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1.
Cell Death Differ ; 23(3): 442-53, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26292756

ABSTRACT

While a great deal of progress has been made in understanding the molecular mechanisms that regulate retino-tectal mapping, the determinants that target retinal projections to specific layers of the optic tectum remain elusive. Here we show that two independent RGMa-peptides, C- and N-RGMa, activate two distinct intracellular pathways to regulate axonal growth. C-RGMa utilizes a Leukemia-associated RhoGEF (LARG)/Rho/Rock pathway to inhibit axonal growth. N-RGMa on the other hand relies on ϒ-secretase cleavage of the intracellular portion of Neogenin to generate an intracellular domain (NeICD) that uses LIM-only protein 4 (LMO4) to block growth. In the developing tectum (E18), overexpression of C-RGMa and dominant-negative LARG (LARG-PDZ) induced overshoots in the superficial tectal layer but not in deeper tectal layers. In younger embryos (E12), C-RGMa and LARG-PDZ prevented ectopic projections toward deeper tectal layers, indicating that C-RGMa may act as a barrier to descending axons. In contrast both N-RGMa and NeICD overexpression resulted in aberrant axonal-paths, all of which suggests that it is a repulsive guidance molecule. Thus, two RGMa fragments activate distinct pathways resulting in different axonal responses. These data reveal how retinal projections are targeted to the appropriate layer in their target tissue.


Subject(s)
Amyloid Precursor Protein Secretases/physiology , Nerve Tissue Proteins/physiology , Rho Guanine Nucleotide Exchange Factors/physiology , Animals , Cell Enlargement , Chick Embryo , Organ Specificity , Retinal Ganglion Cells/physiology , Superior Colliculi/cytology , Superior Colliculi/enzymology , Tissue Culture Techniques
2.
Cell Death Dis ; 6: e1967, 2015 Nov 05.
Article in English | MEDLINE | ID: mdl-26539914

ABSTRACT

Previous studies show that caspase-6 and caspase-8 are involved in neuronal apoptosis and regenerative failure after trauma of the adult central nervous system (CNS). In this study, we evaluated whether caspase-6 or -8 inhibitors can reduce cerebral or retinal injury after ischemia. Cerebral infarct volume, relative to appropriate controls, was significantly reduced in groups treated with caspase-6 or -8 inhibitors. Concomitantly, these treatments also reduced neurological deficits, reduced edema, increased cell proliferation, and increased neurofilament levels in the injured cerebrum. Caspase-6 and -8 inhibitors, or siRNAs, also increased retinal ganglion cell survival at 14 days after ischemic injury. Caspase-6 or -8 inhibition also decreased caspase-3, -6, and caspase-8 cleavage when assayed by western blot and reduced caspase-3 and -6 activities in colorimetric assays. We have shown that caspase-6 or caspase-8 inhibition decreases the neuropathological consequences of cerebral or retinal infarction, thereby emphasizing their importance in ischemic neuronal degeneration. As such, caspase-6 and -8 are potential targets for future therapies aimed at attenuating the devastating functional losses that result from retinal or cerebral stroke.


Subject(s)
Caspase 6/metabolism , Caspase 8/metabolism , Caspase Inhibitors/pharmacology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Reperfusion Injury/drug therapy , Stroke/drug therapy , Animals , Apoptosis/drug effects , Female , Molecular Targeted Therapy , Neurons/pathology , Rats , Rats, Sprague-Dawley , Reperfusion Injury/enzymology , Reperfusion Injury/pathology , Retina/pathology , Stroke/enzymology , Stroke/pathology
3.
Cell Death Dis ; 6: e1744, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25950474

ABSTRACT

The dependence receptor Neogenin and its ligand, the repulsive guidance molecule a (RGMa), regulate apoptosis and axonal growth in the developing and the adult central nervous system (CNS). Here, we show that this pathway has also a critical role in neuronal death following stroke, and that providing RGMa to neurons blocks Neogenin-induced death. Interestingly, the Neogenin pro-death function following ischemic insult depends on Neogenin association with lipid rafts. Thus, a peptide that prevents Neogenin association with lipid rafts increased neuronal survival in several in vitro stroke models. In rats, a pro-survival effect was also observed in a model of ocular ischemia, as well as after middle cerebral artery occlusion (MCAO). Treatments that prevented Neogenin association with lipid rafts improved neuronal survival and the complexity of the neuronal network following occlusion of the middle artery. Toward the development of a treatment for stroke, we developed a human anti-RGMa antibody that also prevents Neogenin association with lipid rafts. We show that this antibody also protected CNS tissue from ischemic damage and that its application resulted in a significant functional improvement even when administrated 6 h after artery occlusion. Thus, our results draw attention to the role of Neogenin and lipid rafts as potential targets following stroke.


Subject(s)
Antibodies, Monoclonal/pharmacology , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Neurons/metabolism , Stroke/metabolism , Stroke/therapy , Animals , Antibodies, Monoclonal/immunology , Cell Survival/physiology , Female , GPI-Linked Proteins/immunology , Humans , Male , Membrane Microdomains/pathology , Mice , Nerve Tissue Proteins/immunology , Neurons/cytology , Random Allocation , Rats , Rats, Sprague-Dawley , Recovery of Function , Stroke/pathology
4.
Neurotox Res ; 22(4): 355-64, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22528248

ABSTRACT

Delineation of how cell death mechanisms associated with Parkinson's disease (PD) interact and whether they converge would help identify targets for neuroprotective therapies. The purpose of this study was to use a cellular model to address these issues. Catecholaminergic SH-SY5Y neuroblastoma cells were exposed to a range of compounds (dopamine, rotenone, 5,8-dihydroxy-1,4-naphtho-107 quinone [naphthazarin], and Z-Ile-Glu(OBut)-Ala-Leu-al [PSI]) that are neurotoxic when applied to these cells for extended periods of times at specific concentrations. At the concentrations used, these compounds cause cellular stress via mechanisms that mimic those associated with causing neurodegeneration in PD, namely oxidative stress (dopamine), mitochondrial dysfunction (rotenone), lysosomal dysfunction (naphthazarin), and proteasomal dysfunction (PSI). The compounds were applied to the SH-SY5Y cells either alone or in pairs. When applied separately, the compounds produced a significant decrease in cell viability confirming that oxidative stress, mitochondrial, proteosomal, or lysosomal dysfunction can individually result in catecholaminergic cell death. When the compounds were applied in pairs, some of the combinations produced synergistic effects. Analysis of these interactions indicates that proteasomal, lysosomal, and mitochondrial dysfunction is exacerbated by dopamine-induced oxidative stress. Furthermore, inhibition of the proteasome or lysosome or increasing oxidative stress has a synergistic effect on cell viability when combined with mitochondrial dysfunction, suggesting that all cell death mechanisms impair mitochondrial function. Finally, we show that there are reciprocal relationships between oxidative stress, proteasomal dysfunction, and mitochondrial dysfunction, whereas lysosome dysfunction appears to mediate cell death via an independent pathway. Given the highly interactive nature of the various cell death mechanisms linked with PD, we predict that effective neuroprotective strategies should target multiple sites in these pathways, for example oxidative stress and mitochondria.


Subject(s)
Apoptosis/drug effects , Mitochondria/drug effects , Neurotoxins/toxicity , Antineoplastic Agents/toxicity , Cell Line, Tumor , Cell Survival/drug effects , Dopamine/toxicity , Dose-Response Relationship, Drug , Drug Synergism , Humans , Lysosomes/drug effects , Naphthoquinones/toxicity , Neuroblastoma/pathology , Proteasome Endopeptidase Complex/metabolism , Rotenone/toxicity
5.
Neuroscience ; 169(1): 495-504, 2010 Aug 11.
Article in English | MEDLINE | ID: mdl-20457227

ABSTRACT

The repulsive guidance molecule, RGMa, and its receptor Neogenin, regulate neuronal cell death during development, but little is known about their expression and roles in the adult CNS. Here, we show that Neogenin is expressed in the adult rodent retina, particularly on retinal ganglion cells. To determine whether the Neogenin/RGMa pathway is important in the fully developed retina, we examined its contribution to damage-induced neurodegeneration. The effects of RGMa on survival of retinal ganglion cells (RGCs) were examined in vitro and in vivo. Using cultured whole-mount retinal explants, we showed that the addition of RGMa increased RGC survival and that this effect was mediated by the Neogenin receptor. Immunohistochemical analysis indicated that the inhibition of cell death by RGMa resulted from reduced caspase-3 activation. Then, using an in vivo model of RGC apoptosis after optic nerve transection, we demonstrated that intraocular injection of RGMa at 3 and 7 days after axotomy greatly reduced RGC death 14 days postaxotomy. This study provides the first evidence that RGMa is a molecular target for neuroprotection in retinal pathologies, and suggests that targeting "dependence receptors" such as Neogenin has therapeutic potential for the treatment of neuropathologies in the adult CNS.


Subject(s)
Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Optic Nerve Injuries/drug therapy , Retinal Ganglion Cells/drug effects , Animals , Antibodies, Neutralizing/pharmacology , Axotomy , Caspase 3/physiology , Cell Survival/drug effects , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cloning, Molecular , Female , GPI-Linked Proteins/physiology , Membrane Proteins/drug effects , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , Nerve Degeneration/genetics , Nerve Degeneration/pathology , Nerve Tissue Proteins/immunology , Optic Nerve Injuries/pathology , Organ Culture Techniques , Protein Binding , Rats , Rats, Sprague-Dawley , Retinal Ganglion Cells/cytology
7.
Dev Biol ; 229(1): 1-14, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11133150

ABSTRACT

We have characterized the antigen recognized by mab10, a monoclonal antibody that has been shown to modify outgrowth of thalamic and cortical axons in vitro, and investigated the influence of this antibody on axonal growth in the chicken retina in vivo. Immunopurification, peptide sequencing, and biochemical characterization proved the epitope recognized by mab10 to be polysialic acid (PSA), associated with the neural cell adhesion molecule (NCAM). Intravitreal injections of antibody-secreting hybridoma cells were combined with whole-mount studies using the fluorescent tracer 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI). Pathfinding at the optic fissure was affected, resulting in a failure of axons to exit into the nerve. Misprojections also occurred in more peripheral areas of the retina; however, axons eventually oriented toward the center. Similar projection errors were observed after enzymatic removal of PSA by injecting endoneuraminidase N (endo N). Quantitative measurements of the optic nerve diameter as well as the width of the optic fiber layer confirmed that many axons failed to leave the retina and grew back in the optic fiber layer of the retina. Our findings suggest that NCAM-linked PSA is involved in guiding ganglion cell axons in the retina and at the optic fissure.


Subject(s)
Axons/physiology , Neural Cell Adhesion Molecules/metabolism , Optic Nerve/embryology , Retina/embryology , Retinal Ganglion Cells/physiology , Sialic Acids/metabolism , Animals , Antibodies, Monoclonal , Antibody Specificity , Brain Chemistry , Cell Communication , Chick Embryo , Epitopes , Glycoside Hydrolases/metabolism , Neural Cell Adhesion Molecules/immunology , Optic Nerve/pathology , Rats , Sialic Acids/immunology , Sialic Acids/isolation & purification
8.
Mol Cell Neurosci ; 18(5): 541-56, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11922144

ABSTRACT

A proteoglycan was identified and isolated from physiological saline extracts of chick embryo brains by using a new monoclonal antibody (hybridoma clone mab Te38). The purified proteoglycan displayed an apparent molecular mass of 2500-3500 kDa, which became reduced to 370 and 600 kDa after digestion with chondroitinase ABC or chondroitinase AC. After additional treatment with keratanase the 600-kDa band was no longer detectable in Western blots. The specific epitope recognized by mab Te38 is an O-linked carbohydrate associated with the core protein. Tenascin-C, an extracellular matrix protein known to associate with several proteoglycans, copurified with the mab Te38 proteoglycan on the immunoaffinity column. Mab Te38 binds to the surface of nonneuronal cells; in sections from the primary visual system, expression is restricted to cells in the optic fissure, the dorsal optic nerve, and the chiasm. No retinal cells were found to express the mab Te38 epitope. The isolated molecule inhibited axon outgrowth from retinal explants when offered bound to a substrate consisting of either matrigel or collagen, chondroitinase treatment did not alter the inhibitory properties. The distribution and in vitro function of the Te38 proteoglycan indicate that it may serve a role in guidance of retinal ganglion cell axons.


Subject(s)
Growth Cones/metabolism , Growth Inhibitors/metabolism , Optic Nerve/metabolism , Proteoglycans/metabolism , Retina/metabolism , Retinal Ganglion Cells/metabolism , Superior Colliculi/metabolism , Animals , Antibodies, Monoclonal , Cell Differentiation/drug effects , Cells, Cultured , Chick Embryo , Epitope Mapping , Growth Cones/drug effects , Growth Cones/ultrastructure , Mice , Optic Nerve/drug effects , Retina/cytology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/drug effects , Superior Colliculi/cytology , Tenascin/metabolism
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