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Objectives: To evaluate differences in coronary microvascular function approximately 12 years after delivery between women who had had early- (EO-PE) or late- (LO-PE) onset pre-eclampsia and those who had had a normotensive pregnancy, and to assess the relationship between microvascular function and myocardial deformation at follow-up in these women.Methods: This was a case-control study of 88 women who had delivered at the Department of Gynecology and Obstetrics, Randers Regional Hospital, Randers, Denmark, between 1998 and 2008. Coronary flow velocity reserve (CFVR) was assessed by Doppler echocardiography approximately 12 years after delivery. Women were grouped according to whether the pregnancy had been complicated by EO-PE (n = 29) or LO-PE (n = 20), or had been normotensive (controls) (n = 39). Study groups were matched for maternal age and time since delivery. CFVR at follow-up was compared between the study groups. Regression analysis was used to assess the association between gestational age at onset of PE and CFVR. The association between left ventricular global longitudinal strain (LV-GLS) and CFVR at follow-up was also evaluated.Results: Resting coronary flow velocity assessed 12 years after delivery was comparable between the study groups (P = 0.55), whereas peak hyperemic flow velocity was significantly lower in the EO-PE group than in the LO-PE group (P < 0.01) and controls (P < 0.0001). As such, mean CFVR at follow-up was significantly lower in the EO-PE group than in the LO-PE group (P < 0.01) and controls (P < 0.0001). CFVR was < 2.5 in 48% of women in the EO-PE group, 25% of those in the LO-PE group and 8% of controls (P < 0.01). There was a significant positive association between gestational age at diagnosis of PE and CFVR at 12-year follow-up (β1 = 1.8 (95% CI, 0.8-2.9); P < 0.01). This relationship remained significant after adjustment for cardiovascular risk factors, namely mean arterial blood pressure, glycated hemoglobin level, body mass index, low-density lipoprotein cholesterol level and smoking status (P < 0.05). There was a significant association between LV-GLS and CFVR in women who had had PE (β1 = -1.5 (95% CI, -2.2 to -0.9); R2  = 0.33, P < 0.0001).Conclusions: Low gestational age at onset of PE, both as a continuous variable and when categorized as early onset, was associated with decreased CFVR 12 years after delivery. Nearly 50% of women who had had EO-PE had CFVR < 2.5 at follow-up. Reduced CFVR in women who had had PE was associated with subclinical myocardial dysfunction in terms of reduced LV-GLS. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

The aim was to use the DETECT algorithm to detect pulmonary arterial hypertension (PAH) in a single-centre Danish cohort of patients with systemic sclerosis (SSc) without known PAH. To assess for pulmonary vascular dysfunction patients referred for right heart catheterization were assessed with invasive hemodynamic exercise testing. 73 patients (aged 60 ± 10 years; 56 (77%) females) diagnosed with SSc for 13.5 (IQR 2.7 - 33.5) years underwent echocardiography, pulmonary funktion test, NT-proBNP and ECG. 19/73 (aged 62 ± 10 years, 12 (63%) females) patients were found to have an elevated STEP 2 score and underwent invasive hemodynamic assessment at rest and during exercise. At rest, 3/19 patients fulfilled the 2022 ESC guidelines PAH criteria (mean pulmonary arterial pressure (mPAP) > 20 mmHg, pulmonary arterial wedge pressure ≤ 15 mmHg and pulmonary vascular resistance > 2 wood units). Using the 2015 ESC guidelines only 1/19 patient had PAH (mPAP>25 mmHg and PAWP < 15 mmHg). With exercise 18/19 experienced mPAP > 30 mmHg and the majority (13/19) had an abnormal pulmonary pressure-flow relationship > 3 mmHg/ml/min (figure 1). Many patients (7/19) had elevated PAWP > 25 mmHg with exercise. In a Danish cohort of 73 prevalent scleroderma patients, the application of the DETECT algorithm identified only a few patients that fulfilled the guideline criteria for PAH. However, a large proportion of patients had an abnormal pulmonary vascular response to exercise and elevated PAWP with exercise. [ABSTRACT FROM AUTHOR]

Background: Wild-type transthyretin amyloid (ATTRwt) cardiomyopathy is increasingly recognized in the development of heart failure. The link between cardiac performance, hemodynamics, and mitochondrial function in disease stages of ATTRwt has not previously been studied but may provide new insights into the pathophysiology and clinical performance of the patients., Methods and Results: The study investigated 47 patients diagnosed with ATTRwt at Aarhus University Hospital, Denmark. Patients were stratified according to the disease stages of the National Amyloidosis Centre (NAC) as NAC I with low levels of NT-proBNP (N-terminal pro-B-type natriuretic peptide) (NAC I-L, n=14), NAC I with high levels NT-proBNP (NAC I-H, n=20), and NAC II-III (n=13). Exercise testing with simultaneous right heart catheterization was performed in all patients. Endomyocardial biopsies were collected from the patients and the mitochondrial oxidative phosphorylation capacity was assessed. All NAC disease groups, even in the NAC I-L group, a significant abnormal increase in biventricular filling pressures were noted during exercise while the filling pressures was normal or near normal at rest. The inotropic response to exercise was reduced with diminished increase in cardiac output which was significantly more pronounced in the NAC I-H (Diff. -2.4, 95% CI (-4.2: -0.7), P =0.00) and the NAC II-III group (Diff: -3.1 L/min, 95% CI (-5.2: -1.1), P =0.00) compared with the NAC I-L group. The pulmonary artery wedge pressure to cardiac output ratio at peak exercise was significantly different between NAC I-L and NAC II-III (Diff: 1.6 mm Hg*min/L, 95% CI (0.01:3.3, P =0.04)). Patients with ATTRwt had a reduced oxidative phosphorylation capacity which correlated to left ventricular mass but not to cardiac output capacity., Conclusions: An abnormal restrictive left ventricle and right ventricle response to exercise was demonstrated, even present in patients with early-stage ATTRwt. In more advanced disease stages a progressive impairment of the pressure-flow relationship was noted. The myocyte energetics is deranged but not associated to the contractile reserve or restrictive filling characteristics in ATTRwt.

Aims: This study aimed to study the prognostic value of myocardial oxygen consumption (MVO 2 ) and myocardial external efficiency (MEE) from 11 C-acetate positron emission tomography (PET) in cardiac amyloidosis (CA) patients., Methods and Results: Forty-eight CA patients, both transthyretin (ATTR) and immunoglobulin light chain (AL) amyloidosis, and 20 controls were included. All subjects were examined with 11 C-acetate PET and echocardiography. MVO 2 , forward stroke volume (FSV), and left ventricular mass (LVM) were derived from 11 C-acetate PET and used to calculate MEE. CA patients were followed for survival and the prognostic impact of clinical, echocardiographic, and 11 C-acetate PET parameters was analysed. MVO 2 and MEE were reduced in CA compared with controls, but without significant difference between deceased and surviving CA patients. The ratio of 11 C-acetate PET-derived FSV and LVM was also reduced in CA and significantly lowered in deceased patients compared with survivors. In univariate analysis, New York Heart Association class, N-terminal pro-brain natriuretic peptide, and the 11 C-acetate PET parameters FSV/LVM and MEE were the strongest prognostic factors. Of the 11 C-acetate PET parameters, FSV/LVM was the strongest survival predictor with hazard ratio of 0.56 per 0.1 mL/g (95% confidence interval 0.39-0.81, P = 0.002) and independently prognostic in a multivariate model. MEE significantly separated deceased from surviving CA patients with the cut-off of 15.7% (P = 0.032). Survival was significantly shorter with FSV/LVM below 0.27 mL/g (P < 0.001), also when separating AL- and ATTR-CA., Conclusions: Reduced MEE was associated with shorter survival in CA patients, but FSV/LVM was the strongest survival predictor and the only independently prognostic 11 C-acetate PET parameter in multivariate analysis., (© 2023 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Background: Cardiac amyloidosis (CA) is a restrictive and infiltrative cardiomyopathy, characterized by increased biventricular filling pressures and low output. Symptoms are predominantly of right heart origin. The role of right ventricular (RV) myocardial blood flow (MBF) in CA has not been studied., Objectives: This study aimed to first associate RV MBF measured by using positron emission tomography (PET) with reference standards of RV pressures and then to explore its prognostic value in CA., Methods: Cardiac PET was performed at rest in 52 patients with CA and 9 healthy control subjects. MBF was quantified from the right and left ventricles by using 11 C-acetate, 15 O-water, or both (n = 25). RV pressure was measured invasively or by echocardiography. Associations between biventricular MBF toward symptoms, RV function, and outcome (death or acute heart failure) were studied in patients with CA., Results: MBF of the right ventricle (MBF RV ) and the ratio of MBF RV and MBF of the left ventricle (MBF RV/LV ) for the 2 tracers were significantly correlated (r > 0.92). MBF RV was directly correlated with RV systolic pressures with both tracers (P ≤ 0.005). MBF LV was inversely correlated with wall thickness (P < 0.0001). MBF RV/LV was significantly associated with N-terminal pro-B-type natriuretic peptide levels, NYHA functional class, RV pressures, and RV systolic function (all; P < 0.001). Twenty-six cardiac events (25 deaths) occurred during follow-up (median 44 months). MBF RV/LV higher than 56% was associated with a diagnosis of pulmonary hypertension (AUC: 0.96 [95% CI: 0.91-1.00]; P < 0.0001); and predicted outcome with HR: 9.0 (95% CI: 4.2-14.5), P < 0.0001)., Conclusions: Measurements of MBF RV using PET are feasible, as confirmed with 2 different tracers. Imbalance between RV and LV myocardial perfusion is associated with increased RV load and adverse events in cardiac amyloidosis., Competing Interests: Funding Support and Author Disclosures This study was supported by grants from Lundbeck foundation (grant number R192-2015-966, awarded to Dr Harms) and Swedish Heart-Lung Foundation (grant numbers 20160746 and 20190593 awarded to Dr Sorensen). Drs Harms, Tolbod, and Sorensen are co-founders and co-owners of MedTrace Pharma A/S. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. All rights reserved.)

Aims: Wild-type transthyretin cardiac amyloidosis (ATTRwt) is an infiltrative cardiomyopathy with a poor prognosis. The condition is associated with carpal tunnel syndrome (CTS), which often precedes the ATTRwt diagnosis by several years. The aim of the study was (i) to screen patients with a recent history of CTS for ATTRwt using red flags, (ii) to determine whether patients with screened ATTRwt had less advanced disease compared with patients with clinical ATTRwt, and (iii) to assess the sensitivity and specificity of known red flags in ATTRwt., Methods and Results: Patients aged ≥60 years at the time of CTS surgery were invited for screening. Red flags were defined as elevated biomarker levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP) or cardiac troponin, an electrocardiogram pattern associated with ATTRwt, left ventricular hypertrophy (LVH), and impaired longitudinal strain with apical sparring. All patients with a red flag were referred for a diagnostic scintigraphy. Patients with ATTRwt diagnosed by screening were compared with patients with clinical ATTRwt (n = 51) matched by age, gender, and CTS surgery. Among the 120 enrolled subjects (mean age 74.5 years, 90% male), the suspicion of ATTR was raised in 67 (55.8%), and 10 (8.3%) were diagnosed with ATTRwt. Patients identified with ATTRwt were predominantly asymptomatic and had mildly elevated NT-proBNP, mildly increased LVH, preserved left ventricular ejection fraction, and systolic longitudinal function, which differed significantly from clinical ATTRwt controls (P < 0.001)., Conclusions: The study found an ATTRwt prevalence of 8.3% in a population of age and gender-selected patients with a recent history of CTS. The identified patients with ATTRwt had less structural and functional cardiac involvement than clinical ATTRwt controls., (© 2022 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

Background: Informal carers of people with dementia report having unmet needs for support and few supportive interventions have been shown to be effective. There is a need to develop needs assessment instruments and supportive interventions with a holistic and person-centred approach to meet the various and complex needs of carers. The aim of this study was to provide an overview of carers' support needs when caring for people with dementia with the objectives to map and synthesise knowledge on key concepts of carers' support needs., Methods: A scoping review methodology was used. A literature search was conducted in PsycINFO, CINAHL, PubMed and EMBASE between January 2007 and October 2019. Three authors independently selected articles meeting the inclusion criteria, and data were extracted using a matrix developed for that purpose. Inductive content analysis was used to synthesise key concepts of carers' support needs., Results: The search identified 2748 articles after removing duplicates, and 122 articles were included in the mapping of carers' support needs. Synthesising carers' support needs indicated that the full extent of support needs emerges in the interaction between the carer and the person cared for and that it is possible to categorise support needs into four key concepts related to: 1) the carer as a person, 2) managing being a carer, 3) providing care, and 4) knowledge of dementia., Conclusion: The findings of this study help to map a framework describing carers' support needs that may guide the development of future needs assessment instruments and supportive interventions., (© 2020 Nordic College of Caring Science.)

Background Despite correction of the atrial septal defect (ASD), patients experience atrial fibrillation frequently and have increased morbidity and mortality. We examined physical capacity, cardiac performance, and invasive hemodynamics in patients with corrected ASD. Methods and Results Thirty-eight corrected patients with isolated secundum ASD and 19 age-matched healthy controls underwent right heart catheterization at rest and during exercise with simultaneous expired gas assessment and echocardiography. Maximum oxygen uptake was comparable between groups (ASD 32.7±7.7 mL O 2 /kg per minute, controls 35.2±7.5 mL O 2 /kg per minute, P =0.3), as was cardiac index at both rest and peak exercise. In contrast, pulmonary artery wedge v wave pressures were increased at rest and peak exercise (rest: ASD 14±4 mm Hg, controls 10±5 mm Hg, P =0.01; peak: ASD 25±9 mm Hg, controls 14±9 mm Hg, P =0.0001). The right atrial v wave pressures were increased at rest but not at peak exercise. The transmural filling pressure gradient (TMFP) was higher at peak exercise among patients with ASD (10±6 mm Hg, controls 7±3 mm Hg, P =0.03). One third of patients with ASD demonstrated an abnormal hemodynamic exercise response defined as mean pulmonary artery wedge pressure ≥25 mm Hg and/or mean pulmonary artery pressure ≥35 mm Hg at peak exercise. These patients had significantly elevated peak right and left atrial a wave pressures, right atrial v wave pressures, pulmonary artery wedge v wave pressures, and transmural filling pressure compared with both controls and patients with ASD with a normal exercise response. Conclusions Patients with corrected ASD present with elevated right and in particular left atrial pressures at rest and during exercise despite preserved peak exercise capacity. Abnormal atrial compliance and systolic atrial function could predispose to the increased long-term risk of atrial fibrillation. Registration Information clinicaltrials.gov. Identifier: NCT03565471.

Objectives: This dual-site study evaluated the diagnostic accuracy of the method., Background: Pittsburgh compound ([ 11 C]PIB) positron emission tomography (PIB-PET) has shown promise as a specific and noninvasive method for the diagnosis of cardiac amyloidosis (CA)., Methods: The study had 2 parts. In the initial study, 51 subjects were included, 36 patients with known CA and increased wall thickness (15 immunoglobulin light chain [AL] and 21 transthyretin [ATTR] amyloidosis) and 15 control patients (7 were nonamyloid hypertrophic and 8 healthy volunteers). Subjects underwent PIB-PET and echocardiography. Sensitivity and specificity of PIB-PET were established for 2 simple semiquantitative approaches, standardized uptake value ratio (SUVR) and retention index (RI). The second part of the study included 11 amyloidosis patients (5 AL and 6 hereditary ATTR) without increased wall thickness to which the optimal cutoff values of SUVR (>1.09) and RI (>0.037 min -1 ) were applied prospectively., Results: The diagnostic accuracy of visual inspection of [ 11 C]PIB uptake was 100% in discriminating CA patients with increased wall thickness from controls. Semiquantitative [ 11 C]PIB uptake discriminated CA from controls with a 94% (95% confidence interval [CI]: 80% to 99%) sensitivity for both SUVR and RI and specificity of 93% (95% CI: 66% to 100%) for SUVR and 100% (95% CI: 75% to 100%) for RI. [ 11 C]PIB uptake was significantly higher in AL-CA than in ATTR-CA patients (p < 0.001) and discriminated AL-CA from controls with 100% (95% CI: 88% to 100%) accuracy for both the semiquantitative measures. In the prospective group without increased wall thickness, RI was elevated compared to controls (p = 0.001) and 5 of 11 subjects were evaluated as [ 11 C]PIB PET positive., Conclusions: In a dual-center setting, [ 11 C]PIB PET was highly accurate in detecting cardiac involvement in the main amyloid subtypes, with 100% accuracy in AL amyloidosis. A proportion of amyloidosis patients without known cardiac involvement were [ 11 C]PIB PET positive, indicating that the method may detect early stages of CA., (Copyright © 2020 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: The aim was to investigate the effect of Electromyography (EMG)-biofeedback guided exercises (BIONEX) on shoulder pain and function in participants with subacromial pain syndrome (SPS)., Methods: Twenty-five women and 24 men (19-67 years), diagnosed with SPS, were randomised to BIONEX or the same exercises without EMG-biofeedback (NEX). Primary outcome was shoulder pain during the past 7 days (Numeric Pain Rating Scale (NPRS)). Secondary outcomes included self-reported (Disability of Arm Shoulder and Hand (DASH), Oxford Shoulder Score (OSS)), and measured shoulder function (surface EMG from upper trapezius, lower trapezius and serratus anterior) in mean and ratios of % of maximum voluntary EMG (%MVE) and onset time (msec), during arm tasks with 0, 1 and 3 kg., Results: There was no group difference (BIONEX versus NEX) in changed shoulder pain (NPRS, mean difference 0.18 (95% CI. -1.56; 1.19)), self-reported or measured shoulder function. Both groups, however, showed significant within-group improvements on self-reported outcomes (NPRS, DASH, OSS), only clinically relevant on NPRS (BIONEX 2.23 (SD 2.47); NEX 2.04 (SD 2.29))., Conclusion: BIONEX and NEX were both effective in reducing pain to a clinically relevant level, while EMG-biofeedback did not make a difference. The current neuromuscular shoulder exercise protocol is recommended., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Protein interacting with C-kinase 1 (PICK1) is a widely expressed scaffold protein known to interact via its PSD-95/discs-large/ZO-1 (PDZ)-domain with several membrane proteins including the dopamine (DA) transporter (DAT), the primary target for cocaine's reinforcing actions. Here, we establish the importance of PICK1 for behavioral effects observed after both acute and repeated administration of cocaine. In PICK1 knock-out (KO) mice, the acute locomotor response to a single injection of cocaine was markedly attenuated. Moreover, in support of a role for PICK1 in neuroadaptive changes induced by cocaine, we observed diminished cocaine intake in a self-administration paradigm. Reduced behavioral effects of cocaine were not associated with decreased striatal DAT distribution and most likely not caused by the ∼30% reduction in synaptosomal DA uptake observed in PICK1 KO mice. The PICK1 KO mice demonstrated preserved behavioral responses to DA receptor agonists supporting intact downstream DA receptor signaling. Unexpectedly, we found a prominent increase in striatal DA content and levels of striatal tyrosine hydroxylase (TH) in PICK1 KO mice. Chronoamperometric recordings showed enhanced DA release in PICK1 KO mice, consistent with increased striatal DA pools. Viral-mediated knock-down (KD) of PICK1 in cultured dopaminergic neurons increased TH expression, supporting a direct cellular effect of PICK1. In summary, in addition to demonstrating a key role of PICK1 in mediating behavioral effects of cocaine, our data reveal a so far unappreciated role of PICK1 in DA homeostasis that possibly involves negative regulation of striatal TH levels.

The majority of renal cell carcinomas (RCCs) occur within the 7th decade of life, uncommonly arising in adults ≤46 years. We reviewed the clinicopathologic features of early onset RCC and evaluated the role of immunohistochemistry (IHC) in potentially identifying diagnoses of newly recognized RCC subtypes that may have been previously misclassified. A retrospective review was performed from 2011-2016 for cases of RCC. Early onset RCC was defined as ≤46 years of age. Clinicopathologic findings and hematoxylin and eosin (H&E) slides were reviewed on early onset RCC patients. IHC was performed on all cases previously diagnosed as unclassified or papillary. Clinicopathologic findings were compared to a control group of RCC patients >46 years over the same time period. We identified 98/598 (16.4%) early onset RCCs. The median age in the early onset RCC and control group was 38.4 and 62.8 years, respectively. The early onset RCC group contained 33/96 (34.3%) females and 63/96 (65.6%) males, including 52/96 (54.2%) whites, 39/96 (40.6%) African Americans, 4/96 (4.2%) Hispanics, and 1/96 (1%) Asian. Nonwhites were significantly more likely to develop early onset RCC (P=.004). Early onset RCCs included 52% clear cell, 28.6% papillary, 8.2% unclassified, 5.1% chromophobe, 3.1% clear cell papillary(CCP), and 3 other rare tumors. Six unclassified and 26 papillary RCCs had tissue available for IHC. Two of 6 (33.3%) unclassified RCCs were reclassified (1 CCP, 1 Xp11 translocation). One of 26 (3.8%) papillary RCCs was reclassified as CCP. Early onset RCCs were more likely to occur in nonwhites (P=.004), be lower stage (P=.03), and undergo partial nephrectomy (P=.002). Few unclassified and papillary tumors were reclassified with IHC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.The dopamine transporter (DAT) has a crucial role in the regulation of neurotransmission. Here, the authors use super-resolution imaging to show that DAT clusters into cholesterol-dependent membrane regions that are reversibly regulated by ionotropic glutamate receptors activation.

Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000-30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1-enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1-eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1-eGFP and wild-type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary targets for antidepressants for therapeutic intervention of mood disorders. Our previous studies have identified the involvement of several signalling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether Akt/PKB (protein kinase) regulates SERT function is not known. In the present study, we made the novel observation that inhibition of Akt resulted in the down-regulation of SERT function through the regulation of SERT trafficking and phosphorylation. Akt inhibitor Akt X {10-(4'-[N-diethylamino)butyl]-2-chlorophenoxazine} reduced the endogenously phosphorylated Akt and significantly decreased 5-HT uptake and 5-HT-uptake capacity. Furthermore, SERT activity is also reduced by siRNA down-regulation of total and phospho-Akt levels. The reduction in SERT activity is paralleled by lower levels of cell-surface SERT protein, reduced SERT exocytosis with no effect on SERT endocytosis and accumulation of SERT in intracellular endocytic compartments with the most prominent localization to late endosomes and lysosomes. Akt2 inhibitor was more effective than Akt1 inhibitor in inhibiting SERT activity. Inhibition of downstream Akt kinase GSK3α/β (glycogen synthase kinase α/β) stimulates SERT function. Akt inhibition leads to a decrease in SERT basal phosphorylation. Our results provide evidence that Akt regulates SERT function and cell-surface expression by regulating the intracellular SERT distribution and plasma membrane availability, which perhaps may be linked to SERT phosphorylation state. Thus any changes in the activation of Akt and/or GSK3α/β could alter SERT-mediated 5-HT clearance and subsequently serotonergic neurotransmission.

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the "long loop" recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells.

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.