The exocyst subunit Sec15 is critical for proper synaptic development and function at the Drosophila NMJ.

The exocyst protein complex is important for targeted vesicle fusion in a variety of cell types, however, its function in neurons is still not entirely known. We found that presynaptic knockdown (KD) of the exocyst component sec15 by transgenic RNAi expression caused a number of unexpected morphological and physiological defects in the synapse. These include the development of active zones (AZ) devoid of essential presynaptic proteins, an increase in the branching of the presynaptic arbor, the appearance of satellite boutons, and a decrease in the amplitude of stimulated postsynaptic currents as well as a decrease in the frequency of spontaneous synaptic vesicle release. We also found the release of extracellular vesicles from the presynaptic neuron was greatly diminished in the Sec15 KDs. These effects were mimicked by presynaptic knockdown of Rab11, a protein known to interact with the exocyst. sec15 RNAi expression caused an increase in phosphorylated Mothers against decapentaplegic (pMad) in the presynaptic terminal, an indication of enhanced bone morphogenic protein (BMP) signaling. Some morphological phenotypes caused by Sec15 knockdown were reduced by attenuation of BMP signaling through knockdown of wishful thinking (Wit), while other phenotypes were unaffected. Individual knockdown of multiple proteins of the exocyst complex also displayed a morphological phenotype similar to Sec15 KD. We conclude that Sec15, functioning as part of the exocyst complex, is critically important for proper formation and function of neuronal synapses. We propose a model in which Sec15 is involved in the trafficking of vesicles from the recycling endosome to the cell membrane as well as possibly trafficking extracellular vesicles for presynaptic release and these processes are necessary for the correct structure and function of the synapse.

Evaluation of oxygen sensitivity of hyperpolarized helium imaging for the detection of pulmonary ischemia.

PURPOSE: In this study, a new model of pulmonary embolism in rats was developed and tested, to examine if hyperpolarized (HP) (3) He MR images can measure impairment of the exchange of oxygen from the airspaces to the blood during pulmonary embolism. METHODS: HP (3) He MRI was used to image six treatment-group rats in which a branch of the pulmonary artery was embolized, and six control-group rats. HP (3) He MR images were used to calculate the initial partial pressure of oxygen (pO ) and the rate of oxygen depletion (R) in rat lungs. RESULTS: The pO was significantly higher in the ischemic lung than in the contralateral normal side, and pO was significantly higher in the ischemic lung than in both sides of the control lungs. Mean R in ischemic lungs was significantly lower than in the contralateral lungs, and mean R in ischemic lungs was also significantly lower than in both control lungs. CONCLUSION: These results demonstrate that pO and R, as measured by the T1 decay of HP (3) He, are sensitive to pulmonary ischemia in rats, confirming the findings in studies performed in large animal models of pulmonary ischemia.

Using hyperpolarized 3He MRI to evaluate treatment efficacy in cystic fibrosis patients.

PURPOSE: To use hyperpolarized (HP) (3)He MR imaging to assess functional lung ventilation in subjects with cystic fibrosis (CF) before and after treatment. MATERIALS AND METHODS: We performed HP (3)He static ventilation MRI scans on three subjects, using a Philips 3.0 Tesla (T) Achieva MRI scanner, before and after 11 days of in-patient treatment with combined intravenous and inhaled therapies for pulmonary exacerbations of CF. We also collected spirometry data. We quantified pulmonary ventilation volume measured with HP (3)He MRI using an advanced semi-automated analysis technique. RESULTS: Following 11 days of treatment with intravenous antibiotics, hypertonic saline, and rhDNase, HP (3)He MR images in one subject displayed a 25% increase in total ventilation volume. Total ventilation volume in the other two subjects slightly decreased. All three subjects showed increases in FEV(1) and FVC following treatment. CONCLUSION: In all subjects, the HP (3)He MR images provided detailed information on precisely where in the lungs gas was reaching. These data provide additional support for the conclusion that HP noble gas MRI can be a powerful tool for evaluating lung ventilation in patients with cystic fibrosis, but also raise important questions about the correlation between spirometry and HP gas MRI measurements.

Rab3-GEF Controls Active Zone Development at the Drosophila Neuromuscular Junction.

Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse.

Regulation of synaptic plasticity and synaptic vesicle dynamics by the PDZ protein Scribble.

The Drosophila tumor suppressor Scribble (Scrib) is a PDZ-containing protein required for maintaining epithelial cell polarity. At the larval neuromuscular junction, Scrib colocalizes and indirectly interacts with another tumor suppressor and PDZ protein, Discs-Large (Dlg). Previous studies demonstrate that Dlg is critical for development of normal synapse structure and function, as well as for normal synaptic Scrib localization. Here we show that Scrib is also an important regulator of synaptic architecture and physiology. The most notable ultrastructural defect in scrib mutants is an increase in the number of synaptic vesicles in an area of the synaptic bouton thought to contain the reserve vesicle pool. Additionally, the number of active zones is reduced in scrib mutants. Functionally, the scrib synapse behaves relatively normally at low-frequency stimulation. However, several forms of plasticity at this synapse are drastically altered in the mutants. Specifically, scrib mutants exhibit loss of facilitation and post-tetanic potentiation, and faster synaptic depression. In addition, FM1-43 imaging of recycling synaptic vesicles shows that vesicle dynamics are impaired in scrib mutants. These results identify Scrib as an essential regulator of short-term synaptic plasticity. Taken together, our results are consistent with a model in which Scrib is required to sustain synaptic vesicle concentrations at their sites of release.

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction.

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

Antibodies and a cysteine-modifying reagent show correspondence of M current in neurons to KCNQ2 and KCNQ3 K+ channels.

1. KCNQ K(+) channels are thought to underlie the M current of neurons. To probe if the KCNQ2 and KCNQ3 subtypes underlie the M current of rat superior cervical ganglia (SCG) neurons and of hippocampus, we raised specific antibodies against them and also used the cysteine-alkylating agent N-ethylmaleimide (NEM) as an additional probe of subunit composition. 2. Tested on tsA-201 (tsA) cells transfected with cloned KCNQ1-5 subunits, our antibodies showed high affinity and selectivity for the appropriate subtype. The antibodies immunostained SCG neurons and hippocampal sections at levels similar to those for channels expressed in tsA cells, indicating that KCNQ2 and KCNQ3 are present in SCG and hippocampal neurons. Some hippocampal regions contained only KCNQ2 or KCNQ3 subunits, suggesting the presence of M currents produced by channels other than KCNQ2/3 heteromultimers. 3. We found that NEM augmented M currents in SCG neurons and KCNQ2/3 currents in tsA cells via strong voltage-independent and modest voltage-dependent actions. Expression of individual KCNQ subunits in tsA cells revealed voltage-independent augmentation of KCNQ2, but not KCNQ1 nor KCNQ3, currents by NEM indicating that this action on SCG M currents likely localizes to KCNQ2. Much of the voltage-independent action is lost after the C242T mutation in KCNQ2. 4. The correspondence of NEM effects on expressed KCNQ2/3 and SCG M currents, along with the antibody labelling, provide further evidence that KCNQ2 and KCNQ3 subunits strongly contribute to the M current of neurons. The site of NEM action may be important for treatment of diseases caused by under-expression of these channels.

Assessment of repeatability of hyperpolarized gas MR ventilation functional imaging in cystic fibrosis.

RATIONALE AND OBJECTIVES: Hyperpolarized (HP) gas magnetic resonance imaging (MRI) is an advanced imaging technique that provides high-resolution regional information on lung function without using ionizing radiation. Before this modality can be considered for assessing clinical or investigational interventions, baseline repeatability needs to be established. We assessed repeatability of lung function measurement using HP helium-3 MRI (HP (3)He MRI) in a small cohort of patients with cystic fibrosis (CF). MATERIALS AND METHODS: We examined repeatability of HP (3)He MR images of five patients with CF in four scanning sessions over a 4-week period. We acquired images on a Philips 3.0 Tesla Achieva MRI scanner using a quadrature, flexible, wrap-around, (3)He radiofrequency coil with a fast gradient-echo pulse sequence. We determined ventilation volume and ventilation defect volume using an advanced semiautomatic segmentation algorithm and also quantified ventilation heterogeneity. RESULTS: There were no significant differences in total ventilation volume, ventilation defect volume, ventilation defect percentage, or mean ventilation heterogeneity (repeated-measures analysis of variance, P = .2116, P = .2825, P = .2871, and P = .7265, respectively) in the patients across the four scanning sessions. CONCLUSIONS: Our results indicate that total ventilation volume, ventilation defect volume, ventilation defect percentage, and mean ventilation heterogeneity as assessed by HP gas MRI in CF patients with stable health are reproducible over time. This repeatability and the technique's capability to provide noninvasive high-resolution data on regional lung function without ionizing radiation make (3)He MRI a potentially useful outcome measure for CF-related clinical trials.

Print media coverage of risk-risk tradeoffs associated with West Nile encephalitis and pesticide spraying.

When mosquito-borne West Nile virus emerged in the United States in 1999 and triggered pesticide spraying, society was faced with a controversy over an important risk-risk tradeoff-the risks of pesticide exposure versus those of West Nile encephalitis. Effective public communication about risk-risk tradeoffs is important because it can assist individuals and society in investing resources optimally. This study examined how effectively major North American print media in the year 2000 provided information on this risk-risk tradeoff. My colleagues and I found that the print media were generally ineffective in providing precise information about pesticide risks and in comparing risks of pesticide exposure with those of West Nile encephalitis. The media were also ineffective in mentioning the efficacy of pesticide spraying or comparing the economic costs of pesticide spraying with those of West Nile encephalitis. We suggest that greater effort in collecting and reporting precise risk information, fostering more active relationships between journalists and scientists/public health professionals, and recognizing biases resulting from preconceptions can help improve reporting by the print media and public health agencies on risk-risk tradeoffs associated with emerging insect-borne infectious diseases. These efforts could help improve public health by improving decision making related to the control of insect-borne diseases.