Neonatal Diet Type and Associations With Adverse Feeding Outcomes in Neonates With Critical Congenital Heart Defects.

BACKGROUND: Neonates with critical congenital heart defects (CCHD neonates) experience high rates of feeding intolerance, necrotizing enterocolitis (NEC), and malnutrition. The benefits of human milk and direct chest/breastfeeding are well known, but research is limited in CCHD neonates. Therefore, the purpose of this study is to examine the impact of neonatal diet and feeding modality on the incidence of feeding intolerance, NEC, and malnutrition among a cohort of CCHD neonates. METHODS: A single-center retrospective study was conducted using electronic health record data of CCHD neonates admitted to a cardiac intensive care unit between April 2016 and April 2020. Regression models were fit to analyze associations between neonatal diet, feed modality, and adverse feeding outcomes. RESULTS: Seventy-four CCHD neonates were included. Increased days of direct chest/breastfeeding were associated with fewer signs of gastrointestinal distress ( P = .047) and bloody stools ( P = .021). Enteral feeding days of "all human milk" were associated with higher growth trajectory ( P < .001). CONCLUSIONS: Human milk and direct chest/breastfeeding may be protective against some adverse feeding outcomes for CCHD neonates. Larger, multicenter cohort studies are needed to continue investigating the effects of neonatal diet type and feeding modality on the development of adverse feeding outcomes in this unique population.

Predictors of Mediastinal Exploration While on Extracorporeal Membrane Oxygenation After Pediatric Cardiac Surgery.

Cardiac surgical patients requiring extracorporeal membrane oxygenation (ECMO) are at increased risk for hemorrhage due to necessary anticoagulation, in-situ cannulas, and disturbed hemostasis. We performed a retrospective, cross-sectional study of patients 0-18 years old in our cardiac intensive care unit (CICU) cannulated to ECMO within 48 h of cardiopulmonary bypass. The 69 patients included in the study were divided into three analysis groups based on serial chest tube output per hour: no bleeding (NB) on admission to the CICU (21/69, 30%), bleeding stopped (BS) with medical management (26/69, 38%), bleeding requiring emergent mediastinal exploration (BME) (22/69, 32%). The NB group had a more favorable coagulation profile upon admission to the CICU (PTT 53 s NB, 105 s BS, 83 s BME p < 0.001, ACT 169 s NB, 225 s BS, 211 s BME, p =0.013). Only chest tube output during the first three postcannulation hours remained associated with the need for mediastinal exploration by multivariable analysis. An average chest-tube output of 11.6 mL/kg/h during the first three hours had the highest percentage of patients classified correctly (84%) for requiring mediastinal exploration during their ECMO run (sensitivity 91%, specificity 81%).

Clinical factors associated with microstructural connectome related brain dysmaturation in term neonates with congenital heart disease.

Objective: Term congenital heart disease (CHD) neonates display abnormalities of brain structure and maturation, which are possibly related to underlying patient factors, abnormal physiology and perioperative insults. Our primary goal was to delineate associations between clinical factors and postnatal brain microstructure in term CHD neonates using diffusion tensor imaging (DTI) magnetic resonance (MR) acquisition combined with complementary data-driven connectome and seed-based tractography quantitative analyses. Our secondary goal was to delineate associations between mild dysplastic structural brain abnormalities and connectome and seed-base tractography quantitative analyses. These mild dysplastic structural abnormalities have been derived from prior human infant CHD MR studies and neonatal mouse models of CHD that were collectively used to calculate to calculate a brain dysplasia score (BDS) that included assessment of subcortical structures including the olfactory bulb, the cerebellum and the hippocampus. Methods: Neonates undergoing cardiac surgery for CHD were prospectively recruited from two large centers. Both pre- and postoperative MR brain scans were obtained. DTI in 42 directions was segmented into 90 regions using a neonatal brain template and three weighted methods. Clinical data collection included 18 patient-specific and 9 preoperative variables associated with preoperative scan and 6 intraoperative (e.g., cardiopulmonary bypass and deep hypothermic circulatory arrest times) and 12 postoperative variables associated with postoperative scan. We compared patient specific and preoperative clinical factors to network topology and tractography alterations on a preoperative neonatal brain MRI, and intra and postoperative clinical factors to network topology alterations on postoperative neonatal brain MRI. A composite BDS was created to score abnormal findings involving the cerebellar hemispheres and vermis, supratentorial extra-axial fluid, olfactory bulbs and sulci, hippocampus, choroid plexus, corpus callosum, and brainstem. The neuroimaging outcomes of this study included (1) connectome metrics: cost (number of connections) and global/nodal efficiency (network integration); (2) seed based tractography methods of fractional anisotropy (FA), radial diffusivity, and axial diffusivity. Statistics consisted of multiple regression with false discovery rate correction (FDR) comparing the clinical risk factors and BDS (including subcortical components) as predictors/exposures and the global connectome metrics, nodal efficiency, and seed based- tractography (FA, radial diffusivity, and axial diffusivity) as neuroimaging outcome measures. Results: A total of 133 term neonates with complex CHD were prospectively enrolled and 110 had analyzable DTI. Multiple patient-specific factors including d-transposition of the great arteries (d-TGA) physiology and severity of impairment of fetal cerebral substrate delivery (i.e., how much the CHD lesion alters typical fetal circulation such that the highest oxygen and nutrient rich blood from the placenta are not directed toward the fetal brain) were predictive of preoperative reduced cost (p < 0.0073) and reduced global/nodal efficiency (p < 0.03). Cardiopulmonary bypass time predicted postoperative reduced cost (p < 0.04) and multiple postoperative factors [extracorporeal membrane oxygenation (ECMO), seizures and cardiopulmonary resuscitation (CPR)] were predictive of postoperative reduced cost and reduced global/nodal efficiency (p < 0.05). Anthropometric measurements (weight, length, and head size) predicted tractography outcomes. Total BDS was not predictive of brain network topology. However, key subcortical components of the BDS score did predict key global and nodal network topology: abnormalities of the cerebellum predicted reduced cost (p < 0.0417) and of the hippocampus predicted reduced global efficiency (p < 0.0126). All three subcortical structures predicted unique alterations of nodal efficiency (p < 0.05), including hippocampal abnormalities predicting widespread reduced nodal efficiency in all lobes of the brain, cerebellar abnormalities predicting increased prefrontal nodal efficiency, and olfactory bulb abnormalities predicting posterior parietal-occipital nodal efficiency. Conclusion: Patient-specific (d-TGA anatomy, preoperative impairment of fetal cerebral substrate delivery) and postoperative (e.g., seizures, need for ECMO, or CPR) clinical factors were most predictive of diffuse postnatal microstructural dysmaturation in term CHD neonates. Anthropometric measurements (weight, length, and head size) predicted tractography outcomes. In contrast, subcortical components (cerebellum, hippocampus, olfactory) of a structurally based BDS (derived from CHD mouse mutants), predicted more localized and regional postnatal microstructural differences. Collectively, these findings suggest that brain DTI connectome and seed-based tractography are complementary techniques which may facilitate deciphering the mechanistic relative contribution of clinical and genetic risk factors related to poor neurodevelopmental outcomes in CHD.

Harmonization of Multi-Center Diffusion Tensor Tractography in Neonates with Congenital Heart Disease: Optimizing Post-Processing and Application of ComBat.

Advanced brain imaging of neonatal macrostructure and microstructure, which has prognosticating importance, is more frequently being incorporated into multi-center trials of neonatal neuroprotection. Multicenter neuroimaging studies, designed to overcome small sample sized clinical cohorts, are essential but lead to increased technical variability. Few harmonization techniques have been developed for neonatal brain microstructural (diffusion tensor) analysis. The work presented here aims to remedy two common problems that exist with the current state of the art approaches: 1) variance in scanner and protocol in data collection can limit the researcher's ability to harmonize data acquired under different conditions or using different clinical populations. 2) The general lack of objective guidelines for dealing with anatomically abnormal anatomy and pathology. Often, subjects are excluded due to subjective criteria, or due to pathology that could be informative to the final analysis, leading to the loss of reproducibility and statistical power. This proves to be a barrier in the analysis of large multi-center studies and is a particularly salient problem given the relative scarcity of neonatal imaging data. We provide an objective, data-driven, and semi-automated neonatal processing pipeline designed to harmonize compartmentalized variant data acquired under different parameters. This is done by first implementing a search space reduction step of extracting the along-tract diffusivity values along each tract of interest, rather than performing whole-brain harmonization. This is followed by a data-driven outlier detection step, with the purpose of removing unwanted noise and outliers from the final harmonization. We then use an empirical Bayes harmonization algorithm performed at the along-tract level, with the output being a lower dimensional space but still spatially informative. After applying our pipeline to this large multi-site dataset of neonates and infants with congenital heart disease (n= 398 subjects recruited across 4 centers, with a total of n=763 MRI pre-operative/post-operative time points), we show that infants with single ventricle cardiac physiology demonstrate greater white matter microstructural alterations compared to infants with bi-ventricular heart disease, supporting what has previously been shown in literature. Our method is an open-source pipeline for delineating white matter tracts in subject space but provides the necessary modular components for performing atlas space analysis. As such, we validate and introduce Diffusion Imaging of Neonates by Group Organization (DINGO), a high-level, semi-automated framework that can facilitate harmonization of subject-space tractography generated from diffusion tensor imaging acquired across varying scanners, institutions, and clinical populations. Datasets acquired using varying protocols or cohorts are compartmentalized into subsets, where a cohort-specific template is generated, allowing for the propagation of the tractography mask set with higher spatial specificity. Taken together, this pipeline can reduce multi-scanner technical variability which can confound important biological variability in relation to neonatal brain microstructure.

Acute Neurologic Injury in Children Admitted to the Cardiac Intensive Care Unit.

BACKGROUND: Children with acquired and congenital heart disease both have low mortality but an increased risk of neurologic morbidity that is multifactorial. Our hypothesis was that acute neurologic injuries contribute to mortality in such children and are an important cause of death. METHODS: All admissions to the pediatric cardiac intensive care unit (CICU) from January 2011 through January 2015 were retrospectively reviewed. Patients were assessed for any acute neurologic events (ANEs) during admission, as defined by radiologic findings or seizures documented on an electroencephalogram. RESULTS: Of the 1,573 children admitted to the CICU, the incidence of ANEs was 8.6%. Mortality of the ANE group was 16.3% compared with 1.5% for those who did not have an ANE. The odds ratio for death with ANEs was 8.55 (95% confidence interval, 4.56 to 16.03). Patients with ANEs had a longer hospital length of stay than those without ANEs (41.4 ± 4 vs 14.2 ± 0.6 days; p < 0.001). Need for extracorporeal membrane oxygenation, previous cardiac arrest, and prematurity were independently associated with the presence of an ANE. CONCLUSIONS: Neurologic injuries are common in pediatric CICUs and are associated with an increase in mortality and hospital length of stay. Children admitted to the CICU are likely to benefit from improved surveillance and neuroprotective strategies to prevent neurologic death.

Associations of Perioperative Renal Oximetry Via Near-Infrared Spectroscopy, Urinary Biomarkers, and Postoperative Acute Kidney Injury in Infants After Congenital Heart Surgery: Should Creatinine Continue to Be the Gold Standard?

OBJECTIVES: Examine the relationship between perioperative renal regional tissue oximetry, urinary biomarkers, and acute kidney injury in infants after congenital cardiac surgery with cardiopulmonary bypass. DESIGN: Prospective, observational. SETTING: Cardiac operating room and cardiac ICU. PATIENTS: Neonates and infants without history of kidney injury or anatomic renal abnormality. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Renal regional tissue oximetry was measured intraoperatively and for 48 hours postoperatively. Urinary levels of neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2 together with insulin-like growth factor-binding protein 7 were measured preoperatively, 2, 12, and 24 hours postoperatively. Patients were categorized as no acute kidney injury, stage 1, or Stage 2-3 acute kidney injury using the Kidney Disease: Improving Global Outcomes criteria with 43 of 70 (61%) meeting criteria for any stage acute kidney injury. Stage 2-3 acute kidney injury patients had higher tissue inhibitor of metalloproteinases 2, insulin-like growth factor-binding protein 7 at 2 hours (0.3 vs 0.14 for stage 1 acute kidney injury and 0.05 for no acute kidney injury; p = 0.052) and 24 hours postoperatively (1.71 vs 0.27 for stage 1 acute kidney injury and 0.19 for no acute kidney injury, p = 0.027) and higher neutrophil gelatinase-associated lipocalin levels at 24 hours postoperatively (10.3 vs 3.4 for stage 1 acute kidney injury and 6.2 for no acute kidney injury, p = 0.019). Stage 2-3 acute kidney injury patients had lower mean cardiac ICU renal regional tissue oximetry (66% vs 79% for stage 1 acute kidney injury and 84% for no acute kidney injury, p = 0.038). Regression analyses showed that tissue inhibitor of metalloproteinases 2, insulin-like growth factor-binding protein 7 at 2 hours postoperatively and nadir intraoperative renal regional tissue oximetry to be independent predictors of postoperative kidney damage as measured by urinary neutrophil gelatinase-associated lipocalin. CONCLUSIONS: We observed modest differences in perioperative renal regional tissue oximetry and urinary biomarker levels compared between acute kidney injury groups classified by creatinine-dependent Kidney Disease: Improving Global Outcomes criteria, but there were significant correlations between renal regional tissue oximetry, tissue inhibitor of metalloproteinases 2, insulin-like growth factor-binding protein 7, and postoperative neutrophil gelatinase-associated lipocalin levels. Kidney injury after infant cardiac surgery may be undetectable by functional assessment (creatinine) alone, and continuous monitoring of renal regional tissue oximetry may be more sensitive to important subclinical acute kidney injury.

Serum Neuronal Biomarkers in Neonates With Congenital Heart Disease Undergoing Cardiac Surgery.

BACKGROUND: Newborns with congenital heart disease have associated brain damage that affects short-and long-term neurodevelopment. Several neuronal biomarkers exist that could predict brain damage. We investigated the pattern of neuron-specific enolase (NSE) and s100B levels after cardiopulmonary bypass surgery in neonates with congenital heart disease. METHODS: We completed a prospective observational study of neonates with congenital heart disease who were undergoing cardiopulmonary bypass surgery. NSE and s100B levels were measured from serum samples obtained preoperatively, immediately postoperatively, and once daily on postoperative days one to seven. Cranial ultrasounds were obtained preoperatively and postoperatively and findings were scored using an internally developed scoring system. RESULTS: Eighteen neonates were included. Immediate postoperative and peak levels of both NSE (58.0 [21.6] and 68.1 [55.7] µg/L) and s100B (0.14 [0.3] and 0.14 [0.3] µg/L) were significantly increased when compared with preoperative levels (34.0 [21.6] µg/L; P < 0.01 and 0.08 [0.1] µg/L; P < 0.02). By postoperative day seven, NSE and s100B levels were lower than preoperative levels: NSE (18 [5.7]; P = 0.09) and s100B (0.03 [0.05]; P < 0.01). Postoperative s100B levels were negatively correlated with age at surgery and positively correlated with circulatory arrest time. Although there was no significant correlation between either NSE or s100B levels and intensive care unit length of stay, hospital length of stay, and pediatric cerebral performance category score, there was a negative correlation between postoperative levels of NSE and ventriculomegaly. CONCLUSIONS: NSE and s100B levels increase after bypass surgery and return below preoperative baseline levels by postoperative day seven. The levels of s100B were positively correlated with circulatory arrest time and negatively correlated with age at time of surgery. This finding may be supportive of pre-existing prenatal brain injury that could be enhanced by longer surgical times but also of some brain protection effect associated with longer wait until surgery.

Bedside Ultrasound for the Diagnosis of Abnormal Diaphragmatic Motion in Children After Heart Surgery.

OBJECTIVE: To assess the utility of bedside ultrasound combining B- and M-mode in the diagnosis of abnormal diaphragmatic motion in children after heart surgery. DESIGN: Prospective post hoc blinded comparison of ultrasound performed by two different intensivists and fluoroscopy results with electromyography. SETTING: Tertiary university hospital. SUBJECTS: Children with suspected abnormal diaphragmatic motion after heart surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Abnormal diaphragmatic motion was suspected in 26 children. Electromyography confirmed the diagnosis in 20 of 24 children (83.3%). The overall occurrence rate of abnormal diaphragmatic motion during the study period was 7.5%. Median patient age was 5 months (range, 16 d to 14 yr). Sensitivity and specificity of chest ultrasound performed at the bedside by the two intensivists (91% and 92% and 92% and 95%, respectively) were higher than those obtained by fluoroscopy (87% and 83%). Interobserver agreement (k) between both intensivists was 0.957 (95% CI, 0.87-100). CONCLUSIONS: Chest ultrasound performed by intensivists is a valid tool for the diagnosis of diaphragmatic paralysis, presenting greater sensitivity and specificity than fluoroscopy. Chest ultrasound should be routinely used after pediatric heart surgery given its reliability, reproducibility, availability, and safety.

The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to Regulate Enzyme Activity and Synaptic Vesicle Localization.

We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis.

Early Initiation of Renal Replacement Therapy in Pediatric Heart Surgery Is Associated with Lower Mortality.

Acute kidney injury (AKI) is frequent in the postoperative period of pediatric heart surgery and leads to significant morbidity and mortality. Renal replacement therapies (RRTs) are often used to treat AKI; however, these therapies have also been associated with higher mortality rates. Earlier initiation of RRT might improve outcomes. This study aims to investigate the relationship between the RRT and morbidity and mortality after pediatric heart surgery. We performed a single-center retrospective study of all children undergoing pediatric heart surgery between April 2010 and December 2012 at a tertiary children's hospital. A total of 480 patients were included. Of those, 109 (23 %) were neonates and 126 patients (26 %) developed AKI within the first 72 postoperative hours. Patients who developed AKI had longer PICU admissions [12 days (4-37.75) vs. 4 (2-11); p < 0.001] and hospital length of stay [27 (11-53) vs. 14 (8-24) p < 0.001] and higher mortality [22/126 (17.5 %) vs. 13/354 (3.7 %); p < 0.001]. RRT techniques were used in 32 (6.6 %) patients [18/109 (16 %) neonates and 14/371 (3.8 %) infants and children; p < 0.01], with 25 (78 %) receiving peritoneal dialysis (PD) and 7 (22 %) continuous RRT (CRRT). Patients who received PD within the first 24 postoperative hours had lower mortality compared with those in whom PD was initiated later [4/16 (25 %) vs. 4/9 (44.4 %)]. Mortality among patients who received CRRT was 28.6 % (2/7). No deaths were reported in patients treated with CRRT within the first 24 postoperative hours. Postoperative AKI is associated with higher mortality in children undergoing cardiac surgery. Early initiation of RRT, both PD in neonates and CRRT in pediatric patients, might improve morbidity and mortality associated with AKI.

Inhibition of dopamine transporter activity by G protein ßγ subunits.

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson's disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the ßγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gßγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gßγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gßγ subunit overexpression, or the Gßγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gßγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gßγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gßγ signaling in the control of dopamine homeostasis.

A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.

Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT(2)) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT(2) physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT(2), whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT(1) and with overexpressed VMAT(2). GST pull-down assays further identified three cytosolic domains of VMAT(2) involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT(2). Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT(2)-containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT(2)/TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT(2)-mediated transport into vesicles.

The molecular chaperone Hsc70 interacts with the vesicular monoamine transporter-2.

Synaptic transmission depends on the efficient loading of transmitters into synaptic vesicles by vesicular neurotransmitter transporters. The vesicular monoamine transporter-2 (VMAT2) is essential for loading monoamines into vesicles and maintaining normal neurotransmission. In an effort to understand the regulatory mechanisms associated with VMAT2, we have embarked upon a systematic search for interacting proteins. Glutathione-S-transferase pull-down assays combined with mass spectrometry led to the identification of the 70-kDa heat shock cognate protein (Hsc70) as a VMAT2 interacting protein. Co-immunoprecipitation experiments in brain tissue and heterologous cells confirmed this interaction. A direct binding was observed between the amino terminus and the third cytoplasmic loop of VMAT2, as well as, a region containing the substrate binding and the carboxy-terminal domains of Hsc70. Furthermore, VMAT2 and Hsc70 co-fractionated with purified synaptic vesicles obtained from a sucrose gradient, suggesting that this interaction occurs at the synaptic vesicle membrane. The functional significance of this novel VMAT2/Hsc70 interaction was examined by performing vesicular uptake assays in heterologous cells and purified synaptic vesicles from brain tissue. Recombinant Hsc70 produced a dose-dependent inhibition of VMAT2 activity. This effect was mimicked by the closely related Hsp70 protein. In contrast, VMAT2 activity was not altered in the presence of previously denatured Hsc70 or Hsp70, as well as the unrelated Hsp60 protein; confirming the specificity of the Hsc70 effect. Finally, a purified Hsc70 fragment that binds VMAT2 was sufficient to inhibit VMAT2 activity in synaptic vesicles. Our results suggest an important role for Hsc70 in VMAT2 function and regulation.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

The orphan transporter Rxt1/NTT4 (SLC6A17) functions as a synaptic vesicle amino acid transporter selective for proline, glycine, leucine, and alanine.

Rxt1/NTT4 (SLC6A17) belongs to a gene family of "orphan transporters" whose substrates and consequently functions remain unidentified. Although Rxt1/NTT4 was previously thought to function as a sodium-dependent plasma membrane transporter, recent studies localized the protein to synaptic vesicles of glutamatergic and GABAergic neurons. Here, we provide evidence indicating that Rxt1/NTT4 functions as a vesicular transporter selective for proline, glycine, leucine, and alanine. Using Western blot, immunoprecipitation, immunocytochemistry, and polymerase chain reaction approaches, we demonstrate that PC12 cells express the Rxt1/NTT4 gene and protein. Small interfering RNA (siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells resulted in selective reductions in uptake levels for proline, glycine, leucine, and alanine. Likewise, gas chromatography analysis of amino acid content in an enriched synaptic vesicle fraction from wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that proline, glycine, leucine, and alanine levels were decreased in siRNA-treated cells compared with wild-type cells. Furthermore, Rxt1/NTT4-transfected Chinese hamster ovary (CHO) cells exhibited significant uptake increases of these amino acids compared with mock-transfected CHO cells. Finally, proline uptake in both PC12 cells and Rxt1/NTT4-transfected CHO cells was dependent on the electrochemical gradient maintained by the vacuolar-type H(+)-ATPase. These data indicate that the orphan Rxt1/NTT4 protein functions as a vesicular transporter for proline, glycine, leucine, and alanine, further suggesting its important role in synaptic transmission.