Excess Bcl-X_L increases the intrinsic growth potential of adult CNS neurons in vitro

Abstract

The regenerative potential of adult mammalian CNS neurons is limited. Recent data suggest that inactivation of major growth inhibitors may not suffice to induce robust regeneration from mature neurons unless the intrinsic growth state is modulated. To investigate a possible role of Bcl-X_L for axon regeneration in the adult mammalian CNS, Bcl-X_L was adenovirally overexpressed in severed rat RGCs. Bcl-X_L overexpression in mature axotomized RGCs in vivo increased both numbers [3.10-fold (±0.20)] and cumulative length [6.72-fold (±0.47)] of neurites regenerated from retinal explants, and this effect was further pronounced in the central retina where specific and dense axoplasmatic transduction occurs. Similarly, delayed Bcl-X_L gene transfer to explanted retinae 12–13 days after lesion increased the numbers and length of emanating neurites by a factor of 5.22 (±0.41) and 8.29 (±0.69), respectively. In vivo, intraretinal sprouting of unmyelinated RGC axons into the nerve fiber layer was increased. However, fiber ingrowth into the optic nerve remained sparse, likely due to myelin inhibitors and scar components. Therefore, Bcl-X_L overexpression may enhance, but may not be sufficient to, restitute functional regeneration in the adult CNS. As assessed by cell quantification analysis, Bcl-X_L overexpression rescued a higher proportion of RGCs in vivo than in vitro. Therefore, Bcl-X_L is capable to induce both neuronal survival and axon regeneration, but these two processes appear to be differentially modified by distinct pathways in vivo.

Introduction

In the adult rodent, transection of the optic nerve (ON) leads to retrograde degeneration of more than 85% of RGCs within 14 days Berkelaar et al., 1994, Isenmann et al., 1997, Villegas-Perez et al., 1993, and the subpopulation of RGCs that survive is almost incapable to regenerate axons (Hüll and Bähr, 1994). Recent work on major growth suppressors of the extracellular environment suggests that intrinsic determinants may fundamentally contribute to enable regeneration in the mature CNS. Thus, elaboration of axons remains sparse even after neutralization or abolishment of myelin-associated inhibitory proteins Caroni and Schwab, 1988, Chierzi et al., 1999, Kim et al., 2003, Simonen et al., 2003, Zheng et al., 2003 or other growth suppressors of the extracellular environment Mc Keon et al., 1995, Niederöst et al., 1999. Even in the presence of peripheral nerve grafts So and Aguayo, 1985, Vidal-Sanz et al., 1987, limited fiber regeneration restricts functional recovery.

The proto-oncogene bcl-2 and its pro- and anti-apoptotic family members are major regulators of cell death and survival. Bcl-2 overexpression inhibits naturally occurring cell death (Martinou et al., 1994) and reduces CNS injury in various paradigms Bonfanti et al., 1996, Chen et al., 1997, Cenni et al., 1996. On the other hand, the fraction of RGCs surviving ON axotomy does not require Bcl-2 (Dietz et al., 2001). Whether Bcl-2 also affects axonal regeneration has remained a controversial issue. Bcl-2 augments neurite outgrowth in embryonic sensory neurons in vitro (Hilton et al., 1997) and in injured postnatal RGCs in vivo and in vitro (Chen et al., 1997). On the other hand, Bcl-2 failed to induce RGC axon restitution of neonatal mice even in the presence of growth-conducting Schwann cell transplants (Lodovichi et al., 2001). In adult rodents, Bcl-2 overexpression was again incapable of enhancing axonal sprouting subsequently to ON transection even if growth was propagated by neutralizing myelin-associated inhibitory molecules (Chierzi et al., 1999) or insertion of PN grafts (Inoue et al., 2002). Bcl-2 is expressed constitutively at high levels in the developing nervous system, but expression declines during maturation of the nervous system Isenmann et al., 1997, Merry et al., 1994. In contrast, Bcl-X_L has been identified as a predominant Bcl-2 member in the adult retina (Levin et al., 1997). Targeted disruption of the bcl-X_L gene increased developmental cell death Motoyama et al., 1995, Shindler et al., 1998, and conditional Bcl-X_L overexpression protected postnatal and adult neurons from traumatic, hypoxic Cao et al., 2002, Parsadanian et al., 1998, and metabolic (Shinoura et al., 2000) injury, a process supposed to underlie caspase-mediated proteolysis and neurotoxicity Fujita et al., 2000, Hu et al., 1998. The role of Bcl-X_L in neurite outgrowth and distant regeneration is unknown.

To investigate the possible role of Bcl-X_L for axon regeneration in the adult CNS, Bcl-X_L was adenovirally overexpressed in severed RGCs in retinal stripe cultures and in axotomized RGCs of adult rats in vivo. Retrograde RGC transduction or transgene delivery ex vivo increased both numbers and total length of emerging neurites in cultured explants by factors of more than three and six, respectively. Neurite regeneration was particularly enhanced in the peripapillary retina following specific retrograde RGC transduction. In vivo, Bcl-X_L overexpression promoted intraretinal axon sprouting and fiber ingrowth into the proximal optic nerve stump. However, newly generated axons were repelled when confronted with myelin epitopes and the lesion intersite, and failed to extend into or beyond the scar-forming region.