Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons

Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors.     

Promotion of Peripheral Nerve Regeneration by Stimulation of the Extracellular Signal-Regulated Kinase (ERK) Pathway

Peripherally projecting neurons undergo significant morphological changes during development and regeneration. This neuroplasticity is controlled by growth factors, which bind specific membrane bound kinase receptors that in turn activate two major intracellular signal transduction cascades. Besides the PI3 kinase/AKT pathway, activated extracellular signal-regulated kinase (ERK) plays a key role in regulating the mode and speed of peripheral axon outgrowth in the adult stage. Cell culture studies and animal models revealed that ERK signaling is mainly involved in elongative axon growth in vitro and long-distance nerve regeneration in vivo. Here, we review ERK dependent morphological plasticity in adult peripheral neurons and evaluate the therapeutic potential of interfering with regulators of ERK signaling to promote nerve regeneration. Anat Rec, 302:1261–1267, 2019. © 2019 Wiley Periodicals, Inc.     

IGF-I specifically enhances axon outgrowth of corticospinal motor neurons

Corticospinal motor neurons (CSMN) are among the most complex CNS neurons; they control voluntary motor function and are prototypical projection neurons. In amyotrophic lateral sclerosis (ALS), both spinal motor neurons and CSMN degenerate; their damage contributes centrally to the loss of motor function in spinal cord injury. Direct investigation of CSMN is severely limited by inaccessibility in the heterogeneous cortex. Here, using new CSMN purification and culture approaches, and in vivo analyses, we report that insulin-like growth factor-1 (IGF-I) specifically enhances the extent and rate of murine CSMN axon outgrowth, mediated via the IGF-I receptor and downstream signaling pathways; this is distinct from IGF-I support of neuronal survival. In contrast, brain-derived neurotrophic factor (BDNF) enhances branching and arborization, but not axon outgrowth. These experiments define specific controls over directed differentiation of CSMN, indicate a distinct role of IGF-I in CSMN axon outgrowth during development, and might enable control over CSMN derived from neural precursors.     

ENU‐3 functions in an UNC‐6/netrin dependent pathway parallel to UNC‐40/DCC/frazzled for outgrowth and guidance of the touch receptor neurons in C. elegans

Results: An enu‐3 mutation enhances defects in ventral guidance of the processes of the AVM and PVM touch receptor neurons, the dorsal guidance of the distal tip cell and causes additional architectural defects in axons in unc‐40 mutant strains in an UNC‐6 dependent manner. These observations suggest that ENU‐3 and UNC‐40 function in parallel pathways dependent on UNC‐6. ENU‐3 depends on the presence of UNC‐40 for its full effect on motor neuron axon outgrowth. 相似文献   

Clinical hypochondroplasia in a family caused by a heterozygous double mutation in FGFR3 encoding GLY380LYS

In classical achondroplasia (Ach), a glycine residue is replaced by an arginine at codon 380 in exon 10 of the fibroblast growth factor receptor 3 gene (FGFR3). Here we report on a mother and daughter with hypochondroplasia (Hch) caused by a new heterozygous double mutation (1138_1139GG > AA) at the same codon 380, but encoding a lysine instead of the usual arginine. Previous functional assays of these codon 380 amino acid substitutions demonstrated a lesser activation of receptor signaling by lysine compared to arginine [Webster and Donoghue, 1996; EMBO J 15:520-527]. This could explain the milder phenotype observed in our patients. Several other rare double mutations were previously described in both FGFR2 and FGFR3 and interpreted as resulting from positive selection of spermatogonial cells owing to gain-of-function in the encoded protein [Goriely et al., 2005; Proc Natl Acad Sci USA 102:6051-6056]. The present case contributes additional support for this hypothesis.     

Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones

Growth cones at the tips of nascent and regenerating axons direct axon elongation. Netrin-1, a secreted molecule that promotes axon outgrowth and regulates axon pathfinding, elevates cyclic AMP (cAMP) levels in growth cones and regulates growth cone morphology and axonal outgrowth. These morphological effects depend on the intracellular levels of cAMP. However, the specific pathways that regulate cAMP levels in response to netrin-1 signaling are unclear. Here we show that 'soluble' adenylyl cyclase (sAC), an atypical calcium-regulated cAMP-generating enzyme previously implicated in sperm maturation, is expressed in developing rat axons and generates cAMP in response to netrin-1. Overexpression of sAC results in axonal outgrowth and growth cone elaboration, whereas inhibition of sAC blocks netrin-1-induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC-generated cAMP, and identify a fundamental role for sAC in axonal development.     

Slit1 promotes regenerative neurite outgrowth of adult dorsal root ganglion neurons in vitro via binding to the Robo receptor

Secreted Slit proteins have previously been shown to signal through Roundabout (Robo) receptors to negatively regulate axon guidance and cell migration. During vertebrate development, Slit proteins have also been shown to stimulate branching and elongation of sensory axons and cortical dendrites. In this study, Slit1/Robo2 mRNA and protein expressions were detected in adult rat dorsal root ganglion (DRG) and in cultured DRG neurons. Treatment of both models with recombinant, soluble Slit1 protein was found to promote neurite outgrowth and elongation. In contrast, treatment with a recombinant human Robo2/Fc chimera inhibited neurite outgrowth and elongation. When adult DRG and cultured DRG neurons were pretreated with soluble recombinant human Robo2/Fc chimera, neurite outgrowth and elongation was not induced. These findings indicate that Slit1/Robo2 signaling may have a role in regulating peripheral nerve regeneration.     

VAMP-2 promotes neurite elongation and SNAP-25A increases neurite sprouting in PC12 cells

Recent studies suggest that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion in the plasma membrane that allows expansion for neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. In this study, we took the opposite approach by overexpressing green fluorescent protein (GFP)-fusion SNAREs, including VAMP-2, SNAP-25A, and syntaxin1A, in PC12 cells to investigate the role of SNAREs in the neurite outgrowth of PC12 cells. Neurite outgrowth analysis demonstrated that: (1) GFP-VAMP-2 increased the length of individual neurites, without changing the number of neurites per cell; (2) GFP-SNAP-25A increased the number of neurites per cell, with no change in the length of the individual neurites. In both cases, the total length of neurites per cell was increased; (3) GFP-syntaxin1A resulted in no significant change, either in neurite length, or in the number of neurites per cell. These findings suggest that when overexpressed in PC12 cells, VAMP-2 can promote neurite elongation, while SNAP-25A can stimulate neurite sprouting. On the other hand, overexpression of syntaxin1A neither promotes nor inhibits neurite outgrowth. Thus VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.     

Effects of ethanol on axon outgrowth and branching in developing rat cortical neurons

Humans exposed prenatally to ethanol can exhibit brain abnormalities and cognitive impairment similar to those seen in patients expressing mutant forms of the L1 cell adhesion molecule (L1CAM). The resemblance suggests that L1CAM may be a target for ethanol, and consistent with this idea, ethanol can inhibit L1CAM adhesion in cell lines and L1CAM-mediated outgrowth and signaling in cerebellar granule neurons. However, it is not known whether ethanol inhibits L1CAM function in other neuron types known to require L1CAM for appropriate development. Here we asked whether ethanol alters L1CAM function in neurons of the rat cerebral cortex. We find that ethanol does not alter axonal polarization, L1CAM-dependent axon outgrowth or branching, or L1CAM recycling in axonal growth cones. Thus, ethanol inhibition of L1CAM is highly dependent on neuronal context.     

Reduced binding of FGF1 to mutant fibroblast growth factor receptor 3

The activating mutation FGFR3-R248C in the D2-D3 linker region of fibroblast growth factor receptor 3 leads as germline mutation to the neonatal lethal syndrome thanatophoric dysplasia type I (TD1). As somatic mutation it has been found in cancer. We introduced into the murine FGFR3 the mutation R242C that is orthologoues to the human mutation R248C. A strong reduction in binding of the 16 and 18 kDa forms of FGF1 to the mutant receptor was found, highlighting the importance of D2-D3 linker region of FGFR3 in determination of binding affinity to ligands. Another mutant, G374R, introduced into the murine FGFR3, is orthologoues to the human mutant FGFR3-G380R, and leads to achondroplasia (ACH). The binding of the 16 kDa and 18 kDa forms of FGF1 to this mutant receptor was the same as for wild-type FGFR3 in a cell-free system, but it was reduced in living cells. The data indicate a minor changes in conformation of FGFR3-G374R receptors at the cell surface that lead to reduced binding to FGF1.     

FGFs,their receptors,and human limb malformations: Clinical and molecular correlations

Fibroblast growth factors (FGFs) comprise a family of 22 distinct proteins with pleiotropic signaling functions in development and homeostasis. These functions are mediated principally by four fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase family, with heparin glycosaminoglycan as an important cofactor. Developmental studies in chick and mouse highlight the critical role of FGF‐receptor signaling in multiple phases of limb development, including the positioning of the limb buds, the maintenance of limb bud outgrowth, the detailed patterning of the limb elements, and the growth of the long bones. Corroborating these important roles, mutations of two members of the FGFR family (FGFR1 and FGFR2) are associated with human disorders of limb patterning; in addition, mutations of FGFR3 and FGF23 affect growth of the limb bones. Analysis of FGFR2 mutations in particular reveals a complex pattern of genotype/phenotype correlation, which will be reviewed in detail. Circumstantial evidence suggests that the more severe patterning abnormalities are mediated by illegitimate paracrine signaling in the mesoderm, mediated by FGF10 or by a related FGF, and this is beginning to gain some experimental support. A further test of this hypothesis is provided by a unique family segregating two FGFR2 mutations in cis (S252L; A315S), in which severe syndactyly occurs in the absence of the craniosynostosis that typically accompanies FGFR2 mutations. © 2002 Wiley‐Liss, Inc.     

Reversible Inhibition of Endocytosis in Cultured Neurons from the Drosophila Temperature-Sensitive Mutant Shibire

The Drosophila mutant, shibire^ts1 (shi^ts1), is paralyzed at restrictive temperatures (> 29^oC) by a reversible block in synaptic transmission. Heat pulses deplete synaptic vesicles in nerve terminals and inhibit endocytic internalization of plasma membrane in garland cells and oocytes. In dissociated cultures of larval central nervous system (CNS), a temperature-sensitive defect is also expressed in shi^ts1 neurons: at 30^oC, growth cone formation is retarded and neurite outgrowth is arrested.

We now report that we have examined constitutive endocytosis in Drosophila CNS culture and have demonstrated directly an endocytic defect in shi^ts1 neurons. At the permissive temperature, 20-22^oC, both shi^ts1and wild-type neurons actively endocytosed fluorescein-labelled dextran (40KD, 5%) or horseradish peroxidase (HRP, 1%). Within 5 min, HRP was seen in vesicles, cup-shaped bodies, tubules and multivesicular bodies in neurites and cell bodies. In contrast, endocytosis was inhibited in cultures derived from the temperature-sensitive paralytic shi by a 15 min heat pulse (30^oC). Even after 30 min of HRP exposure at 30^oC, HRP-containing membranes were absent from almost all shi^ts1 neurites; a minority of cell bodies had a few HRP-containing vesicles. The temperature-dependent block in endocytosis was readily reversed at 20^oC. Interestingly, the block was overcome by high concentration of external cations: shi^ts1 neurons in culture actively took up HRP in numerous vesicles at 30^oC if 18mM Ca²⁺ or Mg² was added to the medium.

Our results support the notion that membrane recycling plays a critical role in regulating neurite outgrowth. This study also provides baseline information for further mutational analysis of the mechanism underlying the membrane cycling process in cultured neurons.     

Sigma-1 Receptor Expression in DRG Neurons During a Carrageenan-Provoked Inflammation

Sigma 1 receptor (σ1R) is a non-opioid receptor that modulates pain perception and is strongly expressed in dorsal root ganglion (DRG) neurons. We studied the changes in the expression of σ1R in different sub-populations of DRG neurons during the first 48 hr in a carrageenan-induced inflammation rat model, with σ1R being a possible base for the development of neuropathic pain after inflammation. Twenty Sprague Dawley rats were divided into five groups (N = 4 in each group): the control (C) group was sacrificed immediately; all other animals received an intraplantar injection of 0.1 mL 2% carrageenan and were sacrificed in 6, 12, 24 or 48 hr after the injection and DRGs were collected and processed for immunohistochemistry. σ1R fluorescence intensity decreased slightly but significantly in up to 24 hr post-carrageenan injection in all sub-populations of DRG neurons (ib4+; ib4− medium, ib4− large and ib4− in total; P < 0.05 – P < 0.001), with the exception of the ib4− small neurons (<25 μm; P > 0.05). This decrement was followed by a subsequent increase in σ1R fluorescence intensity 48 hr after the plantar carrageenan injection (P < 0.05 – P < 0.0001). The same trend was also observed in the CGRP+ population of the DRG neurons, in the total population as well as in the CGRP+ small (<25 μm) and larger CGRP (>25 μm) sub-populations (P < 0.05 – P < 0.001). The presented results may contribute to further understanding of role of σ1R in the development of peripheral sensitization during inflammation. They may also be valuable for the therapeutic application of σ1R antagonists, particularly in the adjustment of the antagonist's dosage in a particular time window. Anat Rec, 302:1620–1627, 2019. © 2019 American Association for Anatomy     

Activation of fibroblast growth factor receptor by axotomy, through downstream p38 in dorsal root ganglion, contributes to neuropathic pain

The possible involvement of fibroblast growth factor receptor (FGFR) activation in the dorsal root ganglion (DRG) was examined following peripheral nerve injury in the rat. Ligation of the sciatic nerve down-regulated FGFR2, -3 and -4 mRNA; however, the expression of FGFR1 mRNA showed no change. Activation of FGFR was examined by immunohistochemistry using an antibody of the phosphorylated form of FGFR1-4. Ligation of the sciatic nerve produced phosphorylation of FGFR in the L4 and 5 DRG ipsilateral to the injury, starting at 3 days after the lesion and persisting for more than 30 days. Substantial activation of FGFR was observed, mainly in unmyelinated small DRG neurons that co-expressed phosphorylated p38 mitogen-activated protein kinase (MAPK). Continuous intrathecal infusion of the FGFR1 inhibitor, 3-[3-(2-carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone, reduced p38 MAPK phosphorylation in the DRG and pain-related behaviors in the partial sciatic nerve model rat without affecting on the activation of spinal glia cells (microglia and astrocyte). In the injured small DRG neurons, activation of FGFR1 may contribute to the generation of neuropathic pain by activating p38 MAPK.     

Involvement of the synaptotagmin/stonin2 system in vesicular transport regulated by semaphorins in Caenorhabditis elegans epidermal cells

Vesicular transport serves as an important mechanism for cell shape regulation during development. Although the semaphorin signaling molecule, a well‐known regulator of axon guidance, induces endocytosis in the growth cone and the axonal transport of vertebrate neurons, the underlying molecular mechanisms remain largely unclear. Here, we show that the Caenorhabditis elegans SNT‐1/synaptotagmin‐UNC‐41/stonin2 system, whose role in synaptic vesicle recycling in neurons has been studied extensively, is involved in semaphorin‐regulated vesicular transport in larval epidermal cells. Mutations in the snt‐1/unc‐41 genes strongly suppressed the cell shape defects of semaphorin mutants. The null mutation in the semaphorin receptor gene, plx‐1, altered the expression and localization pattern of endocytic and exocytic markers in the epidermal cells while repressing the transport of SNT‐1‐containing vesicles toward late endosome/lysosome pathways. Our findings suggest that the nematode semaphorins regulate the vesicular transport in epidermal cells in a manner distinct from that of vertebrate semaphorins in neurons.     

Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). The pharmacological inhibition of InsP₃ receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP₃R1, which may play an important role in the regulation of neurite outgrowth.     

Anosmin-1 stimulates outgrowth and branching of developing Purkinje axons

During development, Purkinje axons elongate along precise trajectories and acquire stereotypic branching patterns to innervate targets in the deep nuclei and cerebellar cortex. These processes are accomplished through cell-intrinsic mechanisms, whose operation is regulated by environmental signaling cues. Here, we show that Anosmin-1, the protein defective in the X-linked form of Kallmann syndrome, is one among such cues. Anosmin-1, that stimulates axon elongation and branching in the olfactory system, is expressed by Purkinje cells and deep nuclear neurons of the rat cerebellum during the ontogenetic period when Purkinje axons acquire their mature pattern. These neurons also express the putative Anosmin-1 receptor, fibroblast growth factor receptor 1. Application of Anosmin-1 to dissociated cultures of embryonic (embryonic day 17, E17) or postnatal (postnatal day 0, P0) rat cerebellar cells enhances neuritic elongation and exerts a strong promoting action on the budding of collateral branches and on the extension of terminal arbors. Opposite effects are observed when neutralizing anti-Anosmin-1 antibodies are applied to the same cultures. Comparable results are obtained by administering the protein or the blocking antibodies to organotypic cultures of postnatal (P0) rat cerebellum. In P10 cerebellar slices, Anosmin-1 does not enhance the spontaneous regenerative capabilities of severed Purkinje axons, but promotes the terminal outgrowth of injured neurites into embryonic neocortical explants apposed to the axotomy site. Although Anosmin-1 is unable to change the overall intrinsic growth competence of Purkinje cells, it exerts a powerful stimulatory action on the budding and extension of collateral branches and terminal plexus, contributing to the patterning of Purkinje axons.     

SPG3A protein atlastin-1 is enriched in growth cones and promotes axon elongation during neuronal development

The hereditary spastic paraplegias (HSPs) (SPG1-29) comprise a group of inherited neurological disorders characterized principally by spastic lower extremity weakness due to a length-dependent, retrograde axonopathy of corticospinal motor neurons. Mutations in the gene encoding the dynamin superfamily member atlastin-1, an oligomeric GTPase highly localized to the Golgi apparatus in the adult brain, are responsible for SPG3A, a common autosomal dominant HSP. A distinguishing feature of SPG3A is its frequent early onset, raising the possibility that developmental abnormalities may be involved in its pathogenesis. Here, we demonstrate that several missense SPG3A mutant atlastin-1 proteins have impaired GTPase activity and thus may act in a dominant-negative, loss-of-function manner by forming mixed oligomers with wild-type atlastin-1. Using confocal and electron microscopies, we have also found that atlastin-1 is highly enriched in vesicular structures within axonal growth cones and varicosities as well as at axonal branch points in cultured cerebral cortical neurons, prefiguring a functional role for atlastin-1 in axonal development. Indeed, knock-down of atlastin-1 expression in these neurons using small hairpin RNAs reduces the number of neuronal processes and impairs axon formation and elongation during development. Thus, the "long axonopathy" in early-onset SPG3A may result from abnormal development of axons because of loss of atlastin-1 function.     

The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.