Molecular characterization of E587 antigen: an axonal recognition molecule expressed in the goldfish central nervous system.

The E587 antigen (Ag) is a 200-Kd membrane glycoprotein originally identified by a monoclonal antibody on new and regenerating retinal ganglion cell axons in the adult goldfish. We report the isolation of cDNAs encoding the E587 Ag and identify it as a member of the L1 family of cell adhesion molecules (CAMs). The predicted amino acid sequence of E587 Ag shows an approximately equal identity (40%) to mouse L1, chick neuron-glia CAM, and chick neuron-glia-related CAM. Although the overall similarity is low, there is a high conservation of structural domains and specific sequence motifs. Wholemount in situ hybridizations were performed on goldfish between 34 hours and 3 days postfertilization (pf). A dramatic increase in E587 Ag mRNA was observed between 34 and 48 hours pf. The expression of E587 Ag mRNA in neurons shortly precedes axonogenesis. A marked decrease in expression occurs by 3 days pf, when the axonal scaffold has already been established. Wholemount immunohistochemistry on embryos demonstrates expression of E587 Ag on all major tracts. E587 Ag is absent from mature retinal ganglion cell axons, but its expression is induced by optic nerve transection. A corresponding induction of E587 Ag mRNA in retinal ganglion cells is shown by in situ hybridization. Furthermore, E587 Ag mRNA was detected in the optic nerve, which suggests that nonneuronal cells also express this molecule. E587 Ag was previously shown to promote retinal axon fasciculation and outgrowth in young fish and to mediate axon-glial interactions in vitro. The expression pattern and developmental regulation of E587 Ag in the central nervous system, its reexpression in retinal ganglion cells following optic nerve transection, and its relation to the L1 family indicate that E587 Ag functions as a cell recognition molecule important during axonal growth and regeneration.

Caspase-9: involvement in secondary death of axotomized rat retinal ganglion cells in vivo.

Recently we have shown that adult rat retinal ganglion cells (RGCs) die by apoptosis following optic nerve (ON) transection, activating caspase-3. In the present study, we report that caspase-9, known to be an important activator of caspase-3, becomes activated in the axotomized adult rat retina as revealed by immunoblot analysis and protease activity assays. Reduction of caspase-9 activity by repeated intraocular injection of specific inhibitors significantly prevented RGC death. Caspase-9 activity was effectively blocked by inhibitor treatment and by application of IGF-I and BDNF, neurotrophic factors which have been shown to be highly neuroprotective in this model. Taken together, our data suggest that caspase-9 plays a critical role in apoptosis induction in axotomized RGCs in vivo and is regulated under treatment with growth and survival factors. Thus, providing more insight into the mechanisms underlying neuronal death and survival following trauma might serve as a basis to improve future therapeutic strategies preventing or at least reducing the severe consequences of neuronal injury.

Degeneration and regeneration of ganglion cell axons.

The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed.

Fate of oligodendrocytes during retinal axon degeneration and regeneration in the goldfish visual pathway.

Retinal axons in goldfish regenerate after optic nerve lesion, restore synaptic connections, and become myelinated by oligodendrocytes. The fate of oligodendrocytes during these events is not known and may require generation of new oligodendrocytes or dedifferentiation and redifferentiation of the existing ones. To determine the reaction of oligodendrocytes to optic nerve lesion, we used the terminal transferase technique to detect apoptosis, bromodeoxyuridine incorporation to reveal mitosis, antibodies to identify myelin and oligodendrocytes, and Lucifer yellow injections to reveal cell morphology. Along with the reappearance of the myelin molecules 36K protein, galactocerebroside, and myelin basic protein, myelinating oligodendrocytes (identified by Lucifer yellow injections) reappear 21 days postlesion. Prior to this time, the dye-filled cells had few processes oriented along the regenerating axons. They resembled oligodendrocytes seen both in vitro and in vivo which express the L1-related E587 antigen and synthesize the 36K myelin protein in coculture with axons. No signs of oligodendrocyte apoptosis were detected after lesion and only few of the oligodendrocytes present had recently arisen. 36K/E587 double-labeled oligodendrocytes which were most likely dedifferentiating oligodendrocytes were identified in 8-day postlesion nerves among E587-positive elongate cells whose numbers increased until 14 days postlesion. These findings suggest that oligodendrocytes dedifferentiate-like Schwann cells-from cells which express myelin molecules to elongate cells which express the L1/E587 antigen. They redifferentiate to myelinate axons from roughly 3 weeks onward. These findings suggest an adaptive plasticity of goldfish oligodendrocytes beneficial to the repair of the visual pathway.

Brain-derived neurotrophic factor-mediated neuroprotection of adult rat retinal ganglion cells in vivo does not exclusively depend on phosphatidyl-inositol-3'-kinase/protein kinase B signaling.

The neurotrophin brain-derived neurotrophic factor (BDNF) serves as a survival, mitogenic, and differentiation factor in both the developing and adult CNS and PNS. In an attempt to identify the molecular mechanisms underlying BDNF neuroprotection, we studied activation of two potentially neuroprotective signal transduction pathways by BDNF in a CNS trauma model. Transection of the optic nerve (ON) in the adult rat induces secondary death of retinal ganglion cells (RGCs). Repeated intraocular injections of BDNF prevent the degeneration of RGCs 14 d after ON lesion most likely by inhibition of apoptosis. Here, we report that BDNF activates both protein kinase B (PKB) via a phosphatidyl-inositol-3'-kinase (PI-3-K)-dependent mechanism and the mitogen-activated protein kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2. Furthermore, we provide evidence that BDNF suppresses cleavage and enzymatic activity of the neuronal cell death effector caspase-3. Distinct from our recent study in which inhibition of the PI-3-K/PKB pathway attenuated the survival-promoting action of insulin-like growth factor-I on axotomized RGCs (Kermer et al., 2000), it does not in the case of BDNF. Thus, we assume that BDNF does not depend on a single signal transduction pathway exerting its neuroprotective effects on lesioned CNS neurons.

E587 antigen is upregulated by goldfish oligodendrocytes after optic nerve lesion and supports retinal axon regeneration.

The properties of glial cells in lesioned nerves contribute quite substantially to success or failure of axon regeneration in the CNS. Goldfish retinal axons regenerate after optic nerve lesion (ONS) and express the L1-like cell adhesion protein E587 antigen on their surfaces. Goldfish oligodendrocytes in vitro also produce E587 antigen and promote growth of both fish and rat retinal axons. To determine whether glial cells in vivo synthesize E587 antigen, in situ hybridizations with E587 antisense cRNA probes and light- and electron microscopic E587 immunostainings were carried out. After lesion, the goldfish optic nerve/tract contained glial cells expressing E587 mRNA, which were few in number at 6 days after ONS, increased over the following week and declined in number thereafter. Also, E587-immunopositive elongated cells with ultrastructural characteristics of oligodendrocytes were found. Thus, glial cells synthesize E587 antigen in spatiotemporal correlation with retinal axon regeneration. To determine the functional contribution of E587 antigen, axon-oligodendrocyte interactions were monitored in co-culture assays in the presence of Fab fragments of a polyclonal E587 antiserum. E587 Fabs in axon-glia co-cultures prevented the normal tight adhesion of goldfish retinal growth cones to oligodendrocytes and blocked the preferential growth of fish and rat retinal axons on the oligodendrocyte surfaces. The ability of glia in the goldfish visual pathway to upregulate the expression of E587 antigen and the growth supportive effect of oligodendrocyte-associated E587 antigen in vitro suggests that this L1-like adhesion protein promotes retinal axon regeneration in the goldfish CNS.

Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons.

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains).