Combined suppression of CASP2 and CASP6 protects retinal ganglion cells from apoptosis and promotes axon regeneration through CNTF-mediated JAK/STAT signalling

Abstract

We have previously shown that crushing the optic nerve induces death of retinal ganglion cells by apoptosis, but suppression of CASP2, which is predominantly activated in retinal ganglion cells, using a stably modified short interfering RNA CASP2, inhibits retinal ganglion cell apoptosis. Here, we report that combined delivery of short interfering CASP2 and inhibition of CASP6 using a dominant negative CASP6 mutant activates astrocytes and Müller cells, increases CNTF levels in the retina and leads to enhanced retinal ganglion cell axon regeneration. In dissociated adult rat mixed retinal cultures, dominant negative CASP6 mutant + short interfering CASP2 treatment also significantly increases GFAP+ glial activation, increases the expression of CNTF in culture, and subsequently increases the number of retinal ganglion cells with neurites and the mean retinal ganglion cell neurite length. These effects are abrogated by the addition of MAB228 (a monoclonal antibody targeted to the gp130 component of the CNTF receptor) and AG490 (an inhibitor of the JAK/STAT pathway downstream of CNTF signalling). Similarly, in the optic nerve crush injury model, MAB228 and AG490 neutralizes dominant negative CASP6 mutant + short interfering CASP2-mediated retinal ganglion cell axon regeneration, Müller cell activation and CNTF production in the retina without affecting retinal ganglion cell survival. We therefore conclude that axon regeneration promoted by suppression of CASP2 and CASP6 is CNTF-dependent and mediated through the JAK/STAT signalling pathway. This study offers insights for the development of effective therapeutics for promoting retinal ganglion cell survival and axon regeneration.

siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms

“…As in uninjured control animals in vivo, retinal cultures obtained from uninjured adult rats demonstrated an absence of GFAP þ activated glia, but treatment with siRTP801 promoted RGC neuroprotection in these cultures, indicating a direct effect on RGC-intrinsic mTOR signaling for cell survival. Neurite outgrowth was not affected by siRTP801 in the absence of activated retinal glia; however, consistent with previous reports, retinal cultures prepared 5 days after ONC contained GFAP þ activated glia, 19,23,25,67 and therefore may be considered more representative of the postinjury in vivo condition. The siRTP801 had no significant effect on initiation of neurite outgrowth, but promoted longer neurite growth, suggesting that additional factors were present in glial-activated cultures that enabled more sustained neurite outgrowth.…”

“…In agreement with our data, mTOR activity has been implicated as an important factor in the maintenance of an axon growth state, 73 whereas initiation of the axogenic program may be dependent on separate, although complementary, pathways such as CNTF/LIF-mediated JAK-STAT activity. [21][22][23]26,73,74 Previous studies investigating the role of PI3K-mTOR signaling on neuronal survival and axon regeneration have focused on RGC responses alone, rather than on the contribution of glial-derived factors. Retinal glial activation/ reactive gliosis has a major influence on RGC axon regeneration through the production and release of neurotrophins and other proregenerative and inflammatory cytokines, which modulate the injury response through autocrine and paracrine mechanisms.…”

“…57 In addition, a recent study from our laboratory reported that the number of GAP43 þ regenerating axons in the distal ON segment correlated with the number of axons detected by the anterograde tracer Rhodamine B. 23 The cross-sectional width of the ON was measured at the point at which axon counts were taken, and used to calculate the number of axons/mm ON width. This value was used to derive Rad, the total number of axons extending distance d in an ON with radius r using the following formula:…”

Increased expression of dedicator-cytokinesis-10, caspase-2 and Synaptotagmin-like 2 is associated with clinical disease activity in multiple sclerosis

“…Results are presented using the name of the TF in the Jaspar database and the p value of the position-specific weight matrix analysis, ranked by the p value expressed in B cells during the plasmablast stage of differentiation [33], and is overexpressed in MS patients during relapses and in response to interferon-beta or intravenous immunoglobulin therapy [34][35][36]. In addition, CASP2 is involved specifically in apoptosis in neuronal cells [36,37]. Together with its modulation of the immune response, this observation strongly suggests that CASP2 contributes to the dampening of lymphocyte proliferation.…”

Ciliary neurotrophic factor-treated astrocyte-conditioned medium increases the intracellular free calcium concentration in rat cortical neurons

“…In addition, it has important roles in triggering neurite outgrowth, preventing neuronal degeneration and attenuating motor deficits (3). Recent evidence suggests that CNTF is a potential activator of astrocytes, as it could induce astrocyte hypertrophy and glial fibrillary acidic protein (GFAP) overexpression (4,5), which is considered as a predominant feature for the activation of astrocytes.…”