N-Terminal Phosphorylation of the Dopamine Transporter Is Required for Amphetamine-Induced Efflux

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake

Indocyanine green fluorescence angiography during liver and pancreas transplantation: a tool to integrate perfusion statement's evaluation

Background: Indocyanine green (ICG) fluorescence imaging is a promising tool for intraoperative decision-making during surgical procedures, in particular to assess organs perfusion. Methods: We used the ICG fluorescence during liver transplantations in six cirrhotic patients to help assessing the graft biliary duct perfusion in order to identify the appropriate level to perform the anastomosis. We also used ICG fluorescence also in five patients receiving kidney-pancreas transplantation to evaluate the perfusion levels of the duodenal stump of the pancreas graft. Results: Follow-up period for the patients was 12 months. The perioperative period was uneventful, no Hilary complications such as leaks or stenosis were reported after liver transplantation, no complications of the entero-enteric anastomoses occurred after pancreatic transplantation. Conclusions: ICG fluorescence seems to safely provide important objectiflable perfusion information during organ transplantation procedures that can integrate surgeon's expertise. In fact, detecting intraoperatively perfusion defects, it allows real time modifications on technical strategies potentially useful to reduce the feared risk of anastomotic leakage and consequent severe complications

Liver transplantation in recipients over 65 yr old: a single center experience

Introduction: The advanced age of the recipient is considered a "relative contraindication'' to liver transplantation (LT). However, recently some studies reported a morbidity rate and an overall survival comparable with those of younger patients. Here, we reported the outcome after LT in recipients aged >65 yr. Methods: Between January 2000 and December 2006, 565 LT was performed in 502 recipients in our institution. Of these, 34 were recipients of >65 yr old (aged group). We focused our study comparing: donor age, co-morbidities, model for end-stage liver disease (MELD) and American Society of Anesthesiologists (ASA) score, duration of operation, transfusions and outcome between the two groups (young/aged). Results: For the group aged >65: the mean donor age was 52.5 (range 16-75) yr and the graft weight 1339 g (890-1880 g). Co-morbidity was recorded in 25 (73.5%), coronary artery disease (CAD) in 17 (50%), diabetes mellitus (DM) and chronic renal insufficiency in four (11.7%) and chronic obstructive pulmonary disease (COPD) in three patients (8.8%). Mean MELD score was 14.9 (range 12-29) and ASA score was two in 15 (44.1%); and three in 19 (55.8%) recipients. Mean operation time was four h 45 min, three patients also received combined kidney transplantation. Twenty-five (73.5%) recipients received blood transfusions (mean 3.2). Morbidity was observed in 20 patients (58.8%); of these two had hepatic artery thrombosis requiring re-LT. Overall survival was 80% (40 months of follow-up), in particularly, at 30-d, one yr, three yr was 91%, 84%, 80%, respectively. The only two statistical differences reported (p = 0.02) are: the lower rate of CAD in the younger group of recipients (12%), compared with the aged group (50%) and the subsequently lower mortality rate secondary to cardiac causes in the younger group (1.4%) compared with aged group (8.8%). Conclusion: Our results suggest that the recipient age should not be considered an absolute contraindication for LT when the graft/recipient matching is optimal and when an adequate cardiac assessment is performed

PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux

The absence of functional peripheral myelin protein 22 (PMP22) is associated with shortened lifespan in rodents and severe peripheral nerve myelin abnormalities in several species including humans. Schwann cells and nerves from PMP22 knock-out (KO) mice show deranged cholesterol distribution and aberrant lipid raft morphology, supporting an unrecognized role for PMP22 in cellular lipid metabolism. To examine the mechanisms underlying these abnormalities, we studied Schwann cells and nerves from male and female PMP22 KO mice. Whole-cell current-clamp recordings in cultured Schwann cells revealed increased membrane capacitance and decreased membrane resistance in the absence of PMP22, which was consistent with a reduction in membrane cholesterol. Nerves from PMP22-deficient mice contained abnormal lipid droplets, with both mRNA and protein levels of apolipoprotein E (apoE) and ATP-binding cassette transporter A1 (ABCA1) being highly upregulated. Despite the upregulation of ABCA1 and apoE, the absence of PMP22 resulted in reduced localization of the transporter to the cell membrane and diminished secretion of apoE. The absence of PMP22 also impaired ABCA1-mediated cholesterol efflux capacity. In nerves from ABCA1 KO mice, the expression of PMP22 was significantly elevated and the subcellular processing of the overproduced protein was aberrant. In wild-type samples, double immunolabeling identified overlapping distribution of PMP22 and ABCA1 at the Schwann cell plasma membrane and the two proteins were coimmunoprecipitated from Schwann cell and nerve lysates. Together, these results reveal a novel role for PMP22 in regulating lipid metabolism and cholesterol trafficking through functional interaction with the cholesterol efflux regulatory protein ABCA1.SIGNIFICANCE STATEMENT Understanding the subcellular events that underlie abnormal myelin formation in hereditary neuropathies is critical for advancing therapy development. Peripheral myelin protein 22 (PMP22) is an essential peripheral myelin protein because its genetic abnormalities account for ∼80% of hereditary neuropathies. Here, we demonstrate that in the absence of PMP22, the cellular and electrophysiological properties of the Schwann cells' plasma membrane are altered and cholesterol trafficking and lipid homeostasis are perturbed. The molecular mechanisms for these abnormalities involve a functional interplay among PMP22, cholesterol, apolipoprotein E, and the major cholesterol-efflux transporter protein ATP-binding cassette transporter A1 (ABCA1). These findings establish a critical role for PMP22 in the maintenance of cholesterol homeostasis in Schwann cells

Early Hepatic Artery Thrombosis After Liver Transplantation: What is the Impact of the Arterial Reconstruction Type?

Objective Hepatic artery thrombosis (HAT) is the most severe vascular complication occurring after liver transplantation, with an incidence ranging from 2 to 9% in adults. Although the ideal arterial reconstruction is often described as a short and non-redundant anastomosis fashioned between the recipient and donor hepatic arteries, there is no strong evidence about this ideal reconstruction in the literature. The aim of this study was to assess the impact of the type of arterial reconstruction on early HAT after primary liver transplantation. Methods We retrospectively reviewed a contemporary MELD era cohort of 282 patients who underwent deceased donor primary liver transplantation from 2007 to 2012. Graft artery was classified as "short'' when the section was located at the proper/common hepatic artery or "long'' when the celiac trunk was used for anastomosis. Recipient arterial sites for arterial anastomosis were classified in three sites: (1) "distal'' (proper hepatic artery or common hepatic artery/gastro-duodenal bifurcation), (2) "intermediate'' (common hepatic artery) and (3) "proximal'' (celiac trunk-splenic artery-aorta). We used univariate and multivariate analyses to assess the impact of different types of arterial reconstruction on early HAT. Results Of 282 primary liver transplantations, 17 patients (6%) developed early HAT. Patients with and without early HAT had comparable demographic and operative data. The main anastomotic combination was short graft artery on the recipient-common hepatic artery (n = 111, 39%). A long graft artery was used in 91 patients (32%) and was associated with hepatic artery variations (56%; n = 51; p = 0.001). Arterial reconstructions using a long graft artery (p = 0.003), a recipient proximal site as celiac trunk-splenic artery-aorta (p = 0.02) and the combination of a long graft artery on the recipient distal hepatic artery (p = 0.02) were significantly associated with early HAT. The early HAT rate in patients with a long graft artery was not significantly different between patients with or without donor arterial variation (respectively, 12% (n = 6/51) vs. 12% (n = 5/40); p = 1). In multivariate analysis, the use of a long graft artery, whatever the recipient anastomosis site, was an independent risk factor of early HAT (OR 3.2; 95% CI 1.2-9; p = 0.02). Conclusion The type of arterial reconstruction used for arterial anastomosis during primary liver transplantation has an impact on the occurrence of early HAT. The use of a long graft artery is an independent risk factor of early HAT. Thereby, we recommend the use of a short graft artery with a direct path when feasible to reduce the occurrence of early HAT after primary liver transplantation