1 |
Glutamatergic Synapse Formation in Developing Zebrafish EmbryosFierro Jr., Javier 14 January 2015 (has links)
In order for a human being to process complex thought, cells within the brain must communicate with each other in a very precise manner. The mechanisms which underlie the development of these connections, however, are poorly understood and thus require a thorough investigation. In this dissertation, we attempt to identify components involved in stabilizing synaptic contacts and the mechanisms by which synaptic proteins are trafficked to newly forming contact sites. Interestingly, we also identify a gene involved in the formation of the myotome.
To identify proteins involved in stabilizing synaptic contacts, we characterized the function of 4.1B in developing zebrafish embryos. 4.1B is a scaffolding molecule involved in stabilizing protein complexes at sites of cell adhesion. We identified two 4.1B genes in the zebrafish genome, 4.1B-a and 4.1B-b, which are differentially expressed and have evolved divergent functions. 4.1B-a is expressed within the central nervous system, specifically within primary motor neurons. Knockdown studies show a reduction in the number of synapses and altered kinetics of touch evoked-responses, suggesting a role in synaptic stabilization. In contrast, 4.1B-b is primarily expressed in muscle cells. Knockdown of 4.1B-b results in severe muscle fiber disorganization as well as altered locomotor behaviors. Together, these data suggest the basic functions of 4.1B are evolutionarily conserved, with new roles described in the development of synapses and muscle fibers.
To determine the mechanisms that underlie protein recruitment to newly forming synapses, we examined the recruitment of three distinct transport packets in the zebrafish spinal cord. During presynaptic assembly, we found synaptic vesicle protein transport vesicles preceded piccolo-containing active zone precursor transport vesicles, which in turn preceded synapsin transport vesicles. We identified the last transport packet as a unique and independent mechanism for the recruitment of synapsin, a protein involved in regulating the reserve pool of synaptic vesicles. Importantly, we found cyclin-dependent kinase 5 regulated the late recruitment of synapsin transport packets to synapses, thus identifying kinases as a key signaling molecule in the formation of synaptic contacts. Together, this work provides new insight into the mechanisms that underlie synaptogenesis.
This dissertation includes both previously published and unpublished co-authored material.
|
2 |
Molecular Mechanisms of Astrocyte Vesicle Fusion at Synaptic InterfacesWolfes, Anne 28 September 2015 (has links)
No description available.
|
3 |
The Presynaptic F-box Protein FSN-1 Regulates Synapse Development via Retrograde Insulin Signaling in Caenorhabditis elegansHwang, Christine 26 July 2010 (has links)
Synaptogenesis entails the development and establishment of functional synapses, which form the fundamental unit of communication in the nervous system. Initially identified in Caenorhabditis elegans (C. elegans), the FSN-1, F-box protein family has emerged as evolutionarily conserved binding partners of PHR family proteins, which regulate synaptogenesis. Previously, we have shown that FSN-1 and RPM-1 form a SCF/FSN-1/RPM-1 ubiquitin ligase complex that negatively regulates synapse growth by downregulating presynaptic targets, like the MAP kinase pathway. For my master’s thesis, I used a combination of both candidate and forward genetic approaches to identify additional components of signaling pathways that are regulated by FSN-1 during synaptogenesis. Our studies are among the first to suggest diverging roles for these partners and provide the first evidence for a mechanism through which the F-box protein regulates synaptogenesis via retrograde insulin/IGF/FOXO signaling and glucosaminidase/O-GlcNAc modifications.
|
4 |
Role of DNedd4 Splice Isoforms in Neuromuscular Synaptogenesis in Drosophila MelanogasterZhong, Yunan 01 June 2011 (has links)
Drosophila Nedd4 (DNedd4), an E3 ubiquitin ligase, is known to be involved in neuromuscular (NM) synaptogenesis during embryogenesis. To further elucidate its mechanism and function in this process, two major splice isoforms, dNedd4 short (dNedd4S) and dNedd4 long (dNedd4L), were studied. My work shows that while dNedd4S positively regulates NM synaptogenesis, dNedd4L plays a negative role in this process. Unique regions in dNedd4L, including the N-terminal 66 amino acid-long sequence (but not the putative dAkt phosphorylation site) and the middle 159 amino acid-long sequence, as well as the catalytic site, are required for its negative function. I proposed one possible mechanism of dNedd4L acting as a negative regulator of dNedd4S. Results from my studies of the putative effect of dNedd4L on the catalytic activity of dNedd4S in vitro, as well as on the function of dNedd4S towards Comm in Drosophila S2 cells, did not support this mechanism.
|
5 |
The Presynaptic F-box Protein FSN-1 Regulates Synapse Development via Retrograde Insulin Signaling in Caenorhabditis elegansHwang, Christine 26 July 2010 (has links)
Synaptogenesis entails the development and establishment of functional synapses, which form the fundamental unit of communication in the nervous system. Initially identified in Caenorhabditis elegans (C. elegans), the FSN-1, F-box protein family has emerged as evolutionarily conserved binding partners of PHR family proteins, which regulate synaptogenesis. Previously, we have shown that FSN-1 and RPM-1 form a SCF/FSN-1/RPM-1 ubiquitin ligase complex that negatively regulates synapse growth by downregulating presynaptic targets, like the MAP kinase pathway. For my master’s thesis, I used a combination of both candidate and forward genetic approaches to identify additional components of signaling pathways that are regulated by FSN-1 during synaptogenesis. Our studies are among the first to suggest diverging roles for these partners and provide the first evidence for a mechanism through which the F-box protein regulates synaptogenesis via retrograde insulin/IGF/FOXO signaling and glucosaminidase/O-GlcNAc modifications.
|
6 |
Role of DNedd4 Splice Isoforms in Neuromuscular Synaptogenesis in Drosophila MelanogasterZhong, Yunan 01 June 2011 (has links)
Drosophila Nedd4 (DNedd4), an E3 ubiquitin ligase, is known to be involved in neuromuscular (NM) synaptogenesis during embryogenesis. To further elucidate its mechanism and function in this process, two major splice isoforms, dNedd4 short (dNedd4S) and dNedd4 long (dNedd4L), were studied. My work shows that while dNedd4S positively regulates NM synaptogenesis, dNedd4L plays a negative role in this process. Unique regions in dNedd4L, including the N-terminal 66 amino acid-long sequence (but not the putative dAkt phosphorylation site) and the middle 159 amino acid-long sequence, as well as the catalytic site, are required for its negative function. I proposed one possible mechanism of dNedd4L acting as a negative regulator of dNedd4S. Results from my studies of the putative effect of dNedd4L on the catalytic activity of dNedd4S in vitro, as well as on the function of dNedd4S towards Comm in Drosophila S2 cells, did not support this mechanism.
|
7 |
Regulation of filopodia dynamics is critical for proper synapse formationGauthier-Campbell, Catherine 05 1900 (has links)
Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts.
We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation.
We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process.
|
8 |
Regulation of filopodia dynamics is critical for proper synapse formationGauthier-Campbell, Catherine 05 1900 (has links)
Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts.
We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation.
We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process.
|
9 |
The biology of microglia in neural development and synaptic maintenance in homeostatic and inflammatory conditionsWoodbury, Maya Ellen 03 November 2016 (has links)
Microglia, the innate immune cells of the brain, are not only immune surveyors, but also play important roles in neural development and maintenance. Microglial aberrations, including changes in morphology, gene expression, and phagocytic activity, have been observed in humans and animal models of pathologies involving cognitive and behavioral consequences. However, the precise contribution of microglial biology is not well characterized. Expression profiling of microglia and neural stem cells, co-culture assays, and transgenic mice were used to identify microglial micro-RNAs and genes, and study their roles in neural development. The results show that a specific micro-RNA, miR-155, participates in the neurogenic deficits induced by inflammation, and microglia-derived Wnt5a is essential for neural differentiation and maturation. This indicates the potential involvement of abnormal microglia in neurodevelopmental disorders such as autism spectrum disorders (ASDs). ASDs are group of debilitating disorders characterized by behavioral symptoms, including social and communication deficits and repetitive or restricted behaviors. I hypothesize that aberrant microglial biology plays a role in neurogenic and behavioral deficits in a mouse model of ASD. I performed a time-course study of microglial gene expression profiling, neural and microglial morphology, neurophysiology, and behavior in the maternal immune activation (MIA) model of ASD induced by the innate immunity ligand polyinosinic:polycytidylic acid. Microglia in MIA offspring displayed altered expression of 22 genes including 14 involved in cell-cell interaction, increased complexity of branching, and increased interactions with dendritic spines of cortical layer V pyramidal neurons. Microglial abnormalities were associated with neurophysiological alterations, measured by whole-cell patch clamp recordings, increased neuronal spine density, and ASD-like behaviors. MIA offspring treated with a colony stimulating factor -1 receptor inhibitor, to deplete and replenish microglia, showed correction of specific behaviors, microglial gene expression and branching, microglia-spine interactions, and spine density, and partial correction of neurophysiology. The data presented here shed new insight into the functional effects of microglia gene and microRNA expression in neurodevelopment. Furthermore, inflammation induces microglial aberrations that lead to altered neurodevelopment; this strongly supports the idea that targeting specific microglial genes and miRNAs will be a worthwhile approach to pursue for molecular intervention in ASD and related disorders. / 2018-11-02T00:00:00Z
|
10 |
Regulation of filopodia dynamics is critical for proper synapse formationGauthier-Campbell, Catherine 05 1900 (has links)
Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts.
We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation.
We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process. / Medicine, Faculty of / Graduate
|
Page generated in 0.1054 seconds