Altered dopamine transporter function and immunoregulation in PD macrophages

Abstract

Parkinson's Disease (PD), the second‐most common neurodegenerative disorder, has three key hallmarks: dysregulated dopamine (DA) signaling in part due to degeneration of Substantia Nigra neurons, intracellular aggregates of a‐synulcein, and neuroinflammation. However, the origin and interplay of these features remains unclear. DA substitution therapy, the standard pharmacological treatment, only alleviates symptoms with limited efficacy. Interestingly, plasma DA levels are altered in PD, and DA can regulate immune cell function in the periphery. Additionally, the dopamine transporter (DAT), a potent regulator of DA tone in the CNS, also exists on leukocytes. However, although it is characterized in the CNS and is a therapeutic target for some neurological disorders, its function in the periphery is largely unknown. Our study asks how DAT regulation of plasma DA modulates the peripheral immune system. To begin to address this hypothesis, we first used biochemical and microscopy approaches to examine DAT distribution and function in the healthy monocyte‐derived human macrophages (MDM). We found DAT constitutively exists in human MDM and is localized to the plasma membrane and in the intracellular space (n=10 biological replicates). We demonstrated the DAT molecules on the human MDM are functional as measured by uptake of IDT307, a fluorescent DAT substrate, and of 3H‐DA (Km=3.178). In addition we found DAT is subjected to canonical trafficking such as PKC‐dependent endocytosis as measured by Total Internal Reflection Florescence Microscopy (TIRF‐M). These data are consistent with the overarching hypothesis that immuno‐activation of human MDM regulates DAT activity and thus dopamine transmission. We discovered lipopolysaccharide (LPS)‐immuno‐activation of human macrophages recruits DAT to the membrane leading to increased surface DAT localization (n= 4 biological replicates P < 0.05). Contrary to our prediction, our preliminary data suggests a decrease in nomifensine‐sensitive, DAT‐mediated substrate uptake. Furthermore, we have also found that basal DAT‐dependent uptake in PD macrophages is elevated (p<0.0001, n=5 biological replicates), and our preliminary data suggests reduced sensitivity to LPS‐induced trafficking. Using simultaneous whole cell patch clamp electrophysiology and amperometry, we are currently testing the hypothesis that LPS‐induced TLR4 activation of macrophages stimulates DAT trafficking to the membrane and stabilizes the transporter in an efflux promoting conformation. We then plan to test the hypothesis that PD macrophages are immunocompromised, exhibiting an altered DAT response to LPS‐stimulation.Support or Funding InformationR21NS103108 R21DA043895This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.