21 research outputs found

    Role of inhibitors of apoptosis (IAPs) in retinal ganglion cell death and dendrite remodelling.

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    Neuronal viability and connectivity is essential for neuronal function in heath and disease. The aim of this study was to elucidate factors and mechanisms that govern the maintenance and remodelling of RGC dendrites, as well as neuronal cell death in ageing and neurodegenerative diseases. Using PCR technique, the expression pattern of caspases 3,6-9 and inhibitors of apoptosis (lAPs), namely neuronal IAP (NIAP), cellular IAP1 and 2 (clAP1 and 2), X-chromosome linked IAP (XIAP), Survivin, Bruce and Livin was determined in young adult (6 weeks), mature (24-52 weeks), old (88 weeks) and diseased retinae of Wistar albino and Brown Norway (BN) rats. Caspase expression was not altered during maturation and ageing in both strains. In ageing Wistar, NIAP, clAP2 and XIAP and clAP1 were decreased in 88 compared to 24-52 weeks, while Survivin, Bruce and Livin were slightly increased with age. lAPs expression was generally decreased in mature (24- 52 weeks) BN retinae compared to younger (6 weeks). Furthermore, validation of the expression of these molecules at protein level was carried out using western blotting and immunofluorescence techniques. clAP1 protein levels were downregulated in RGCL of BN rats. Reduction of clAP1 did not alter caspase activity but led to impairment in the survival pathway. Older BN retinae demonstrated compromised RGC morphology, but there was no retinal cell loss. Microbead experimental glaucoma model demonstrated no alteration in caspase expression upon induction of glaucoma. NIAP, clAP2, Survivin and Livin were up-regulated, while Bruce was down-regulated in glaucomatous eyes. clAP1 and XIAP expression remain similar between control and experimental eye. In conclusion, reduction in lAPs, which may lead to impairment in survival pathways might be the underlaying cause of reduction in dendrite complexity. Compromised RGC morphology makes a preparatory platform for neurodegenerative process observed in glaucoma disease where age is a major risk factor

    T-type Ca 2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling

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    Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca2+ influx, and highly selective T-type Ca2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca2+ channels, prevented enhanced axon growth. T-type Ca2+ channel-specific inhibitors eliminated Ca2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca2+ channels along sympathetic axons, which is required for enhanced axon growth

    Regional differences in the contributions of TNF reverse and forward signaling to the establishment of sympathetic innervation

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    Members of the TNF and TNF receptor superfamilies acting by both forward and reverse signaling are increasingly recognized as major physiological regulators of axon growth and tissue innervation in development. Studies of the experimentally tractable superior cervical ganglion (SCG) neurons and their targets have shown that only TNF reverse signaling, not forward signaling, is a physiological regulator of sympathetic innervation. Here, we compared SCG neurons and their targets with prevertebral ganglion (PVG) neurons and their targets. Whereas all SCG targets were markedly hypoinnervated in both TNF‐deficient and TNFR1‐deficient mice, PVG targets were not hypoinnervated in these mice and one PVG target, the spleen, was significantly hyperinnervated. These in vivo regional differences in innervation density were related to in vitro differences in the responses of SCG and PVG neurons to TNF reverse and forward signaling. Though TNF reverse signaling enhanced SCG axon growth, it did not affect PVG axon growth. Whereas activation of TNF forward signaling in PVG axons inhibited growth, TNF forward signaling could not be activated in SCG axons. These latter differences in the response of SCG and PVG axons to TNF forward signaling were related to TNFR1 expression, whereas PVG axons expressed TNFR1, SCG axons did not. These results show that both TNF reverse and forward signaling are physiological regulators of sympathetic innervation in different tissues

    A novel method for the induction of experimental glaucoma using magnetic microspheres

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    Purpose. The development of a method for the sustained elevation of intraocular pressure in experimental glaucoma based on the anterior chamber injection of paramagnetic microbeads. Methods. Unilateral glaucoma was induced in adult male Norwegian Brown rats by the injection of paramagnetic polystyrene microspheres. A handheld 0.45 Tesla magnet was used to draw the beads into the iridocorneal angle to impede aqueous drainage via the trabecular meshwork. Results. Elevated intraocular pressures (IOPs) were induced in 61 rats, resulting in a mean elevation of 5.8 mm Hg ± 1.0 (SEM) relative to the contralateral control eye. The mean duration of sustained IOP elevation (defined as >5 mm Hg relative to the control eye for at least 7 consecutive days) after a single injection was 12.8 days ± 0.9 (SEM, maximum duration 27 days). In all eyes, the visual axis remained clear from the time of injection, with minimal inflammation after injection. Retinal ganglion cell loss was determined in 21 animals (mean integral IOP, 194.5 mm Hg days ± 87.5 [SEM]) as 36.4% ± 2.4 (SEM) compared with the contralateral, untreated eye. Conclusions. The use of paramagnetic microbeads for the occlusion of the iridocorneal angle produces a sustained elevation of IOP with fewer injections and avoids the risk of visual axis occlusion. It represents a simple and effective method for the induction of experimental glaucoma

    Retinal ganglion cell death postponed: giving apoptosis a break?

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    Glaucoma is characterised by the preferential death of retinal ganglion cells (RGCs). However, mammalian models indicate that neurons pass through a period in which they manifest signs of neuronal damage, but have yet to fully commit to death. Mounting evidence suggests that one of the clearest indications of this process is the reduction in RGC dendritic arborisation, resulting in functional compromise. The extent to which this may be reversible is unclear, since the molecular events that precede changes in dendritic structure have received little attention. Furthermore, there are likely to be many factors involved in this process potentially acting in different individual cells at different times. Recent work in Drosophila shows that dendritic reorganisation/remodelling involves local activation and tight regulation of caspase activity. Here, we propose a model in which the balance between caspases and inhibitors of apoptosis (IAPs) contributes towards the regulation of dendritic remodelling. Thus, RGC dendrite reorganisation and cell death represent opposite ends of a spectrum of events regulated by apoptosis signalling pathways. We summarise relevant events in apoptosis, focusing on caspases and IAPs. We also discuss mechanisms of dendrite development, structure and reorganisation and the implications for early diagnosis and treatment of glaucoma and neurodegenerative diseas

    Cellular inhibitor of apoptosis (cIAP1) is down-regulated during retinal ganglion cell (RGC) maturation

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    Apoptosis, is the main type of cell death that occurs in ageing and neurodegenerative disease, such as glaucoma. This study therefore characterises the expression profile of caspases (pro-apoptosis) and inhibitors of apoptosis (IAPs; anti-apoptosis) during maturation of the Brown Norway rat retina between 6 weeks and >24 weeks and also examines concomitant changes in expression of tumor necrosis factor receptor associated factor 2 (TRAF2). The expression profiles of caspases (initiator caspases 8, 9 and effector caspases 6, 7, 3) and inhibitors of apoptosis (IAPs) (Neuronal IAP), cellular IAP1 and 2 (cIAP1/2), X-chromosome linked IAP (XIAP), Survivin, Bruce and Livin) were examined in retinae from 6 weeks and >24 weeks old BN rats using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, Western blotting and immunofluoroscence analysis. Caspase expression was not altered significantly during the study interval. IAP expression showed a general reduction during maturation of BN retina, which was statistically significant for cIAP1. cIAP1 reduction was confirmed by Western blotting and immunoflouroscence and was restricted to cells in the retinal ganglion cell layer (RGCL). Accumulation of TRAF2 was observed in the RGCL accompanying the down-regulation of cIAP1 observed. Our results suggest that cells in the mature RGCL may have a greater susceptibility to cell death compared to their younger counterparts and this may be due in part to a reduction in activation of survival pathways involving IAPs and TRAFs. Research highlights ► Caspase expression was not altered significantly during ageing of the Brown Norway rat retina. ► IAP expression showed a general reduction during maturation of BN retina, which was statistically significant for cIAP1. ► cIAP1 reduction was confirmed by Western blotting and immunoflouroscence and was restricted to cells in the retinal ganglion cell layer (RGCL). ► Accumulation of TRAF2 was observed in the RGCL accompanying the downregulation of cIAP1 observed. ► The results suggest that cells in the mature RGCL may have a greater susceptibility to cell death compared to their younger counterparts and this may be due in part to a reduction in activation of survival pathways involving IAPs and TRAFs

    Regulation of neurite growth by tumour necrosis superfamily member RANKL

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    RANKL (receptor-activator of NF-κB ligand, TNFSF11) is a member of the TNF superfamily that regulates bone remodelling and the development of the thymus, lymph nodes and mammary glands. While RANKL and its membrane bound receptor RANK (TNFRSF11A) are expressed in the adult central nervous system and have been implicated in thermoregulation, the potential function of RANK signalling in the developing nervous system remains unexplored. Here, we show that RANK is expressed by sympathetic and sensory neurons of the developing mouse peripheral nervous system and that activating RANK signalling in these neurons during perinatal development by either treating cultured neurons with soluble RANKL or overexpressing RANK in the neurons inhibited neurotrophin-promoted neurite growth without affecting neurotrophin-promoted neuronal survival. RANKL is expressed in tissues innervated by these neurons, and studies in compartment cultures demonstrated that RANKL is capable of acting directly on neurites to inhibit growth locally. Enhancing RANK signalling in cultured neurons resulted in NF-κB activation and phosphorylation of the p65 NF-κB subunit on serine 536. Transfecting neurons with a series of mutated signalling proteins showed that NF-κB activation and p65 phosphorylation occurred by an IKKβ-dependent mechanism and that blockade of this signalling pathway prevented neurite growth inhibition by RANKL. These findings reveal that RANKL is a novel negative regulator of neurite growth from developing PNS neurons and that it exerts its effects by IKKβ-dependent activation of NF-κB
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