Who Knew? Dopamine Transporter Activity Is Critical in Innate and Adaptive Immune Responses.

The dopamine transporter (DAT) regulates the dimension and duration of dopamine transmission. DAT expression, its trafficking, protein-protein interactions, and its activity are conventionally studied in the CNS and within the context of neurological diseases such as Parkinson's Diseases and neuropsychiatric diseases such as drug addiction, attention deficit hyperactivity and autism. However, DAT is also expressed at the plasma membrane of peripheral immune cells such as monocytes, macrophages, T-cells, and B-cells. DAT activity via an autocrine/paracrine signaling loop regulates macrophage responses to immune stimulation. In a recent study, we identified an immunosuppressive function for DAT, where blockade of DAT activity enhanced LPS-mediated production of IL-6, TNF-α, and mitochondrial superoxide levels, demonstrating that DAT activity regulates macrophage immune responses. In the current study, we tested the hypothesis that in the DAT knockout mice, innate and adaptive immunity are perturbed. We found that genetic deletion of DAT (DAT-/-) results in an exaggerated baseline inflammatory phenotype in peripheral circulating myeloid cells. In peritoneal macrophages obtained from DAT-/- mice, we identified increased MHC-II expression and exaggerated phagocytic response to LPS-induced immune stimulation, suppressed T-cell populations at baseline and following systemic endotoxemia and exaggerated memory B cell expansion. In DAT-/- mice, norepinephrine and dopamine levels are increased in spleen and thymus, but not in circulating serum. These findings in conjunction with spleen hypoplasia, increased splenic myeloid cells, and elevated MHC-II expression, in DAT-/- mice further support a critical role for DAT activity in peripheral immunity. While the current study is only focused on identifying the role of DAT in peripheral immunity, our data point to a much broader implication of DAT activity than previously thought. This study is dedicated to the memory of Dr. Marc Caron who has left an indelible mark in the dopamine transporter field.

The complex role of inflammation and gliotransmitters in Parkinson's disease.

Our understanding of the role of innate and adaptive immune cell function in brain health and how it goes awry during aging and neurodegenerative diseases is still in its infancy. Inflammation and immunological dysfunction are common components of Parkinson's disease (PD), both in terms of motor and non-motor components of PD. In recent decades, the antiquated notion that the central nervous system (CNS) in disease states is an immune-privileged organ, has been debunked. The immune landscape in the CNS influences peripheral systems, and peripheral immunological changes can alter the CNS in health and disease. Identifying immune and inflammatory pathways that compromise neuronal health and survival is critical in designing innovative and effective strategies to limit their untoward effects on neuronal health.

Functional characterization of the biogenic amine transporters on human macrophages.

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

In Parkinson's patient-derived dopamine neurons, the triplication of α-synuclein locus induces distinctive firing pattern by impeding D2 receptor autoinhibition.

Pathophysiological changes in dopamine neurons precede their demise and contribute to the early phases of Parkinson's disease (PD). Intracellular pathological inclusions of the protein α-synuclein within dopaminergic neurons are a cardinal feature of PD, but the mechanisms by which α-synuclein contributes to dopaminergic neuron vulnerability remain unknown. The inaccessibility to diseased tissue has been a limitation in studying progression of pathophysiology prior to degeneration of dopamine neurons. To address these issues, we differentiated induced pluripotent stem cells (iPSCs) from a PD patient carrying the α-synuclein triplication mutation (AST) and an unaffected first-degree relative (NAS) into dopaminergic neurons. In human-like dopamine neurons α-synuclein overexpression reduced the functional availability of D2 receptors, resulting in a stark dysregulation in firing activity, dopamine release, and neuronal morphology. We back-translated these findings into primary mouse neurons overexpressing α-synuclein and found a similar phenotype, supporting the causal role for α-synuclein. Importantly, application of D2 receptor agonist, quinpirole, restored the altered firing activity of AST-derived dopaminergic neurons to normal levels. These results provide novel insights into the pre-degenerative pathophysiological neuro-phenotype induced by α-synuclein overexpression and introduce a potential mechanism for the long-established clinical efficacy of D2 receptor agonists in the treatment of PD.

Radiographic appearance of extracorporeal membrane oxygenations versus left ventricular assist device.

Chest radiography compares left ventricular decompression in the same patient supported with extracorporeal membrane oxygenation with atrial septal fenestration and subsequently supported with left ventricular assist device with apical cannulation.

A novel approach to study markers of dopamine signaling in peripheral immune cells.

Human monocytes express known markers of dopamine synthesis, storage and clearance, including dopamine transporter (DAT), tyrosine hydroxylase (TH), all subtypes of dopamine receptors and vesicular monoamine transporter 2 (VMAT2). Immunohistochemical and immunofluorescent methodologies have traditionally been employed to determine DAT and TH expression in the CNS, their detection in the blood and specifically in the peripheral monocytes has not been studied by flow cytometry. Flow cytometry assays are widely used in medicine and in basic, preclinical or clinical research to quantify physical and chemical characteristics of target cell populations. Here, we have established a highly sensitive and reproducible flow cytometry panel to detect and quantify DAT and TH expression in freshly isolated or cryopreserved human peripheral monocytes. In healthy humans (n = 41 biological replicates), we show baseline DAT and TH expressing monocytes constitute ~12% of the peripheral blood mononuclear cell (PBMC) fraction when examined in fresh isolation from whole blood. Using an identical flow cytometry panel, we found that cryopreservation of PBMCs using multiple techniques resulted in altered PBMC populations as compared to fresh isolation and relative to one another. Among these, we identified an optimum cryopreservation method for detecting TH and DAT in cryopreserved PBMCs. Our data provide a sensitive and reproducible approach to examine dopamine signaling in peripheral human immune cells. This approach can be applied to study peripheral dopamine signaling under healthy and potentially under disease conditions. The use of dopamine signaling could also be explored as a technique to monitor therapeutic interventions particularly those targeting DAT and TH in the periphery.

Clustered Kv2.1 decreases dopamine transporter activity and internalization.

The dopamine transporter (DAT) regulates dopamine neurotransmission via reuptake of dopamine released into the extracellular space. Interactions with partner proteins alter DAT function and thereby dynamically shape dopaminergic tone important for normal brain function. However, the extent and nature of these interactions are incompletely understood. Here, we describe a novel physical and functional interaction between DAT and the voltage-gated K+ channel Kv2.1 (potassium voltage-gated channel subfamily B member 1 or KCNB1). To examine the functional consequences of this interaction, we employed a combination of immunohistochemistry, immunofluorescence live-cell microscopy, co-immunoprecipitation, and electrophysiological approaches. Consistent with previous reports, we found Kv2.1 is trafficked to membrane-bound clusters observed both in vivo and in vitro in rodent dopamine neurons. Our data provide evidence that clustered Kv2.1 channels decrease DAT's lateral mobility and inhibit its internalization, while also decreasing canonical transporter activity by altering DAT's conformational equilibrium. These results suggest that Kv2.1 clusters exert a spatially discrete homeostatic braking mechanism on DAT by inducing a relative increase in inward-facing transporters. Given recent reports of Kv2.1 dysregulation in neurological disorders, it is possible that alterations in the functional interaction between DAT and Kv2.1 affect dopamine neuron activity.