Postsynaptic neuronal activity promotes regeneration of retinal axons

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Cyclic AMP and the regeneration of retinal ganglion cell axons

In this paper we present a brief review of studies that have reported therapeutic benefits of elevated cAMP on plasticity and regeneration after injury to the central nervous system (CNS). We also provide new data on the cellular mechanisms by which elevation of cyclic adenosine monophosphate (cAMP) promotes cytokine driven regeneration of adult CNS axons, using the visual system as the experimental model. cAMP is a second messenger for many intracellular signalling pathways. Elevation of cAMP in the eye by intravitreal injection of the cell permeant analogue (8-(4-chlorophenylthio)-adenosine-3′,5′-cyclic monophosphate; CPT-cAMP), when added to recombinant ciliary neurotrophic factor (rCNTF), significantly enhances rCNTF-induced regeneration of adult rat retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafted onto transected optic nerve. This effect is mediated to some extent by protein kinase A (PKA) signalling, but CPT-cAMP also acts via PI3K/Akt signalling to reduce suppressor of cytokine signalling protein 3 (SOCS3) activity in RGCs. Another target for cAMP is the exchange protein activated by cAMP (Epac), which can also mediate cAMP-induced axonal growth. Here we describe some novel results and discuss to what extent the pro-regenerative effects of CPT-cAMP on adult RGCs are mediated via Epac as well as via PKA-dependent pathways. We used the established PN–optic nerve graft model and quantified the survival and regenerative growth of adult rat RGCs after intravitreal injection of rCNTF in combination with a selective activator of PKA and/or a specific activator of Epac. Viable RGCs were identified by βIII-tubulin immunohistochemistry and regenerating RGCs retrogradely labelled and quantified after an injection of fluorogold into the distal end of the PN grafts, 4 weeks post-transplantation. The specific agonists of either PKA or Epac were both effective in enhancing the effects of rCNTF on RGC axonal regeneration, but interestingly, injections that combined rCNTF with both agonists were significantly less effective. The results are discussed in relation to previous CPT-cAMP studies on RGCs, and we also consider the need to modulate cAMP levels in order to obtain the most functionally effective regenerative response after CNS trauma.This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration.

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stained for E 21. In adult fish, however, E 21 immunoreactivity exhibited a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen was absent from older axons, except the terminal arbor layer in the tectum, the Stratum fibrosum et griseum superficiale where it was uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquired E 21 positivity as they regenerated throughout their path to the tectum. Several months after ONS, however, E 21 staining disappeared from the regenerated axons over most of their lengths but reappeared as in normal fish in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion molecules of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion molecule DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion molecule during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth.     

The sorting behaviour of olfactory and vomeronasal axons during regeneration

Summary In order to begin to understand how primary olfactory and vomeronasal organ (VNO) axons target specific regions of the olfactory bulb, we examined the sorting behaviour of these axons following neonatal unilateral olfactory bulbectomy. Bulbectomy induced widespread ipsilateral death of the primary olfactory and VNO neurons. After 4 weeks, many new sensory axons had re-grown into the cranial cavity and established a prominent plexus with evidence of dense tufts that were similar in gross appearance to glomeruli. Axons expressing the cell adhesion molecule OCAM, which normally innervate the ventrolateral and rostral halves of the main and accessory olfactory bulbs, respectively, sorted out and segregated from those axons not expressing this molecule within the plexus. In addition, VNO axons formed large discrete bundles that segregated from main olfactory axons within the plexus. Thus, VNO and primary olfactory axons as well as discrete subpopulations of both are able to sort out and remain segregated in the absence of the olfactory bulb. Sorting and convergence of axons therefore occur independently of the olfactory bulb and are probably attributable either to inherent properties of the axons themselves or to interactions between the axons and accompanying glial ensheathing cells.     

Prediction of the beta Bp crystallin dimer structure by the complementary surfaces (CS) method

The method of searching for complementary surfaces of proteins was used to evaluate the modes of beta Bp crystallin autoassociation. The dimer structure of beta Bp is predicted and characterized.     

Interleukin 1 beta (IL-1 beta) action in porcine thyroid cells involves the ceramide signalling pathway

Interleukin 1 beta (IL-1beta) is often associated with thyroidal autoimmune diseases. This cytokine has been largely described to trigger an important biological signalling pathway: the sphingomyelin/ceramide pathway. In this report we show that IL-1beta induces ceramide formation and sphingomyelin degradation in porcine thyroid cells via the activation of a neutral sphingomyelinase. Among the potential targets of IL-1beta and ceramides action, we have investigated the role of an atypical protein kinase C (PKC), the PKC zeta. We show that both IL-1beta and ceramides lead to an increase of PKCzeta activity. All these results suggest an important role for ceramides and IL-1beta in regulation of thyroid function, leading to cell survival or to apoptosis.     

Induction of S-100b (beta beta) protein in human teratocarcinoma cells

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of alpha S-100 and beta S-100 proteins (alpha and beta subunits of S-100 proteins), and three subunits (alpha, beta and gamma) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of beta S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of alpha S-100 was undetectably low, indicating that the S-100b (beta beta) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to beta S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.     

Changes in goldfish retinal ganglion cells during axonal regeneration

Recent work suggests that mammalian retinal ganglion cells may become more like developing ganglion cells in form while regenerating through a peripheral nerve graft. We have injected Lucifer Yellow into regenerating ganglion cells of goldfish to look for similar changes. Within three weeks of injury, we saw dye-coupling to nearby cells, which is a common developmental feature in many species. Dendrites and axons, which in most mature ganglion cells are smooth, became varicose and hairy, like those examined in mammalian development. Secondary axons arose later, not only as side-branches of the primary axon but also from the soma, as in mammalian development and regeneration. Since, in fish, these responses are clearly an intrinsic part of functional regeneration, their equivalence in fish and mammals strengthens the view that a similar regenerative competence may exist in the retinal ganglion cells of all vertebrates.     

Variation of (2''-5'')oligo(adenylate) synthetase activity during rat-liver regeneration

The inhibitory effect of fructose 2,6-biphosphate on fructose 1,6-bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly noncompetitive inhibitor. The effect of fructose 2,6-bisphosphate was compared to that of other ligands of the enzyme, which, like the substrate and methyl (alpha + beta)fructofuranoside 1,6-bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, lithium or higher concentrations of magnesium as well as partial proteolysis by subtilisin increased [I]0.5 for fructose 2,6-bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (alpha + beta)fructofuranoside 1,6-bisphosphate. These observations can be explained by assuming that fructose 2,6-bisphosphate has no affinity for the active site of fructose 1,6-bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6-bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.     

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.     

An essential role for talin during alpha(M)beta(2)-mediated phagocytosis

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.     

The Retinal Homeobox (Rx) gene is necessary for retinal regeneration

The Retinal Homeobox (Rx) gene is essential for vertebrate eye development. Rx function is required for the specification and maintenance of retinal progenitor cells (RPCs). Loss of Rx function leads to a lack of eye development in a variety of species. Here we show that Rx function is also necessary during retinal regeneration. We performed a thorough characterization of retinal regeneration after partial retinal resection in pre-metamorphic Xenopus laevis. We show that after injury the wound is repopulated with retinal progenitor cells (RPCs) that express Rx and other RPC marker genes. We used an shRNA-based approach to specifically silence Rx expression in vivo in tadpoles. We found that loss of Rx function results in impaired retinal regeneration, including defects in the cells that repopulate the wound and the RPE at the wound site. We show that the regeneration defects can be rescued by provision of exogenous Rx. These results demonstrate for the first time that Rx, in addition to being essential during retinal development, also functions during retinal regeneration.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

Inactivation of BK channels mediated by the NH(2) terminus of the beta3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade

A family of auxiliary beta subunits coassemble with Slo alpha subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The beta subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The beta3b auxiliary subunit, when coexpressed with the Slo alpha subunit, results in a particularly rapid ( approximately 1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the beta3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism (C(n) <---> O(n) <---> O(*)(n) <---> I(n)) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.     

atp2b1a regulates Ca(2+) export during differentiation and regeneration of mechanosensory hair cells in zebrafish

The molecular mechanisms of development of mechanosensory hair cells have been tackled successfully due to in vivo studies in the zebrafish lateral line. The enhancer trap (ET) transgenic line, SqET4 was instrumental in these studies even despite a lack of a link of its GFP expression pattern to a particular gene(s). We mapped the Tol2 transposon insertion of the SqET4 transgenics onto Chr. 4 next to a gene encoding Atp2b1a (Pmca1) - one of the four PMCAs acting to export Ca(2+) from a cell. atp2b1a expression recapitulates that of GFP during the development of mechanoreceptors of the inner ear and lateral line. atp2b1a expression correlates with the regeneration of these cells. Thus, SqET4 represents the Tg:atp2b1a-GFP line, which links Ca(2+) metabolism and the differentiation of mechanoreceptors. The morpholino-mediated knockdown of atp2b1a blocks Ca(2+) export and affects the division of hair cell progenitors, resulting in their accumulation. Under the control of a master gene of hair cells, Atoh1a, Atp2b1a functions during progenitor cell proliferation and hair cell differentiation. Given the similarity between the phenotypes of atp2b1a morphants and embryos treated with the pan-PMCA inhibitor 5(6)-carboxyeosin, Atp2b1a emerges as member of the Atp2b family responsible for Ca(2+) export during the development of hair cells.