Brain damage and disease involve activation of microglia and production of potentially neurotoxic molecules, but there are no treatments that effectively target their harmful properties. We present evidence that the small-conductance Ca 2ϩ /calmodulin-activated K ϩ channel KCNN4/ KCa3.1/SK4/IK1 is highly expressed in rat microglia and is a potential therapeutic target for acute brain damage. Using a Transwell cell-culture system that allows separate treatment of the microglia or neurons, we show that activated microglia killed neurons, and this was markedly reduced by treating only the microglia with a selective inhibitor of KCa3.1 channels, triarylmethane-34 (TRAM-34). To assess the role of KCa3.1 channels in microglia activation and key signaling pathways involved, we exploited several fluorescence plate-reader-based assays. KCa3.1 channels contributed to microglia activation, inducible nitric oxide synthase upregulation, production of nitric oxide and peroxynitrite, and to consequent neurotoxicity, protein tyrosine nitration, and caspase 3 activation in the target neurons. Microglia activation involved the signaling pathways p38 mitogen-activated protein kinase (MAPK) and nuclear factor B (NF-B), which are important for upregulation of numerous proinflammatory molecules, and the KCa3.1 channels were functionally linked to activation of p38 MAPK but not NF-B. These in vitro findings translated into in vivo neuroprotection, because we found that degeneration of retinal ganglion cells after optic nerve transection was reduced by intraocular injection of TRAM-34. This study provides evidence that KCa3.1 channels constitute a therapeutic target in the CNS and that inhibiting this K ϩ channel might benefit acute and chronic neurodegenerative disorders that are caused by or exacerbated by inflammation.

To promote functional recovery after CNS injuries, it is crucial to develop strategies that enhance both neuronal survival and regeneration. Here, we report that caspase-6 is upregulated in injured retinal ganglion cells and that its inhibition promotes both survival and regeneration in these adult CNS neurons. Treatment of rat retinal whole mounts with Z-VEID-FMK, a selective inhibitor of caspase-6, enhanced ganglion cell survival. Moreover, retinal explants treated with this drug extended neurites on myelin. We also show that caspase-6 inhibition resulted in improved ganglion cell survival and robust axonal regeneration following optic nerve injury in adult rats. The effects of Z-VEID-FMK were similar to other caspase inhibitory peptides including Z-LEHD-FMK and Z-VAD-FMK. In searching for downstream effectors for caspase-6, we identified caspase-8, whose expression pattern resembled that of caspase-6 in the injured eye. We then showed that caspase-8 is activated downstream of caspase-6 in the injured adult retina. Furthermore, we investigated the role of caspase-8 in RGC apoptosis and regenerative failure both in vitro and in vivo. We observed that caspase-8 inhibition by Z-IETD-FMK promoted survival and regeneration to an extent similar to that obtained with caspase-6 inhibition. Our results indicate that caspase-6 and caspase-8 are components of a cellular pathway that prevents neuronal survival and regeneration in the adult mammalian CNS.

Recent evidence suggests that approximately 90% of retinal ganglion cells (RGCs) die by the process of apoptosis within 14 days of optic nerve transection. RGCs begin to disappear from the retina between 5 and 7 days postaxotomy when the highest percentage of RGCs show characteristics typical of apoptosis. A single intraocular injection of glial cell-line derived neurotrophic factor (GDNF) given at the time of axotomy resulted in a delay in the initiation of RGC death and increased the densities of surviving RGCs at 7, 10 and 14 days postaxotomy. The mean RGC densities in GDNF treated retinas at 7 (2381 +/- 144), 10 (1561 +/- 117) and 14 (1123 +/- 116) days postaxotomy were significantly higher than that of controls (1835 +/- 82, 835 +/- 272 and 485 +/- 39, respectively). The loss of RGCs was paralleled by increases in TUNEL positive staining in control retinas and a lower percentage of TUNEL positive cells in GDNF treated retinas at 5, 7 and 10 days postaxotomy. These results suggest that GDNF is capable of promoting RGC survival following injury, possibly by interfering with an essential step in apoptosis.

Transection of the optic nerve induces an apoptotic degeneration of retinal ganglion cells (RGC) in the rat retina. The immediate early gene c-Jun, the proapoptotic Bcl-2 family member Bax and the apoptosome constituent Apaf-1 have been shown previously to play major roles in the induction or execution of the apoptosis cascade. In this study we have designed and generated short interfering RNAs (siRNAs) against c-Jun, Bax and Apaf-1, which were injected into the optic nerve stump in order to inhibit axotomy-induced apoptosis. siRNAs were first tested in vitro to ensure silencing efficiency. In vivo, a clear neuronal localization of Cy3-labelled siRNA could be visualized in retinal flat mounts. Retinas that were injected with anti-Apaf-1- and anti-c-Jun-siRNA showed significantly more surviving RGC than non-injected or anti-EGFP-injected controls (approximately 2- to 3-fold, respectively). Anti-Bax-siRNA-injected retinas showed a trend towards an increased RGC number (not significant). Regulation of target proteins in situ could be visualized by immunohistochemical stainings. We conclude that (i) c-Jun and Apaf-1 play major roles in the apoptotic cascade of RGC and may represent useful targets for antiapoptotic strategies in RGC in vivo, and (ii) injection of siRNAs into the optic nerve stump is a new method to down-regulate target genes specifically in RGC.

No abstract