Role of Nogo-A in neuronal survival in the reperfused ischemic brain

Abstract

Nogo-A is an oligodendroglial neurite outgrowth inhibitor, the deactivation of which enhances brain plasticity and functional recovery in animal models of stroke. Nogo-A's role in the reperfused brain tissue was still unknown. By using Nogo-A(-/-) mice and mice in which Nogo-A was blocked with a neutralizing antibody (11C7) that was infused into the lateral ventricle or striatum, we show that Nogo-A inhibition goes along with decreased neuronal survival and more protracted neurologic recovery, when deactivation is constitutive or induced 24 h before, but not after focal cerebral ischemia. We show that in the presence of Nogo-A, RhoA is activated and Rac1 and RhoB are deactivated, maintaining stress kinases p38/MAPK, SAPK/JNK1/2 and phosphatase-and-tensin homolog (PTEN) activities low. Nogo-A blockade leads to RhoA deactivation, thus overactivating Rac1 and RhoB, the former of which activates p38/MAPK and SAPK/JNK1/2 via direct interaction. RhoA and its effector Rho-associated coiled-coil protein kinase2 deactivation in turn stimulates PTEN, thus inhibiting Akt and ERK1/2, and initiating p53-dependent cell death. Our data suggest a novel role of Nogo-A in promoting neuronal survival by controlling Rac1/RhoA balance. Clinical trials should be aware of injurious effects of axonal growth-promoting therapies. Thus, Nogo-A antibodies should not be used in the very acute stroke phase.

Figure 1

Nogo-A deactivation by genetic knockout and by pre-ischemic, but not post-ischemic delivery of 11C7 antibody compromises neuronal survival after focal cerebral ischemia. The LDF during and after 30 mins MCA occlusion (A, D, G, J); density of DNA-fragmented, i.e., injured cells in the ischemic striatum, basal forebrain, and cortex, revealed by TUNEL staining (B, E, H, K); percentage of surviving neurons in the same structures, evaluated by NeuN immunohistochemistry (C, F, I, L, N) and volumetry of the ischemic striatum (O). The WT and Nogo-A^−/− animals (A–C), animals treated with control IgG or 11C7 antibody that was delivered into the lateral ventricle (D–F, J–L, N, O) or ischemic striatum (G–I) starting immediately after reperfusion onset (D–I) or 24 h before stroke (J–L, N, O) are shown, which were assessed by conventional histochemical analysis at 96 or 72 h after reperfusion (A–L; regions of interest shown in M) or by computer-based stereological analysis at 7 days after reperfusion (N, O). Representative microphotographs of NeuN+ neurons are also shown (N). Note the absence of differences in LDF recordings between groups (A, D, G). Data are means±s.d. (B–C: n=8 animals/group [WT mice] n=5 animals/group [Nogo-A^−/− mice] A, D–L, N, O: n=8 animals/group [all groups]). ^*P<0.05 compared with WT/control IgG-treated groups.

Figure 2

Nogo-A deactivation by neutralizing 11C7 antibody delays neurologic recovery when initiated 24 h before MCA occlusion. Body weight (A); grip strength in the paretic right forelimb, as revealed by a spring balance (B); motor coordination, as examined on the RotaRod (C); and spontaneous locomotor behavior, as evaluated by elevated plus maze (D) and open field (E, F) tests. Note the more protracted recovery of grip strength (B) and motor coordination (C) in Nogo-A antibody treated as compared with control mice. No differences were seen in spontaneous locomotor and exploration behavior (D–F). Note that the animals used for this more detailed behavioral analysis differed from those for which behavioural results are presented in the text. Data are means±s.d. (n=8 animals per group). ^*P<0.05 compared with control IgG-treated animals. Condition × time interaction effects are also shown.

Figure 3

Nogo-A blockade inhibits the small GTPase RhoA (A), at the same time overactivating the small GTPases Rac1 and RhoB in animals exhibiting exacerbated neuronal injury (B, C). Western blots for Nogo-A, RhoA, Rac1, and RhoB, as well as pull down assays for RhoA, Rac1, and RhoB of ischemic mice exhibiting genetic NogoA-/- as well as animals treated with the Nogo-A antibody 11C7 either immediately after or 24 h before stroke. Note the absence of Nogo-A in NogoA-/- animals (A). Blots were normalized with β-actin. RhoA, Rac1, and RhoB activation state was expressed as percent of total protein expression. C, contralateral; I, ischemic. Two blots, of eight samples each, were simultaneously processed and developed, and merged into one figure for further data analysis and presentation. Data are means±s.d. (n=3 experiments per group). ^*P<0.05; ^**P<0.01 compared with corresponding samples of WT/control IgG-treated animals.

Figure 4

Nogo-A inhibition activates the p38/MAPK and SAPK/JNK1/2 pathways via direct interaction with active Rac1. Western blots using antibodies detecting either phosphorylated (p-p38, p-JNK1/2) or total (=overall; i.e., phosphorylated and non-phosphorylated) p38/MAPK (A) and SAPK/JNK1/2 (B, C), as well as immunoprecipitation and co-precipitation studies examining protein interactions of total p38/MAPK and total SAPK/JNK1/2 with Rac1, and of phosphorylated p38/MAPK and phosphorylated SAPK/JNK1/2 with constitutively active GST-Rac1 (L61) (D). Blots were normalized with β-actin and expressed as ratio between phosphorylated and total stress kinases. C, contralateral; I, ischemic. Two blots, of eight samples each, were simultaneously processed and developed, and merged for further data analysis and presentation. Data are means±s.d. (n=3 experiments per group). ^**P<0.01 compared with corresponding samples of WT/control IgG-treated animals.

Figure 5

Nogo-A blockade deactivates Rock2, decreasing PTEN phosphorylation and activating it, thus dephosphorylating and deactivating Akt and ERK1/2 pathways and inducing the p53 death pathway. Western blots using antibodies detecting either phosphorylated (p-PTEN, p-Akt, p-ERK1/2) or total (i.e., phosphorylated and non-phosphorylated) PTEN, Akt, and ERK1/2 (A–D), immunoprecipitation studies examining protein interactions of total and phosphorylated PTEN with Rock2 (E) and western blots for the death protein p53 (F). Blots were normalized with β-actin and expressed as ratio between phosphorylated and total signal proteins. C, contralateral; I, ischemic. Two blots, of eight samples each, were simultaneously processed and developed, and merged for further data analysis and presentation. Data are means±s.d. (n=3 experiments per group). ^*P<0.05; ^**P<0.01 compared with corresponding samples of WT/control IgG-treated animals.

Figure 6

Scheme outlining the novel role of Nogo-A in controlling neuronal survival in the ischemic brain. When Nogo-A signaling pathway is active, through the putative receptor and/or directly through its N-terminal inhibitory region (NiG) (as shown in A), the small GTPase RhoA is active, whereas the Rho GTPases members Rac1 and RhoB are inactive. As a consequence of Rac1 inactivation, the p38/MAPK and SAPK/JNK1/2 pathways are inhibited, and PTEN activity is controlled and maintained low by increasing its phosphorylation via Rock2, and the PI3K/Akt and ERK1/2 pathways are active, thus enhancing p53 degradation and promoting neuronal survival. Nogo-A signaling inhibition, on the other hand (as shown in B), deactivates RhoA, at the same time overactivates Rac1 and RhoB, the former of which mediates the activation of the p38/MAPK and SAPK/JNK1/2 stress response via direct interaction. Deactivation of RhoA, as a consequence of Nogo-A inhibition, inactivates its direct downstream effector Rock2, therefore the latter looses PTEN phosphorylation control, whereby the PTEN phosphatase is activated, which in turn deactivates PI3K/Akt and ERK1/2 pathways and induces death signaling by stabilizing p53. Black complete lines were used for direct signaling pathways, and black dashed lines were used for indirect signaling pathways.