Differential subcellular distribution of endosomal compartments and the dopamine transporter in dopaminergic neurons

Abstract

Dopamine (DA) transporter (DAT) functions at the surface of dopaminergic neurons to clear extracellular DA. DAT surface levels are regulated by endocytosis. However, the endosomelysosome system is not well characterized in dopaminergic neurons and the endocytic trafficking of endogenous DAT is poorly studied. Hence we analyzed the distribution of endocytic compartments and DAT localization in cultured rat embryonic and postnatal neurons using fluorescence microscopy. Early Rab5 and EEA.1 containing endosomes were mostly found in somatodendritic regions of neurons, whereas endosomes containing recycling markers were primarily found in axons. In axons, DAT was located mainly in recycling endosomes and plasma membrane whereas in cell bodies and dendrites DAT was detected in early, late and recycling endosomal compartments. Subcellular fractionation of adult rat striatal synaptosomes demonstrated that DAT is enriched in fractions containing plasma membrane and recycling endosomes. This pattern of DAT distribution was not altered upon activation of protein kinase C in postnatal DA neurons. Altogether, our data suggest that axonal DAT mainly shuttles between the plasma membrane and recycling endosomes, whereas in the somatodendritic region of neurons DAT traffics through all conventional endosomal pathways.

Prolonged High Fat Diet Reduces Dopamine Reuptake without Altering DAT Gene Expression

“…DAT is an N -linked glycoprotein with an apparent molecular weight of between 50 and 80 kDa due to increasing levels of glycosylation as the protein matures [32]. Fractionation was confirmed by enriched expression of the NR2B subunit of the NMDA receptor in synaptosomal membrane fraction and of the transferrin receptor in the endosomal fraction (for example blot see Fig.…”

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

“…As described above, studies performed on DAT indicate that the sorting pattern observed in neuronal cell lines is representative of the sorting pattern of endogenous DAT in dopaminergic cultures. Nevertheless, we should note that a study by Sorkin and co-workers (47) revealed that DAT might be sorted differentially in axonal or somatodendritic parts of the dopaminergic neurons, thus emphasizing the importance of future studies of SERT trafficking in neuronal cultures.…”

Mechanisms of dopamine transporter regulation in normal and disease states

“…Acute endocytotic regulation of DAT controls the number of transporter surface copies available to translocate DA [40], whereas extended activation of PKC funnels DAT into lysosomal degradation for long-term regulation of transporter levels [25, 41, 42]. Most studies on acute endocytosis support clathrin-dependent processes [41-43], and targeting to early, late, and recycling endosomes [25, 44]. Multiple mechanisms have been linked to PKC-stimulated DAT endocytosis, including N-terminal ubiquitylation [22], Rin1 binding to the C-terminal endocytosis motif [27], and possibly Flot1 interactions [35, 42].…”