Forskolin and dopamine D-1 receptor activation increase huntingtin's association with endosomes in immortalized neuronal cells of striatal origin

Huntingtin is a cytoplasmic protein of unknown function that associates wit h vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this stud y we sought to identify factors that regulate the transport of huntingtin i n striatal neurons, which are the cells most affected in Huntington's disea se. In clonal striatal cells derived from fusions of neuroblastoma and embr yonic striatal neurons, huntingtin localization is diffuse and slightly pun ctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein i nvolved in endocytosis. Immunoblot analysis of subcellular membrane fractio ns separated by differential centrifugation confirmed that huntingtin immun oreactivity in differentiated neurons markedly increased in membrane fracti ons enriched with clathrin and with huntingtin-interacting protein 1. Dopam ine treatment altered the subcellular localization of huntingtin and increa sed its expression in clathrin-enriched membrane fractions. The dopamine-in duced changes were blocked by the D-1 antagonist SCH 23390 and were absent in a clonal cell line lacking D-1 receptors. Results suggest that the transport of huntingtin and its co-expression in c lathrin and huntingtin-interacting protein 1-enriched membranes is influenc ed by activation of adenylyl cyclase and stimulation of dopamine D-1 recept ors. (C) 1999 IBRO. Published by Elsevier Science Ltd.