Glial Control of Synapse Assembly at the \u3cem\u3eDrosophila\u3c/em\u3e Neuromuscular Junction: A Dissertation

Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ

Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. Here we use chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, we identified wingless (wg), a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), as a potential Repo target gene. We demonstrate that Repo regulates wg expression in vivo and that local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ

Depressive Symptom Trajectories Among Girls in the Juvenile Justice System: Findings from an RCT of Multidimensional Treatment Foster Care

Youth depression is a significant and growing international public health problem. Youth who engage in high levels of delinquency are at particularly high risk for developing problems with depression. The present study examined the impact of a behavioral intervention designed to reduce delinquency (Multidimensional Treatment Foster Care; MTFC) compared to a group care intervention (GC; i.e., services as usual) on trajectories of depressive symptoms among adolescent girls in the juvenile justice system. MTFC has documented effects on preventing girls’ recidivism, but its effects on preventing the normative rise in girls’ depressive symptoms across adolescence have not been examined. This indicated prevention sample included 166 girls (13–17 years at T1) who had at least one criminal referral in the past 12 months and who were mandated to out-of-home care; girls were randomized to MTFC or GC. Intent-to-treat analyses examined the main effects of MTFC on depression symptoms and clinical cut-offs, and whether benefits were greatest for girls most at risk. Depressive symptom trajectories were specified in hierarchical linear growth models over a two year period using five waves of data at six month intervals. Depression clinical cut-off scores were specified as nonlinear probability growth models. Results showed significantly greater rates of deceleration for girls in MTFC versus GC for depressive symptoms and for clinical cut-off scores. The MTFC intervention also showed greater benefits for girls with higher levels of initial depressive symptoms. Possible mechanisms of effect are discussed, given MTFC’s effectiveness on targeted and nontargeted outcomes.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Springer and can be found at: http://link.springer.com/journal/11121.Keywords: randomized controlled trial, juvenile justice, maltreatment, MTFC (Multidimensional Treatment Foster Care), girls, depressio

Depressive symptom trajectories among girls in the juvenile justice system: 24-month outcomes of an RCT of Multidimensional Treatment Foster Care

Youth depression is a significant and growing international public health problem. Youth who engage in high levels of delinquency are at particularly high risk for developing problems with depression. The present study examined the impact of a behavioral intervention designed to reduce delinquency (Multidimensional Treatment Foster Care; MTFC) compared to a group care intervention (GC; i.e., services as usual) on trajectories of depressive symptoms among adolescent girls in the juvenile justice system. MTFC has documented effects on preventing girls' recidivism, but its effects on preventing the normative rise in girls' depressive symptoms across adolescence have not been examined. This indicated prevention sample included 166 girls (13-17 years at T1) who had at least one criminal referral in the past 12 months and who were mandated to out-of-home care; girls were randomized to MTFC or GC. Intent-to-treat analyses examined the main effects of MTFC on depression symptoms and clinical cut-offs, and whether benefits were greatest for girls most at risk. Depressive symptom trajectories were specified in hierarchical linear growth models over a 2 year period using five waves of data at 6 month intervals. Depression clinical cut-off scores were specified as nonlinear probability growth models. Results showed significantly greater rates of deceleration for girls in MTFC versus GC for depressive symptoms and for clinical cut-off scores. The MTFC intervention also showed greater benefits for girls with higher levels of initial depressive symptoms. Possible mechanisms of effect are discussed, given MTFC's effectiveness on targeted and nontargeted outcomes. © 2013 Society for Prevention Research

WldS requires Nmnat1 enzymatic activity and N16–VCP interactions to suppress Wallerian degeneration

Slow Wallerian degeneration (WldS) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of WldS remains controversial. In this study, we characterize WldS-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than WldS, and enzyme-dead versions of Nmnat1 and WldS exhibit severely reduced axon-protective function. Interestingly, a 16–amino acid N-terminal domain of WldS (termed N16) accounts for the differences in axon-sparing activity between WldS and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in WldS requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, WldS-mediated suppression of Wallerian degeneration results from VCP–N16 interactions and Nmnat1 activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from WldS. Thus, nuclear Nmnat activity does not appear to be essential for WldS-like axon protection

Anticitrullinated protein antibody (ACPA) in rheumatoid arthritis: influence of an interaction between HLA-DRB1 shared epitope and a deletion polymorphism in glutathione s-transferase in a cross-sectional study

Abstract Introduction A deletion polymorphism in glutathione S-transferase Mu-1 (GSTM1-null) has previously been implicated to play a role in rheumatoid arthritis (RA) risk and progression, although no prior investigations have examined its associations with anticitrullinated protein antibody (ACPA) positivity. The purpose of this study was to examine the associations of GSTM1-null with ACPA positivity in RA and to assess for evidence of interaction between GSTM1 and HLA-DRB1 shared epitope (SE). Methods Associations of GSTM1-null with ACPA positivity were examined separately in two RA cohorts, the Veterans Affairs Rheumatoid Arthritis (VARA) registry (n = 703) and the Study of New-Onset RA (SONORA; n = 610). Interactions were examined by calculating an attributable proportion (AP) due to interaction. Results A majority of patients in the VARA registry (76%) and SONORA (69%) were positive for ACPA with a similar frequency of GSTM1-null (53% and 52%, respectively) and HLA-DRB1 SE positivity (76% and 71%, respectively). The parameter of patients who had ever smoked was more common in the VARA registry (80%) than in SONORA (65%). GSTM1-null was significantly associated with ACPA positivity in the VARA registry (odds ratio (OR), 1.45; 95% confidence interval (CI), 1.02 to 2.05), but not in SONORA (OR, 1.00; 95% CI, 0.71 to 1.42). There were significant additive interactions between GSTM1 and HLA-DRB1 SE in the VARA registry (AP, 0.49; 95% CI, 0.21 to 0.77; P < 0.001) in ACPA positivity, an interaction replicated in SONORA (AP, 0.38; 95% CI, 0.00 to 0.76; P = 0.050). Conclusions This study is the first to show that the GSTM1-null genotype, a common genetic variant, exerts significant additive interaction with HLA-DRB1 SE on the risk of ACPA positivity in RA. Since GSTM1 has known antioxidant functions, these data suggest that oxidative stress may be important in the development of RA-specific autoimmunity in genetically susceptible individuals

DNA Methylation Changes in Atypical Adenomatous Hyperplasia, Adenocarcinoma In Situ, and Lung Adenocarcinoma

BACKGROUND:Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation. METHODOLOGY/PRINCIPAL FINDINGS:We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer. CONCLUSIONS/SIGNIFICANCE:The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions

The Role of Trust in Public Attitudes toward Invasive Species Management on Guam: A Case Study

Public attitudes toward invasive alien species management and trust in managers’ ability to effectively manage non-native species can determine public support for conservation action. Guam has experienced widespread species loss and ecosystem transformation due to invasive species. Despite Guam’s long history with invasives and efforts to eradicate them, we know little about the sociological context of invasive species. Using focused group discussions, we explore public attitudes toward invasive species management. Respondents expressed support for management activities and a desire to participate directly in conservation actions. Participants also expressed frustration with government institutions and lack of confidence in managers’ abilities to control invasive species. Perceptions of managers’ trustworthiness, communication with managers, and positive personal experiences with managers were related to positive attitudes about management and support for existing initiatives

Dynamic Switch of Negative Feedback Regulation in Drosophila Akt–TOR Signaling

Akt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)–dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K–independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt–TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo

The NANOGrav 15-year Data Set: Evidence for a Gravitational-Wave Background

We report multiple lines of evidence for a stochastic signal that is correlated among 67 pulsars from the 15-year pulsar-timing data set collected by the North American Nanohertz Observatory for Gravitational Waves. The correlations follow the Hellings-Downs pattern expected for a stochastic gravitational-wave background. The presence of such a gravitational-wave background with a power-law-spectrum is favored over a model with only independent pulsar noises with a Bayes factor in excess of $10^{14}$, and this same model is favored over an uncorrelated common power-law-spectrum model with Bayes factors of 200-1000, depending on spectral modeling choices. We have built a statistical background distribution for these latter Bayes factors using a method that removes inter-pulsar correlations from our data set, finding $p = 10^{-3}$ (approx. $3\sigma$) for the observed Bayes factors in the null no-correlation scenario. A frequentist test statistic built directly as a weighted sum of inter-pulsar correlations yields $p = 5 \times 10^{-5} - 1.9 \times 10^{-4}$ (approx. $3.5 - 4\sigma$). Assuming a fiducial $f^{-2/3}$ characteristic-strain spectrum, as appropriate for an ensemble of binary supermassive black-hole inspirals, the strain amplitude is $2.4^{+0.7}_{-0.6} \times 10^{-15}$ (median + 90% credible interval) at a reference frequency of 1/(1 yr). The inferred gravitational-wave background amplitude and spectrum are consistent with astrophysical expectations for a signal from a population of supermassive black-hole binaries, although more exotic cosmological and astrophysical sources cannot be excluded. The observation of Hellings-Downs correlations points to the gravitational-wave origin of this signal.Comment: 30 pages, 18 figures. Published in Astrophysical Journal Letters as part of Focus on NANOGrav's 15-year Data Set and the Gravitational Wave Background. For questions or comments, please email [email protected]