Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family

Like naturally occurring neuronal cell death, stereotyped pruning of long axon branches to temporary targets is a widespread regressive phenomenon in the developing mammalian brain that helps sculpt the pattern of neuronal connections. The mechanisms controlling stereotyped pruning are, however, poorly understood. Here, we provide evidence that semaphorins, activating the Plexin-A3 receptor, function as retraction inducers to trigger-stereotyped pruning of specific hippocampal mossy fiber and pyramidal axon branches. Both pruning events are defective in Plexin-A3 mutants, reflecting a cell-autonomous requirement for Plexin-A3. The distribution of mRNAs for Sema3F and Sema3A makes them candidates for triggering the pruning. In vitro, hippocampal neurons respond to semaphorins by retracting axon branches. These results implicate semaphorins as retraction inducers controlling stereotyped pruning in the mammalian brain.     

Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system

Axon pruning is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neurodegenerative diseases. However, little is known about the cellular and molecular mechanisms of axon pruning. We use the stereotyped pruning of gamma neurons of the Drosophila mushroom bodies (MB) during metamorphosis to investigate these mechanisms. Detailed time course analyses indicate that MB axon pruning is mediated by local degeneration rather than retraction and that the disruption of the microtubule cytoskeleton precedes axon pruning. In addition, multiple lines of genetic evidence demonstrate an intrinsic role of the ubiquitin-proteasome system in axon pruning; for example, loss-of-function mutations of the ubiquitin activating enzyme (E1) or proteasome subunits in MB neurons block axon pruning. Our findings suggest that some forms of axon pruning during development may share similarities with degeneration of axons in response to injury.     

Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus

Mounting evidence suggests that prolonged exposure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis relies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part dependent on proper homeostasis of neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and pruning and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotoxicity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclusions: a surge in BDNF protein expression “signals” intense IPB pruning in control animals and ketamine-induced downregulation of BDNF synthesis and maturation could contribute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal circuitry formation.     

Rho proteins: their function in neurons

Neurons possess a polarized morphology. In general, each neuron has several dendrites but only one axon. Such morphology is the basis for directionalized rapid signaling, information flowing from the short dendrites to the long axon. The mechanisms involved in the establishment of the neuronal polarity remain largely unknown. However, recently, members of Rho family proteins have been implicated in the regulation of neuronal morphology especially development of neuronal polarity, axon outgrowth and guidance, dendritic tree elaboration and synapse formation. Moreover, the Rho GTPases have been reported to be directly or indirectly involved in some neurological conditions such as X-linked mental retardation as well as Alzheimer's and Parkinson's diseases. These findings demonstrate the importance of Rho GTPases in the development, maintenance and function of the nervous system.     

Glia engulf degenerating axons during developmental axon pruning

Developmental axon pruning is widely used in constructing the nervous system. Accordingly, diverse mechanisms are likely employed for various forms of axon pruning. In the Drosophila mushroom bodies (MB), gamma neurons initially extend axon branches into both the dorsal and medial MB axon lobes in larvae. Through a well-orchestrated set of developmental events during metamorphosis, axon branches to both lobes degenerate prior to the formation of adult connections. Here, we analyze ultrastructural changes underlying axon pruning by using a genetically encoded electron microscopic (EM) marker to selectively label gamma neurons. By inhibiting axon pruning in combination with the use of this EM marker, we demonstrate a causal link between observed cellular events and axon pruning. These events include changes in axon ultrastructure, synaptic degeneration, and engulfment of degenerating axon fragments by glia for their subsequent breakdown via the endosomal-lysosomal pathway. Interestingly, glia selectively invade MB axon lobes at the onset of metamorphosis; this increase in cell number is independent of axon fragmentation. Our study reveals a key role for glia in the removal of axon fragments during developmental axon pruning.     

Astrocytes Play a Key Role in Drosophila Mushroom Body Axon Pruning

Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-β ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. Here we show that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, we show that astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, we found that blocking the expression of the cell engulfment receptor Draper in astrocytes only affects axonal debris clearance. Thereby we uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. Our study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning.     

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.     

Wallerian degeneration of zebrafish trigeminal axons in the skin is required for regeneration and developmental pruning

Fragments of injured axons that detach from their cell body break down by the molecularly regulated process of Wallerian degeneration (WD). Although WD resembles local axon degeneration, a common mechanism for refining neuronal structure, several previously examined instances of developmental pruning were unaffected by WD pathways. We used laser axotomy and time-lapse confocal imaging to characterize and compare peripheral sensory axon WD and developmental pruning in live zebrafish larvae. Detached fragments of single injured axon arbors underwent three stereotyped phases of WD: a lag phase, a fragmentation phase and clearance. The lag phase was developmentally regulated, becoming shorter as embryos aged, while the length of the clearance phase increased with the amount of axon debris. Both cell-specific inhibition of ubiquitylation and overexpression of the Wallerian degeneration slow protein (Wld(S)) lengthened the lag phase dramatically, but neither affected fragmentation. Persistent Wld(S)-expressing axon fragments directly repelled regenerating axon branches of their parent arbor, similar to self-repulsion among sister branches of intact arbors. Expression of Wld(S) also disrupted naturally occurring local axon pruning and axon degeneration in spontaneously dying trigeminal neurons: although pieces of Wld(S)-expressing axons were pruned, and some Wld(S)-expressing cells still died during development, in both cases detached axon fragments failed to degenerate. We propose that spontaneously pruned fragments of peripheral sensory axons must be removed by a WD-like mechanism to permit efficient innervation of the epidermis.     

神经元轴突生长的力生物学

神经系统作为一个复杂的体系,在其发育过程中轴突需要延伸较长的距离才能与下一级神经元或靶细胞形成突触。在这个复杂的移动过程中,神经元轴突在空间分布上形成了精确有序的结构。过去认为这种有序结构的形成主要由形态发生素的化学浓度梯度来指导,而最近的研究发现力学因素对调控轴突的延伸速度与方向发挥着重要的作用。因此,轴突的延伸本质上是一个力化学耦合过程。本文将结合自己过去的工作论述力学因素对轴突延伸的调控机制及相关的信号转导。这一领域的研究将为认识对神经系统疾病的发生以及神经再生提供重要的参考。     

Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1

The regulated degeneration of axons or dendrites (pruning) and neuronal apoptosis are widely used during development to determine the specificity of neuronal connections. Pruning and apoptosis often share similar mechanisms; for example, developmental dendrite pruning of Drosophila class IV dendritic arborization (da) neurons is induced by local caspase activation triggered by ubiquitin-mediated degradation of the caspase inhibitor DIAP1. Here, we examined the function of Valosin-containing protein (VCP), a ubiquitin-selective AAA chaperone involved in endoplasmic reticulum-associated degradation, autophagy and neurodegenerative disease, in Drosophila da neurons. Strong VCP inhibition is cell lethal, but milder inhibition interferes with dendrite pruning and developmental apoptosis. These defects are associated with impaired caspase activation and high DIAP1 levels. In cultured cells, VCP binds to DIAP1 in a ubiquitin- and BIR domain-dependent manner and facilitates its degradation. Our results establish a new link between ubiquitin, dendrite pruning and the apoptosis machinery.     

A little nip and tuck: axon refinement during development and axonal injury

While building the nervous system, regions of some developing axons are eliminated; this can also happen as a result of axonal injury. During development, many axon branches that are formed in excess of an organism's needs are fated for removal in a process called axon pruning. By contrast, when axons are injured the axon segment distal to the injury site is compartmentalized and eliminated. In both cases, the end result is similar -- a region of an axon is selected for removal. Recent evidence suggests that there are some similarities in the cellular and molecular mechanisms that regulate axon elimination in development and during axonal injury.     

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis

BACKGROUND: Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. RESULTS: Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppressed not only axon pruning but also the infiltration and engulfing action of the surrounding glia. CONCLUSIONS: These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. We therefore propose that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis.     

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.     

Wired on hormones: endocrine regulation of hypothalamic development

The hormones that regulate the hypothalamic circuits that control essential functions, such as reproduction and energy homeostasis, also specify brain architecture by regulating key developmental events. The cellular mechanisms underlying the developmental actions of testosterone and estrogen to determine patterns of neuronal cell death, synaptogenesis and axon guidance are being identified. Recent neuroanatomical evidence indicates that the adipocyte-derived hormone leptin may direct the development of hypothalamic pathways involved in energy homeostasis by promoting axonal projections from the arcuate nucleus of the hypothalamus to other hypothalamic sites that mediate the effects of leptin on food intake and body weight. Understanding how sex steroids and leptin regulate hypothalamic development will enable us to identify hormonally directed signaling events essential to the specification of neural circuitry that is optimized for sustained homeostasis.     

Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes, as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both: a distinction that is vital for engineering intervention strategies. To address this crucial issue, we have used the Drosophila FraX model to investigate the developmental function of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and is required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement during late brain development.     

C. elegans as a genetic model to identify novel cellular and molecular mechanisms underlying nervous system regeneration

Research into conditions that improve axon regeneration has the potential to open a new door for treatment of brain injury caused by stroke and neurodegenerative diseases of aging, such as Alzheimer, by harnessing intrinsic neuronal ability to reorganize itself. Elucidating the molecular mechanisms of axon regeneration should shed light on how this process becomes restricted in the postnatal stage and in the CNS and therefore could provide therapeutic targets for developing strategies to improve axon regeneration in the adult CNS. In this review, we first discuss the general view about nerve regeneration and the advantages of using C. elegans as a model system to study axon regeneration. We then compare the conserved regeneration patterns and molecular mechanisms between C. elegans and vertebrates. Lastly, we discuss the power of femtosecond laser technology and its application in axon regeneration research.Key words: axon regeneration, C. elegans, genetics, femtosecond laser, neuronal circuits     

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, we analyzed da neuron axon projections in embryonic and larval stages. We found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. We have identified a class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development, which could allow different classes of da neurons to develop differential connectivity to second-order neurons. We have examined the effect of Robo family members on class-specific axon lamination, and have also taken a forward genetic approach to identify new genes involved in axon and dendrite development. For the latter, we screened the third chromosome at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology.     

The specific targeting of guidance receptors within neurons: Who directs the directors?

Guidance molecules present in both axonal and dendritic growth cones mediate neuronal responses to extracellular cues thereby ensuring correct neurite pathfinding and development of the nervous system. Little is known though about the mechanisms employed by neurons to deliver these receptors, specifically and efficiently, to the extending growth cone. A deeper understanding of this process is crucial if guidance receptors are to be manipulated to promote nervous system repair. Studies in other polarised cells, notably epithelial, have elucidated fundamental routes to the intracellular segregation of molecules mediated by endosomal pathways. Due to their extreme complexity and specialisation, neurons appear to have built upon these generic systems to evolve sophisticated trafficking networks. A striking feature is the axon initial segment which acts like a valve to tightly regulate the flux of molecules both entering and leaving the axon. Once in the growth cone, further controls operate to enhance the retention or rejection, as appropriate, of membrane receptors. We discuss the current state of knowledge regarding the intracellular trafficking of axon guidance receptors and how this relates to their developmental roles. We highlight the various facets still to be properly elucidated and by building on existing data regarding neuronal polarity and intracellular sorting mechanisms suggest ways to fill these gaps.     

C. elegans as a genetic model to identify novel cellular and molecular mechanisms underlying nervous system regeneration

Research into conditions that improve axon regeneration has the potential to open a new door for treatment of brain injury caused by stroke and neurodegenerative diseases of aging, such as Alzheimer, by harnessing intrinsic neuronal ability to reorganize itself. Elucidating the molecular mechanisms of axon regeneration should shed light on how this process becomes restricted in the postnatal stage and in CNS and therefore could provide therapeutic targets for developing strategy to improve axon regeneration in adult CNS. In this review, we first discuss the general view about nerve regeneration and the advantages of using C. elegans as a model system to study axon regeneration. We then compare the conserved regeneration patterns and molecular mechanisms between C. elegans and vertebrates. Lastly, we discuss the power of femtosecond laser technology and its application in axon regeneration research.     

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.