Doctor of Philosophy

dissertationSynapses are the places where neurons communicate with their targets. At chemical synapses, neurotransmitters are contained in synaptic vesicles and are released into the synaptic cleft upon fusion with the plasma membrane. This event happens at high frequency at synapses and thus synaptic vesicles need to be regenerated locally to prevent vesicle depletion. The popular model for synaptic vesicle endocytosis is to re-sort vesicle proteins left in the plasma membrane into an invaginated vesicle by clathrin-mediated endocytosis. However, some pieces of evidence suggest that clathrin-independent endocytosis might also contribute to synaptic vesicle recycling. In this dissertation, we present the studies on endocytic accessory proteins from clathrin-mediated endocytosis and focus primarily on their potential roles in neurotransmission by using the genetic model organism Caenorhabditis elegans. The proteins investigated include the major adaptor complex AP2, the synaptotagmin adaptor UNC-41 and the membrane bending protein, Epsin. We demonstrate that, one; AP2 is responsible for 70% synaptic vesicle recycling in C. elegans. Second, synaptic recycling of synaptotagmin requires UNC-41. Third, Epsin is not required for curvature acquisition in clathrin-mediated endocytosis at synapses. Thus these studies push forward our understanding towards synaptic vesicle recycling at synapses and demonstrate clathrinmediated endocytosis is likely the major mechanism for synaptic vesicle endocytosis in C. elegans

μ2 adaptin facilitates but is not essential for synaptic vesicle recycling in Caenorhabditis elegans

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (μ2) of AP2 binds to cargo proteins and phosphatidylinositol-4,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only μ2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 μ2 facilitates but is not essential for synaptic vesicle recycling

Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2

Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a β strand within the μ–homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 β strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding–defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals

The role of miRNAs in Behçet’s disease

The symptoms of Behçet’s disease (BD), a multisystemic condition with autoimmune and inflammation as hallmarks, include arthritis, recurring oral and vaginal ulcers, skin rashes and lesions, and involvement of the nervous, gastrointestinal, and vascular systems. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), may be important regulators of inflammation and autoimmune disease. These ncRNAs are essential to the physiological and pathophysiological disease course, and miRNA in particular has received significant attention for its role and function in BD and its potential use as a diagnostic biomarker in recent years. Although promising as therapeutic targets, miRNAs must be studied further to fully comprehend how miRNAs in BD act biologically

A Cost-Effectiveness Analysis of Comprehensive Smoking-Cessation Interventions Based on the Community and Hospital Collaboration

BackgroundThe prevalence of cigarette smoking in China is high and the utilization of smoking cessation clinics is very low. Multicomponent smoking cessation interventions involving community and hospital collaboration have the potential to increase the smoking cessation rate. However, the cost-effectiveness of this intervention model is unknown.MethodsWe conducted a smoking cessation intervention trial in 19 community health service centers in Beijing, China. A cost-effectiveness analysis was performed from a societal perspective to compare three strategies of smoking cessation: no intervention (NI), pharmacological intervention (PI), and comprehensive intervention (CI) (PI plus online health promotion). A Markov model, with a time horizon of 20 years, was used to simulate the natural progression of estimated 10,000 male smokers. A cross-sectional survey was conducted to obtain data on costs and quality-adjusted life years (QALYs) by using the five-level EuroQol-5-dimension (EQ-5D-5L) questionnaire. Probabilistic sensitivity analysis was performed to explore parameters of uncertainty in the model.ResultsA total of 680 participants were included in this study, including 283 in the PI group and 397 in the CI group. After 6 months of follow-up, the smoking cessation rate reached 30.0% in the CI group and 21.2% in the PI group. Using the Markov model, compared with the NI group, the intervention strategies of the PI group and the CI group were found to be cost-effective, with an incremental cost-effectiveness ratio (ICER) of $535.62/QALY and$366.19/QALY, respectively. The probabilistic sensitivity analysis indicated that the CI strategy was always the most cost-effective intervention.ConclusionCI for smoking cessation, based in hospital and community in China, is more cost-effective than PI alone. Therefore, this smoking cessation model should be considered to be implemented in healthcare settings

The Membrane-Associated Proteins FCHo and SGIP Are Allosteric Activators of the AP2 Clathrin Adaptor Complex

The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for C. elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the mu-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope