Elongation of Axons during Regeneration Involves Retinal Crystallin β b2 (crybb2)

Abstract

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...

Time-dependent Gene Expression Analysis of the Developing Superior Olivary Complex

“…These proteins promote axon regeneration of injured optic nerve fibers (31). Consistent with this, the related crystallin-␤ subunit b2 accumulates in axons and growth cones of retinal ganglion cells (63). Our immunohistochemical analysis, which demonstrated the presence of crystallin-␥ proteins in fiber tracts and the neuropil of the SOC (Fig.…”

Functional Whole-genome Analysis Identifies Polo-like Kinase 2 and Poliovirus Receptor as Essential for Neuronal Differentiation Upstream of the Negative Regulator αB-crystallin

“…However, whether the specific role of Cryab is beneficial or, rather, detrimental to this pathology is still a matter of debate (22)(23)(24)(25)(26). Furthermore, a potential role of crystallins during axonal regeneration has also been recently reported (27,28). In the present study we uncovered a novel function for Cryab as an inhibitor of neuronal differentiation.…”

“…Recently, however, a protective role of Cryab during experimental autoimmune encephalomyelitis, the animal model mirroring MS, has been published (26). In addition, ␤-and ␥-crystallin family members, have been suggested to play a role during axonal regeneration (27,28).…”

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development

“…Various β,γ-crystallins are present in low abundance in tissues other than the lens, most prominently in the retina (Andley, 2007) where their expression is reported to be upregulated following various injuries or stresses (Sakaguchi et al, 2003;Vazquez-Chona et al, 2004;Crabb et al, 2002). βB2-crystallin has recently been reported to be secreted by the retina in culture where it promotes the regrowth of retinal ganglion cell axons (Liedtke et al, 2007). βB2-crystallin is also expressed in sperm and its mutation in the Philly mouse is reported to cause reduced fertility (DuPrey et al, 2007).…”