SPHINGOLIPIDS AS SIGNALING MOLECULES: THEIR INVOLVEMENT IN HEALTH AND DISEASE.

Sphingolipids (SLs) are minor cell membrane amphyphilic components, residing in the external layer of the plasma membrane (PM), with the hydrophobic moiety, the ceramide (Cer), inserted into the membrane layer and the hydrophilic head group protruding toward the extracellular environment. They are a family of several compounds with different structural properties: the phospholipid, sphingomyelin (SM), the glycosphingolipids (GSLs) characterized for containing a complex oligosaccharide chain as hydrophilic moiety and gangliosides, GSLs containing sialic acid. As membrane components, SLs participate to modulate several cell processes, such as cell growth, differentiation, morphogenesis, cell to matrix interaction and cell to cell communication. From this, it follows that a defect in SL metabolism can obviously lead to a great number of dysfunctions, ranging from neurodegeneration to cancer. Along my Ph.D. course I considered the different faces of SLs roles: from their involvement in physiology to that in pathology. i.SPHINGOLIPIDS and HEALT. SLs cluster to form SL-enriched domains on cellular PM (lipid rafts, caveolae, and glycosynapses) providing a microenvironment within the PM for reciprocal interaction between lipids and proteins. In particular biochemical analyses have demonstrated that GSLs-enriched microdomains contain several transducer molecules, especially membrane-anchored signal transduction molecules, such as tyrosine kinases belonging to the Src family. Although it has been speculated that GSLs are involved in cell differentiation, proliferation and functions such as phagocytosing, there are quite few evidence that GSLs, by themselves, directly mediate signal transduction, which lead to these cell functions. LACTOSYLCERAMIDE-ENRICHED MICRODOMAIN in NEUTROPHILIS. Lactosylceramide (LacCer), a neutral GSL, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. It has been previously demonstrated that LacCer forms PM lipid domains, that can be separated by detergent treatment of cells followed by ultracentrifugation, and that these lipid domains are coupled with Lyn, a Src family kinase. Ligand binding to LacCer activates Lyn, resulting in neutrophils functions, such as superoxide generation, phagocytosis and migration. The presence of LacCer molecular species with Cer containing a very long fatty acid chain is necessary for the association of Lyn with LacCer-enriched PM domains and LacCer-mediated functions. Lyn is associated by a palmitoyl anchor to the cytoplasmic leaflet, while LacCer is inserted into the outer layer of membrane bilayer. So the question is: how does LacCer interact with signal transducer molecules? The GSL-protein interactions in neutrophilis have been investigated by photoactivable GSLs. These molecules have been administered and taken up by the cell PM. When cells are illuminated, the photoactivable group, linked to the terminal portion of Cer, yields a very reactive intermediate, that covalently binds the molecules in the close environment. For the first time, at the best of our knowledge, we show a direct connection, across the PM, between GSLs and palmitoylated proteins: these results suggest that LacCer with a long fatty acid chain in Cer moiety could be the key-player of the transduction of information across the PM, modulating membrane interdigitation, through the long acyl chain, and forming specific PM microdomains. ii. SPHINGOLIPIDS and DISEASES. IMPLICATION IN NEURODEGENERATIVE DISORDERS. SLs are particularly abundant in the nervous system where they play crucial roles regulating signaling events. Severe neurodegeneration is the prominent pathological hallmark of the most sphingolipidoses, inherited metabolic diseases characterized by a defect in the lysosomal GSL catabolism. Most of sphingolipidosis are caused by deficiencies of a specific lysosomal hydrolases, resulting in the accumulation of undegraded lipid metabolite in many cells. SECONDARY ACCUMULATIONS OF GANGLIOSIDES: THE CASE PF NIEMMAN-PICK TYPE Niemann-Pick Disease (NPD) is a rare, autosomal recessive, lysosomal storage disease resulting from a deficiency in acid sphingomyelinase (ASM) activity. If ASM is absent or not functioning properly, SM cannot be metabolized properly and is accumulated within the cell, eventually causing cell death and the malfunction of major organ systems. In NPD type A, residual enzyme activity is very low (<5%) and in NPD-A patients, storage pathology is severe within neurons and glial cells throughout the nervous system resulting in progressive, global deterioration of neurological function and death by 3 years of age. To better understand the secondary biochemical mechanism underling the pathogenesis and the importance of these secondary alterations of SL metabolism on lysosomal diseases, we analyzed lipid composition of CNS and extraneural tissues from the acid sphingomyelinase-deficient (ASMKO) mouse, the animal model of NPD type A, that has been developed using gene targeting and embryo transfer techniques. Our data show an unexpected tissue specific selection of the accumulated molecular species of SM, and an accumulation of GM3 and GM2 gangliosides in both neural and extraneural tissues, that cannot be solely explained by the lack of ASM. In ASMKO mice, we observed the preferential accumulation of SM molecular species with shorter acyl chains in the nervous system, but not in extraneural tissues. The unbalance toward C18/C16-fatty acid containing SM species was detectable as early as SM accumulation started, and monosialoganglioside accumulation followed immediately afterwards. These changes in SL patterns should thus represent the effect of secondary biochemical pathways altered as a consequence of a non-related primary cause. The mechanism underlying these changes still remains to be elucidated and is probably the result of changes in the expression and/or activity of more than one single enzyme, and of anomalies in the traffic of the substrate/product concentrations in multiple cellular compartments. Several pieces of evidence suggest that altered SL metabolism results in a non-physiological PM composition and organization, leading to altered PM-originated signalling pathways that could be relevant to the onset of cellular damage and of tissue pathology. CHAPERONE THERAPY FOR GM2 GANGLIOSIDOSIS: PYRIMETHAMINE and SANDHOFF DISEASE. Sandhoff disease is an autosomal recessive neurodegenerative disease characterized by the intralysosomal accumulation of GM2 ganglioside. This is due to mutations in the \u3b2-hexosaminidases \u3b2-chain gene, resulting in a \u3b2-hexosaminidases A (\u3b1\u3b2) and B (\u3b2\u3b2) deficiency. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER), and most of them are transported to the Golgi apparatus for glycosylation and taggeting for lysosomes, by the addition of mannose-6-phosphate. The ER contains a highly conserved degradation pathway, to protect cells from misfolding and potentially toxic proteins. Thus, the protein turnover ensures integrity and biological functions of cells. Treatment of sphingolipidoses, such as Sandhoff disease, have received increasing interest despite the low incidence. However, the replacing enzyme therapy meets the problem to be ineffective over the blood brain barrier and thus new approaches are now under investigation. One of these is the use of pharmacological chaperones. Pyrimethamine, a drug used to treat or prevent serious parasite infections, such as toxoplasmosis and malaria, has been described to act as chaperone for the \u3b2-hexosaminidase. Fibroblasts from two cases of juvenile Sandhoff disease, displaying the new mutations C1082+5G>A (#1) and C446-13A>G (#2) in the HEX B gene, were treated with pyrimethamine. The two examined cell lines, after treatment with pyrimethamine, showed an increased total \u3b2-hexosaminidase activity, so that the residual value was around 10%. This value should be enough to limit the accumulation of GM2. In contrast to this, cells treated with pyrimethamine and fed with isotopically tritium labeled ganglioside GM1 did not show any GM2 lysosomal catabolism. This negative result was explained after fractionation of the total cell proteins by ion exchange DEAE column chromatography. Clearly, we found that the increased \u3b2-hexosaminidase activity on the artificial substrate was due to the increase of the activity of \u3b2-hexosaminidase isoform S. Unlikely, the isoform S, even if it hydrolyzes the artificial compound, does not recognize the natural substrate GM2 and, therefore, the pyrimethamine is unable to modify cell lysosomal activity in the two cell lines showing the new mutations C1082+5G>A and C446-13A>G, and, moreover, cannot be used as a therapeutic drug for the these two patients. Our results are in agreement with a previous information on the selective activity of pyrimethamine depending on the mutation and confirm the advantages deriving from the use of patients\u2019 fibroblasts for the decision on the use of pyrimethamine as therapeutic drug. IMPLICATION IN CANCER Tumor cells are characterized by aberrant glycosylation processes responsible to increase proliferation and motility. A precise content and molar ratio between SLs is necessary in cells to have the correct membrane organization and the correct interaction processes between membrane components and membranes of adjacent cells. CELL SURFACE MODULATION DURING TUMOR IRRADIATIONS. Radiotherapy has actually been used clinically for a number of years, with very positive results on tumor reduction. Irradiation of cancer cells leads to cell death by different mechanism. DNA damage, mitochondrial damage and oxidative stress bring mainly to necrosis, whereas the PM production of ceramide leads to apoptosis. The ceramide mediated apoptosis has been reported to depend on activation of cell surface sphingomyelinase and the following activation of the ceramide dependent phosphorylation cascade. Other enzymes of the SL metabolism have been recently found associated to the external leaflet of the PMs, like the \u3b1-sialidase, the \u3b2-hexosaminidase, the \u3b2-galactosidase and the \u3b2-glucosidase. These glycohydrolases lead to ceramide from complex GSLs and suggest a new way to trigger the ceramide-induced apoptosis. The PM enzymatic activities display optimal pH under mild acidic condition. This is found in specific membrane domains, known as lipid rafts, where GSLs are highly enriched together with ion exchanger proteins such as proton pumps. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of \u3b2-glucosidase and \u3b2-galactosidase was parallel to the increase of irradiation dose up to 60 Gy and continued with time, at least up to 72 hr from irradiation. \u3b2-glucosidase increased up to 17 times and \u3b2-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20 Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of PM ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the PMs and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells. We are characterizing the role of PM glycohydrolases in the process of apoptosis activated by cell irradiation and developing new protocols for radiation therapy combined with pharmacological treatments capable to exert enhanced antitumor activity through synergic action. Increase of the activities of the PM glycohydrolases, and of the apoptotic process, cold be obtained by i) activating with specific drugs the PM proton pumps to decrease the local extracellular pH; ii) the use of recombinant glycohydrolases; iii) using drugs or chaperones capable to modulate directly the glycohydrolase activities. Concerning this last opportunity, molecules acting as chaperones able to up-regulate the activity of lysosomal enzymes are known. Also if no information is available, considering the nature of PM enzymes, we believe that these regulators should be effective also on PM enzymes. \u200

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

GM1 Ganglioside Is A Key Factor in Maintaining the Mammalian Neuronal Functions Avoiding Neurodegeneration

Many species of ganglioside GM1, differing for the sialic acid and ceramide content, have been characterized and their physico\u2010chemical properties have been studied in detail since 1963. Scientists were immediately attracted to the GM1 molecule and have carried on an ever\u2010increasing number of studies to understand its binding properties and its neurotrophic and neuroprotective role. GM1 displays a well balanced amphiphilic behavior that allows to establish strong both hydrophobic and hydrophilic interactions. The peculiar structure of GM1 reduces the fluidity of the plasma membrane which implies a retention and enrichment of the ganglioside in specific membrane domains called lipid rafts. The dynamism of the GM1 oligosaccharide head allows it to assume different conformations and, in this way, to interact through hydrogen or ionic bonds with a wide range of membrane receptors as well as with extracellular ligands. After more than 60 years of studies, it is a milestone that GM1 is one of the main actors in determining the neuronal functions that allows humans to have an intellectual life. The progressive reduction of its biosynthesis along the lifespan is being considered as one of the causes underlying neuronal loss in aged people and severe neuronal decline in neurodegenerative diseases. In this review, we report on the main knowledge on ganglioside GM1, with an emphasis on the recent discoveries about its bioactive component

Characterization of the GM1 oligosaccharide transport across the blood-brain-barrier

Ganglioside GM1 has demonstrated to attenuate Parkinson Disease (PD) symptoms in clinical and preclinical trials. Nevertheless, the GM1 efficacy revealed in vitro is critically reduced in vivo, because of the amphiphilic behavior that limits the passage across the blood brain barrier (BBB). In vitro and in vivo experiments showed that GM1 exerts neurotrophic functions by interacting with plasma membrane (PM) proteins throughout its oligosaccharide portion (OligoGM1). Furthermore, OligoGM1 intravenously or subcutaneously injected into mice is absorbed and taken up by different organs and tissues, including brain. In order to take advantage of GM1 oligosaccharide properties and to overcome GM1 pharmacological limitation, this study has been aimed by the investigation of the OligoGM1 transportthrough the BBB, by using a human in vitro model for human brain-like endothelial cells (hBLEC). Ruled out the toxicity of OligoGM1 on hBLEC, the OligoGM1 transport across the hBBB has been analyzed, finding out a 20 fold higher rate than GM1 and a time and concentration dependence. In order to characterize the OligoGM1 passage, a direct evaluation of the OligoGM1 interaction with the ABC-transporters was carried on, leaving out this way for OligoGM1 transport. Moreover, inverse- and 4\ub0C-transport experiments were performed excluding the implication of the active transport for OligoGM1 passage across the hBLEC, leading to consider the passive-paracellular route. Furthermore, after the hBLEC transport, OligoGM1 maintained its stability and capacity to induce neuritogenesis in the mouse neuroblastoma cells line Neuro2a. This preliminary study has improved the knowledge about the GM1 pharmacological potential by proving that OligoGM1 can cross advantageously the BBB, offering a new promising therapeutic strategy

GM1 promotes TrkA-mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA\u2013GM1 oligosaccharide complex at the cell surface. To study the TrkA\u2013GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross-linked TrkA\u2013GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA\u2013GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent-resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by \u2018flopping\u2019 down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1. (Figure presented.)

IL-17 induces an expanded range of downstream genes in reconstituted human epidermis model

Background: IL-17 is the defining cytokine of the Th17, Tc17, and γδ T cell populations that plays a critical role in mediating inflammation and autoimmunity. Psoriasis vulgaris is an inflammatory skin disease mediated by Th1 and Th17 cytokines with relevant contributions of IFN-γ, TNF-α, and IL-17. Despite the pivotal role IL-17 plays in psoriasis, and in contrast to the other key mediators involved in the psoriasis cytokine cascade that are capable of inducing broad effects on keratinocytes, IL-17 was demonstrated to regulate the expression of a limited number of genes in monolayer keratinocytes cultured in vitro. Methodology/Principal Findings: Given the clinical efficacy of anti-IL-17 agents is associated with an impressive reduction in a large set of inflammatory genes, we sought a full-thickness skin model that more closely resemble in vivo epidermal architecture. Using a reconstructed human epidermis (RHE), IL-17 was able to upregulate 419 gene probes and downregulate 216 gene probes. As possible explanation for the increased gene induction in the RHE model is that C/CAATenhancer- binding proteins (C/EBP) -β, the transcription factor regulating IL-17-responsive genes, is expressed preferentially in differentiated keratinocytes. Conclusions/Significance: The genes identified in IL-17-treated RHE are likely relevant to the IL-17 effects in psoriasis, since ixekizumab (anti-IL-17A agent) strongly suppressed the «RHE» genes in psoriasis patients treated in vivo with this IL-17 antagonist. © 2014 Chiricozzi et al

Parkinson&apos;s disease recovery by GM1 oligosaccharide treatment in the B4galnt1+/- mouse model

Given the recent in vitro discovery that the free soluble oligosaccharide of GM1 is the bioactive portion of GM1 for neurotrophic functions, we investigated its therapeutic potential in the B4galnt1+/- mice, a model of sporadic Parkinson's disease. We found that the GM1 oligosaccharide, systemically administered, reaches the brain and completely rescues the physical symptoms, reduces the abnormal nigral \u3b1-synuclein content, restores nigral tyrosine hydroxylase expression and striatal neurotransmitter levels, overlapping the wild-type condition. Thus, this study supports the idea that the Parkinson's phenotype expressed by the B4galnt1+/- mice is due to a reduced level of neuronal ganglioside content and lack of interactions between the oligosaccharide portion of GM1 with specific membrane proteins. It also points to the therapeutic potential of the GM1 oligosaccharide for treatment of sporadic Parkinson's disease

Similar levels of efficacy of two different maintenance doses of adalimumab on clinical severity and quality of life of patients with hidradenitis suppurativa

Adalimumab is the only biologic agent approved for the treatment of moderate-to-severe hidradenitis suppurativa (HS) patients (i.e., with Hurley II or III), which is recommended in two different maintenance doses (i.e., 40 mg weekly or 80 mg every two weeks). We conducted a prospective multicentric study to measure outcomes related to the severity of disease and quality of life (QoL) of patients affected by moderate-to-severe HS, treated with adalimumab at a maintenance dosing of 40 mg or 80 mg. Assessments were performed at baseline (T0) and after 32 weeks of treatment (T32). We enrolled 85 moderate-to-severe HS Italian patients, 43 men (50.6%) and 42 women, aged between 16 and 62 years (median 31 years, interquartile range 24.4-43.8). Statistically significant improvements were observed for clinical status (with a mean reduction of 7.1 points for the International Hidradenitis Suppurativa Severity Score System (IHS4)), pain levels (3.1 mean decrease in VAS), and QoL (3.4 mean improvement in DLQI score). Patients with no comorbidities, and those with higher levels of perceived pain showed significantly greater improvement in QoL than their counterpart from T0 to T32. As for the proportion of patients who at follow-up reached the minimal clinical important difference (MCID) in QoL, significantly higher proportions of success were observed for age (patients in the 29-39 category), pain (patients with higher reported pain), and Hurley stage III. While both treatment regimen groups (i.e., 40 vs. 80 mg) improved significantly, no statistical differences were observed when comparing the two treatment dosages

Analysis of predictive factors influencing dupilumab continuation rate in adult patients with atopic dermatitis. results from an italian multicenter study

Objectives: The purpose of this study was to analyze the drug survival rate of dupilumab up to 2 years in a large real-world cohort of adult patients affected by moderate/severe atopic dermatitis (AD), and to investigate the clinical, demographic and predictive factors influencing the patients’ treatment persistence. Material and methods: This study included adult patients affected by moderate-to-severe AD treated with dupilumab for at least 16 weeks who visited 7 dermatologic outpatient clinics in Lazio, Italy, from January 2019 until August 2021. Results: A total of 659 adult patients (345 male [52.3%], mean age: 42.8 years) with an average treatment duration of 23.3 months were enrolled in the study. Overall, 88.6% and 76.1% of patients were still on treatment after 12 and 24 months, respectively. The drug survival rate for discontinuation due to AEs and dupilumab ineffectiveness was 95.0% at 12 months and 90.0% at 24 months. The main reasons for drug discontinuation included inefficacy (29.6%), failed compliance (17.4%), persistent efficacy (20.4%) and adverse events (7.8%). Adult AD onset (≥18 years) and EASI score severity measured at the last follow-up visit were the only factors significantly associated with lower drug survival. Conclusion: This study revealed an increased cumulative probability of dupilumab survival at 2 years, reflected by a sustained effectiveness and a favorable safety profile of the drug