Drosophila GSK3ß promotes microtubule disassembly and dendrite pruning in sensory neurons.

The evolutionarily conserved Glycogen Synthase Kinase 3ß (GSK3ß), a negative regulator of microtubules, is crucial for neuronal polarization, growth and migration during animal development. However, it remains unknown whether GSK3ß regulates neuronal pruning, which is a regressive process. Here, we report that the Drosophila GSK3ß homologue Shaggy (Sgg) is cell-autonomously required for dendrite pruning of ddaC sensory neurons during metamorphosis. Sgg is necessary and sufficient to promote microtubule depolymerization, turnover and disassembly in the dendrites. Although Sgg is not required for the minus-end-out microtubule orientation in dendrites, hyperactivated Sgg can disturb the dendritic microtubule orientation. Moreover, our pharmacological and genetic data suggest that Sgg is required to promote dendrite pruning at least partly via microtubule disassembly. We show that Sgg and Par-1 kinases act synergistically to promote microtubule disassembly and dendrite pruning. Thus, Sgg and Par-1 might converge on and phosphorylate a common downstream microtubule-associated protein(s) to disassemble microtubules and thereby facilitate dendrite pruning.

A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila.

Drosophila class IV ddaC neurons selectively prune all larval dendrites to refine the nervous system during metamorphosis. During dendrite pruning, severing of proximal dendrites is preceded by local microtubule (MT) disassembly. Here, we identify an unexpected role of Mini spindles (Msps), a conserved MT polymerase, in governing dendrite pruning. Msps associates with another MT-associated protein TACC, and both stabilize each other in ddaC neurons. Moreover, Msps and TACC are required to orient minus-end-out MTs in dendrites. We further show that the functions of msps in dendritic MT orientation and dendrite pruning are antagonized by the kinesin-13 MT depolymerase Klp10A. Excessive MT depolymerization, which is induced by pharmacological treatment and katanin overexpression, also perturbs dendritic MT orientation and dendrite pruning, phenocopying msps mutants. Thus, we demonstrate that the MT polymerase Msps is required to form dendritic minus-end-out MTs and thereby promotes dendrite pruning in Drosophila sensory neurons.

Polycomb group genes are required for neuronal pruning in Drosophila.

BACKGROUND: Pruning that selectively eliminates unnecessary or incorrect neurites is required for proper wiring of the mature nervous system. During Drosophila metamorphosis, dendritic arbourization sensory neurons (ddaCs) and mushroom body (MB) γ neurons can selectively prune their larval dendrites and/or axons in response to the steroid hormone ecdysone. An ecdysone-induced transcriptional cascade plays a key role in initiating neuronal pruning. However, how downstream components of ecdysone signalling are induced remains not entirely understood. RESULTS: Here, we identify that Scm, a component of Polycomb group (PcG) complexes, is required for dendrite pruning of ddaC neurons. We show that two PcG complexes, PRC1 and PRC2, are important for dendrite pruning. Interestingly, depletion of PRC1 strongly enhances ectopic expression of Abdominal B (Abd-B) and Sex combs reduced, whereas loss of PRC2 causes mild upregulation of Ultrabithorax and Abdominal A in ddaC neurons. Among these Hox genes, overexpression of Abd-B causes the most severe pruning defects, suggesting its dominant effect. Knockdown of the core PRC1 component Polyhomeotic (Ph) or Abd-B overexpression selectively downregulates Mical expression, thereby inhibiting ecdysone signalling. Finally, Ph is also required for axon pruning and Abd-B silencing in MB γ neurons, indicating a conserved function of PRC1 in two types of pruning. CONCLUSIONS: This study demonstrates important roles of PcG and Hox genes in regulating ecdysone signalling and neuronal pruning in Drosophila. Moreover, our findings suggest a non-canonical and PRC2-independent role of PRC1 in Hox gene silencing during neuronal pruning.

The membrane protein Raw regulates dendrite pruning via the secretory pathway.

Neuronal pruning is essential for proper wiring of the nervous systems in invertebrates and vertebrates. Drosophila ddaC sensory neurons selectively prune their larval dendrites to sculpt the nervous system during early metamorphosis. However, the molecular mechanisms underlying ddaC dendrite pruning remain elusive. Here, we identify an important and cell-autonomous role of the membrane protein Raw in dendrite pruning of ddaC neurons. Raw appears to regulate dendrite pruning via a novel mechanism, which is independent of JNK signaling. Importantly, we show that Raw promotes endocytosis and downregulation of the conserved L1-type cell-adhesion molecule Neuroglian (Nrg) prior to dendrite pruning. Moreover, Raw is required to modulate the secretory pathway by regulating the integrity of secretory organelles and efficient protein secretion. Mechanistically, Raw facilitates Nrg downregulation and dendrite pruning in part through regulation of the secretory pathway. Thus, this study reveals a JNK-independent role of Raw in regulating the secretory pathway and thereby promoting dendrite pruning.

A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila.

It has long been thought that microtubule disassembly, one of the earliest cellular events, contributes to neuronal pruning and neurodegeneration in development and disease. However, how microtubule disassembly drives neuronal pruning remains poorly understood. Here, we conduct a systematic investigation of various microtubule-destabilizing factors and identify exchange factor for Arf6 (Efa6) and Stathmin (Stai) as new regulators of dendrite pruning in ddaC sensory neurons during Drosophila metamorphosis. We show that Efa6 is both necessary and sufficient to regulate dendrite pruning. Interestingly, Efa6 and Stai facilitate microtubule turnover and disassembly prior to dendrite pruning without compromising the minus-end-out microtubule orientation in dendrites. Moreover, our pharmacological and genetic manipulations strongly support a key role of microtubule disassembly in promoting dendrite pruning. Thus, this systematic study highlights the importance of two selective microtubule destabilizers in dendrite pruning and substantiates a causal link between microtubule disassembly and neuronal pruning.

Protein phosphatase PP2A regulates microtubule orientation and dendrite pruning in Drosophila.

Pruning that selectively eliminates inappropriate projections is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, ddaC sensory neurons undergo dendrite-specific pruning in response to the steroid hormone ecdysone. However, the understanding of the molecular mechanisms underlying dendrite pruning remains incomplete. Here, we show that protein phosphatase 2A (PP2A) is required for dendrite pruning. The catalytic (Microtubule star/Mts), scaffolding (PP2A-29B), and two regulatory subunits (Widerborst/Wdb and Twins/Tws) play important roles in dendrite pruning. Functional analyses indicate that PP2A, via Wdb, facilitates the expression of Sox14 and Mical prior to dendrite pruning. Furthermore, PP2A, via Tws, governs the minus-end-out orientation of microtubules (MTs) in the dendrites. Moreover, the levels of Klp10A, a MT depolymerase, increase when PP2A is compromised. Attenuation of Klp10A fully rescues the MT orientation defects in mts or pp2a-29b RNAi ddaC neurons, suggesting that PP2A governs dendritic MT orientation by suppressing Klp10A levels and/or function. Taken together, this study sheds light on a novel function of PP2A in regulating dendrite pruning and dendritic MT polarity in sensory neurons.

Drosophila CLASP regulates microtubule orientation and dendrite pruning by suppressing Par-1 kinase.

The evolutionarily conserved CLASPs (cytoplasmic linker-associated proteins) are microtubule-associated proteins that inhibit microtubule catastrophe and promote rescue. CLASPs can regulate axonal elongation and dendrite branching in growing neurons. However, their roles in microtubule orientation and neurite pruning in remodeling neurons remain unknown. Here, we identify the Drosophila CLASP homolog Orbit/MAST, which is required for dendrite pruning in ddaC sensory neurons during metamorphosis. Orbit is important for maintenance of the minus-end-out microtubule orientation in ddaC dendrites. Our structural analysis reveals that the microtubule lattice-binding TOG2 domain is required for Orbit to regulate dendritic microtubule orientation and dendrite pruning. In a genetic modifier screen, we further identify the conserved Par-1 kinase as a suppressor of Orbit in dendritic microtubule orientation. Moreover, elevated Par-1 function impairs dendritic microtubule orientation and dendrite pruning, phenocopying orbit mutants. Overall, our study demonstrates that Drosophila CLASP governs dendritic microtubule orientation and dendrite pruning at least partly via suppressing Par-1 kinase.

Patronin governs minus-end-out orientation of dendritic microtubules to promote dendrite pruning in Drosophila.

Class IV ddaC neurons specifically prune larval dendrites without affecting axons during Drosophila metamorphosis. ddaCs distribute the minus ends of microtubules (MTs) to dendrites but the plus ends to axons. However, a requirement of MT minus-end-binding proteins in dendrite-specific pruning remains completely unknown. Here, we identified Patronin, a minus-end-binding protein, for its crucial and dose-sensitive role in ddaC dendrite pruning. The CKK domain is important for Patronin's function in dendrite pruning. Moreover, we show that both patronin knockdown and overexpression resulted in a drastic decrease of MT minus ends and a concomitant increase of plus-end-out MTs in ddaC dendrites, suggesting that Patronin stabilizes dendritic minus-end-out MTs. Consistently, attenuation of Klp10A MT depolymerase in patronin mutant neurons significantly restored minus-end-out MTs in dendrites and thereby rescued dendrite-pruning defects. Thus, our study demonstrates that Patronin orients minus-end-out MT arrays in dendrites to promote dendrite-specific pruning mainly through antagonizing Klp10A activity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that minor issues remain unresolved (see decision letter).