The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2alpha. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons. Elimination of dunc-13 expression abolished synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Transmitter release remained impaired under elevated calcium influx or application of hyperosmotic saline. Ultrastructurally, mutant terminals accumulated docked vesicles at presynaptic release sites. We conclude that Dunc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion.

Taken together, these studies demonstrate that the UPS functions locally within synaptic boutons to acutely control levels of presynaptic protein and that the rate of UPS-dependent protein degradation is a primary determinant of neurotransmission strength.

The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyper-reactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

Airway diseases such as asthma involve increased airway smooth muscle (ASM) contractility and remodeling via enhanced proliferation. Neurotrophins (NTs) such as brain derived neurotrophic factor (BDNF), well-known in the nervous system, can regulate Ca2+ signaling, and interact with cytokines in contributing to airway hyperreactivity. In this study, we determined whether and how BDNF regulates human ASM cell proliferation in the presence of inflammation, thus testing its potential role in airway remodeling. Cells were treated with 10nM BDNF, 25ng/ml tumor necrosis factor (TNFα) or interleukin-13 (IL-13), or 10ng/ml platelet derived growth factor (PDGF). Proliferation was measured using CyQuant dye, with immunoblotting of cell cycle proteins predicted to change with proliferation. 48h of BDNF enhanced ASM proliferation to ~50% of that by PDGF or cytokines. Transfection with siRNAs targeting high-affinity TrkB receptor (tropomyosin related kinase B) abolished BDNF effects on proliferation, while low-affinity 75 KD neurotrophin receptor (p75NTR) siRNA had no effect. Systematic pharmacologic inhibition of different components of ERK1/2 and PI3K/Akt1 pathways blunted BDNF or TNFα induced proliferation. BDNF also induced IκB phosphorylation and nuclear translocation of p50 and p65 NFκB subunits, with electron mobility shift assay confirmation of NFκB binding to consensus DNA sequence. These results demonstrate that neurotrophins such as BDNF can enhance human ASM cell proliferation by activating proliferation-specific signaling pathways and a versatile transcription factor such as NFκB, which are common to cytokines and growth factors involved in asthma.