Nitric Oxide as a Switching Mechanism between Axon Degeneration and Regrowth during Developmental Remodeling.

During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), we found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. We found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.

Transneuronal Dpr12/DIP-δ interactions facilitate compartmentalized dopaminergic innervation of Drosophila mushroom body axons.

The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB γ-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in γ-neurons for their developmental regrowth into the distal γ4/5 zones, where both Dpr12 and its interacting protein, DIP-δ, are enriched. DIP-δ functions in a subset of dopaminergic neurons that wire with γ-neurons within the γ4/5 zone. During metamorphosis, these dopaminergic projections arrive to the γ4/5 zone prior to γ-axons, suggesting that γ-axons extend through a prepatterned region. Thus, Dpr12/DIP-δ transneuronal interaction is required for γ4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution.

The caloric value of food intake structurally adjusts a neuronal mushroom body circuit mediating olfactory learning in Drosophila.

Associative learning enables the adaptive adjustment of behavioral decisions based on acquired, predicted outcomes. The valence of what is learned is influenced not only by the learned stimuli and their temporal relations, but also by prior experiences and internal states. In this study, we used the fruit fly Drosophila melanogaster to demonstrate that neuronal circuits involved in associative olfactory learning undergo restructuring during extended periods of low-caloric food intake. Specifically, we observed a decrease in the connections between specific dopaminergic neurons (DANs) and Kenyon cells at distinct compartments of the mushroom body. This structural synaptic plasticity was contingent upon the presence of allatostatin A receptors in specific DANs and could be mimicked optogenetically by expressing a light-activated adenylate cyclase in exactly these DANs. Importantly, we found that this rearrangement in synaptic connections influenced aversive, punishment-induced olfactory learning but did not impact appetitive, reward-based learning. Whether induced by prolonged low-caloric conditions or optogenetic manipulation of cAMP levels, this synaptic rearrangement resulted in a reduction of aversive associative learning. Consequently, the balance between positive and negative reinforcing signals shifted, diminishing the ability to learn to avoid odor cues signaling negative outcomes. These results exemplify how a neuronal circuit required for learning and memory undergoes structural plasticity dependent on prior experiences of the nutritional value of food.

Cofilin regulates axon growth and branching of Drosophila γ-neurons.

The mechanisms that control intrinsic axon growth potential, and thus axon regeneration following injury, are not well understood. Developmental axon regrowth of Drosophila mushroom body γ-neurons during neuronal remodeling offers a unique opportunity to study the molecular mechanisms controlling intrinsic growth potential. Motivated by the recently uncovered developmental expression atlas of γ-neurons, we here focus on the role of the actin-severing protein cofilin during axon regrowth. We show that Twinstar (Tsr), the fly cofilin, is a crucial regulator of both axon growth and branching during developmental remodeling of γ-neurons. tsr mutant axons demonstrate growth defects both in vivo and in vitro, and also exhibit actin-rich filopodial-like structures at failed branch points in vivo Our data is inconsistent with Tsr being important for increasing G-actin availability. Furthermore, analysis of microtubule localization suggests that Tsr is required for microtubule infiltration into the axon tips and branch points. Taken together, we show that Tsr promotes axon growth and branching, likely by clearing F-actin to facilitate protrusion of microtubules.

Mechanisms of developmental neurite pruning.

The precise wiring of the nervous system is a combined outcome of progressive and regressive events during development. Axon guidance and synapse formation intertwined with cell death and neurite pruning sculpt the mature circuitry. It is now well recognized that pruning of dendrites and axons as means to refine neuronal networks, is a wide spread phenomena required for the normal development of vertebrate and invertebrate nervous systems. Here we will review the arising principles of cellular and molecular mechanisms of neurite pruning. We will discuss these principles in light of studies in multiple neuronal systems, and speculate on potential explanations for the emergence of neurite pruning as a mechanism to sculpt the nervous system.

Cis inhibition of co-expressed DIPs and Dprs shapes neural development.

In Drosophila , two interacting adhesion protein families, Dprs and DIPs, coordinate the assembly of neural networks. While intercellular DIP/Dpr interactions have been well characterized, DIPs and Dprs are often co-expressed within the same cells, raising the question as to whether they also interact in cis . We show, in cultured cells and in vivo, that DIP-α and DIP-δ can interact in cis with their ligands, Dpr6/10 and Dpr12, respectively. When co-expressed in cis with their cognate partners, these Dprs regulate the extent of trans binding, presumably through competitive cis interactions. We demonstrate the neurodevelopmental effects of cis inhibition in fly motor neurons and in the mushroom body. We further show that a long disordered region of DIP-α at the C-terminus is required for cis but not trans interactions, likely because it alleviates geometric constraints on cis binding. Thus, the balance between cis and trans interactions plays a role in controlling neural development.

piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning.

Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body gamma neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as being essential for axon pruning. The cohesin complex maintains sister-chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1(-/-) clones is rescued by expressing SMC1 in neurons, revealing a postmitotic function. SMC1(-/-) clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and is enhanced by a reduced dose of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis.

Cell-type-specific TEV protease cleavage reveals cohesin functions in Drosophila neurons.

Cohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the alpha kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS.

A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. Here we show that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins, is an essential mechanistic link in this compensatory interaction. We found that compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensated for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 was sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Morover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy.

Neuronal excitability as a regulator of circuit remodeling.

Postnatal remodeling of neuronal connectivity shapes mature nervous systems.1,2,3 The pruning of exuberant connections involves cell-autonomous and non-cell-autonomous mechanisms, such as neuronal activity. Indeed, experience-dependent competition sculpts various excitatory neuronal circuits.4,5,6,7,8,9 Moreover, activity has been shown to regulate growth cone motility and the stability of neurites and synaptic connections.10,11,12,13,14 However, whether inhibitory activity influences the remodeling of neuronal connectivity or how activity influences remodeling in systems in which competition is not clearly apparent is not fully understood. Here, we use the Drosophila mushroom body (MB) as a model to examine the role of neuronal activity in the developmental axon pruning of γ-Kenyon cells. The MB is a neuronal structure in insects, implicated in associative learning and memory,15,16 which receives mostly olfactory input from the antennal lobe.17,18 The MB circuit includes intrinsic neurons, called Kenyon cells (KCs), which receive inhibitory input from the GABAergic anterior paired lateral (APL) neuron among other inputs. The γ-KCs undergo stereotypic, steroid-hormone-dependent remodeling19,20 that involves the pruning of larval neurites followed by regrowth to form adult connections.21 We demonstrate that silencing neuronal activity is required for γ-KC pruning. Furthermore, we show that this is mechanistically achieved by cell-autonomous expression of the inward rectifying potassium channel 1 (irk1) combined with inhibition by APL neuron activity likely via GABA-B-R1 signaling. These results support the Hebbian-like rule "use it or lose it," where inhibition can destabilize connectivity and promote pruning while excitability stabilizes existing connections.

Actin-dependent astrocytic infiltration is a key step for axon defasciculation during remodeling.

Astrocytes are essential for synapse formation, maturation, and plasticity; however, their function during developmental neuronal remodeling is largely unknown. To identify astrocytic molecules required for axon pruning of mushroom body (MB) γ neurons in Drosophila, we profiled astrocytes before (larva) and after (adult) remodeling. Focusing on genes enriched in larval astrocytes, we identified 12 astrocytic genes that are required for axon pruning, including the F-actin regulators Actin-related protein 2/3 complex, subunit 1 (Arpc1) and formin3 (form3). Interestingly, perturbing astrocytic actin dynamics does not affect their gross morphology, migration, or transforming growth factor ß (TGF-ß) secretion. In contrast, actin dynamics is required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitates infiltration by Arpc1 knockdown (KD) astrocytes and promotes axon pruning. Conversely, increased axonal adhesion reduces lobe infiltration by wild-type (WT) astrocytes. Together, our findings suggest that actin-dependent astrocytic infiltration is a key step in axon pruning, thus promoting our understanding of neuron-glia interactions during remodeling.

Spatiotemporal Control of Neuronal Remodeling by Cell Adhesion Molecules: Insights From Drosophila.

Developmental neuronal remodeling is required for shaping the precise connectivity of the mature nervous system. Remodeling involves pruning of exuberant neural connections, often followed by regrowth of adult-specific ones, as a strategy to refine neural circuits. Errors in remodeling are associated with neurodevelopmental disorders such as schizophrenia and autism. Despite its fundamental nature, our understanding of the mechanisms governing neuronal remodeling is far from complete. Specifically, how precise spatiotemporal control of remodeling and rewiring is achieved is largely unknown. In recent years, cell adhesion molecules (CAMs), and other cell surface and secreted proteins of various families, have been implicated in processes of neurite pruning and wiring specificity during circuit reassembly. Here, we review some of the known as well as speculated roles of CAMs in these processes, highlighting recent advances in uncovering spatiotemporal aspects of regulation. Our focus is on the fruit fly Drosophila, which is emerging as a powerful model in the field, due to the extensive, well-characterized and stereotypic remodeling events occurring throughout its nervous system during metamorphosis, combined with the wide and constantly growing toolkit to identify CAM binding and resulting cellular interactions in vivo. We believe that its many advantages pose Drosophila as a leading candidate for future breakthroughs in the field of neuronal remodeling in general, and spatiotemporal control by CAMs specifically.

Pruning deficits of the developing Drosophila mushroom body result in mild impairment in associative odour learning and cause hyperactivity.

The principles of how brain circuits establish themselves during development are largely conserved across animal species. Connections made during embryonic development that are appropriate for an early life stage are frequently remodelled later in ontogeny via pruning and subsequent regrowth to generate adult-specific connectivity. The mushroom body of the fruit fly Drosophila melanogaster is a well-established model circuit for examining the cellular mechanisms underlying neurite remodelling. This central brain circuit integrates sensory information with learned and innate valences to adaptively instruct behavioural decisions. Thereby, the mushroom body organizes adaptive behaviour, such as associative learning. However, little is known about the specific aspects of behaviour that require mushroom body remodelling. Here, we used genetic interventions to prevent the intrinsic neurons of the larval mushroom body (γ-type Kenyon cells) from remodelling. We asked to what degree remodelling deficits resulted in impaired behaviour. We found that deficits caused hyperactivity and mild impairment in differential aversive olfactory learning, but not appetitive learning. Maintenance of circadian rhythm and sleep were not affected. We conclude that neurite pruning and regrowth of γ-type Kenyon cells is not required for the establishment of circuits that mediate associative odour learning per se, but it does improve distinct learning tasks.

Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning.

Axon pruning by degeneration remodels exuberant axonal connections and is widely required for the development of proper circuitry in the nervous system from insects to mammals. Developmental axon degeneration morphologically resembles injury-induced Wallerian degeneration, suggesting similar underlying mechanisms. As previously reported for mice, we show that Wlds protein substantially delays Wallerian degeneration in flies. Surprisingly, Wlds has no effect on naturally occurring developmental axon degeneration in flies or mice, although it protects against injury-induced degeneration of the same axons at the same developmental age. By contrast, the ubiquitin-proteasome system is intrinsically required for both developmental and injury-induced axon degeneration. We also show that the glial cell surface receptor Draper is required for efficient clearance of axon fragments during developmental axon degeneration, similar to its function in injury-induced degeneration. Thus, mechanistically, naturally occurring developmental axon pruning by degeneration and injury-induced axon degeneration differ significantly in early steps, but may converge onto a common execution pathway.

With a little help from my friends: how intercellular communication shapes neuronal remodeling.

Developmental neuronal remodeling shapes the mature connectivity of the nervous system in both vertebrates and invertebrates. Remodeling often combines degenerative and regenerative events, and defects in its normal progression have been linked to neurological disorders. Here we review recent progress that highlights the roles of cell-cell interactions during remodeling. We propose that these are fundamental to elucidating how spatiotemporal control of remodeling and coordinated circuit remodeling are achieved. We cover examples spanning various neuronal circuits in vertebrates and invertebrates and involving interactions between neurons and different cell types.

Developmental axon regrowth and primary neuron sprouting utilize distinct actin elongation factors.

Intrinsic neurite growth potential is a key determinant of neuronal regeneration efficiency following injury. The stereotypical remodeling of Drosophila γ-neurons includes developmental regrowth of pruned axons to form adult specific connections, thereby offering a unique system to uncover growth potential regulators. Motivated by the dynamic expression in remodeling γ-neurons, we focus here on the role of actin elongation factors as potential regulators of developmental axon regrowth. We found that regrowth in vivo requires the actin elongation factors Ena and profilin, but not the formins that are expressed in γ-neurons. In contrast, primary γ-neuron sprouting in vitro requires profilin and the formin DAAM, but not Ena. Furthermore, we demonstrate that DAAM can compensate for the loss of Ena in vivo. Similarly, DAAM mutants express invariably high levels of Ena in vitro. Thus, we show that different linear actin elongation factors function in distinct contexts even within the same cell type and that they can partially compensate for each other.

Role and regulation of starvation-induced autophagy in the Drosophila fat body.

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Here, we show that signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor.

Glial Derived TGF-ß Instructs Axon Midline Stopping.

A fundamental question that underlies the proper wiring and function of the nervous system is how axon extension stops during development. However, our mechanistic understanding of axon stopping is currently poor. The stereotypic development of the Drosophila mushroom body (MB) provides a unique system in which three types of anatomically distinct neurons (γ, α'/ß', and α/ß) develop and interact to form a complex neuronal structure. All three neuronal types innervate the ipsi-lateral side and do not cross the midline. Here we find that Plum, an immunoglobulin (Ig) superfamily protein that we have previously shown to function as a TGF-ß accessory receptor, is required within MB α/ß neurons for their midline stopping. Overexpression of Plum within MB neurons is sufficient to induce retraction of α/ß axons. As expected, rescue experiments revealed that Plum likely functions in α/ß neurons and mediates midline stopping via the downstream effector RhoGEF2. Finally, we have identified glial-derived Myoglianin (Myo) as the major TGF-ß ligand that instructs midline stopping of MB neurons. Taken together, our study strongly suggests that TGF-ß signals originating from the midline facilitate midline stopping of α/ß neuron in a Plum dependent manner.

Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila.

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.