Adult retinal ganglion cells (RGCs) can regenerate their axons in vitro. Using proteomics, we discovered that the supernatants of cultured retinas contain isoforms of crystallins with crystallin  b2 (crybb2) being clearly up-regulated in the regenerating retina. Immunohistochemistry revealed the expression of crybb within the retina, including in filopodial protrusions and axons of RGCs. Cloning and overexpression of crybb2 in RGCs and hippocampal neurons increased axonogenesis, which in turn could be blocked with antibodies against -crystallin. Conditioned medium from crybb2-transfected cell cultures also supported the growth of axons. Finally real time imaging of the uptake of green fluorescent protein-tagged crybb2 fusion protein showed that this protein becomes internalized. These data are the first to show that axonal regeneration is related to crybb2 movement. The results suggest that neuronal crystallins constitute a novel class of neurite-promoting factors that likely operate through an autocrine mechanism and that they could be used in neurodegenerative diseases. Molecular & Cellular Proteomics 6:895-907, 2007. Adult retinal ganglion cells (RGCs)1 exhibit only a short and transient sprouting reaction after injury, and they fail to extend axons throughout the interior of the optic nerve (1). The failure of regeneration is commonly attributed to inhibitory factors associated with myelin components and/or the glial scar that includes cells and extracellular matrix proteins (2-7). The inhibitory myelin proteins have been shown to include NogoA, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, all of which act through the Nogo receptor, NgR (8 -11). Expanding on this pathway of inhibition, blockage of signaling through NgR, applying antibodies against NogoA, and inactivation of RhoA (a downstream effector of NgR) signaling result in only modest axonal regeneration (12-14).There are several experimental conditions that permit the regrowth of RGC axons, including (i) replacing the distal segment of the cut optic nerve with a sciatic nerve segment (15), (ii) injuring the optic nerve and delayed culturing of retinal explants in vitro (16) or dissociation of RGCs and culturing of primary cells mainly in the postnatal retina (17), and (iii) injuring the lens (18 -21) or implanting a peripheral nerve fragment directly into the vitreous (22). Expanding on the mutual inflammatory mechanism of lens injury, stimulation of ocular macrophages with intravitreal injections of zymosan (19, 21, 23) also supports axonal growth. Explorations of alternative noninflammatory mechanisms have revealed that coculturing dissociated RGCs with injured lens tissue devoid of macrophages also improves the growth of axons (24). In an attempt to localize the growth factors within the lens, a coculture of retinal stripes with intact lens epithelium cells was shown to also support axonal growth in vitro (25), suggesting that independent mechanisms can result in the successful growth of axons. Consequently axon regene...
The interactions between migrating glioma cells and myelinated fiber tracts are poorly understood. We identified that C6 glioma cells can migrate along myelinated chicken retinal axons in a novel coculture, thereby expressing small GTPases of the Rho family and serine/threonine Rho-associated kinases (ROCKs). We found that the ROCK1 isoform is also highly expressed in native human high-grade gliomas. Glioma cells migrated faster in vitro along myelinated axons than on laminin-1, with the former but not the latter being specifically and reversibly blocked by the ROCK inhibitor Y27632. These data suggest that the mechanisms underlying the migration of glioma cells on myelinated axons differ from those underlying the migration on extracellular matrix molecules such as laminin-1.
The ability of neurons to form axons requires the choreographed assembly of growth cones. We show that there is a time window from postnatal day 14 (P14) until P21/22 when axons of rat retinal ganglion cells will regenerate under serum-free culture conditions. In contrast, no outgrowth occurred before P13, and growth declined from P22 and ceased after P30. Using proteomics, we have identified translin-associated factor X (Trax), a DNA-binding factor that is expressed during this period of postnatal development. Trax is shown to coexpress with growth-associated protein GAP-43. Small interfering RNA-mediated inhibition of Trax expression resulted in downregulation of both Trax and GAP-43 transcripts and protein both before and during the period of regeneration (P8) and (P16). In contrast, silencing of Trax at P30 resulted in significant upregulation of the GAP-43 transcript and protein and induced outgrowth of axons. These data suggest that Trax regulates GAP-43 transcription and regeneration-promoting effects during the postnatal maturation period. Trax may represent a new potent therapeutic target gene for optic nerve and spinal cord injuries.
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