Electrical stimulation promotes peripheral axon regeneration by enhanced neuronal neurotrophin signaling

Electrical stimulation of cut peripheral nerves at the time of their surgical repair results in an enhancement of axon regeneration. Regeneration of axons through nerve allografts was used to evaluate whether this effect is due to an augmentation of cell autonomous neurotrophin signaling in the axons or signaling from neurotrophins produced in the surrounding environment. In the thy-1-YFP-H mouse, a single 1 h application of electrical stimulation at the time of surgical repair of the cut common fibular nerve results in a significant increase in the proportion of YFP+ dorsal root ganglion neurons, which were immunoreactive for BDNF or trkB, as well as an increase in the length of regenerating axons through allografts from wild type litter mates, both 1 and 2 weeks later. Axon growth through allografts from neurotrophin-4/5 knockout mice or grafts made acellular by repeated cycles of freezing and thawing is normally very poor, but electrical stimulation results in a growth of axons through these grafts, which is similar to that observed through grafts from wild type mice after electrical stimulation. When cut nerves in NT-4/5 knockout mice were electrically stimulated, no enhancement of axon regeneration was found. Electrical stimulation thus produces a potent enhancement of the regeneration of axons in cut peripheral nerves, which is independent of neurotrophin production by cells in their surrounding environment but is dependent on stimulation of trkB and its ligands in the regenerating axons themselves.     

Initial axon growth rate from embryonic sensory neurons is correlated with birth date

Axon growth rate from different populations of sensory neurons is correlated with the distance they have to grow to reach their targets in development: neurons with more distant targets extend axons at intrinsically faster rates. With growth of the embryo, later‐born neurons within each population have further to extend their axons to reach their targets than early‐born neurons. Here we examined whether the axon growth rate is related to birth date by studying the axon growth from neurons that differentiate in vitro from precursor cells isolated throughout the period of neurogenesis. We first showed that neurons that differentiated in vitro from different precursor cell populations exhibited differences in axon growth rate related to in vivo target distance. We then examined the axon growth rate from neurons that differentiate from the same precursor population at different stages throughout the period of neurogenesis. We studied the epibranchial placode precursors that give rise to nodose ganglion neurons in the chicken embryo. We observed a highly significant, threefold difference in axon growth rate from neurons that differentiate from precursor cells cultured early and late during the period of neurogenesis. Our findings suggest that intrinsic differences in axon growth rate are correlated with the neuronal birth date.     

Neuronal Galectin‐4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin‐4 (Gal‐4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule‐ and sulfatide‐dependent manner. Gal‐4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal‐4 addition. This Gal‐4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth‐promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal‐4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N‐glycans. Impairing the maturation of these N‐glycans precludes Gal‐4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal‐4 sorts to axon plasma membrane segments by binding to sulfatide‐containing microtubule‐associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.     

Notch‐1 signaling is lost in prostate adenocarcinoma and promotes PTEN gene expression

Prostate tumorigenesis is associated with loss of PTEN gene expression. We and others have recently reported that PTEN is regulated by Notch‐1 signaling. Herein, we tested the hypothesis that alterations of the Notch‐1 signaling pathway are present in human prostate adenocarcinoma and that Notch‐1 signaling regulates PTEN gene expression in prostate cells. Prostate adenocarcinoma cases were examined by immunohistochemistry for ligand cleaved (activated) Notch‐1 protein. Tumor foci exhibited little cleaved Notch‐1 protein, but expression was observed in benign tissue. Both tumor and benign tissue expressed total (uncleaved) Notch‐1. Reduced Hey‐1 expression was seen in tumor foci but not in benign tissue, confirming loss of Notch‐1 signaling in prostate adenocarcinoma. Retroviral expression of constitutively active Notch‐1 in human prostate tumor cell lines resulted in increased PTEN gene expression. Incubation of prostate cell lines with the Notch‐1 ligand, Delta, resulted in increased PTEN expression indicating that endogenous Notch‐1 regulates PTEN gene expression. Chromatin immunoprecipitation demonstrated that CBF‐1 was bound to the PTEN promoter. These data collectively indicate that defects in Notch‐1 signaling may play a role in human prostate tumor formation in part via a mechanism that involves regulation of the PTEN tumor suppressor gene. J. Cell. Biochem. 107: 992–1001, 2009. © 2009 Wiley‐Liss, Inc.     

Cis interaction between Semaphorin6A and Plexin‐A4 modulates the repulsive response to Sema6A

The correct navigation of axons to their targets depends on guidance molecules in the extra‐cellular environment. Differential responsiveness to a particular guidance cue is largely an outcome of disparity in the expression of its receptors on the reacting axons. Here, we show that the differential responsiveness of sympathetic and sensory neurons to the transmembrane Semaphorin Sema6A is mainly determined by its co‐expression in the responding neurons. Both sympathetic and sensory neurons express the Sema6A receptor Plexin‐A4, but only sympathetic neurons respond to it. The expression of Sema6A counteracts this responsiveness and is detected only in sensory neurons. Remarkably, sensory neurons that lack Sema6A gain sensitivity to it in a Plexin‐A4‐dependent manner. Using heterologus systems, we show that the co‐expression of Sema6A and Plexin‐A4 hinders the binding of exogenous ligand, suggesting that a Sema6A–Plexin‐A4 cis interaction serves as an inhibitory mechanism. Finally, we provide evidence for differential modes of interaction in cis versus in trans. Thus, co‐expression of a transmembrane cue together with its receptor can serve as a guidance response modulator.     

Postsynaptic neuronal activity promotes regeneration of retinal axons

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Low‐level laser therapy 810‐nm up‐regulates macrophage secretion of neurotrophic factors via PKA‐CREB and promotes neuronal axon regeneration in vitro

Macrophages play key roles in the secondary injury stage of spinal cord injury (SCI). M1 macrophages occupy the lesion area and secrete high levels of inflammatory factors that hinder lesion repair, and M2 macrophages can secrete neurotrophic factors and promote axonal regeneration. The regulation of macrophage secretion after SCI is critical for injury repair. Low‐level laser therapy (810‐nm) (LLLT) can boost functional rehabilitation in rats after SCI; however, the mechanisms remain unclear. To explore this issue, we established an in vitro model of low‐level laser irradiation of M1 macrophages, and the effects of LLLT on M1 macrophage polarization and neurotrophic factor secretion and the related mechanisms were investigated. The results showed that LLLT irradiation decreased the expression of M1 macrophage‐specific markers, and increased the expression of M2 macrophage‐specific markers. Through forward and reverse experiments, we verified that LLLT can promote the secretion of various neurotrophic factors by activating the PKA‐CREB pathway in macrophages and finally promote the regeneration of axons. Accordingly, LLLT may be an effective therapeutic approach for SCI with clinical application prospects.     

Axonal outgrowth from adult mouse nodose ganglia in vitro is stimulated by neurotrophin‐4 in a Trk receptor and mitogen‐activated protein kinase‐dependent way

The actions of neurotrophic factors on sensory neurons of the adult nodose ganglion were studied in vitro. The ganglia were explanted in an extracellular matrix–based gel that permitted observation of the growing axons. Neurotrophin‐4 (NT‐4) was a very efficient stimulator of outgrowth of axons from the nodose ganglion and had almost doubled the outgrowth length when this was analyzed after 2 days in culture. Brain‐derived neurotrophic factor also stimulated outgrowth, but to a lesser degree, whereas NT‐3 gave only weak stimulatory tendencies. Nerve growth factor and glial cell line–derived neurotrophic factor both lacked stimulatory effects. NT‐4 is known to act via TrkB receptors, and the presence of these on growing nodose neurons was demonstrated immunohistochemically. In line with a Trk‐mediated growth effect, the NT‐4 stimulation was abolished by K252a, a selective inhibitor of neurotrophin receptor–associated tyrosine kinase activity. K252a had no effect on the unstimulated preparation. NT‐4 treatment led to activation of the mitogen‐activated protein kinase and inhibition of the latter pathway by PD98059 significantly reduced the NT‐4 stimulated outgrowth, whereas the drug had no effect on the unstimulated growth. In conclusion, the data suggest that NT‐4 can serve as a powerful growth factor for neurons of adult nodose ganglia and that the growth stimulation involves TrkB‐ and mitogen‐activated protein kinase. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 142–151, 2000     

Synapse‐dependent and independent mechanisms of thalamocortical axon branching are regulated by neuronal activity

Axon branching and synapse formation are critical processes for establishing precise circuit connectivity. These processes are tightly regulated by neural activity, but the relationship between them remains largely unclear. We use organotypic coculture preparations to examine the role of synapse formation in the activity‐dependent axon branching of thalamocortical (TC) projections. To visualize TC axons and their presynaptic sites, two plasmids encoding DsRed and EGFP‐tagged synaptophysin (SYP‐EGFP) were cotransfected into a small number of thalamic neurons. Time‐lapse imaging of individual TC axons showed that most branches emerged from SYP‐EGFP puncta, indicating that synapse formation precedes emergences of axonal branches. We also investigated the effects of neuronal activity on axon branching and synapse formation by manipulating spontaneous firing activity of thalamic cells. An inward rectifying potassium channel, Kir2.1, and a bacterial voltage‐gated sodium channel, NaChBac, were used to suppress and promote firing activity, respectively. We found suppressing neural activity reduced both axon branching and synapse formation. In contrast, increasing neural activity promoted only axonal branch formation. Time‐lapse imaging of NaChBac‐expressing cells further revealed that new branches frequently appeared from the locations other than SYP‐EGFP puncta, indicating that enhancing activity promotes axonal branch formation due to an increase of branch emergence at nonsynaptic sites. These results suggest that presynaptic locations are hotspots for branch emergence, and that frequent firing activity can shift branch emergence to a synapse‐independent process. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 323–336, 2016     

Reduced number of unmyelinated sensory axons in peripherin null mice

Peripherin is a type III intermediate filament (IF) abundantly expressed in developing neurons, but in the adult, it is primarily found in neurons extending to the peripheral nervous system. It has been suggested that peripherin may play a role in axonal elongation and/or cytoskeletal stabilization during development and regeneration. To further clarify the function of peripherin, we generated and characterized mice with a targeted disruption of the peripherin gene. The peripherin null mice were viable, reproduced normally and did not exhibit overt phenotypes. Microscopic analysis revealed no gross morphological defects in the ventral and dorsal roots, spinal cord, retina and gut, but protein analyses showed increased levels of the type IV IF alpha-internexin in ventral roots of peripherin null mice. Whereas the number and caliber of myelinated motor and sensory axons in the L5 roots remained unchanged in peripherin knockout mice, there was a substantial reduction ( approximately 34%) in the number of L5 unmyelinated sensory fibers that correlated with a decreased binding of the lectin IB4. These results demonstrate a requirement of peripherin for the proper development of a subset of sensory neurons.     

Synthesis of 5‐(4′‐carboxyphenyl)‐10,15,20‐tris‐(4 pyridyl)‐porphyrin and its peptidyl phosphonate derivatives

The synthesis and characterization of three new 4‐pyridyl porphyrin‐peptidyl‐phosphonate compounds, containing a diphenyl 3‐pyridylmethyl‐phosphonate moiety, is described in this article. Nitrogen atoms in the pyridine rings of the obtained compounds were alkylated using methyl iodide, to give additional three, water soluble derivatives of these peptidyl‐porphyrin conjugates. All the synthesized compounds could serve as potential photosensitizers for the photodynamic therapy (PDT) method of tumor therapy and displayed activity as inhibitors of aminopeptidase N. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.