Mutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher's disease (GD), are the strongest genetic risk factor for Parkinson's disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using fluorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and α-synuclein levels as well as autophagic and lysosomal defects. Quantitative proteomic profiling reveals an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. Mutant neurons show a dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium. Importantly, correction of the mutations rescues such pathological phenotypes. These findings provide evidence for a link between GBA1 mutations and complex changes in the autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNF␣-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR ␤-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.adipocyte ͉ caveolae microdomain ͉ lipid rafts ͉ live cell imaging ͉ type 2 diabetes

Cell membrane components are organized as specialized domains involved in membrane-associated events such as cell signaling, cell adhesion, and protein sorting. These membrane domains are enriched in sphingolipids and cholesterol but display a low protein content. Theoretical considerations and experimental data suggest that some properties of gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. Gangliosides are glycolipids with strong amphiphilic character and are particularly abundant in the plasma membranes, where they are inserted into the external leaflet with the hydrophobic ceramide moiety and with the oligosaccharide chain protruding into the extracellular medium. The geometry of the monomer inserted into the membrane, largely determined by the very large surface area occupied by the oligosaccharide chain, the ability of the ceramide amide linkage to form a network of hydrogen bonds at the water-lipid interface of cell membranes, the Delta(4) double bond of sphingosine proximal to the water-lipid interface, the capability of the oligosaccharide chain to interact with water, and the absence of double bonds into the double-tailed hydrophobic moiety are the ganglioside features that will be discussed in this review, to show how gangliosides are responsible for the formation of cell lipid membrane domains characterized by a strong positive curvature.

Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83(+)CCR7(-) DCs within human tumors. Mice injected with tumors expressing the LXR ligand-inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-alpha (Nr1h3(-/-) mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer.

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine- The increasing body of evidence, obtained by several different experimental approaches from both artificial and cellular models (1-23), suggests that lipid and protein components in the cell membrane are not randomly or homogeneously distributed but rather organized in domains with peculiar physicochemical and functional properties, different from those of the surrounding membrane environment, confirming the original prediction of Singer and Nicholson (24).Sphingolipid-enriched domains that are reported to be enriched in gangliosides, sphingomyelin, and cholesterol (1, 17-23) are emerging as membrane compartments with relevant biological functions. They are rich in proteins involved in the mechanisms of signal transduction (1, 18, 21-23, 25-31) and cell adhesion molecules (34). Thus, sphingolipid-enriched microdomains could represent a site within the plasma membrane where different molecules (both lipids and proteins) involved in signal transduction and/or cell adhesion and cell-cell interactions are specifically sorted and concentrated, allowing reciprocal interactions of functional significance. Recent studies have revealed that gangliosides in membrane sphingolipidenriched domains associate closely and specifically with single or multiple signal transducer molecules. Ganglioside GM3 1 is closely associated with c-Src, Rho, FAK, and Ras in B16 melanoma cells (1, 21), with c-Src and Csk in neuroblastoma Neuro2a cells (22), and GD3 is associated with Src-family kinase Lyn and the neural cell adhesion molecule TAG-1 in rat brain (33,35). Such structural units seem to be involved in signal transduction in response to glycosphingolipid-mediated stimulation; GM3-mediated cell adhesion of melanoma B16 cells induces c-Src and FAK phosphorylation and Rho and Ras activation (1); treatment of neuroblastoma Neuro2a cells with exogenous gangliosides induces c-Src and mitogen-activated protein kinase activation, leading to neuronal differentiation (22); and treatment of primary cultured rat cerebellar neurons with anti-GD3 antibody induces Lyn activation with consequent phosphorylation of mitogen-activated protein kinases (33). Neurotrophin-induced p75 NTR -dependent sphingomyelin hydrolysis is also localized in a caveolar domain (27).Procedures...