Identification of the Lipid Antigens Recognized by rHIgM22, a Remyelination-Promoting Antibody.

Failure of the immune system to discriminate myelin components from foreign antigens plays a critical role in the pathophysiology of multiple sclerosis. In fact, the appearance of anti-myelin autoantibodies, targeting both proteins and glycolipids, is often responsible for functional alterations in myelin-producing cells in this disease. Nevertheless, some of these antibodies were reported to be beneficial for remyelination. Recombinant human IgM22 (rHIgM22) binds to myelin and to the surface of O4-positive oligodendrocytes, and promotes remyelination in mouse models of chronic demyelination. Interestingly, the identity of the antigen recognized by this antibody remains to be elucidated. The preferential binding of rHIgM22 to sulfatide-positive cells or tissues suggests that sulfatide might be part of the antigen pattern recognized by the antibody, however, cell populations lacking sulfatide expression are also responsive to rHIgM22. Thus, we assessed the binding of rHIgM22 in vitro to purified lipids and lipid extracts from various sources to identify the antigen(s) recognized by this antibody. Our results show that rHIgM22 is indeed able to bind both sulfatide and its deacylated form, whereas no significant binding for other myelin sphingolipids has been detected. Remarkably, binding of rHIgM22 to sulfatide in lipid monolayers can be positively or negatively regulated by the presence of other lipids. Moreover, rHIgM22 also binds to phosphatidylinositol, phosphatidylserine and phosphatidic acid, suggesting that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to oligodendrocytes and to other cell types not expressing sulfatide.

A pathogenic HEXA missense variant in wild boars with Tay-Sachs disease.

Gangliosidoses are inherited lysosomal storage disorders caused by reduced or absent activity of either a lysosomal enzyme involved in ganglioside catabolism, or an activator protein required for the proper activity of a ganglioside hydrolase, which results in the intra-lysosomal accumulation of undegraded metabolites. We hereby describe morphological, ultrastructural, biochemical and genetic features of GM2 gangliosidosis in three captive bred wild boar littermates. The piglets were kept in a partially-free range farm and presented progressive neurological signs, starting at 6 months of age. Animals were euthanized at approximately one year of age due to their poor conditions. Neuropathogens were excluded as a possible cause of the signs. Gross examination showed a reduction of cerebral and cerebellar consistency. Central (CNS) and peripheral (PNS) nervous system neurons were enlarged and foamy, with severe and diffuse cytoplasmic vacuolization. Transmission electron microscopy (TEM) of CNS neurons demonstrated numerous lysosomes, filled by parallel or concentric layers of membranous electron-dense material, defined as membranous cytoplasmic bodies (MCB). Biochemical composition of gangliosides analysis from CNS revealed accumulation of GM2 ganglioside; furthermore, Hex A enzyme activity was less than 1% compared to control animals. These data confirmed the diagnosis of GM2 gangliosidosis. Genetic analysis identified, at a homozygous level, the presence of a missense nucleotide variant c.1495C > T (p Arg499Cys) in the hexosaminidase subunit alpha gene (HEXA), located within the GH20 hexosaminidase superfamily domain of the encoded protein. This specific HEXA variant is known to be pathogenic and associated with Tay-Sachs disease in humans, but has never been identified in other animal species. This is the first report of a HEXA gene associated Tay-Sachs disease in wild boars and provides a comprehensive description of a novel spontaneous animal model for this lysosomal storage disease.

Lipid rafts and neurodegeneration: structural and functional roles in physiologic aging and neurodegenerative diseases.

Lipid rafts are small, dynamic membrane areas characterized by the clustering of selected membrane lipids as the result of the spontaneous separation of glycolipids, sphingolipids, and cholesterol in a liquid-ordered phase. The exact dynamics underlying phase separation of membrane lipids in the complex biological membranes are still not fully understood. Nevertheless, alterations in the membrane lipid composition affect the lateral organization of molecules belonging to lipid rafts. Neural lipid rafts are found in brain cells, including neurons, astrocytes, and microglia, and are characterized by a high enrichment of specific lipids depending on the cell type. These lipid rafts seem to organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating the homeostasis of the brain. The progressive decline of brain performance along with physiological aging is at least in part associated with alterations in the composition and structure of neural lipid rafts. In addition, neurodegenerative conditions, such as lysosomal storage disorders, multiple sclerosis, and Parkinson's, Huntington's, and Alzheimer's diseases, are frequently characterized by dysregulated lipid metabolism, which in turn affects the structure of lipid rafts. Several events underlying the pathogenesis of these diseases appear to depend on the altered composition of lipid rafts. Thus, the structure and function of lipid rafts play a central role in the pathogenesis of many common neurodegenerative diseases.jlr;61/5/636/F1F1f1.

Construction and reconstruction of brain circuits: normal and pathological axon guidance.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.

Paradoxical Psoriasis Induced by Anti-TNFα Treatment: Evaluation of Disease-Specific Clinical and Genetic Markers.

Paradoxical psoriasis (PP) may occur during treatment with anti-tumor necrosis factor-alpha (TNF-α) drugs in various chronic immune-mediated diseases, mainly inflammatory bowel diseases (IBD) and psoriasis. In this study, clinical and genetic characteristics of PP arising in IBD and psoriatic patients were investigated to identify disease-specific markers of the paradoxical effect. A total of 161 IBD and psoriatic patients treated with anti-TNF-α drugs were included in the study. Of these patients, 39 developed PP. All patients were characterized for the main clinical-pathologic characteristics and genotyped for six candidate single nucleotide polymorphisms (SNPs) selected for their possible role in PP susceptibility. In IBD patients, the onset of PP was associated with female sex, presence of comorbidities, and use of adalimumab. IBD patients with PP had a higher frequency of the TNF-α rs1799964 rare allele (p = 0.006) compared with cases without the paradoxical effect, and a lower frequency of the human leucocyte antigen (HLA)-Cw06 rs10484554 rare allele (p = 0.03) compared with psoriatic patients with PP. Overall, these findings point to specific clinical and genetic characteristics of IBD patients with PP and provide data showing that genetic variability may be related to the paradoxical effect of anti-TNF-α drugs with possible implications into clinical practice.

Sphingolipids and neuronal degeneration in lysosomal storage disorders.

Ceramide, sphingomyelin, and glycosphingolipids (both neutral and acidic) are characterized by the presence in the lipid moiety of an aliphatic base known as sphingosine. Altogether, they are called sphingolipids and are particularly abundant in neuronal plasma membranes, where, via interactions with the other membrane lipids and membrane proteins, they play a specific role in modulating the cell signaling processes. The metabolic pathways determining the plasma membrane sphingolipid composition are thus the key point for functional changes of the cell properties. Unnatural changes of the neuronal properties are observed in sphingolipidoses, lysosomal storage diseases occurring when a lysosomal sphingolipid hydrolase is not working, leading to the accumulation of the substrate and to its distribution to all the cell membranes interacting with lysosomes. Moreover, secondary accumulation of sphingolipids is a common trait of other lysosomal storage diseases. This article is part of the Special Issue "Lysosomal Storage Disorders".

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 PDGFαR 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 h 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 PDGFαR. 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.

Neuronal membrane dynamics as fine regulator of sphingolipid composition.

Sphingolipid metabolism is an intricate network of several interdependent and co-regulated pathways. In addition to the mainstream biosynthetic and catabolic pathways, several processes, even if less important in contributing to the final tissue sphingolipid composition from the quantitative point of view, might become relevant when sphingolipid metabolism is for any reason dysregulated and concur to the onset of neuronal pathologies. The main subcellular sites involved in the mainstream metabolic pathway are represented by the Golgi apparatus (for the biosynthesis) and by the lysosomes (for catabolism). On the other hand, the minor collateral pathways are associated with the plasma membrane and membranes of other organelles, and likely play important roles in the local regulation of membrane dynamics and contribute to maintain a perfect membrane organization functional to the physiology of the cell. In this review, we will consider few aspects of the sphingolipid metabolic pathway depending by the dynamic of the membranes that seems to become relevant in neurodegenerative diseases.

Lipid membrane domains in the brain.

The brain is characterized by the presence of cell types with very different functional specialization, but with the common trait of a very high complexity of structures originated by their plasma membranes. Brain cells bear evident membrane polarization with the creation of different morphological and functional subcompartments, whose formation, stabilization and function require a very high level of lateral order within the membrane. In other words, the membrane specialization of brain cells implies the presence of distinct membrane domains. The brain is the organ with the highest enrichment in lipids like cholesterol, glycosphingolipids, and the most recently discovered brain membrane lipid, phosphatidylglucoside, whose collective behavior strongly favors segregation within the membrane leading to the formation of lipid-driven membrane domains. Lipid-driven membrane domains function as dynamic platforms for signal transduction, protein processing, and membrane turnover. Essential events involved in the development and in the maintenance of the functional integrity of the brain depend on the organization of lipid-driven membrane domains, and alterations in lipid homeostasis, leading to deranged lipid-driven membrane organization, are common in several major brain diseases. In this review, we summarize the forces behind the formation of lipid membrane domains and their biological roles in different brain cells. This article is part of a Special Issue entitled Brain Lipids.

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's very likely that sulfatide might represent not only a critical player in the pathogenesis of several diseases, including multiple sclerosis and Alzheimer's disease, but also a potentially promising therapeutic target.

Narrow-band UVB phototherapy and psoralen-ultraviolet A photochemotherapy in the treatment of cutaneous mastocytosis: a study in 20 patients.

BACKGROUND: In mastocytosis, the skin is almost invariably involved, and cutaneous symptoms deeply affect patients' quality of life. METHODS: A retrospective observational analysis of patients affected by cutaneous mastocytosis (CM) and indolent systemic mastocytosis (ISM) treated with phototherapy/photochemotherapy (PUVA or NB-UVB) has been conducted. For each patient, total numbers of PUVA or NB-UVB exposures, the cumulative UV dose (J/cm2 ), serum tryptase profile, and pruritus, before and after treatment, according to the visual analogue scale (VAS) were considered. Skin lesions of each patient were assessed, before and after treatment, according to a cutaneous scale score. RESULTS: Twenty patients affected by CM and ISM were studied; in particular, 10 patients received NB-UVB therapy, and other 10 patients received PUVA. A statistically significant mean reduction of pruritus in both groups (P < 0.01) was observed. The number of treatments necessary to obtain symptom relief was significantly lower in the PUVA group, but the mean exposure dose was significantly higher, if compared to the NB-UVB group. Serum tryptase levels showed a downward trend. The cutaneous score improved in both groups. LIMITATIONS: This study was a retrospective study with a small sample size and without a control group. CONCLUSION: This work provides evidence that both NB-UVB and PUVA represent a safe and useful second-line therapy of the cutaneous symptoms in mastocytosis.

Adult-onset tufted angiomas associated with an arteriovenous malformation in a renal transplant recipient: case report and review of the literature.

Tufted angioma (TA) is a rare benign vascular neoplasm characterized histopathologically by the proliferation of endothelial cells arranged in lobules in the dermis and subcutaneous fat. To date, about 200 cases have been reported, most of which are of Japanese ethnicity. TA predominantly affects children and young adults, developing in 80% of patients younger than 10 years. A white 72-year-old renal transplant recipient presented with 2 asymptomatic dusky red papules on his right leg. The lesions appeared 5 years after the start of immunosuppressive treatment. Histopathologic examination showed a proliferation of poorly canalized capillary-sized vascular structures with typical "cannonball" pattern in the dermis and subcutaneous fat. Eccrine glands were also evident focally in the stroma of capillary lobules. On immunohistochemistry, endothelial cells in the vascular tufts stained positive for CD31 and CD34 but were negative for factor VIII-related antigen, human herpes virus 8, and podoplanin (clone D2-40); α-smooth muscle actin stained pericytes disposed in a single layer in capillary-sized vessels and in 2-3 or more layers in vessels of larger size, respectively. The microscopic findings were suggestive of TA. In the deep dermis, venules with smooth muscle wall and arterioles, as shown by Van Gieson staining, normally not found at that level, were present and appeared surrounded by capillary lobules. Onset of TA in adulthood is rare and may be associated with pregnancy, varicella zoster virus infection, and pharmacological immunosuppression. A case of acquired adult-onset TA associated with an arteriovenous malformation in an elderly transplanted patient is described.

Glomuvenous malformations with smooth muscle and eccrine glands: unusual histopathologic features in a familial setting.

Glomuvenous malformations (OMIM 138000) are hamartomas presenting in childhood as multiple, bluish papules and nodules in the skin, which are characterized histopathologically by irregular vascular spaces surrounded by typical glomus cells. Glomuvenous malformations are caused by autosomal dominant mutations of the GLMN gene. A 34-year-old woman and her 16-year-old son presented with bluish papules and nodules since childhood. Biopsy specimens from both patients showed histopathologic features of glomuvenous malformations, unusually in consistent and close association with smooth muscle, hair follicles and eccrine glands. Sequencing of the GLMN gene revealed the p.C36X (c.108C>A) mutation in germline DNA from both patients. This is probably the first report describing the hamartomatous features of familial glomuvenous malformations consistently associated with a prominent smooth muscle component and eccrine glands.

Atopic dermatitis-derived Staphylococcus aureus strains: what makes them special in the interplay with the host.

Background: Atopic dermatitis (AD) is a chronic inflammatory skin condition whose pathogenesis involves genetic predisposition, epidermal barrier dysfunction, alterations in the immune responses and microbial dysbiosis. Clinical studies have shown a link between Staphylococcus aureus and the pathogenesis of AD, although the origins and genetic diversity of S. aureus colonizing patients with AD is poorly understood. The aim of the study was to investigate if specific clones might be associated with the disease. Methods: WGS analyses were performed on 38 S. aureus strains, deriving from AD patients and healthy carriers. Genotypes (i.e. MLST, spa-, agr- and SCCmec-typing), genomic content (e.g. virulome and resistome), and the pan-genome structure of strains have been investigated. Phenotypic analyses were performed to determine the antibiotic susceptibility, the biofilm production and the invasiveness within the investigated S. aureus population. Results: Strains isolated from AD patients revealed a high degree of genetic heterogeneity and a shared set of virulence factors and antimicrobial resistance genes, suggesting that no genotype and genomic content are uniquely associated with AD. The same strains were characterized by a lower variability in terms of gene content, indicating that the inflammatory conditions could exert a selective pressure leading to the optimization of the gene repertoire. Furthermore, genes related to specific mechanisms, like post-translational modification, protein turnover and chaperones as well as intracellular trafficking, secretion and vesicular transport, were significantly more enriched in AD strains. Phenotypic analysis revealed that all of our AD strains were strong or moderate biofilm producers, while less than half showed invasive capabilities. Conclusions: We conclude that in AD skin, the functional role played by S. aureus may depend on differential gene expression patterns and/or on post-translational modification mechanisms rather than being associated with peculiar genetic features.

Insulin receptor and glucose transporters mRNA expression throughout the menstrual cycle in human endometrium: a physiological and cyclical condition of tissue insulin resistance.

The expression of insulin receptor (IR), together with that of glucose transporters 1 and 4 (GLUT1-4) and of Insulin Growth Factor-I and -II (IGF-I,-II) in the endometrium of healthy and young women in both phases of menstrual cycle was assessed. Sixteen out of 20 healthy and normal menstruating volunteers were studied. Endometrial samplings were performed in every subject, twice in the same cycle, during the follicular and luteal phase respectively. The mRNA expression of IR, GLUT1-4, IGF-I and -II were evaluated by real-time quantitative RT-PCR and immunostaining reactions. Our results indicate that IR, GLUT1-4, IGF-I and -II mRNAs were expressed in both phases of the endometrial cycle: GLUT4 and IGF-I mRNA expression were significantly higher in the follicular phase and localized at the epithelial and stromal cell level, respectively, whereas IR, GLUT1 and IGF-II mRNA expression were mostly present in the secretory phase and mainly localized at the stromal level. An inverse tendency of IR and GLUT4 mRNA expression was respectively observed from follicular to luteal phase. In conclusion our data suggest that IR, glucose transporters and IGFs are significantly and differently expressed at the endometrial level throughout the menstrual cycle and that human endometrium cyclically undergoes through a transitory condition from normal to an insulin-resistance state.

A Possible Link between Gut Microbiome Composition and Cardiovascular Comorbidities in Psoriatic Patients.

Cardiovascular disease (CVD) is one of the most common comorbidities that may affect psoriatic patients. Several exogenous and endogenous factors are involved in the etiology and progression of both psoriasis and CVD. A potential genetic link between the two diseases has emerged; however, some gaps remain in the understanding of the CVD prevalence in psoriatic patients. Recently, the role of the gut microbiome dysbiosis was documented in the development and maintenance of both diseases. To investigate whether gut microbiome dysbiosis might influence the occurrence of CVD in psoriatic patients, 16S rRNA gene sequencing was performed to characterize the gut microbiome of 28 psoriatic patients, including 17 patients with and 11 without CVD. The comparison of the gut microbiome composition between patients with and without CVD showed a higher prevalence of Barnesiellaceae and Phascolarctobacterium in patients with CVD. Among patients with CVD, those undergoing biologic therapy had lower abundance levels of Barnesiellaceae, comparable to those found in patients without CVD. Overall, these findings suggest that the co-occurrence of psoriasis and CVD might be linked to gut microbiome dysbiosis and that therapeutic strategies could help to restore the intestinal symbiosis, potentially improving the clinical management of psoriasis and its associated comorbidities.

Lipid rafts as platforms for sphingosine 1-phosphate metabolism and signalling.

Spontaneous segregation of cholesterol and sphingolipids as a liquid-ordered phase leads to their clustering in selected membrane areas, the lipid rafts. These specialized membrane domains enriched in gangliosides, sphingomyelin, cholesterol and selected proteins involved in signal transduction, organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating cell homeostasis. Sphingosine 1-phosphate, an important biologically active mediator, is involved in several signal transduction processes regulating a plethora of cell functions and, not only several of its downstream effectors tend to localize in lipid rafts, some of the enzymes involved in its pathway, of receptors involved in its signalling and its transporters have been often found in these membrane microdomains. Considering this, in this review we address what is currently known regarding the relationship between sphingosine 1-phosphate metabolism and signalling and plasma membrane lipid rafts.

Isolation and Analysis of Lipid Rafts from Neural Cells and Tissues.

Lipid rafts are membrane areas characterized by the clustering of selected membrane lipids, as the result of their phase separation forming a liquid-ordered phase floating in the lipid-disordered bulk membrane. van Meer and Simons hypothesized the existence of lipid rafts to explain the differential composition of the apical and basolateral domains of polarized epithelial cells and proposed that association of given proteins with lipid rafts along the traffic route might represent an important mechanism for protein sorting. However, great attention was paid to the lipid raft theory after Simons and Ikonen highlighted the enrichment of several proteins involved in signal transduction in "detergent-insoluble, glycolipid-enriched complexes," and postulated that lipid rafts might serve as hubs in regulating intracellular signaling. Most notably, the feature of detergent-insolubility was incorporated in the definition of lipid rafts used in 1997 by these authors. "Lipid rafts" and "detergent-resistant membranes" became almost synonymous after the publication, in 1992, of the seminal paper by Brown and Rose, describing the separation of a low-density, Triton X-100-insoluble fraction from epithelial cells, enriched in GSL and apical GPI-anchored proteins and depleted of basolateral membrane marker proteins. This paper provided a working definition of lipid rafts and a putative biochemical method for their separation. More than 2000 papers have been published using "the Triton method." Evidences obtained by the use of alternative biochemical methods for the isolation of lipid rafts and of methods enabling to analyze the dynamics of lipid rafts in intact cells highlighted the several limitations of the Triton X-100 method. On the other hand, the main findings obtained by this method have not been confuted, and the method is still widely used.In this chapter, we will discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, with a special focus on neural cells and tissues.