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.

Methamphetamine is a widely abused psychostimulant and one of the main targets of dopamine transporter (DAT). Methamphetamine reduces DAT‐mediated dopamine uptake and stimulates dopamine efflux leading to increased synaptic dopamine levels many folds above baseline. Methamphetamine also targets DAT‐expressing peripheral immune cells, reduces wound healing and increases infection susceptibility. Peripheral immune cells such as myeloid cells, B cells and T cells express DAT. DAT activity on monocytes and macrophages exhibits immune suppressive properties via an autocrine paracrine mechanism, where deletion or inhibition of DAT activity increases inflammatory responses. In this study, utilizing a mouse model of daily single dose of methamphetamine administration, we investigated the impact of the drug on DAT expression in peripheral immune cells. We found in methamphetamine‐treated mice that DAT expression was down‐regulated in most of the innate and adaptive immune cells. Methamphetamine did not increase or decrease the total number of innate and adaptive immune cells but changed their immunophenotype to low‐DAT‐expressing phenotype. Moreover, serum cytokine distributions were altered in methamphetamine‐treated mice. Therefore, resembling its effect in the CNS, in the periphery, methamphetamine regulates DAT expression on peripheral immune cell subsets, potentially describing methamphetamine regulation of peripheral immunity.

Background: Ring sideroblasts (RS) are erythroblasts with iron-loaded mitochondria, appears as blue granules when stained with Prussian blue, and are characteristic of myelodysplastic syndromes (MDS) subgroups refractory anemia with ring sideroblasts (RARS), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), and RARS with thrombocytosis (RARS-T) but are also infrequently found in other MDS groups. The etiology of RS remains unclear. Molecular genetic analysis of MDS patients with RS (MDS-RS) using exome and targeted sequencing identified mutations in several genes including SF3B1. While the RS phenotype was shown to be strongly associated with SF3B1 mutations, the association of SF3B1 mutations with better prognosis remained controversial. Also, there remained several patients who do not show any mutations in SF3B1 suggesting there are additional genes associated with RS phenotype. In addition to SF3B1 mutations, RARS-T patients show mutations in JAK2 (V617F and Exon 12), MPL and CALR genes, frequently mutated in Essential Thrombocytopenia (ET). But there remained a high number of patients who do not show any mutation in JAK2, MPL and CALR genes suggesting additional genes are associated with thrombocytosis. In order to gain further insight into the molecular genetics of MDS-RS patients and to elucidate their clinical significance we screened a large cohort of patients with RS (RARS/RCMD-RS) and a subgroup with thrombocytosis (RARS-T). Methods: This study is approved by the Institutional Review Board of Columbia University and informed consent was obtained from all the individuals participated in the study. Bone marrow mono nuclear cells were isolated from bone marrow aspirate and DNA was extracted using DNeasy Blood and Tissue kit. To screen for most frequent mutations in SF3B1 (exon 14 and 15), MPL (exon 10), JAK2 (exon 12), and CALR (exon 9) genes, primers were designed to amplify the exons and the exon-intron junctions using PCR and the amplified and purified PCR products were sequenced using Sanger sequencing on both strands. To screen for mutations V617F in JAK2, primers were designed to carry out allele specific PCR. Demographic and clinical data is collected from patient’s charts/reports. Statistical analysis including survival curve (Kaplan–Meier method) was performed using GraphPad Prism Software. Results: A total of 209 patients with RS phenotype were screened for SF3B1 mutations. Mutations in SF3B1 gene were detected in 62% (130/209) of RS patients, including 85% (23/27) of RARS-T and 59% (107/182) of RARS/RCMD-RS patients. Among all the SF3B1 mutations, K700E was the most frequent mutation (60%) followed by mutations at H662. Clinical significance of SF3B1 mutations on overall survival, using Kaplan-Meier method, showed SF3B1 mutations were associated with better prognosis (Fig 1). The median survival of patients with SF3B1 mutation is 110 months compared to those without mutations, 70 months (p value < 0.034). Studies to screen SF3B1 negative patients for additional mutations are being carried out using exome sequencing to identify genes associated with RS phenotype. In addition to SF3B1, RARS-T patients were also screened for JAK2 V617F and exon 12 mutations. JAK2 V617F mutations were detected in 11% of RARS-T patients and no mutations were found in JAK2 exon 12. Patients negative for JAK2 were screened for mutations in MPL and CALR genes. No mutations were found in MPL but 8% (2/23) of those negative for JAK2 were found to have CALR mutations. Both CALR mutations were frameshift mutations that alter the C-terminus thus abolishing the KDEL sequence required for CALR function. There are still 80% RARS-T patients in our cohort who do not show any mutations in JAK2, MPL, CALR genes. Conclusions: The association of SF3B1 mutation with prognosis is controversial with some studies suggesting a better prognosis while other reports no effect. Our study, using a large cohort of well-characterized RS patients provides an Independent verification of the observation that SF3B1 mutations are associated with better overall survival. There remains a large group of patients (40% in RARS/RCMD-RS groups) without SF3B1 mutation but with a RS phenotype suggesting yet other gene(s) is associated with RS phenotype. Similarly, there remain patients who do not show any mutations in JAK2, MPL and CALR suggesting other genes are involved in the etiology of thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

ID 52323Poster Board 370 Dopamine transporter (DAT) is a master regulator of dopamine transmission in the brain. Mutation in DAT structure is implicated in familial Parkinson's disease. In addition, DAT is one of the main targets of psychostimulants, therefore, DAT is conventionally studied in central nervous system (CNS) and in the context of neurological and neuropsychiatric diseases. But, in addition to the brain, DAT is also expressed at the plasma membrane of myeloid and lymphoid cells in the periphery. We found DAT activity reduces macrophage responses to immune stimulation through autocrine/paracrine signaling. In human monocyte-derived macrophages, we identified an immunosuppressive role for DAT, where inhibition of DAT activity exaggerated macrophages' phagocytic response and enhanced production of inflammatory mediators such as IL-6, CCL2, and TNFa. Consistent with our ex-vivo studies, our in vivo data in a mouse model with genetic deletion of DAT (DAT KO) also showed an immunosuppressive role for DAT. Unlike WT littermates, LPS-induced immune stimulation in DAT KO mice resulted in elevated baseline inflammation, exaggerated phagocytosis, increased B-cell and T-cell responses. In addition, our studies in 96 PD patients and mouse models of PD revealed the disease relevance of increased DAT activity as measured by increased number of DAT expressing monocytic suppressor cells. These data collectively point to the importance of DAT activity in peripheral immune responses. Our current and ongoing studies seek to determine the impact of DAT deletion and its restoration on the CNS immune landscape and the ensuing changes in peripheral immunity. We will first determine whether DAT deletion and/or LPS-mediated immune stimulation alters microglial number, morphology, and their contact with dopaminergic nuclei in DAT-expressing brain regions such as midbrain, striatum, median forebrain bundle and hypothalamus. To determine which dopaminergic nuclei is critical for top-down (CNSperiphery) regulation of peripheral immunity, we will virally restore DAT in each brain region and monitor peripheral immunity, as described above. The completion of this study will reveal the contribution of DAT activity in the regulation of CNS-to-periphery neuroimmune interactions, providing a key opening into a new field for dopamine transporter biology.