The Molecular Mechanisms of Activity-Dependent Wingless (Wg)/Wnt Signaling at a Drosophila Glutamatergic Synapse: a Dissertation

Synaptic plasticity, the ability of synapses to change in strength, underlies complex brain functions such as learning and memory, yet little is known about the precise molecular mechanisms and downstream signaling pathways involved. The major goal of my doctoral thesis was to understand these molecular mechanisms and cellular processes underlying synaptic plasticity using the Drosophilalarval neuromuscular junction (NMJ) as a model system. My work centered on a signaling pathway, the Wg/Wnt signaling pathway, which was found to be crucial for activity-driven synapse formation. The Wg/Wnt family of secreted proteins, besides its well-characterized roles in embryonic patterning, cell growth and cancer, is beginning to be recognized as a pivotal player during synaptic differentiation and plasticity in the brain. At the DrosophilaNMJ, the Wnt-1 homolog Wingless (Wg) is secreted from presynaptic terminals and binds to Frizzled-2 (DFz2) receptors in the postsynaptic muscle. Perturbations in Wg signaling lead to poorly differentiated NMJs, containing synaptic sites that lack both neurotransmitter release sites and postsynaptic structures. In collaboration with other members of the Budnik lab, I set out to unravel the mechanisms by which Wg regulates synapse differentiation. We identified a novel transduction pathway that provides communication between the postsynaptic membrane and the nucleus, and which is responsible for proper synapse development. In this novel Frizzled Nuclear Import (FNI) pathway, the DFz2 receptor is internalized and transported towards the nucleus. The C-terminus of DFz2 is subsequently cleaved and imported into the postsynaptic nucleus for potential transcriptional regulation of synapse development (Mathews, Ataman, et al. Science (2005) 310:1344). My studies also centered on the genetic analysis of Glutamate Receptor (GluR) Interacting Protein (dGRIP), which in mammals has been suggested to regulate the localization of GluRs and more recently, synapse development. I generated mutations in the gene, transgenic strains carrying a dGRIP-RNAi and fluorescently tagged dGRIP, and antibodies against the protein. Remarkably, I found dgrip mutants had synaptic phenotypes that closely resembled those in mutations altering the FNI pathway. Through the genetic analysis of dgrip and components of the FNI pathway, immunoprecipitation studies, electron microscopy, in vivotrafficking assays, time-lapse imaging, and yeast two-hybrid assays, I demonstrated that dGRIP had a hitherto unknown role as an essential component of the FNI pathway. dGRIP was found in trafficking vesicles that contain internalized DFz2. Further, DFz2 and dGRIP likely interact directly. Through the use of pulse chase experiments I found that dGRIP is required for the transport of DFz2 from the synapse to the nucleus. These studies thus provided a molecular mechanism by which the Wnt receptor, DFz2, is trafficked from the postsynaptic membrane to the nucleus during synapse development and implicated dGRIP as an essential component of the FNI pathway (Ataman et al. PNAS (2006) 103:7841). In the final part of my dissertation, I concentrated on understanding the mechanisms by which neuronal activity regulates synapse formation, and the role of the Wnt pathway in this process. I found that acute changes in patterned activity lead to rapid modifications in synaptic structure and function, resulting in the formation of undifferentiated synaptic sites and to the potentiation of spontaneous neurotransmitter release. I also found that these rapid modifications required a bidirectional Wg transduction pathway. Evoked activity induced Wg release from synaptic sites, which stimulated both the postsynaptic FNI pathway, as well as an alternative presynaptic Wg pathway involving GSK-3ß/Shaggy. I suggest that the concurrent activation of these alternative pathways by the same ligand is employed as a mechanism for the simultaneous and coordinated assembly of the pre- and postsynaptic apparatus during activity-dependent synapse remodeling (Ataman et al. Neuron (2008) in press). In summary, my thesis work identified and characterized a previously unrecognized synaptic Wg/Wnt transduction pathway. Further, it established a mechanistic link between activity-dependent synaptic plasticity and bidirectional Wg/Wnt signaling. These findings provide novel mechanistic insight into synaptic plasticity

Fasciclin II signals new synapse formation through amyloid precursor protein and the scaffolding protein dX11/Mint

Cell adhesion molecules (CAMs) have been universally recognized for their essential roles during synapse remodeling. However, the downstream pathways activated by CAMs have remained mostly unknown. Here, we used the Drosophila larval neuromuscular junction to investigate the pathways activated by Fasciclin II (FasII), a transmembrane CAM of the Ig superfamily, during synapse remodeling. We show that the ability of FasII to stimulate or to prevent synapse formation depends on the symmetry of transmembrane FasII levels in the presynaptic and postsynaptic cell and requires the presence of the fly homolog of amyloid precursor protein (APPL). In turn, APPL is regulated by direct interactions with the PDZ (postsynaptic density-95/Discs large/zona occludens-1)-containing protein dX11/Mint/Lin-10, which also regulates synapse expansion downstream of FasII. These results provide a novel mechanism by which cell adhesion molecules are regulated and provide fresh insights into the normal operation of APP during synapse development

Glia and Muscle Sculpt Neuromuscular Arbors by Engulfing Destabilized Synaptic Boutons and Shed Presynaptic Debris

As synapses grow at the Drosophila neuromuscular junction, they shed membrane material in an activity-dependent manner. Glia and postsynaptic muscle cells are required to engulf this debris to ensure new synaptic growth

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase-independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease

A Chemical Genetic Approach Reveals Distinct Mechanisms of EphB Signaling During Brain Development

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, as EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knockin mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly, and specifically blocked. Using these mice we demonstrate that the tyrosine kinase activity of EphBs is required for axon guidance in vivo. By contrast, EphB-mediated synaptogenesis occurs normally when the kinase activity of EphBs is inhibited suggesting that EphBs mediate synapse development by an EphB tyrosine kinase-independent mechanism. Taken together, these experiments reveal that EphBs control axon guidance and synaptogenesis by distinct mechanisms, and provide a new mouse model for dissecting EphB function in development and disease

Assessment of the requisites of microbiology based infectious disease training under the pressure of consultation needs

<p>Abstract</p> <p>Background</p> <p>Training of infectious disease (ID) specialists is structured on classical clinical microbiology training in Turkey and ID specialists work as clinical microbiologists at the same time. Hence, this study aimed to determine the clinical skills and knowledge required by clinical microbiologists.</p> <p>Methods</p> <p>A cross-sectional study was carried out between June 1, 2010 and September 15, 2010 in 32 ID departments in Turkey. Only patients hospitalized and followed up in the ID departments between January-June 2010 who required consultation with other disciplines were included.</p> <p>Results</p> <p>A total of 605 patients undergoing 1343 consultations were included, with pulmonology, neurology, cardiology, gastroenterology, nephrology, dermatology, haematology, and endocrinology being the most frequent consultation specialties. The consultation patterns were quite similar and were not affected by either the nature of infections or the critical clinical status of ID patients.</p> <p>Conclusions</p> <p>The results of our study show that certain internal medicine subdisciplines such as pulmonology, neurology and dermatology appear to be the principal clinical requisites in the training of ID specialists, rather than internal medicine as a whole.</p

Whole-Exome Sequencing and Homozygosity Analysis Implicate Depolarization-Regulated Neuronal Genes in Autism

Although autism has a clear genetic component, the high genetic heterogeneity of the disorder has been a challenge for the identification of causative genes. We used homozygosity analysis to identify probands from nonconsanguineous families that showed evidence of distant shared ancestry, suggesting potentially recessive mutations. Whole-exome sequencing of 16 probands revealed validated homozygous, potentially pathogenic recessive mutations that segregated perfectly with disease in 4/16 families. The candidate genes (UBE3B, CLTCL1, NCKAP5L, ZNF18) encode proteins involved in proteolysis, GTPase-mediated signaling, cytoskeletal organization, and other pathways. Furthermore, neuronal depolarization regulated the transcription of these genes, suggesting potential activity-dependent roles in neurons. We present a multidimensional strategy for filtering whole-exome sequence data to find candidate recessive mutations in autism, which may have broader applicability to other complex, heterogeneous disorders

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

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