Psidin is required for neuron survival and axon targeting through two distinct molecular mechanisms in Drosophila

Stephan, Daniel (2012): Psidin is required for neuron survival and axon targeting through two distinct molecular mechanisms in Drosophila. Dissertation, LMU München: Fakultät für Biologie

Abstract

The formation of neuronal networks depends on the proper development and targeting of the neurons within the network. One key challenge during the development of such networks is the correct cross linking of axons and dendrites. Only correct synapse formation between dendrites and axons will allow neurons to contribute to the entire network. Therefore further insights into axon targeting mechanisms will help to understand the underlying developmental processes and contribute to future cures for a number of related diseases. Generally, once a neuron forms an axon, it starts growing towards a certain “target zone”. The underlying axon targeting mechanisms are controlled by a large number of extracellular cues provided by the extracellular matrix and neighboring cells. Depending on the neuron type, axons travel different distances towards their future synaptic partner. During that journey the neurons, more specifically the growth cone, constantly comes into contact with guidance cues. The growth cone symbolizes the forefront of an axon and is responsible to integrate different guidance signals. Depending on their nature, they trigger the local assembly or disassembly of the cytoskeleton and ultimately force the axon to turn into a certain direction. Although different guidance cues activate different signaling pathways, all of these cascades will eventually converge down on the cytoskeleton. These cytoskeletal rearrangements and changes in actin dynamics within the growth cone will promote the turning of the entire axon. In a series of events different guidance cues, attractive and repulsive, will guide the growth cone to its respective target. In this study I used the olfactory system, more specifically the olfactory receptor neurons (ORNs), of Drosophila melanogaster to investigate the mechanisms of axon targeting. The olfactory system of the fruit fly proved to be a very powerful model organism for a number of reasons: First, the number of genetic tools available for Drosophila allows the manipulation of many cellular aspects. Second, ORNs have an extremely stereotyped targeting pattern that proved to be a good system to investigate axon targeting mechanisms. The work presented in this thesis studied the role of the highly conserved actin binding protein Psidin during the development and targeting of ORNs. Herein, I was able to demonstrate that Psidin uses two independent molecular mechanisms to control ORN targeting and survival. To elucidate Psidin’s role in the aforementioned processes, I analyzed two predicted null alleles psidin1 (Brennan et al., 2007) and psidin55D4 (Kim et al., 2011), and one hypomorphic allele psidinIG978 (this study). The new hypomorphic allele psidinIG978 was mapped during this study and found to have a single point mutation within Psidin’s coding region (E320K). The data shown in this study demonstrate that Psidin is required at two different time points during the development of the olfactory system. During ORN development, Psidin is required as non-catalytic part of the N-acetyltransferase complex (NatB) to ensure ORN survival. At later stages during development, Psidin functions as an actin binding protein to regulate actin dynamics to ultimately ensure proper ORN axon targeting. I was able to show for the first time that Psidin’s previously reported function as actin binding protein in oocytes (Kim et al., 2011), is also true for neurons. The loss of Psidin leads to significantly reduced lamellipodia in growth cones of primary neurons in vitro. In agreement with Psidin’s role in actin dynamics is the finding that the parallel removal of the actin stabilizer Tropomyosin rescues the lamellipodia defect in psidin1 primary neurons. This strongly argues for Psidin being an actin destabilizing protein and antagonist of Tropomyosin. In general, psidin1 and psidin55D4 mutant axons showed severe mistargeting defects in vivo – e.g. defasciculation in Or59c and Or42a neurons or ectopic synapse formation in Or47a neurons. However, axons mutant for psidinIG978 displayed a less severe phenotype compared to the null alleles. In agreement with in vitro data, the parallel removal of Tropomyosin rescued the targeting defect in Or59c neurons in vivo. The growth cone and the lamellipodia are both important structures that keep axons responsive towards guidance cues. Therefore the lamellipodia reduction in psidin mutants is likely the cause for the observed targeting defects. Nevertheless, Psidin is required differentially among the ORN classes – the ones that project to dorsolateral or ventromedial glomeruli within the antennal lobe (AL) are more affected than centrally projecting classes. ORN classes that are more affected in psidin mutants have to turn upon entry of the AL. Therefore those classes (dorsolateral and ventromedial) have a higher requirement of Psidin, which has to maintain the lamellipodium, so that the axon can respond to cues in the first place. In addition, I overexpressed different isoforms of LimK and Cofilin to artificially create conditions that favor actin stabilization or destabilization. More generally, conditions that promoted actin destabilization and actin stabilization were able to rescue and aggravate the psidin1 phenotype, respectively. In addition to the targeting defect, psidin1 and psidin55D4 mutants showed a strong reduction in ORN cell numbers. In contrast, cell numbers were not affected in psidinIG978 mutant flies. Again, ORN classes were affected differently – e.g. Or42a neuron number was reduced by 83%, but Or59c number was only reduced by 46%. Indicating Psidin’s function in ORN survival, the expression of the anti-apoptotic protein p35 in psidin mutant neurons selectively rescued the cell number, but failed to rescue the targeting defects. Interestingly, the Psidin/Tropomyosin double mutant showed the opposite effect; here the targeting was rescued, but not the cell number. These findings gave strong indications that Psidin has two independent functions during ORN targeting and development. Psidin is predicted to be the non-catalytic part of the N-acteyltransferase complex B (NatB) in Drosophila (Brennan et al., 2007). Here, Psidin (non-catalytic) forms the NatB-complex together with dNAA20 (catalytic). This complex is thought to acetylate nascent protein chains N-terminally. In this study I demonstrated for the first time that both proteins interact in vivo and in vitro. Indicating that the NatB-complex is involved in ORN survival, the knock-down of dNAA20 in psidinIG978 mutants led to a reduction of ORN cell number that is reminiscent of the cell number in psidin1 or psidin55D4 background. At the same time, the knock-down of dNAA20 had no effect on the targeting of ORNs. Furthermore I was able to show that wild type Psidin and PsidinIG978 interact with dNAA20 at comparable levels in vitro. This is in agreement with the finding that the psidinIG978 allele selectively affects ORN targeting, but not ORN survival. In addition, I was able to map the interaction domain between Psidin and dNAA20. This revealed that the point mutation found in psidinIG978 is just outside of the minimal interaction domain. Deletion of the entire interaction domain led to a complete abolishment of the Psidin/dNAA20 interaction. Furthermore I was able to demonstrate that the interaction of Psidin and dNAA20 is regulated by the phosphorylation of a highly conserved serine residue (S678). Expression of the non-phosphorylatable Psidin isoform (S678A) rescued the targeting and cell number phenotype in vivo. Contrary expression of the phosphomimetic isoform (S678D) only rescued the targeting phenotype, but failed to restore ORN cell number in vivo. In line with this observation is the finding that the S678D isoform is unable to bind dNAA20 in vitro. At the same time the S678A isoform binds dNAA20 at normal levels in vitro. Taken together, the data presented in this work demonstrate that Psidin has two functions during the development and targeting of ORNs using two independent molecular mechanisms: First, during axon targeting Psidin is required as an actin destabilizing molecule and antagonist of Tropomyosin. Psidin maintains the lamellipodia size in growth cones and keeps the cytoskeleton in a dynamic and responsive state. This ensures that growing axons can respond properly to various guidance cues. Second, to ensure ORN survival, Psidin is required as non-catalytic part of the NatB-complex. Here, Psidin interacts with the catalytic subunit dNAA20. The formation of the NatB-complex is regulated by phosphorylation of a conserved serine. In its unphosphorylated state Psidin binds dNAA20 and ensures ORN survival, whereas phosphorylation causes the abolishment of this interaction which results in a reduction of ORN cell number. Concluding, this thesis unambiguously shows that Psidin is required at different time points during the formation of the olfactory system of Drosophila. It utilizes two different pathways to ensure (i) ORN survival as part of the NatB-complex and (ii) ORN targeting as actin binding protein. Due to its strong conservation in higher organisms, the here presented data provide important insights into the function of Psidin’s mammalian homologues.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Neurobiology, Axon Guidance, Actin, Nervous System Development, Drosophila melanogaster
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. Dezember 2012
• 1. Bericht­erstatter:in: Klein, Rüdiger