Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration

Abstract

CNS neurons, such as retinal ganglion cells (RGCs), do not normally regenerate injured axons, but instead undergo apoptotic cell death. Regenerative failure is due to inhibitory factors in the myelin and forming glial scar as well as due to an insufficient intrinsic capability of mature neurons to regrow axons. Nevertheless, RGCs can be transformed into an active regenerative state upon inflammatory stimulation (IS) in the inner eye, for instance by lens injury, enabling these RGCs to survive axotomy and to regenerate axons into the lesioned optic nerve. The beneficial effects of IS are mediated by various factors, including CNTF, LIF and IL-6. Consistently, IS activates various signaling pathways, such as JAK/STAT3 and PI3K/AKT/mTOR, in several retinal cell types. Using a conditional knockdown approach to specifically delete STAT3 in adult RGCs, we investigated the role of STAT3 in IS-induced neuroprotection and axon regeneration. Conditional STAT3 knockdown in RGCs did not affect the survival of RGCs after optic nerve injury compared with controls, but significantly reduced the neuroprotective effects of IS. STAT3 depletion significantly compromised CNTF-stimulated neurite growth in culture and IS-induced transformation of RGCs into an active regenerative state in vivo. As a consequence, IS-mediated axonal regeneration into the injured optic nerve was almost completely abolished in mice with STAT3 depleted in RGCs. In conclusion, STAT3 activation in RGCs is involved in neuroprotection and is a necessary prerequisite for optic nerve regeneration upon IS.

Figure 1

AAV2-mediated conditional STAT3 depletion in RGCs. (a) Retinal flat-mount from a STAT3-floxed mouse 2 weeks after intravitreal application of AAV2-Cre. HA-tagged Cre recombinase (cre, red) was detected in the nuclei of ∼90% of βIII tubulin-positive RGCs (green) using an anti-HA-antibody. Scale bar: 50 μm. (b) Quantification of βIII tubulin-positive RGCs per mm² in flat-mounted retinae 3 weeks after intravitreal injection of either AAV2-GFP (control) or AAV2-Cre. Virus-mediated knockdown of STAT3 (cre) did not affect the survival of uninjured RGCs compared with AAV2-GFP-treated control retinae (gfp). Values represent the mean of three retinae per group. (c) Immunohistochemical staining of cross sections of untreated control retinae (con) and retinae 5 days after optic nerve crush (onc) or onc+inflammatory stimulation (onc/is) from either AAV2-GFP- (gfp) or AAV2-Cre- (cre) injected mice with antibodies against phosphorylated STAT3 (pSTAT3, red) and βIII tubulin (tubulin, green). Virus-mediated STAT3 knockdown reduced IS-induced activation of STAT3 specifically in RGCs, but not in cells of the fiber and inner nuclear layer. Scale bar: 50 μm; GCL, ganglion cell layer; INL, inner nuclear layer. (d) Western blot analysis of retinal lysates from animals treated as described in c with an antibody against phosphorylated STAT3 (pSTAT3). Tubulin served as loading control. Activation of STAT3 strongly increased after ONC+IS compared with untreated (con) and optic nerve crushed (ONC) AAV2-GFP control mice (gfp). Virus-mediated STAT3 knockdown (cre) strongly diminished STAT3 phosphorylation upon ONC+IS. (e) Photometric quantification of western blots as in d. Virus-mediated STAT3 knockdown (cre) reduced STAT3 activation by 78%. Values represent the mean of four independent experiments. Treatment effects: ***P<0.001

Figure 2

STAT3 depletion in RGCs compromises the IS-induced switch into a regenerative state. (a) βIII tubulin-positive RGCs of STAT3-floxed mice either exposed to vehicle (con) or CNTF (200 ng/ml) after 3 days in culture. Animals were intravitreally injected with either AAV2-GFP or AAV2-Cre 14 days prior to preparing cultures. Scale bar: 50 μm. (b) Quantification of neurite length per RGC in retinal cultures as described in a. Values represent the mean of 12 wells from three independent retinae per group. Depletion of STAT3 in RGCs significantly compromised CNTF-induced neurite growth. (c) Quantification of RGCs per well in cultures as described in a. STAT3 knockdown did not affect the survival of mature RGCs in these cultures. (d) In vivo pre-conditioned, βIII tubulin-positive RGCs after 24 h in culture. Pre-treatment: STAT3-floxed mice received intravitreal injections of either AAV2-GFP (gfp) or AAV2-Cre (cre). Two weeks later, animals were subjected either to optic nerve crush (onc) or onc+inflammatory stimulation (onc/is). Retinal cell cultures were prepared 5 days thereafter. Scale bar: 50 μm. (e) Quantification of neurite length per cultured RGC from mice treated as described in d. Values represent the mean of 16 wells from four independent retinae per group. STAT3 depletion significantly compromised IS-induced neurite growth. (f) Quantification of RGCs per well in retinal cultures as described in d. Virus-mediated STAT3 depletion did not affect RGC survival in retinal cell cultures. (g, h) Quantitative real-time PCR: retinal Gap43 (g) and Sprr1a (h) expression was quantified relative to GAPDH expression in animals as described in d. Values represent the mean of four retinae per group. ONC- and IS-induced upregulation of GAP43 and Sprr1a expression was impeded upon STAT3 depletion. (i) Western blot analysis of retinal lysates from animals treated as described in d using antibodies against GAP43, CNTF and pSTAT3. Tubulin served as loading control. Expression of GAP43 and CNTF strongly increased after ONC+IS compared with untreated AAV2-GFP control mice (gfp). IS-induced upregulation of GAP43 expression was compromised upon STAT3 depletion (cre), whereas the increase in CNTF expression was not affected. Treatment effects: n.s., non-significant; **P<0.01; ***P<0.001

Figure 3

STAT3 depletion impairs IS-induced neuroprotection. (a) Immunohistochemical staining of surviving RGCs in retinal flat-mounts 14 days after optic nerve crush (onc) or onc+inflammatory stimulation (onc/is) using an antibody against βIII tubulin. Scale bar: 50 μm. Animals were intravitreally injected with either AAV2-GFP (gfp) or AAV2-Cre (cre) 2 weeks before surgery. (b) Quantification of surviving RGCs per mm² in retinal flat-mounts of animals treated as described in a. Conditional STAT3 knockdown partially reduced the neuroprotective effect of IS. Treatment effects: n.s., non-significant; *P<0.05; ***P<0.001. Values represent the mean of at least five retinae per treatment

Figure 4

STAT3 depletion in RGCs compromises IS-stimulated axon regeneration in the optic nerve. (a) Regenerating axons crossing the lesion site (asterisk) in longitudinal sections of optic nerves visualized with an antibody against GAP43. Scale bar: 100 μm. Animals were pre-treated by intravitreal injection of either AAV2-GFP or AAV2-Cre 2 weeks before optic nerve crush (onc) or onc+inflammatory stimulation (onc/is). Animals were killed 2 weeks later and optic nerves were dissected. (b) Quantification of regenerating axons at 0.5, 1, 1.5 and 2 mm beyond the lesion site in ONC/IS-treated animals injected either with AAV2-GFP (gfp) or AAV2-Cre (cre). STAT3 depletion in RGCs significantly compromised IS-induced axonal regeneration. Treatment effects: ***P<0.001. Values represent the mean of five animals per treatment