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    • We found that the STX R Q variant has

    2018-11-15

    We found that the STX1 R/Q variant has decreased phosphorylation of Ser14, a key residue involved in the functional regulation of STX1 (Khelashvili et al., 2012; Dubois et al., 2002). STX1 in this hypo-phosphorylated state fails to support DAT-mediated reverse transport of DA without inhibiting DA uptake function. Interestingly, in hDAT cells expressing STX1 R/Q, we observe a significantly increased Vmax of DA uptake. In parallel experiments in mouse striatal slices, cleaving STX1 with BoNT/C promotes a decrease in reverse transport of DA with a trend towards increased DA uptake. These data suggest that STX1 function asymmetrically regulates reverse transport of DA and DA uptake. Therefore, to define how hypo-phosphorylation of STX1 R/Q impairs DA reverse transport, we studied the regulatory effects of CK2 function on DA efflux. CK2 phosphorylates STX1 at Ser14 to regulate STX1 function and protein interactions (Hirling and Scheller, 1996; Foletti et al., 2000; Dubois et al., 2002). Here, we discovered that CK2-mediated phosphorylation of STX1 at Ser14 increases the direct association between STX1 and the hDAT N-terminus. Consistently, pharmacological inhibition of CK2 strongly reduces reverse transport of DA without altering DA uptake. Thus, CK2 is a key player involved in DA efflux. It is possible that this mechanism is important in a broader array of DA-associated neuropsychiatric disorders, since the expression of CK2, as well as the phosphorylation state of STX1 at Ser14, is decreased in post-mortem gsk3b inhibitor tissue from patients with schizophrenia (Castillo et al., 2010; Aksenova et al., 1991). To have a more complete understanding of how genetic variants within the DA network discovered in ASD patients alter behaviors, we translated our molecular discoveries in vivo. First, we inhibited CK2 function by selectively expressing CK2DN specifically in DA neurons of WT flies. Drosophila expressing CK2DN exhibited a robust reduction in AMPH-induced hyperlocomotion as compared to WT flies. These data underscore the importance of CK2 function and STX1 phosphorylation in regulating behaviors sustained by reverse transport of DA. Interestingly, inhibiting CK2 function did not regulate basal locomotion or DA uptake in intact Drosophila brains. Parallel to STX1 R/Q, we found that the hDAT R/W variant displays inhibited reverse transport of DA without impairments in uptake function. It is important to note that CK2 function, in addition to phosphorylating STX1 at Ser14, also promotes STX1/DAT interactions. Here, we show that hDAT R/W has reduced association with STX1, resulting in reduced reverse transport of DA and DA-related behaviors. Drosophila expressing hDAT R/W selectively in DA neurons demonstrate reduced sensitivity to the psychomotor effects of AMPH. Interestingly, basal locomotion remained unaltered in hDAT R/W flies, indicating normal DAT-mediated DA clearance as supported by our uptake data. These data suggest that the phosphorylation state of STX1 at Ser14 and STX1/DAT interaction asymmetrically regulate reverse transport of DA and DAT-mediated uptake. Mounting evidence demonstrates that reverse transport of DA and associated behaviors can be promoted by changes in the association between the N-terminus of DAT and STX1 (Binda et al., 2008). Additionally, it has been suggested that reverse transport of DA might participate in shaping DA neurotransmission (Leviel, 2011). Here, we used AMPH as a tool to induce reverse transport of DA to determine whether ASD-associated variants disrupt this event. We show that AMPH promotes phosphorylation of STX1 at Ser14 and, as a consequence, STX1/DAT interaction to cause reverse transport of DA. Therefore, we felt that it was important to demonstrate these discoveries at the level of a single active site, the SCG bouton. Several ASD-associated hDAT variants have now been found to impact reverse transport of DA. The STX1 R/Q variant reported here ablates AMPH-induced efflux similarly to hDAT R/W. Interestingly, two other variants were previously found to cause dysregulation of DA efflux, including a de novo DAT T356M variant and the recurrent DAT A559V variant seen in two boys with ASD, as well as in individuals with bipolar disorder and ADHD. These findings demonstrate diverging mechanisms by which reverse transport of DA can be disrupted. They align with other examples of neurodevelopmental risk emerging from genetic variants causing opposite effects on gene expression or signaling cascades (Sanders et al., 2011; Cook et al., 1997).