Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching

Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, we studied the growth of single sensory axons in an in vitro collagen assay system and identified an activity in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits.     

Reactive astrocytes are substrates for the growth of adult CNS axons in the presence of elevated levels of nerve growth factor.

To assess the necessary parameters for the growth of axons within the adult rat CNS, we have used intracerebral grafts of primary fibroblasts genetically engineered to express nerve growth factor (NGF). Following the implantation of NGF-producing primary fibroblasts within the striatum, cholinergic axons arising from the nucleus basalis grow toward and penetrate these grafts between 1 and 8 weeks. Grafts of noninfected control cells do not elicit axon sprouting at any time. Unmyelinated axons grow into grafts of NGF-producing cells only on reactive astrocytic processes, which contribute to a surrounding glial border. From our data concerning axon growth within the adult rat CNS, we conclude that reactive astrocytes can act as conducive substrates for growing axons; and only in the presence of elevated levels of NGF will permissive substrates (e.g., astrocytes) support axon growth by NGF-sensitive neurons.     

Regulation of immediate-early gene expression in rat retinal ganglion cells after axotomy and during regeneration through a peripheral nerve graft

To determine mechanisms of structural plasticity in adult CNS neurons, we investigated the expression of immediate early genes (IEGs) in the rat retina. Gene products of different IEG families (JUN and FOS proteins) and cAMP-responsive element binding protein (CREBP) were examined by immunohistochemistry under three different paradigms. Normal rats which were not axotomized were compared with axotomized animals, where retinal ganglion cells (RGCs) were axotomized by intraorbital optic nerve cut and retrogradely labeled with fluorogold (FG). Under these circumstances, RGCs show only transient sprouting, followed by continuous retrograde RGC degeneration. In the third group, after the optic nerve lesion, adult rats additionally received a sciatic nerve graft to the transected optic nerve stump. This allows some RGCs to regenerate an axon into the grafted nerve. In both groups, the time course of RGC survival and JUN, CREB, and FOS protein expression was monitored. In normal animals, JUN-Immunoreactivity (JUN-Ir) was not detectable in the retinal ganglion cell layer. JUN-Ir was induced in about 70% of all FG-positive RGCs 5 days after axotomy. The expression of JUN-Ir started to decline 8 days after axotomy. Only a few JUN-Ir-positive RGCs were found after 2 weeks. In transplanted animals, however, the numbers of JUN-Ir-positive RGCs were significantly higher 2 and 3 weeks after transplantation compared to animals that exclusively received axotomy. Furthermore, in grafted rats about 70% of the regenerating RGCs expressed JUN-Ir 2 weeks after grafting as compared to only 38% JUN-positive RGCs among the surviving but not regenerating RGCs. In normal animals CREBP-Ir was constitutively expressed in nearly all cells of the retinal ganglion cell layer. The decline in number of CREBP-Ir-positive cells paralleled the axotmy-induced RGC death. FOS-Ir-positive cells were not found in the ganglion cell layer at any time. These results demonstrate a selective and transient JUN expression of RGCs after axotomy which is sustained during axonal regeneration. This suggests that sciatic nerve grafts are able to regulate the expression of JUN proteins in axotomized RGCs of adult rats. 1994 John Wiley & Sons, Inc.     

Novel Caenorhabditis elegans unc-119 axon outgrowth defects correlate with behavioral phenotypes that are partially rescued by nonneural unc-119

UNC-119 function is necessary for the correct development of the Caenorhabditis elegans nervous system. Worms mutant for unc-119 exhibit nervous system structural defects, including supernumerary axon branches, defasciculated nerve fibers, and choice point errors. Axons of both mechanosensory (ALM) and chemo- sensory (ASI) neurons have elongation defects within the nerve ring. Expressing unc-119 cDNA in mechanosensory neurons rescues the elongation defect of ALM axons, but expression in ASI neurons does not rescue ASI axon elongation defects. Neither gross movement nor dauer larva formation defects are rescued in either case. However, expressing a construct including introns under the control of the same promoters results in substantial rescue of phenotypic defects. In these cases reporter expression expands to tissues outside those specified by the promoter, notably into head muscles. Surprisingly, expressing an unc-119 cDNA construct under the control of a muscle-specific promoter fully rescues the dauer formation defect and substantially rescues movement. Thus, although UNC-119 normally acts in a cell-autonomous fashion, the cell-nonautonomous rescue of neural function suggests that it either acts at the cell surface or that it can be transported into the cell from the extracellular environment and play its normal role.     

Intrinsic Neuronal Determinants Locally Regulate Extrasynaptic and Synaptic Growth at the Adult Neuromuscular Junction

Long-term functional plasticity in the nervous system can involve structural changes in terminal arborization and synaptic connections. To determine whether the differential expression of intrinsic neuronal determinants affects structural plasticity, we produced and analyzed transgenic mice overexpressing the cytosolic proteins cortical cytoskeleton–associated protein 23 (CAP-23) and growth-associated protein 43 (GAP-43) in adult neurons.

Like GAP-43, CAP-23 was downregulated in mouse motor nerves and neuromuscular junctions during the second postnatal week and reexpressed during regeneration. In transgenic mice, the expression of either protein in adult motoneurons induced spontaneous and greatly potentiated stimulus-induced nerve sprouting at the neuromuscular junction. This sprouting had transgene-specific features, with CAP-23 inducing longer, but less numerous sprouts than GAP-43. Crossing of the transgenic mice led to dramatic potentiation of the sprout-inducing activities of GAP-43 and CAP-23, indicating that these related proteins have complementary and synergistic activities. In addition to ultraterminal sprouting, substantial growth of synaptic structures was induced. Experiments with pre- and postsynaptic toxins revealed that in the presence of GAP-43 or CAP-23, sprouting was stimulated by a mechanism that responds to reduced transmitter release and may be independent of postsynaptic activation.

These results demonstrate the importance of intrinsic determinants in structural plasticity and provide an experimental approach to study its role in nervous system function.

     

Contrasting developmental axon regrowth and neurite sprouting of Drosophila mushroom body neurons reveals shared and unique molecular mechanisms

The molecular mechanisms regulating intrinsic axon growth potential during development or following injury remain largely unknown despite their vast importance. Here, we have established a neurite sprouting assay of primary cultured mushroom body (MB) neurons. We used the MARCM technique to both mark and manipulate MB neurons, enabling us to quantify the sprouting abilities of single WT and mutant neurons originating from flies at different developmental stages. Sprouting of dissociated MB neurons was dependent on wnd, the DLK ortholog, a conserved gene that is required for axon regeneration. Next, and as expected, we found that the sprouting ability of adult MB neurons was significantly decreased. In contrast, and to our surprise, we found that pupal‐derived neurons exhibit increased sprouting compared with neurons derived from larvae, suggesting the existence of an elevated growth potential state. We then contrasted the molecular requirements of neurite sprouting to developmental axon regrowth of MB ? neurons, a process that we have previously shown requires the nuclear receptor UNF acting via the target of rapamycin (TOR) pathway. Strikingly, we found that while TOR was required for neurite sprouting, UNF was not. In contrast, we found that PTEN inhibits sprouting in adult neurons, suggesting that TOR is regulated by the PI3K/PTEN pathway during sprouting and by UNF during developmental regrowth. Interestingly, the PI3K pathway as well as Wnd were not required for developmental regrowth nor for initial axon outgrowth suggesting that axon growth during circuit formation, remodeling, and regeneration share some molecular components but differ in others. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 262–276, 2016     

Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems

In an effort to determine whether the “growth state” and the “mature state” of a neuron are differentiated by different programs of gene expression, we have compared the rapidly transported (group I) proteins in growing and nongrowing axons in rabbits. We observed two polypeptides (GAP-23 and GAP-43) which were of particular interest because of their apparent association with axon growth. GAP-43 was rapidly transported in the central nervous system (CNS) (retinal ganglion cell) axons of neonatal animals, but its relative amount declined precipitously with subsequent development. It could not be reinduced by axotomy of the adult optic nerves, which do not regenerate; however, it was induced after axotomy of an adult peripheral nervous system nerve (the hypoglossal nerve, which does regenerate) which transported only very low levels of GAP-43 before axotomy. The second polypeptide, GAP-23 followed the same pattern of growth-associated transport, except that it was transported at significant levels in uninjured adult hypoglossal nerves and not further induced by axotomy. These observations are consistent with the “GAP hypothesis” that the neuronal growth state can be defined as an altered program of gene expression exemplified in part by the expression of GAP genes whose products are involved in critical growth-specific functions. When interpreted in terms of GAP hypothesis, they lead to the following conclusions: (a) the growth state can be subdivided into a “synaptogenic state” characterized by the transport of GAP-23 but not GAP-43, and an “axon elongation state” requiring both GAPs; (b) with respect to the expression of GAP genes, regeneration involves a recapitulation of a neonatal state of the neuron; and (c) the failure of mammalian CNS neurons to express the GAP genes may underly the failure of CNS axons to regenerate after axon injury.     

Oligodendrocytes as regulators of neuronal networks during early postnatal development

Oligodendrocytes are the glial cells responsible for myelin formation. Myelination occurs during the first postnatal weeks and, in rodents, is completed during the third week after birth. Myelin ensures the fast conduction of the nerve impulse; in the adult, myelin proteins have an inhibitory role on axon growth and regeneration after injury. During brain development, oligodendrocytes precursors originating in multiple locations along the antero-posterior axis actively proliferate and migrate to colonize the whole brain. Whether the initial interactions between oligodendrocytes and neurons might play a functional role before the onset of myelination is still not completely elucidated. In this article, we addressed this question by transgenically targeted ablation of proliferating oligodendrocytes during cerebellum development. Interestingly, we show that depletion of oligodendrocytes at postnatal day 1 (P1) profoundly affects the establishment of cerebellar circuitries. We observed an impressive deregulation in the expression of molecules involved in axon growth, guidance and synaptic plasticity. These effects were accompanied by an outstanding increase of neurofilament staining observed 4 hours after the beginning of the ablation protocol, likely dependent from sprouting of cerebellar fibers. Oligodendrocyte ablation modifies localization and function of ionotropic glutamate receptors in Purkinje neurons. These results show a novel oligodendrocyte function expressed during early postnatal brain development, where these cells participate in the formation of cerebellar circuitries, and influence its development.     

Impaired function of HDAC6 slows down axonal growth and interferes with axon initial segment development

The development of morphological neuronal polarity starts by the formation and elongation of an axon. At the same time the axon initial segment (AIS) is generated and creates a diffusion barrier which differentiate axon and somatodendritic compartment. Different structural and functional proteins that contribute to the generation of neuronal action potential are concentrated at the axon initial segment. While axonal elongation is controlled by signalling pathways that regulate cytoskeleton through microtubule associated proteins and tubulin modifications, the microtubule cytoskeleton under the AIS is mostly unknown. Thus, understanding which proteins modify tubulin, where in the neuron and at which developmental stage is crucial to understanding how morphological and functional neuronal polarity is achieved. In this study performed in mice and using a well established model of murine cultured hippocampal neurons, we report that the tubulin deacetylase HDAC6 is localized at the distal region of the axon, and its inhibition with TSA or tubacin slows down axonal growth. Suppression of HDAC6 expression with HDAC6 shRNAs or expression of a non-active mutant of HDAC6 also reduces axonal length. Furthermore, HDAC6 inhibition or suppression avoids the concentration of ankyrinG and sodium channels at the axon initial segment (AIS). Moreover, treatment of mouse cultured hippocampal neurons with detergents to eliminate the soluble pool of microtubules identified a pool of detergent resistant acetylated microtubules at the AIS, not present at the rest of the axon. Inhibition or suppression of HDAC6 increases acetylation all along the axon and disrupts the specificity of AIS cytoskeleton, modifying the axonal distal gradient localization of KIF5C to a somatodendritic and axonal localization. In conclusion, our results reveal a new role of HDAC6 tubulin deacetylase as a regulator of microtubule characteristics in the axon distal region where axonal elongation takes place, and allowing the development of acetylated microtubules microdomains where HDAC6 is not concentrated, such as the axon initial segment.     

Why do mature CNS neurons of mammals fail to re-establish connections following injury--functions of bcl-2

Factors inside and outside neurons control the process of axonal growth and regeneration. Recently, it has become apparent that neurons are determined intrinsically for their ability to grow axons. In the mammalian CNS, the intrinsic machinery of neurons that triggers the growth of axons during early embryonic stages is shut down at a certain point in development; as a consequence, axon elongation and regeneration cannot occur in postnatal life. The proto-oncogene Bcl-2 has been recognized to act as a key regulator for the program of axon elongation inside neurons. However, expressing the gene Bcl-2 in CNS neurons is not sufficient to induce nerve regeneration in the adult CNS, eliminating the inhibitory mechanism in the mature CNS environment is still required. Recently, the formation of glia scar has been reported to be the major limiting factor in the CNS environment that blocks nerve regeneration. These new discoveries challenge the classical view of nerve regeneration in the mammalian CNS. It opens up a new dimension in the study of the cellular and molecular mechanisms underlying neurodevelopmental and neurodegenerative diseases.     

RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

This study shows that p90 ribosomal S6 kinase 1 (RSK1) responds differentially to nerve injury in the peripheral and central nervous systems, and identifies it as a crucial regulator of axonal regeneration; mechanistically, RSK1 preferentially induces the synthesis of regeneration-related proteins via the RSK1-eEF2K-eEF2 axis.     

Enhanced axon outgrowth and improved long‐distance axon regeneration in sprouty2 deficient mice

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal‐regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2^+/? neuron cultures, whereas homozygous Spry2^?/? neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2^+/? mice recovered faster in motor but not sensory testing paradigms (Spry2^?/? mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP‐43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2^?/? mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4‐positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long‐distance axon regeneration in injured peripheral nerves. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 217–231, 2015     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.     

Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.     

Is astrocyte laminin involved in axon guidance in the mammalian CNS?

This paper provides evidence for the expression of laminin on glia in correlation with axon elongation and nerve pathway formation during embryonic development of the mouse optic nerve and other parts of the central nervous system (CNS). We show that punctate deposits of laminin on immature glial cells precede the entrance of the first optic axons into the nerve, and remain in close association with growing axons. Furthermore, we show that in one particular region of the optic pathway that the retinal ganglion cell axons avoid in normal animals (i.e., the pigmented area of the distal nerve) the punctate laminin matrix is missing. As the optic nerve matures punctate laminin deposits disappear, and laminin is reduced in the astroglial cytoplasm. The close correlation of the punctate form of laminin with early axonal growth is true not only in the optic nerve but also in some other parts of the CNS. We demonstrate such punctate laminin deposits in a model of astrocyte-induced regeneration of the corpus callosum in acallosal mice (G. Smith, R. Miller, and J. Silver, 1986, J. Comp. Neurol. 251, 23-43), and in glia associated with several normal developing axon trajectories, such as the corpus callosum, fornix, and pathways in the embryonic hindbrain. In all of these regions punctate laminin deposits are found on astroglia that are associated with early growing axons. Our results indicate that the punctate form of laminin, produced by astrocytes, may be an important factor involved in axon elongation and nerve pathway formation in the mammalian CNS.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

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。     

Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.