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Languages : en
Pages : 0
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Synapses are the basic units of neural structure and function. Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. In my dissertation work, I took a combined approach of genetics, molecular biology and biochemistry to identify genes and molecular pathways that regulate synaptic growth in Drosophila, and discovered neuropeptide signaling as a novel mechanism in this process. Neuropeptides have been known to affect neuronal excitability and the strength of synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Through forward genetics, I discovered a cholecystokinin-like receptor (CCKLR) and its predicted ligand drosulfakinin (DSK), as components of a signaling pathway that strongly promote growth of the Drosophila larval neuromuscular junction (NMJ). Loss-of-function mutations of CCKLR or dsk produce severe NMJ undergrowth, whereas over-expression of CCKLR leads to NMJ overgrowth. Through targeted RNAi expression and transgenic rescue experiments I show that presynaptic expression of CCKLR in motor neurons is necessary and sufficient for regulation of NMJ growth. Through analysis of double mutants, I have dissected the signaling pathway downstream of the receptor. I show that this pathway involves G-protein-dependent stimulation of adenyl cyclase to activate PKA, which in turn activates CREB, the cAMP-response element binding protein. In addition, I demonstrate that DSK activates CCKLR biochemically, and that DSK/CCKLR signaling affects synaptic transmission and larval locomotor behavior. To discover novel genes that interact with the CCKLR-signaling pathway to regulate NMJ growth, I performed a modifier screen using chromosomal deficiencies. This pilot screen has identified several regions on the second and third chromosomes that dominantly enhance or suppress the NMJ phenotype of CCKLR mutants. Intriguingly, a number of neuropeptide signaling molecules were found among the candidate interacting genes. Specifically, I have found that leucokinin (DLK) and leucokinin receptor (LKR) negatively regulate NMJ growth, and strongly interact with CCKLR. The results of the modifier screen suggest that neuropeptide signaling may be a more common mechanism for regulating NMJ growth than previously realized and many of the molecules are yet to be uncovered.
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Languages : en
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Book Description
Synapses are the basic units of neural structure and function. Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. In my dissertation work, I took a combined approach of genetics, molecular biology and biochemistry to identify genes and molecular pathways that regulate synaptic growth in Drosophila, and discovered neuropeptide signaling as a novel mechanism in this process. Neuropeptides have been known to affect neuronal excitability and the strength of synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Through forward genetics, I discovered a cholecystokinin-like receptor (CCKLR) and its predicted ligand drosulfakinin (DSK), as components of a signaling pathway that strongly promote growth of the Drosophila larval neuromuscular junction (NMJ). Loss-of-function mutations of CCKLR or dsk produce severe NMJ undergrowth, whereas over-expression of CCKLR leads to NMJ overgrowth. Through targeted RNAi expression and transgenic rescue experiments I show that presynaptic expression of CCKLR in motor neurons is necessary and sufficient for regulation of NMJ growth. Through analysis of double mutants, I have dissected the signaling pathway downstream of the receptor. I show that this pathway involves G-protein-dependent stimulation of adenyl cyclase to activate PKA, which in turn activates CREB, the cAMP-response element binding protein. In addition, I demonstrate that DSK activates CCKLR biochemically, and that DSK/CCKLR signaling affects synaptic transmission and larval locomotor behavior. To discover novel genes that interact with the CCKLR-signaling pathway to regulate NMJ growth, I performed a modifier screen using chromosomal deficiencies. This pilot screen has identified several regions on the second and third chromosomes that dominantly enhance or suppress the NMJ phenotype of CCKLR mutants. Intriguingly, a number of neuropeptide signaling molecules were found among the candidate interacting genes. Specifically, I have found that leucokinin (DLK) and leucokinin receptor (LKR) negatively regulate NMJ growth, and strongly interact with CCKLR. The results of the modifier screen suggest that neuropeptide signaling may be a more common mechanism for regulating NMJ growth than previously realized and many of the molecules are yet to be uncovered.
Author: Josh Dubnau
Publisher: Cambridge University Press
ISBN: 1107009030
Category : Medical
Languages : en
Pages : 309
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A comprehensive portrayal of the behaviour genetics of the fruit fly (Drosophila melanogaster) and the methods used in these studies.
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Languages : en
Pages : 278
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Molecular and genetic analysis of synaptic signaling in Drosophila has yielded many insights into nervous system development, properties of synaptic transmission, and how long-lasting changes in neurons occur. Synaptic signaling components required for synaptic transmission and pathways leading to nervous system plasticity are typically conserved from insects to humans. The role of proteins and genes in synaptic function in flies can be analyzed from the level of a single synapse to complex behaviors in the whole organism. Because of a fully sequenced genome and the ease of mutagenesis in flies, genetic screens have been useful in identifying novel regulators of synaptic transmission and long-term memory. In flies, conditional mutations affecting synaptic transmission at nerve terminals often lead to temperature sensitive paralysis. In a screen for mutations that interact with Drosophila shibirets mutants, the stoned gene was identified as a regulator of synaptic vesicle cycling. Stoned encodes two neuronally expressed proteins, stonedA and B, which are required for synaptic vesicle recycling and normal synaptic transmission. However, the exact functions of the two stoned proteins are not fully understood. We investigate distinct roles of the stoned proteins here and show that stoned has a novel role in synaptic growth. Memory in flies can be divided into genetically distinct phases based on the requirement for protein synthesis and activation of the transcription factor CREB. Novel regulators of long-term olfactory avoidance memory were isolated in a mutant screen in flies. Mutants in the Drosophila gene lk6, homologous to the translational regulator MNK, have defects in long-term olfactory avoidance memory. We find that lk6 is highly expressed in the fly nervous system, and is activated by and functions downstream of Ras/ERK signaling in fly neurons. Insights provided here from Drosophila add to the evidence that MNK may be the link between ERK signaling and the regulation of translation in long-term plasticity. Ultimately, understanding synaptic function has therapeutic potential to aid in alleviation of nervous system dysfunction. Insight into the molecular pathways underlying plasticity and long-term memory gained from studies in flies, mollusks, and rodents has been pivotal in the development of potential drugs to aid in memory deficits in humans.
Author: Hong Iris Wan
Publisher:
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Category :
Languages : en
Pages : 392
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Author: Aline Dorret Blunk
Publisher:
ISBN:
Category :
Languages : en
Pages : 177
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Neuronal communication requires a spatially organized synaptic apparatus to coordinate neurotransmitter release from synaptic vesicles and activation of postsynaptic receptors. Structural remodeling of synaptic connections can strengthen neuronal communication and synaptic efficacy during development and behavioral plasticity. Here, I describe experimental approaches that have revealed how the actin cytoskeleton participates in transynaptic signaling to control synapse assembly. I also describe my studies on how regulation of endocytic trafficking controls synaptic growth during neuronal development. To identify regulators of synapse assembly, I carried out a large-scale EMS mutagenesis screen of the second chromosome. From this screen I identified a mutation in actin 57B that disrupts synaptic morphology and presynaptic active zone organization. Actin 57B is one of six actin genes in Drosophila and is expressed in body wall muscle during larval development. The isolated allele harbors a point mutation disrupting a highly conserved amino acid present throughout the actin family. Homozygous mutant larvae show impaired alignment and spacing of presynaptic active zones. Additionally, disruption of the organization of the postsynaptic density is observed, with mislocalization of the Spectrin cytoskeleton and the PSD-homolog Disc-Large. Phallodin staining reveals a severe disruption of postsynaptic actin surrounding presynaptic boutons, with the formation of aberrant large actin swirls. Based on these results, we hypothesize that the loss of a synaptic interaction mediated by actin 57B leads to disruption of postsynaptic cytoskeletal organization and dysregulation of signals required to organize presynaptic active zones. Additionally, I present data that provide new insights into the mechanisms controlling synaptic growth signaling during transit through the endocytic pathway. Nervous Wreck (Nwk) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila. Here, I show that Nwk acts through a physical interaction with Sorting Nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated BMP receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwkcontaining recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by ESCRT-mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX116, NWK and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments. Together, these experiments have expanded our understanding of the molecular mechanisms that control synaptic growth and assembly, highlighting the role of the postsynaptic actin cytoskeleton and the presynaptic endosomal trafficking pathway as key regulators.
Author: Seung-Hoon Choi
Publisher:
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Category :
Languages : en
Pages : 240
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Neuropeptides regulate diverse physiological processes, including homeostatic metabolism, behavior, reproduction, and development. The neuropeptide Corazonin (Crz), was first isolated from American cockroach, P. americana, as a potent cardioactive substance, and has been shown to exert diverse functions in different insects. In Drosophila, Crz expression is limited to three groups of neurons; totaling only 26 neurons out of ~10,000 neurons in a third instar larval central nervous system (CNS). In adults, Crz is expressed in 6-8 pairs of protocerebral neurons and 2 pairs of male specific abdominal ganglion. To gain insight into such tight regulatory mechanisms of Crz gene transcription, Crz promoter activity was dissected in vivo. The promoter bashing experiments yielding various 5'-upstream sequences show that there are separate cis-acting elements that are highly conserved phylogenetically, which speaks to its functional significance in the activation of Crz transcription. In larval stage, a 504-bp upstream region is sufficient to activate Crz in all endogenous neurons. Further dissection revealed two important regions; one between -419-bp and -504-bp region for the expression in dorsal medial neuron (DM). The other located between -241-bp and -380-bp is responsible for dorsal lateral (DL) and ventral nerve cord (VNC) expression. The latter region can be subdivided into three DL-specific and two VNC-specific cis-acting elements. For DL-specific expression, two out of any three combination were needed; however, VNC needed two elements altogether. Interestingly, basal transcription factor binding site TATA box showed minor role for Crz expression. In contrast to the larval expression, 321-bp upstream region is sufficient to activate Crz in all adult neurons. For the male-specific abdominal ganglion (ms-aCrz) expression, the cis-acting element was found to be in a region between -250-bp and -290-bp. Overall, the data show that transcriptional regulatory mechanisms for Crz expression are not uniformed among Crz-containing neurons, which further indicates that their neuronal functions might be different. To identify the roles of Crz in Drosophila, several fly behaviors were tested; ethanol-related responses, olfactory sensing responses and circadian rhythmic behaviors. Crz cell deficient (Crz-CD) flies and Crz receptor knock down (CrzR-KD) flies showed significantly delayed recovery from ethanol-induced sedation compared to control flies. Such hangover phenotype was ethanol specific. This result suggests that Drosophila Crz involves in ethanol-related responses. Further analyses suggest that Crz-CD, CrzR-KD and CrzR mutation did not affect aldehyde dehydrogenase (ALDH) at transcription level, but reduced ALDH enzyme activity. Crz is also associated with olfactory signaling, as Crz-CD and CrzR-KD flies are unable to find odor source, such as live yeast paste. Previously, Crz neurons located in the vicinity of nerve terminals originated from circadian pacemaker Pdf-expressing neurons, which indicate Crz neurons as part of circadian circuit. However, circadian locomotor rhythmic behavior of Crz-CD, Crz over-expression and CrzR-KD flies show normal circadian rhythmic behavior.
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Languages : en
Pages : 218
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Drosophila has been used for decades to identify and analyze molecular pathways underlying processes such as neurodevelopment and the innate immune response and for modeling complex diseases. In this thesis, I describe the use of Drosophila in two different projects. The first is characterization of a novel signaling pathway regulating growth of the larval neuromuscular junction. The second is the development of an experimental model to investigate neurotoxic effects of radiation exposure during development. Using the larval neuromuscular junction as a model synapse we have identified the lipocalin GLaz, the ortholog of human Apolipoprotein D (hApoD) as a novel regulator of synaptic growth. We have shown that postsynaptic insulin signaling promotes growth of the presynaptic terminal and that insulin signaling is increased in GLaz mutants. Overexpression of GLaz or hApoD in glia results in NMJ undergrowth, which is suppressed by simultaneously increasing insulin signaling in muscle. These studies uncover a novel role for lipocalins in regulation of synaptic growth and support a model in which GLaz is secreted from glia and antagonizes postsynaptic insulin signaling to restrict NMJ growth. We have also used Drosophila to model the neurotoxic side effects of radiation exposure during development as in the treatment for pediatric central nervous system malignancies. We have shown that many of the side effects observed in human patients treated with radiation during development can be modeled in Drosophila. Adult flies exposed to radiation during larval development display reduced survival to adulthood, early death, impaired locomotor behavior, and neurodegeneration. These phenotypes are consistent with premature aging. One hallmark of premature aging is chronic inflammation. Similarly, we find persistent activation of the innate immune system in adult flies that were exposed to radiation during larval development. We further demonstrate that the innate immune response is protective acutely following radiation exposure. Together these data demonstrate that the innate immune pathway is a potential therapeutic target for reducing the side-effects of CRT. The use of this experimental model in genetic screens should facilitate identification of additional radioprotective or radiosensitizing pathways, which may be of further therapeutic value.
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Languages : en
Pages : 292
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Synaptic development and neural maintenance are essential for proper neural function. In my dissertation, I investigated the molecular and genetic mechanisms of synaptic development and neural maintenance using Drosophila as an experimental model. In part one of my thesis work, I focused on the regulation of synaptic development. To identify novel regulators of synaptic development, we used the Drosophila larval NMJ as a model system and conducted a forward genetic screen for variants affecting NMJ morphology in a collection of isogenic lines isolated from natural populations of Drosophila melanogaster from Africa. I successfully identified the lipocalin family member, Neural Lazarillo (NLaz) as a negative regulator of NMJ development. I extended this discovery by examining the effect of other family members on NMJ development. Analysis of Glial Lazarillo (GLaz) and its human ortholog, Apolipoprotein D (ApoD), uncovered a novel mechanism regulating NMJ development, in which glial expression of GLaz (or ApoD) negatively controls NMJ development by down-regulating a retrograde insulin signaling mechanism that promotes NMJ growth. This finding has identified a novel role for lipocalin and insulin signaling in the regulation of NMJ development, furthering our understanding of the complex mechanisms that mediate synaptic development and whose impairment in humans could lead to neurological disease. The goal of the second part of my work was to identify novel mechanisms for age-dependent neural maintenance. In a forward genetic screen for neurodegeneration mutants in Drosophila, we have identified a mutation of defense repressor 1 (dnr1) that encodes an inhibitor of the immune deficiency (Imd) pathway. Further experiments revealed that loss of dnr1 causes shortened lifespan and age-dependent neurodegeneration associated with activation of the Imd pathway and elevated expression of antimicrobial peptides (AMP). Furthermore, we demonstrated that over-activation of innate immune response in the brain by bacterial infection in the brain or neural overexpression of AMP genes, is responsible for neurodegeneration. Following this work, we have found that mutations of trabid, encoding another negative regulator of Imd pathway also causes neurodegeneration owing to over-activation of the innate immune response. These results may have important implications for the role of neuroinflammation in human neurodegenerative diseases.
Author: Mikhail Soloviev
Publisher: John Wiley & Sons
ISBN: 0470196491
Category : Science
Languages : en
Pages : 432
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The definitive guide to peptidomics- a hands-on lab reference The first truly comprehensive book about peptidomics for protein and peptide analysis, this reference provides a detailed description of the hows and whys of peptidomics and how the techniques have evolved. With chapters contributed by leading experts, it covers naturally occurring peptides, peptidomics methods and new developments, and the peptidomics approach to biomarker discovery. Explaining both the principles and the applications, Peptidomics: Methods and Applications: * Features examples of applications in diverse fields, including pharmaceutical science, toxicity biomarkers, and neuroscience * Details the successful peptidomic analyses of biological material ranging from plants to mammals * Describes a cross section of analytical techniques, including traditional methodologies, emerging trends, and new techniques for high throughput approaches An enlightening reference for experienced professionals, this book is sufficiently detailed to serve as a step-by-step guide for beginning researchers and an excellent resource for students taking biotechnology and proteomics courses. It is an invaluable reference for protein chemists and biochemists, professionals and researchers in drug and biopharmaceutical development, analytical and bioanalytical chemists, toxicologists, and others.
Author: Timothy G. Geary
Publisher: Springer Science & Business Media
ISBN: 1441969020
Category : Medical
Languages : en
Pages : 246
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Book Description
The need to continually discover new agents for the control or treatment of invertebrate pests and pathogens is undeniable. Agriculture, both animal and plant, succeeds only to the extent that arthropod and helminth consumers, vectors and pathogens can be kept at bay. Humans and their companion animals are also plagued by invertebrate parasites. The deployment of chemical agents for these purposes inevitably elicits the selection of resistant populations of the targets of control, necessitating a regular introduction of new kinds of molecules. Experience in other areas of chemotherapy has shown that a thorough understanding of the biology of disease is an essential platform upon which to build a discovery program. Unfortunately, investment of research resources into understanding the basic physiology of invertebrates as a strategy to illuminate new molecular targets for pesticide and parasiticide discovery has been scarce, and the pace of introduction of new molecules for these indications has been slowed as a result. An exciting and so far unexploited area to explore in this regard is invertebrate neuropeptide physiology. This book was assembled to focus attention on this promising field by compiling a comprehensive review of recent research on neuropeptides in arthropods and helminths, with contributions from many of the leading laboratories working on these systems.
