Title: The role of PI(4,5)P2 and PI(3,4,5)P3 in synaptic function and development
Other Titles: De rol van PI(4,5)P2 en PI(3,4,5)P3 in de functie en ontwikkeling van de synaps
Authors: Khuong Manh, Thang; S0175557
Issue Date: 23-May-2012
Abstract: SCIENTIFIC SUMMARYSynapses are specialized connections between nerve cells or between a nerve and a muscle cell that are important for correct functioning of the nervous system, controlling many of our body functions including perception, thought, sensation and muscle activity. Defects in development and function of synapses have been linked to pathologies of neurological disorders such as mental retardation and <SPAN style="FONT-SIZE: 13pt; COLOR: windowtext; LINE-HEIGHT: 150%; FONT-FAMILY: 'Times New Roman','serif'; TEXT-DECORATION: none; text-underline: none">Alzheimer's disease</SPAN> (Berman et al., 2008) and may be caused by abnormal composition and structure of specific synaptic lipids. A special phosphorylated lipid, PI(4,5)P2, has been known to play various roles in regulation of synaptic transmission, including synaptic vesicle endocytosis (Wenk et al., 2001). However, little is known about its role in synaptic development. In addition, a related, but structurally different lipid, PI(3,4,5)P3, present at a very low abundance, is thought to play important roles in neurons as well, but strong evidence is lacking (Martin-Pena et al., 2006; Tohda et al., 2006). Currently, there are few available tools to visualize and adequately localize PI(3,4,5)P3 at synapses, and its precise roles and exact mechanisms of action at the synapse have not all been uncovered. Therefore, in this thesis, by taking advantage of Drosophila genetics, I aimed to study these different issues in vivo using multidisciplinary approaches.To study the roles of PI(4,5)P2 in synaptic development, we tested the effect of PI(4,5)P2 on the growth and development of the Drosophila neuromuscular junction (NMJ). Interestingly, reduced levels of PI(4,5)P2 using a variety of different tools, including overexpression of a PI(4,5)P2-binding protein domain, knockdown of the kinases mediating the formation of PI(4,5)P2, and tweek mutants known to harbor less synaptic PI(4,5)P2, consistently cause the formation of larger NMJs characterized by an increase in the total synaptic length and an increased number of boutons. Restoring PI(4,5)P2 levels by mutating a PI phosphatase, synaptojanin, rescued these phenotypes, confirming that PI(4,5)P2 is important for regulation of synaptic growth.Investigating this regulation mechanistically, we found that PI(4,5)P2 acts in the same pathway with the actin branching activator, Wiskott-Aldrich syndrome protein (WSP) through its PI(4,5)P2 binding domain. At NMJs with reduced PI(4,5)P2 levels, we observed an increased number of actin patches visualized by actin binding protein Moesin-GFP, and this phenotype is very similar to that seen in wsp mutants. These actin patches correlate with NMJ length and the number of boutons, suggesting that altered PI(4,5)P2-WSP signaling induces unbranched actin causing new bouton sprouting.It is thought that to restrict NMJ growth, WSP cooperates with NWK, a presynaptic scaffold protein that negatively regulates WIT, a BMP receptor firmly linked to promoting NMJ growth. To determine if PI(4,5)P2-mediated control of NMJ morphology is NWK-dependent, we performed numerous genetic interactions of WSP, NWK, and WIT. We found that PI(4,5)P2-WSP acts in a parallel pathway to NWK and WIT to restrict NMJ growth. Similar genetic experiments using tweek mutants known to harbor less PI(4,5)P2, suggest that NWK, WSP and PI(4,5)P2 act in a Tweek-dependent pathway. Thus, our work lays down a genetic network controlled by Tweek that mediates local actin dynamics in a WSP/PI(4,5)P2 dependent manner and nuclear signaling in an NWK/BMP-dependent manner. To study the role of PI(3,4,5)P3 at synapses, we have developed a novel transgenic imaging probe based on split Venus to visualize and adequately localize PI(3,4,5)P3. Furthermore, we also generated transgenic fruit flies that allowed us to acutely manipulate PI(3,4,5)P3 levels in neurons using a rapamycin heterodimerization approach. We find the lipid to concentrate in the presynaptic membrane in discrete foci that colocalize with Syntaxin1A. We show PI(3,4,5)P3 is necessary and sufficient for Syntaxin1A clustering. Reduction of PI(3,4,5)P3 levels in neurons by overexperssion of PI(3,4,5)P3 -binding protein domain PH-GRP1 approach results in dispersion of Syntaxin1A clusters, while PI(3,4,5)P3 present in giant unilamellar vesicles is sufficient to cluster Syntaxin1A. This effect is dependent on positively charged residues in the Syntaxin1A polybasic linker, indicating electrostatic interactions control clustering. Functionally, we find that reduced PI(3,4,5)P3 results in temperature sensitive paralysis of adult flies and reduced neurotransmitter release, similar to weak Syntaxin1A loss of function mutants. Thus, my work uncovers a novel role for PI(3,4,5)P3 in the regulation of neurotransmitter release.In conclusion, my work has advanced our understanding of the roles of phosphoinositides in synaptic development and function. We demonstrate that presynaptic PI(4,5)P2 activates WSP to regulate NMJ size by controlling actin dynamics at synaptic boutons, while presynaptic PI(3,4,5)P3 controls Syntaxin1A clustering and neurotransmitter release. Given that synaptic development and function have been linked to pathologies of neurological disorders, our findings support future studies on using phosphoinositide metabolism as a potential therapeutic approach for the treatment of these disorders.
ISBN: 9789461650443
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Department of Human Genetics - miscellaneous
Laboratory of Neuronal Communication

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