betadex and Gangliosidosis--GM1

The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.

Topics: Actins; Animals; beta-Cyclodextrins; Cell Line, Transformed; Cholera Toxin; Cholesterol; Cyclic GMP; Cytochalasin D; Enzyme Inhibitors; Gangliosidosis, GM1; Imidazoles; Ligands; Membrane Microdomains; Microscopy, Confocal; Neurons; Normal Distribution; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Protein Transport; Pyridines; Quantum Dots; Rats; Serotonin; Serotonin Plasma Membrane Transport Proteins; Thionucleotides

The assembly of amyloid beta-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid beta-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid beta-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.

Topics: Amyloid beta-Peptides; Animals; Bacterial Proteins; Bacterial Toxins; beta-Cyclodextrins; cdc42 GTP-Binding Protein; Cell Differentiation; Clathrin Heavy Chains; Dose-Response Relationship, Drug; Endocytosis; Gangliosidosis, GM1; Immunoprecipitation; Mutation; PC12 Cells; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; rac1 GTP-Binding Protein; Rats; rhoA GTP-Binding Protein; RNA, Small Interfering; Time Factors; Transfection

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl beta-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed.

Topics: Aggregatibacter actinomycetemcomitans; Antigens, CD; Bacterial Toxins; beta-Cyclodextrins; Cell Cycle; Cholesterol; Gangliosidosis, GM1; Humans; Jurkat Cells; Membrane Microdomains; Protein Subunits; Receptors, Transferrin