Cocaine-regulated trafficking of dopamine transporters in cultured neurons revealed by a pH sensitive reporter

Parkinson’s disease gene, Synaptojanin1, dysregulates the basal maintenance of the dopamine transporter

“…While we did not observe these clusters in the brain slices of Synj1+/mice, large varicosities are not uncommon in cultured ventral MB neurons. In our previous study, we showed that large varicosities exhibit 25% of sDAT compared to adjacent axons that exhibits 75% in wildtype neurons [42]. We next sought to determine if the reduced axonal sDAT could be due to a higher density of large varicosities in the Synj1+/-axons.…”

“…Second, we expressed a pH sensitive DAT sensor, CAGp-hDAT-pHluorin (Fig. 3E) [42] in cultured ventral MB neurons. Sequential perfusion of a membrane impermeable MES solution (pH 5.5) and a pH neutral NH 4Cl solution (pH 7.4) (Fig.…”

“…Sequential perfusion of a membrane impermeable MES solution (pH 5.5) and a pH neutral NH 4Cl solution (pH 7.4) (Fig. 3E) allowed us to calculate the surface fraction and vesicular pH of the intracellular DAT-pHluorin using the Henderson-Hasselbalch equation [42,[50][51][52]. The TH immunoreactivity of the neuron was determined by post hoc analysis (raw data in Supplemental Fig.…”

“…Both clathrin-dependent and independent endocytosis of DAT have been reported [32][33][34][35][36][37][38][39][40][41]. Our previous work suggested that Synj1 may participate in the cocaine-induced DAT trafficking [42], however, whether Synj1 regulates DAT recycling in physiological conditions has not been explored. Neither do we understand the interactions between DA signaling and DAT surface availability in Synj1 deficient conditions.…”

“…Neither do we understand the interactions between DA signaling and DAT surface availability in Synj1 deficient conditions. We now use a combination of sophisticated imaging tools including dLight [43,44] and DAT-pHluorin [42] to dissect the Synj1's impact on DA homeostasis and the surface maintenance of DAT. Our results revealed an unexpected role of Synj1's 5'-phosphatase domain in maintaining the surface maintenance of DAT by suppressing the PI(4,5)P 2-PKCβ pathway, and we provided essential insights for dysregulation of DA signaling via the dynamic availability of DAT.…”