Post-translational regulation of an Aplysia glutamate transporter during long-term facilitation.

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 (Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum-Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-co-precipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (rough endoplasmic reticulum-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.

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.

Inputs to parasol ganglion cells in the primate retina

Retinal ganglion cells carry signals from the eye to the brain. One of the most common types of ganglion cells is parasol cells. They have larger dendritic trees, somas and axons than other ganglion cells. While much was known about parasol cell light responses, little was known about how these responses are formed. One possibility is that they receive input from a unique set of local circuit neurons that have similar responses. The goal was to identify these presynaptic neurons and study their synaptic connectivity.^ Ganglion cells receive input from bipolar and amacrine cells, but there are numerous subtypes of each. To determine which of these were most likely to provide input to parasol cells, the parasol cells were intracellularly-injected and then various bipolar and amacrine cells were immunolabeled and the tissue analyzed using a confocal microscope. DB3 bipolar cells labeled with antibodies to calbindin made extensive contacts with OFF parasol cells. Antibodies to recover in labeled flat midget bipolar cells (FMB). They made only random contacts with OFF parasol cells, and they are not expected to provide significant input. Type DB2 bipolar cells and FMB cells labeled with antibodies to excitatory amino acid transporter-2 made extensive contacts with OFF parasol cells. This suggests that DB2 bipolar cells are likely to provide input to parasol cells.^ Two types of amacrine cells were labeled in material containing injected parasol cells. Cholinergic amacrine cells were labeled with antibodies to choline acetyltransferase, and they made extensive contacts with ON parasol cells. The large amacrine cells labeled with antibodies to a precursor of cholecystokinin were among the amacrine cells that are tracer-coupled to parasol cells.^ From electron microscopic (EM) analysis, most of the synapses made by DB3 axons were found on varicosities. Some postsynaptic and presynaptic amacrine cells resembled AII amacrine cells. Others were relatively electron-lucent and may be cholinergic amacrine cells or cholecystokinin-containing amacrine cells. Gap junctions were found between neighboring DB3 axons. They occurred whenever two axons contacted each other, and the junctions were as large as the area of contact. In double-label EM experiments, DB3 axons made synapses onto OFF parasol cells. ^

NOVEL AMINO ACID TRANSPORTER-TARGETED RADIOTRACERS FOR BREAST CANCER IMAGING

Breast cancer is the most common malignancy among women in the world. Its 5-year survival rate ranges from 23.4% in patients with stage IV to 98% in stage I disease, highlighting the importance of early detection and diagnosis. 18F-2-Fluoro-2-deoxy-glucose (18F-FDG), using positron emission tomography (PET), is the most common functional imaging tool for breast cancer diagnosis currently. Unfortunately, 18F-FDG-PET has several limitations such as poorly differentiating tumor tissues from inflammatory and normal brain tissues. Therefore, 18F-labeled amino acid-based radiotracers have been reported as an alternative, which is based on the fact that tumor cells uptake and consume more amino acids to sustain their uncontrolled growth. Among those radiotracers, 18F-labeled tyrosine and its derivatives have shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma. They enter the tumor cells via L-type amino acid transporters (LAT), which were reported to be highly expressed in many cancer cell lines and correlate positively with tumor growth. Nevertheless, the low radiosynthesis yield and demand of an on-site cyclotron limit the use of 18F-labeled tyrosine analogues. In this study, four Technetium-99m (99mTc) labeled tyrosine/ AMT (α-methyl tyrosine)-based radiotracers were successfully synthesized and evaluated for their potentials in breast cancer imaging. In order to radiolabel tyrosine and AMT, the chelators N,N’-ethylene-di-L-cysteine (EC) and 1,4,8,11-tetra-azacyclotetradecane (N4 cyclam) were selected to coordinate 99mTc. These chelators have been reported to provide stable chelation ability with 99mTc. By using the chelator technology, the same target ligand could be labeled with different radioisotopes for various imaging modalities for tumor diagnosis, or for internal radionuclide therapy in future. Based on the in vitro and in vivo evaluation using the rat mammary tumor models, 99mTc-EC-AMT is considered as the most suitable radiotracer for breast cancer imaging overall, however, 99mTc-EC-Tyrosine will be more preferred for differential diagnosis of tumor from inflammation.

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

External assessment of myocardial glucose metabolism with $\sp{18}$F-2 -deoxy-2 -fluoro-D-glucose

Despite the popularity of the positron emitting glucose analog, ($\sp{18}$F) -2-deoxy-2-fluoro-D-glucose (2FDG), for the noninvasive "metabolic imaging" of organs with positron emission tomography (PET), the physiological basis for the tracer has not been tested, and the potential of 2FDG for the rapid kinetic analysis of altered glucose metabolism in the intact heart has not been fully exploited. We, therefore, developed a quantitative method to characterize metabolic changes of myocardial glucose metabolism noninvasively and with high temporal resolution.^ The first objective of the work was to provide direct evidence that the initial steps in the metabolism of 2FDG are the same as for glucose and that 2FDG is retained by the tissue in proportion to the rate of glucose utilization. The second objective was to characterize the kinetic changes in myocardial glucose transport and phosphorylation in response to changes in work load, competing substrates, acute ischemia and reperfusion, and the addition of insulin. To assess changes in myocardial glucose metabolism isolated working rat hearts were perfused with glucose and 2FDG. Tissue uptake of 2FDG and the input function were measured on-line by external detection. The steady state rate of 2FDG phosphorylation was determined by graphical analysis of 2FDG time-activity curves.^ The rate of 2FDG uptake was linear with time and the tracer was retained in its phosphorylated form. Tissue accumulation of 2FDG decreased within seconds with a reduction in work load, in the presence of competing substrates, and during reperfusion after global ischemia. Thus, most interventions known to alter glucose metabolism induced rapid parallel changes in 2FDG uptake. By contrast, insulin caused a significant increase in 2FDG accumulation only in hearts from fasted animals when perfused at a sub-physiological work load. The mechanism for this phenomenon is not known but may be related to the existence of two different glucose transporter systems and/or glycogen metabolism in the myocardial cell.^ It is concluded that (1) 2FDG traces glucose uptake and phosphorylation in the isolated working rat heart; and (2) early and transient kinetic changes in glucose metabolism can be monitored with high temporal resolution with 2FDG and a simple positron coincidence counting system. The new method has revealed transients of myocardial glucose metabolism, which would have remained unnoticed with conventional methods. These transients are not only important for the interpretation of glucose metabolic PET scans, but also provide insights into mechanisms of glucose transport and phosphorylation in heart muscle. ^