Mathias Bähr

The c-jun transcription factor. bipotential mediator of neuronal death, survival, and regeneration

Axon interruption elicits a complex neuronal response that leaves neurons poised precariously between death and regeneration. The signals underlying this dichotomy are not fully understood. The transcription factor c-Jun is one of the earliest and most consistent markers for neurons that respond to nerve-fiber transection, and its expression can be related to both degeneration and survival including target re-innervation. In vitro experiments have demonstrated that expression of c-Jun can kill neonatal neurons but, in the adult nervous system, c-Jun might also be involved in neuroprotection and regeneration. The functional characteristics of c-Jun offer a model for the ability of a single molecule to serve as pivotal regulator for death or survival, not only in the response of the cell body to axonal lesions but also following neurodegenerative disorders. In this model, the fate of neurons is determined by a novel transcriptional network comprising c-Jun, ATF-2 (activating transcription factor-2) and JNKs (c-Jun N-terminal kinases).

Insulin-Like Growth Factor-I Protects Axotomized Rat Retinal Ganglion Cells from Secondary Death via PI3-K-Dependent Akt Phosphorylation and Inhibition of Caspase-3 In Vivo

Recently we have shown that the majority of retinal ganglion cells (RGCs) dies via activation of caspase-3 after transection of the optic nerve (ON) in the adult rat. In the present study we investigated whether insulin-like growth factor-I (IGF-I), an important factor in retinal development, prevents secondary death of RGCs after axotomy. Moreover, we studied potential intracellular mechanisms of IGF-mediated neuroprotection in more detail. Our results indicate that intraocular application of IGF-I protects RGCs from death after ON transection in a dose-dependent manner. We show reduced caspase-3 activity as one possible neuroprotective mechanism of IGF-I treatment in vivo. Caspase-3 mRNA expression remained unchanged. Because caspase inhibition can be mediated by Akt in vitro, we examined phosphorylation of Akt after axotomy and under IGF treatment. Western blot analysis revealed decreased Akt phosphorylation after axotomy without treatment and an increased phosphorylation of Akt under treatment with IGF-I. This strong increase could be reduced by simultaneous injection of wortmannin (WM), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-K). To prove the pathway suggested by these experiments as relevant for the in vivo situation, we assessed the number of RGCs 14 d after ON transection under a combined treatment strategy of IGF-I and WM. As expected, WM significantly reduced the neuroprotective effects of IGF-I. In summary, we show for the first time in vivothat IGF is neuroprotective via PI3-K-dependent Akt phosphorylation and by inhibition of caspase-3.

Live or let die – retinal ganglion cell death and survival during development and in the lesioned adult CNS

Programmed cell death or apoptosis is a common and widespread phenomenon that is important for proper development of the nervous system. In the adult CNS, however, apoptosis contributes to secondary cell loss after various types of lesions. The retino-tectal system has been successfully used as a convenient model system to study the molecular mechanisms of neuronal apoptosis and survival during development and in the lesioned adult CNS. This review describes the current knowledge about the interactions of cell death and survival pathways in general and for retinal ganglion cells specifically.

ACTIVATION OF CASPASE-3 IN AXOTOMIZED RAT RETINAL GANGLION CELLS IN VIVO

Recently, we have shown that inhibition of caspase‐3‐like caspases is the most effective treatment strategy to protect adult rat retinal ganglion cells from secondary death following optic nerve transection. In the present study, we localized active caspase‐3 in axotomized retinal ganglion cells in vivo and demonstrated a co‐localization of the active p20 fragment and TUNEL‐staining in some of these cells. In line with this, we detected an enhanced cleavage and activity of caspase‐3 protein in retinal tissue after lesion, while caspase‐3 mRNA expression remained unchanged. These data suggest caspase‐3 as an important mediator of secondary retinal ganglion cell death following axotomy in vivo.

Apoptosis in the developing visual system.

Abstract Programed cellular death is a widespread phenomenon during development of the nervous system. Two classes of molecules are particularly important in the context of apoptosis control in the nervous system: intracellular effectors homologous to the Caenorhabditis elegans Ced-3, -4, and -9 proteins, which in mammals correspond to the proteases of the caspase family, Apaf-1, and the members of the Bcl-2 protein family, and neurotrophic factors. Retinal ganglion cells lend a convenient model system with which to investigate apoptosis in central neurons during development as well as after injury. In this review, we discuss the role of these molecules in the control of programed cellular death in the retinotectal system. Transgenic animal models and expression studies have shown that caspases, Bcl-2, Bax, and possibly Bcl-X are necessary players for the control of programed cellular death in retinal ganglion cells. Bax and caspase 3 expression in retinal ganglion cells is upregulated after injury, and inhibition of Bax or caspase 3 increases the survival of injured retinal ganglion cells. Neurotrophins can support the survival of injured retinal ganglion cells, but this effect is transient. The physiological role of neurotrophins in the development of the retinocollicular system seems more related to the topographic refinement of retinocollicular projections, a process that is mediated, at least partially, by selective elimination of retinal ganglion cells making inappropriate topographic projections.

Long-term effect of inhibition of ced 3-like caspases on the survival of axotomized retinal ganglion cells in vivo

There is growing evidence that caspase inhibition exerts neuroprotective effects in various models of neuronal injury in vivo. However, whether caspase inhibition provides long-term neuroprotection is not known yet. In the present study, we therefore investigated the effects of prolonged caspase inhibition on the survival of adult rat retinal ganglion cells (RGCs) following optic nerve (ON) transection. Four weeks following ON transection the number of surviving RGCs in untreated animals declined to 11% of controls. Treatment for the initial 2 weeks with z-DEVD-cmk, an irreversible inhibitor of ced 3-like caspases, increased the number of surviving RGCs 4 weeks postlesion to 24%. Z-DEVD-cmk treatment over the entire experimental period of 4 weeks had no additional effect. Thus, we still found a neuroprotective effect of caspase inhibition on axotomized RGCs after extended survival time. However, in comparison to our recent observations 2 weeks after optic nerve transection, in which z-DEVD-cmk rescued 46% of RGCs (P. Kermer, N. Klöcker, M. Labes, and M. Bähr, 1998, J. Neurosci. 18(12), 4656-4662) the positive effect clearly decreased. In conclusion, our results indicate that the therapeutical approach presented here results in a significant delay of secondary death rather than providing a permanent and complete rescue of axotomized RGCs.

Immunocytochemical characterization of reactive optic nerve astrocytes and meningeal cells

Regeneration in the adult central nervous system (CNS) is thought to be hampered by the lesion‐induced activation of astrocytes and meningeal cells and the consecutive formation of a glial scar. The substrate properties of reactive astrocytes differ significantly from their neonatal counterparts, which promote axon growth, but in spite of intensive studies the underlying molecular changes are still not fully understood. We have used two cell culture systems to compare the expression of certain surface molecules on neonatal astrocytes, reactive astrocytes and meningeal cells in vitro.

Transection of the optic nerve in rats: studying neuronal death and survival in vivo.

Transection of the optic nerve (ON) in the adult rat, as a model of fiber tract lesion in the adult mammalian CNS, results in delayed, mainly apoptotic death of 80--90% of retinal ganglion cells (RGCs) within 14 days post-lesion. Because of good surgical accessibility of the retina and the optic nerve, the retino-tectal projection represents not only a convenient model to study the molecular mechanisms underlying neuronal death but also serves as a suitable system for investigating potential neuroprotective agents in vivo. In the present report, we provide a detailed protocol for this model including retrograde labeling of RGCs, ON lesion, assessment of the number of surviving neurons, and tissue preparation for several standard techniques like immunohistochemistry, reverse transcription--polymerase chain reaction (RT--PCR), enzyme assays and Immunoblot.

Retinal ganglion cell loss after the period of naturally occurring cell death in bcl-2-/- mice.

Over-expression of Bcl-2 is known to reduce the extent of retinal ganglion cell death during development as well as after axotomy. Here we investigated whether retinal ganglion cell (RGC) numbers are reduced in mice with a targeted inactivation of the bcl-2 gene. Compared with wild-type mice, adult bcl-2 null mutants have lost 29% of the retinal ganglion cell axons in the optic nerve. This reduction was almost fully established at P15, but not present at P10, which marks the end of the period of naturally occurring cell death. These observations, together with the previously reported late loss of primary motoneurons and peripheral neurons, point to a general physiological requirement for Bcl-2 soon after the period of naturally occurring cell death.

Inhibition of experimental rat glioma growth by decorin gene transfer is associated with decreased microglial infiltration

Decorin gene therapy for experimental malignant glioma is thought to involve antagonism of immunosuppression induced by glioma-derived transforming growth factor-beta (TGF-beta). TGF-beta is chemotactic for cells of the monocyte macrophage lineage but inhibits their functional activity in many in vitro paradigms. Here, we examined changes in the patterns of microglial infiltration of rat C6 gliomas expressing a decorin transgene. We find that the number of OX42/ED-1-positive microglial cells is reduced rather than enhanced in the presence of decorin. Decorin-expressing gliomas contain lower numbers of MHC class II antigen-expressing microglial cells whereas the relative frequency of MHC I immunoreactivity among microglial cells is increased. Interestingly, the reduction of TGF-beta levels in the tumors by decorin is associated with the de novo expression of inducible nitric oxide synthase (iNOS) in a minority of microglial cells. These data suggest that microglial cells do not participate in the regression of decorin-expressing rat C6 gliomas.