliosidosis in vivo. Still, the reac-tion is stimulated by anionic BMP �100-fold (Werth et al., 2001) (Fig. 4B). Alsodownstream reactions (Fig. 1) are stimu-lated by anionic membrane lipids. At con-centrations of anionic phospholipids �10mol% in the liposomal membranes, thecleavage of GlcCer by �-glucosidase(Wilkening et al., 1998) and that of cer-amide by acid ceramidase (Linke et al.,2001) proceeds even in the absence of aSAP.

Therapeutic approachesCurrently, there is no treatment availablefor patients with ganglioside storage dis-eases (van Gelder et al., 2012). A main ob-stacle is the blood– brain barrier, whichprevents the passage of therapeutic en-zymes and proteins into the brain. For theprevention of substrate storage, substrateinflux into the lysosomal compartmentmust not exceed this compartment’s cat-abolic capacity (see Protracted clinicalforms of gangliosidoses and the thresholdtheory, above; Conzelmann and Sand-hoff, 1983, 1991; Leinekugel et al., 1992).This means that therapeutic methodsshould either increase the catabolic rateabove the threshold or decrease the influxrate of substrates below the maximumcatabolic capacity of the affected cells, e.g.,by reducing the cellular substrate biosyn-thetic rate (Fig. 6). Therapeutic strategies


in use such as enzyme replacement therapy(ERT) for adult Gaucher disease and thoseunder study in animal models such as BMTyielded so far no substantial and long-lasting improvements in mouse modelswith GM1- and GM2-gangliosidosis. How-ever, intracerebroventricular administra-tion of a modified recombinant HexB(Matsuoka et al., 2011) and a highly phos-phomannosylated recombinant humanHexA reduced the level of storage com-pounds (Tsuji et al., 2011) in the HexA- andHexB-deficient mouse brain efficiently.

Additional approaches using lowmolecular compounds penetrating theblood– brain barrier have been evaluatedin murine models of gangliosidoses, butso far without substantial success. Theyinclude the use of chemical chaperons,substrate analogs, or active site inhibitors(Matsuda et al., 2003; Tropak et al., 2004)to stabilize variant patient hydrolases dur-ing their biosynthesis and folding, andthereby enhance their hydrolytic activityas introduced by Fan et al. (1999).

Another approach is the substrate reduc-tion therapy. It uses inhibitors of the biosyn-

thetic pathway, such as iminosugars of thenojirimycin type (Butters et al., 2005; Fanturet al., 2012; Suzuki et al., 2012) and reducesthe influx of substrates into the lysosomes(Kolter and Sandhoff, 1999). However,treatment of two Tay–Sachs patients withN-butyldeoxynojirimycin (Miglustat) didnot arrest their clinical deterioration (Bembiet al., 2006), whereas a stable neurologicalpicture was reported for two patients withSandhoff disease after Miglustat treatment(Tallaksen and Berg, 2009; Wortmann et al.,2009).

However, reducing the ganglioside bio-synthesis with inhibitors of the ceramideglucosyltransferase, N-butyldeoxynojirimy-cin or N-butyldeoxygalactonojirimycin, ledto an increased survival of Sandhoff mice upto 2 months (Andersson et al., 2004; Jeyaku-mar et al., 2004).

In a clinical Phase I/II trial, treatment oflate onset GM2-gangliosidosis patients witha presumed chaperon of �-hexosamini-dases, pyrimethamine, for a 4 month periodenhanced their leukocyte HexA levels, butclinical efficacy could not be assessed(Clarke et al., 2011).

A combination of different methods forpatients with significant residual enzymaticactivity, e.g., substrate reduction togetherwith enzyme enhancement therapy, non-steroidal anti-inflammatory drugs, and an-tioxidants is expected to yield better results.

Gene therapyTo circumvent the blood– brain barrier,functional copies of the mutant gene weredirectly inserted into murine brain cellswith the help of retroviral and lentiviralvectors (Biffi and Naldini, 2005). Thefunctional enzyme should be stably over-expressed in the transfected mutant cellsand secreted, thereby cross-correcting ad-jacent cells (Arfi et al., 2005; Martino et al.,2005).

Intracerebral injection of an adenovi-ral vector encoding the �-subunit ofHexA increased HexA activity to nearlynormal levels in HexB-deficient mice(Bourgoin et al., 2003). Also, the intracra-nial inoculation of recombinant adeno-associated viral (AAV) vectors encodingboth, human �- and � Hex-subunit genes,resulted in an almost generalized correc-

Figure 6. Intracellular metabolic flux of sphingolipids. vi � the influx rate of the substrate into the lysosome; for further information see the text. Modified from Kolter and Sandhoff, 1999).


tion of the catabolic lysosomal defect inthe same mice (Cachón-González et al.,2006). Animals survived more than a yearwith delayed onset of the disease, reducedganglioside storage and inflammation,preserved motor function, and rescue ofacute neurodegenerative illness (Sargeantet al., 2012).

AAV-mediated neonatal gene deliveryby intracerebroventricular injection seemsto be an effective approach to treat GM1-gangliosidoses. It resulted in a widespreadappearance of the missing �-galactosidaseactivity in the murine brain and a normal-ization of all GSL levels (Broekman et al.,2007; Baek et al., 2010).

Though these results are promising, itwill take many more investigations andprogress to develop a real gene therapy forGSL and sphingolipid storage diseases.

Conclusions and outlookTo identify the molecular basis of amau-rotic idiocy of the lipid accumulating type,it was important to isolate the storagecompounds and clarify their chemicalstructures. This allowed a search for thedefective catabolic steps to identify the en-zymes and lipid binding proteins in-volved, their genomic structures, and themutations causing the different gangli-osidoses. The fact that the main storagecompounds (gangliosides GM1 andGM2) are preferentially synthesized inneuronal cells is crucial for the start ofneurodegenerative diseases and compli-cates all therapeutic approaches. So far,efficient ERT is not feasible, since theblood– brain barrier prevents cerebral up-take of therapeutic proteins. Also geneticapproaches that appeared to be promisingin mouse models are still not reliable(Cachón-González et al., 2006).

There is no simple model to under-stand the complex pathogenesis of mono-genetic gangliosidoses. The primary lipidstorage affects the endolysosomal systemof neurons and obviously does not triggeran effective feedback inhibition of the stor-age compounds’ biosynthesis. However, thestorage may well impair the turnover ofmembranes and macromolecules, affectingneuronal metabolism and function.

Degradation of gangliosides requiresthe interaction of rather unspecific hydro-lases and lipid binding proteins at surfacesof intraluminal vesicles and membranestructures. These complex interactions,including the substantial dependency ofmany catabolic steps on lipid binding co-factors (SAPs) and the lipid compositionof the intraluminal, lysosomal mem-branes, seem to prevent an exact and

quantitative understanding of the degra-dative sphingolipid pathways.

We also do not know to what extentthe lipid load of the affected lysosomesdisturbs their primary functions as cellu-lar stomachs (Tamboli et al., 2011) to de-grade macromolecules and release theproducts into the cytosol as nutrientsfor biosynthetic pathways and energy me-tabolism. One should also keep in mindthat the cellular uptake of essential factors,for example, the uptake of vitamin B12involving several proteins, and that ofFe 2� via transferrin and the transferrinreceptor, may well be impaired by thelipid load causing a traffic jam in the en-dolysosomal system (Jeyakumar et al.,2009). The storage of GSLs in gangli-osidoses also triggers side effects such asthe accumulation of hydrophobic mate-rial including phospholipids and hydro-phobic protein fragments in the storagegranules (“membranous cytoplasmicbodies”) (Samuels et al., 1963), but alsotransmembrane spans of mitochondrialATP synthase and amyloid-�-peptide(Keilani et al., 2012). As in multiple sclero-sis, functionally impaired neurons giverise to an immune response, and areprone to digestion by microglia and in-vaded macrophages (Wada et al., 2000;Wekerle, 2005).

Although we have learned a lot aboutthe function of gangliosides, their bio-chemistry, enzymology, and cell biology,we are still far away from a real under-standing of the molecular and cellularpathogenesis of ganglioside storage dis-eases and the development of therapeuticapproaches. However, the successful dis-section of the clinical picture of amauroticidiocy into 5 different ganglioside storagediseases involving 4 different genes, and agroup of 10 so far not extensively analyzedneuronal ceroidlipofuscinoses, may en-courage the search for similar clinical en-tities of monogenetic diseases but withcausative diversity, among the high num-ber of endemic inherited diseases, the mo-lecular basis of which still has to beelucidated.

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Gangliosides and Gangliosidoses: Principles of Molecular and Metabolic PathogenesisIntroductionSummary outline of clinical symptomatologyPathway of ganglioside degradationOur view of the topology of lysosomal ganglioside digestionProtracted clinical forms of gangliosidoses and the threshold theoryMany proteins involved in ganglioside metabolism are nonspecificMembrane lipids are regulators of ganglioside catabolismTherapeutic approaches

Gene therapyConclusions and outlookReferences

GangliosidesandGangliosidoses:PrinciplesofMolecular … · 2013. 7. 16. · 2000). Gangliosides...

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