IDENTIFICATION OF THE ANTIGEN RECOGNIZED BY RHIGM22, A REMYELINATION-PROMOTING HUMAN MONOCLONAL ANTIBODY

Tutte le cellule eucariotiche sono circondate da una membrana cellulare che funge da barriera tra i compartimenti subcellulari e tra la cellula e il suo ambiente. Oltre alle proteine, le membrane cellulari sono composte da tre diversi gruppi di lipidi: glicerolipidi, sfingolipidi e steroli. I glicerolipidi sono i principali componenti delle membrane cellulari e sono prodotti utilizzando acido fosfatidico (PtdOH) come precursore centrale. Invece, gli sfingolipidi (SL), i componenti cellulari minori, hanno la sfingosina come componente base; l\u2019aggiunta di oligosaccaridi alla sfingosina da origine agli glicosfingolipidi (GSL). SL e GSL non si distribuiscono in modo omogeneo nella membrana plasmatica esterna. Formano domini piccoli, eterogenei, altamente dinamici, arricchiti con steroli e sfingolipidi, chiamati "zattere lipidiche"; in questi microdomini semiordinati, gli SL sono coinvolti i diversi processi, quali quelli di adesione, riconoscimento e trasduzione del segnale. L'organo con il pi\uf9 alto arricchimento di lipidi, come colesterolo e glicosfingolipidi, \ue8 il cervello. La mielina, la sostanza isolante che circonda l'assone delle cellule nervose, \ue8 caratterizzata da un elevato rapporto lipidi-proteine, in cui i lipidi rappresentano l'80% del suo peso secco. Questa membrana contiene un alto livello di due galattosfingolipidi in particolare, il galattosilceramide (GalCer) e il 3-O-sulfogalattosilceramide (solfatide), che rappresentano rispettivamente circa il 20 e il 5% dei lipidi mielinici. I ruoli specifici di GalCer e solfatide sembrano essere legati alla loro capacit\ue0 di formare e stabilizzare specifici domini laterali nella membrana mielinica, che regolano il corretto smistamento, traffico, co-clustering e distribuzione laterale delle principali proteine della mielina. Studi recenti hanno suggerito che alcuni lipidi specifici della mielina potrebbero essere i principali responsabili del meccanismo patogeno della sclerosi multipla (SM), la malattia demielinizzante pi\uf9 comune nel sistema nervoso centrale. Gli individui affetti da SM hanno un\u2019alterata composizione lipidica della mielina e elevati livelli di anticorpi anti-solfatide, nei fluidi biologici, rispetto agli individui sani. D'altro canto, gli anticorpi anti-mielina potrebbero rappresentare un importante strumento immunologico per il trattamento di malattie neurologiche che coinvolgono lesioni mieliniche. In particolare, \ue8 stato dimostrato che gli anticorpi monoclonali umani che legano la mielina e gli oligodendrociti (OL), come rHIgM22, possono stimolare un meccanismo di rimielinizzazione nei modelli animali di demielinizzazione. Ad oggi, l'esatto meccanismo d'azione di rHIgM22 resta da chiarire, ma alcune prove suggeriscono che il meccanismo \ue8 correlato all'organizzazione delle zattere lipidiche presenti sulla superficie della mielina e degli OL. Lo scopo degli esperimenti descritti in questa tesi \ue8 quello di individuare i target molecolari dell'anticorpo e caratterizzare il microambiente di membrana in cui si trova, al fine di comprendere meglio l\u2019 attivit\ue0 rimielinizzante di rHIgM22. Il legame di rHIgM22 ai lipidi purificati e agli estratti lipidici da varie fonti \ue8 stato testato usando saggi di TLC Immunostaining e saggi SPR. I risultati ottenuti mostrano che rHIgM22 si lega al solfatide e, in misura minore, al lisosolfatide in vitro, mentre non si lega ad altri sfingolipidi della mielina. L'affinit\ue0 di legame sia per il solfatide che per il suo derivato deacilato \ue8 bassa, anche se il legame \ue8 specifico. In aggiunta, i nostri dati mostrano che l'affinit\ue0 di legame di rHIgM22 per solfatide pu\uf2 essere modulata dalla presenza di altri lipidi, suggerendo un possibile ruolo del microambiente di membrana nel riconoscimento dell'antigene da parte di rHIgM22. Inoltre, rHIgM22 reagisce anche con acido fosfatidico, fosfatidilinositolo (PI) e fosfatidilserina (PS). Per verificare la capacit\ue0 di rHIgM22 di legare sulfatide o altri lipidi, il legame di rHIgM22 \ue8 stato testato anche su estratti lipidici purificati da diversi campioni: cervelli di topo WT, ASM (- / -), CST (+/-) e CST (- / -), cellule gliali miste di topo (MGC), astrociti di topo, rHIgM22+ OLs di ratto, microglia di ratto e mielina di topo. Negli esperimenti di TLC Immunostaining per le fasi acquose di questi campioni, abbiamo osservato bande immunoreattive a rHIgM22 che co-migrano con lo standard di solfatide puro e, inaspettatamente, una seconda banda immunoreattiva a rHIgM22 co-migrante con lo standard fosfatidilinositolo e fosfatidilserina puro, confermando i dati di TLC Immunostaining sui lipidi purificati. L'identit\ue0 delle specie fosfolipidiche riconosciute da rHIgM22 \ue8 stata confermata tramite spettrometria di massa; gli spettri mostrano che la specie molecolare del fosfatidilinositolo \ue8 18:0/20:4, mentre per la fosfatidilserina \ue8 18:0/22:6 e 18:0/18:1. Inoltre, l'analisi tramite spettrometria di massa per le frazioni arricchite in solfatide, mostra che rHIgM22 pu\uf2 legare diverse specie solfatate, suggerendo che questo legame non \ue8 specifico alle diverse specie degli acidi grassi. Tutti questi dati suggeriscono che non solo il solfatide, ma anche altri lipidi di membrana possono avere un ruolo nel legame di rHIgM22 agli OL o ad altri tipi di cellule. Inoltre, l'antigene riconosciuto da rHIgM22 potrebbe essere associato a zattere lipidiche di membrana plasmatica e incluso in un complesso multimolecolare. L'identificazione dei target riconosciuti da rHIgM22 e la caratterizzazione del microambiente di membrana in cui si trovano, potrebbe contribuire notevolmente a chiarire i meccanismi di segnalazione coinvolti nell'attivit\ue0 di promozione della rimielinizzazione di questo anticorpo, ma anche di quelli coinvolti nell'eziologia della SM, permettendo di identificare nuove potenziali strategie terapeutiche.All eukaryotic cells are surrounded by a cellular membrane that functions as a barrier between subcellular compartments and between the cell and its environment. In addition to proteins, they are composed by three different set of lipids: glycerolipids, sphingolipids and sterols. Glycerolipids are the major components of cell membranes and they are produced using phosphatidic acid (PtdOH) as a central precursor. Instead, sphingolipids (SLs), the minor cell components, have sphingosine as basic building block; the additions of oligosaccharides to sphingosine giving rise glycosphingolipids (GSLs). SLs and GSLs are not distribute homogeneously in the outer plasma membrane. They form small, heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains, called \u201clipid rafts\u201d; in these semiordered lipid microdomains, SLs are involved in cell adhesion/recognition processes and signal transduction pathway. The organ with the highest enrichment in lipids such as cholesterol e glycosphingolipids, is the brain. Myelin, the fatty white substance that surrounds the axon of nerve cells, is characterized by a high lipid-to-protein ratio, where lipids representing 80% of its dry weight. The myelin membrane contains a high level of two galactosphingolipids, galactosylceramide (GalCer) and 3-O-sulfogalactosylceramide (sulfatide), account for about 20 and 5 % of myelin lipids respectively. The specific roles of GalCer and sulfatide seem to be linked to their ability to form and to stabilize specific lateral domains in the membrane of myelin forming-cells and in the myelin sheath, that regulate the correct sorting, trafficking, co-clustering and lateral distribution of the major myelin proteins. Recent reports have suggested that some of the myelin-specific lipids may be key contributors to the pathogenic mechanism of multiple sclerosis (MS), the most common demyelinating disease in the CNS. Individuals with MS disease are reported to have different myelin lipid compositions and elevated levels of anti-sulfatide Ab in biological fluids compared with healthy individuals. On the other hand, anti-myelin antibodies might represent an important immunological tool for the treatment of neurological diseases involving myelin lesions. In particular, it has been shown that human monoclonal antibodies that bind myelin and oligodendrocytes (OLs), as rHIgM22, can initiate dramatic increase in remyelination in animal models of demyelination. Nowadays, the exact mechanism of action of rHIgM22 remains to be elucidated, but some evidence suggest that the mechanism is correlated with the organization of lipid rafts on the surface of myelin and OLs. The experiments described in this thesis were aimed at the individuation of the molecular target(s) of the antibody and at the characterization of its membrane microenvironment, in order to better understand the characteristics of rHIgM22 and its remyelinating activity. The binding of rHIgM22 to purified lipids and to lipid extracts from various sources were tested using TLC immunostaining assays and SPR assays. The results obtained show that rHIgM22 binds to sulfatide, and, to a lesser extent, to lysosulfatide in vitro, while it does not bind to other myelin sphingolipids. The binding affinity for both sulfatide and its deacylated derivate is low, even if the binding is specific. On the other hand, our data shows that the binding affinity of rHIgM22 for sulfatide can be modulated by the presence of other lipids suggesting a possible role of the membrane microenvironment in the recognition of the antigen by rHIgM22. In addition, rHIgM22 also reacts with phosphatidic acid, phosphatidylinositol (PI) and phosphatidylserine (PS). To verify whether rHIgM22 can bind sulfatide or other lipids, the binding of rHIgM22 was tested not only to purified lipids, but also to partially purified lipid extracts obtained from wild type, ASM (-/-), CST (+/-) and CST (-/-) mice brains, mouse mixed glial cells (MGC), mouse astrocytes, rat rHIgM22+ OLs, rat microglia, and mouse myelin. In TLC immunostaining experiments for aqueous phases, we observed rHIgM22-immunoreactive bands co-migrating with the pure sulfatide standard and, unexpectedly, a second rHIgM22-immunoreactive band migrating below sulfated, co-migrating with the pure phosphatidylinositol and phosphatidylserine standard, confirming the TLC immunostraining data to purified lipids. The identity of phospholipid species has been confirmed by ESI mass spectrometry experiments; they show that phosphatidylinositol was an 18:0/20:4-PI and phosphatidylserine was 18:0/22:6-PS and 18:0/18:1-PS. In addition, MS analysis for the fractions enriched in sulfatide, shows that rHIgM22 can bind different sulfated species suggesting that this binding is not fatty acid species-specific for the sulfatide. All these data suggest that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to OLs or to other cell types. Moreover, the antigen recognized by rHIgM22 could be associated with plasma membrane lipid rafts in these cells and this target could be including in a multimolecular complex. The identification of the binding target(s) of a rHIgM22, and the characterization of their membrane microenvironment, could greatly contribute to the elucidation of the signaling mechanisms underlying the remyelination promoting activity of this antibody and also of those involved in MS etiology, allowing to define new potential therapeutic strategies

Sphingosine 1-Phosphate Receptors and Metabolic Enzymes as Druggable Targets for Brain Diseases

The central nervous system is characterized by a high content of sphingolipids and by a high diversity in terms of different structures. Stage- and cell-specific sphingolipid metabolism and expression are crucial for brain development and maintenance toward adult age. On the other hand, deep dysregulation of sphingolipid metabolism, leading to altered sphingolipid pattern, is associated with the majority of neurological and neurodegenerative diseases, even those totally lacking a common etiological background. Thus, sphingolipid metabolism has always been regarded as a promising pharmacological target for the treatment of brain disorders. However, any therapeutic hypothesis applied to complex amphipathic sphingolipids, components of cellular membranes, has so far failed probably because of the high regional complexity and specificity of the different biological roles of these structures. Simpler sphingosine-based lipids, including ceramide and sphingosine 1-phosphate, are important regulators of brain homeostasis, and, thanks to the relative simplicity of their metabolic network, they seem a feasible druggable target for the treatment of brain diseases. The enzymes involved in the control of the levels of bioactive sphingoids, as well as the receptors engaged by these molecules, have increasingly allured pharmacologists and clinicians, and eventually fingolimod, a functional antagonist of sphingosine 1-phosphate receptors with immunomodulatory properties, was approved for the therapy of relapsing-remitting multiple sclerosis. Considering the importance of neuroinflammation in many other brain diseases, we would expect an extension of the use of such analogs for the treatment of other ailments in the future. Nevertheless, many aspects other than neuroinflammation are regulated by bioactive sphingoids in healthy brain and dysregulated in brain disease. In this review, we are addressing the multifaceted possibility to address the metabolism and biology of bioactive sphingosine 1-phosphate as novel targets for the development of therapeutic paradigms and the discovery of new drugs

Human Remyelination Promoting Antibody Stimulates Astrocytes Proliferation Through Modulation of the Sphingolipid Rheostat in Primary Rat Mixed Glial Cultures

Remyelination promoting human IgMs effectively increase the number of myelinated axons in animal models of multiple sclerosis. Hence, they ultimately stimulate myelin production by oligodendrocytes (OLs); however, their exact mechanism of action remains to be elucidated, and in particular, it remains unclear whether they are directly targeting OLs, or their action is mediated by effects on other cell types. We assessed the effect of remyelination promoting antibody rHIgM22 on the proliferative response and on the ceramide/sphingosine 1-phosphate rheostat in mixed glial cell cultures (MGCs). rHIgM22 treatment caused a time-dependent increase in PDGF\u3b1R protein in MGCs. Forty-eight hours of treatment with rHIgM22 induced a dose-dependent proliferative response (evaluated as total cell number and as EdU(+) cell number) in MGCs. When the proliferation response of MGCs to rHIgM22 was analyzed as a function of the cell types, the most significant proliferative response was associated with GLAST(+) cells, i.e., astrocytes. In many cell types, the balance between different sphingolipid mediators (the "sphingolipid rheostat"), in particular ceramide and sphingosine 1-phosphate, is critical in determining the cell fate. rHIgM22 treatment in MGCs induced a moderate but significant inhibition of total acidic sphingomyelinase activity (measured in vitro on cell lysates), the main enzyme responsible for the stimulus-mediated production of ceramide, when treatment was performed in serum containing medium, but no significant differences were observed when antibody treatment was performed in the absence of serum. Moreover, rHIgM22 treatment, either in the presence or in absence of serum, had no effects on ceramide levels. On the other hand, rHIgM22 treatment for 24\ua0h induced increased production and release of sphingosine 1-phosphate in the extracellular milieu of MGC. Release of sphingosine 1-phosphate upon rHIgM22 treatment was strongly reduced by a selective inhibitor of PDGF\u3b1R. Increased sphingosine 1-phosphate production does not seem to be mediated by regulation of the biosynthetic enzymes, sphingosine kinase 1 and 2, since protein levels of these enzymes and phosphorylation of sphingosine kinase 1 were unchanged upon rHIgM22 treatment. Instead, we observed a significant reduction in the levels of sphingosine 1-phosphate lyase 1, one of the key catabolic enzymes. Remarkably, rHIgM22 treatment under the same experimental conditions did not induce changes in the production and/or release of sphingosine 1-phosphate in pure astrocyte cultures. Taken together, these data suggest that rHIgM22 indirectly influences the proliferation of astrocytes in MGCs, by affecting the ceramide/sphingosine 1-phosphate balance. The specific cell population directly targeted by rHIgM22 remains to be identified, however our study unveils another aspect of the complexity of rHIgM22-induced remyelinating effect

The role of 3-O-sulfogalactosylceramide, sulfatide, in the lateral organization of myelin membrane

Sulfatide (3-O-sulfogalactosylceramide, SM4s) was isolated by Thudichum from the human brain in 1884. Together with galactosylceramide, its direct metabolic precursor in the biosynthetic pathway, sulfatide is highly enriched in myelin in the central and peripheral nervous system, and it has been implicated in several aspects of the biology of myelin-forming cells. Studies obtained using galactolipid-deficient mice strongly support the notion that sulfatide plays critical roles in the correct structure and function of myelin membrane. A number of papers are suggesting that these roles are mediated by a specific function of sulfatide in the lateral organization of myelin membrane, thus affecting the sorting, lateral assembly, membrane dynamics and also the function of specific myelin proteins in different substructures of the myelin sheath. The consequences of altered sulfatide metabolism and sulfatide-mediated myelin organization with respect to myelin diseases are still poorly understood, but it\u2019s very likely that sulfatide might represent not only a critical player in the pathogenesis of several diseases, including multiple sclerosis and Alzheimer\u2019s disease, but also a potentially promising therapeutic target

Hexa-associated GM2 gangliosidosis in a family of wild boars

Gangliosidosis are inherited lysosomal storage disorders caused by defective activity of a lysosomal hydrolase required for ganglioside catabolism, resulting in the intra-lysosomal accumulation of undegraded metabolites. The molecular mechanisms linking the lysosomal accumulation to the pathology are still obscure. We report on a novel form of GM2 gangliosidosis in wild boar (Sua scrofa). Three littermate wild boards, from a free ranging farm, presented neurological signs (dysmetria, ataxia, quadriplegia and lateral decubitus) at 6 months of age. Viral, bacterial and toxicological analysis were performed to esclude possible exogenous causes of symptoms. Animale were euthanazed at approximately one year of age. Necropsy revealed in all affected animali reduced consistency of cerebral and cerebellar parenchyma. Histology revealed enlarged foamy neuroni, with diffusely severely vacuolated cytoplasm in brain, cerebellum, spinal cord, peripheral ganglia and retina. EM revealed the presente in neurons of numerous lysosomes, filled by membranous material. Biochemical studies revealed the presente of an elevate amount of GM2 ganglioside, confirming the diagnosis of GM2 gangliosidosis. In addition, genetic analysis revealed the presence of a recessively inherited missense variano (p.Arg499Cys) in the hexo.saminidase subiinit alpha (UW2 gene located within the GH20 hexosaminidase superfamily domain of the encoded protein. In man and other species, pathogenic HEXA variante are known to be associated with the disease. In conclusion, this HEXA-associated form ot GM2 gangliosidosis, described for the first time in wild boars, is thus very similar to human disease