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. 2011 Dec;34(11):1737-46.
doi: 10.1111/j.1460-9568.2011.07891.x. Epub 2011 Nov 18.

Altered Apoptotic Responses in Neurons Lacking RhoB GTPase

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Free PMC article

Altered Apoptotic Responses in Neurons Lacking RhoB GTPase

Sara Barberan et al. Eur J Neurosci. .
Free PMC article

Abstract

Caspase 3 activation has been linked to the acute neurotoxic effects of central nervous system damage, as in traumatic brain injury or cerebral ischaemia, and also to the early events leading to long-term neurodegeneration, as in Alzheimer's disease. However, the precise mechanisms activating caspase 3 in neuronal injury are unclear. RhoB is a member of the Rho GTPase family that is dramatically induced by cerebral ischaemia or neurotrauma, both in preclinical models and clinically. In the current study, we tested the hypothesis that RhoB might directly modulate caspase 3 activity and apoptotic or necrotic responses in neurons. Over-expression of RhoB in the NG108-15 neuronal cell line or in cultured corticohippocampal neurons elevated caspase 3 activity without inducing overt toxicity. Cultured corticohippocampal neurons from RhoB knockout mice did not show any differences in sensitivity to a necrotic stimulus - acute calcium ionophore exposure - compared with neurons from wild-type mice. However, corticohippocampal neurons lacking RhoB exhibited a reduction in the degree of DNA fragmentation and caspase 3 activation induced by the apoptotic agent staurosporine, in parallel with increased neuronal survival. Staurosporine induction of caspase 9 activity was also suppressed. RhoB knockout mice showed reduced basal levels of caspase 3 activity in the adult brain. These data directly implicate neuronal RhoB in caspase 3 activation and the initial stages of programmed cell death, and suggest that RhoB may represent an attractive target for therapeutic intervention in conditions involving elevated caspase 3 activity in the central nervous system.

Figures

Fig. 1
Fig. 1
Effect of elevated RhoB activity on morphology of corticohippocam-pal neurons. Neurons were transfected with vectors expressing GFP (A, C) or caRhoB-GFP (B, D). (A, B) caRhoB induces excessive dendritic arborisation. (C, D) Nuclear morphology, as assessed by DAPI staining (blue); note that elevated RhoB does not compromise nuclear morphology. Scale bars = 20 lm. For interpretation of color references in figure legend, please refer to the Web version of this article.
Fig. 2
Fig. 2
Effect of elevated RhoB activity on neuronal caspase 3 activity. (A–D) caRhoB and STS induce caspase 3 activation. Upper images in each pair show GFP-labelled neuronal soma (A–D), and lower images in each pair show the corresponding active caspase 3-immunoreactivity (red) (A′–D′). Scale bar = 10 µm. (E) Effect of RhoB expression and STS on active caspase 3-immunoreactivity in corticohippocampal neurons. Results are shown as percentage of signal in control (vehicle-treated, GFP-transfected) neurons, and are mean ± SEM, n = 8–16 per group. Effect of vector: F1,53 = 10.67, P < 0.01; effect of STS: F1,53 = 15.90, P < 0.001 (anova). *P < 0.05 vs. control (Veh-GFP) (Tukey’s post-hoc test); #P < 0.05 vs. Veh-RhoB. (F) Effect of elevated RhoB activity on caspase activity in response to STS exposure in NG108-15 cells. Results are shown as mean ± SEM, n = 6 per group. Effect of vector: F1,17 = 4.89, P < 0.05; effect of STS: F1,17 = 610.17, P < 0.001 (anova – transformed data). #P < 0.05 vs. control (Veh-GFP) (Mann–Whitney test). For interpretation of color references in figure legend, please refer to the Web version of this article.
Fig. 3
Fig. 3
Effect of RhoB deletion on nuclear morphology in corticohippocampal neurons following exposure to STS. (A) DAPI staining (blue) of corticohip-pocampal neurons from WT or RhoB KO mice 24 h following exposure to 500 nm STS. Scale bar = 10 µm. Note the higher proportion of nuclei showing condensation in the neurons lacking RhoB. (B,C) Nuclear morphology in corticohippocampal neurons from WT or RhoB KO mice 6 h (B) or 24 h (C) following exposure to STS. Results are shown as mean ± SEM, n = 3–5 per group. (B) Two-way anova: effect of STS concentration – F3,39 = 48.71, P < 0.001; effect of genotype – F3,39 = 39.71, P < 0.001. (C) Two-way anova: effect of STS dose – F3,39 = 254.38, P < 0.001; effect of genotype – F1,39 = 159.21, P < 0.001; **P < 0.01; post-hoc Tukey’s test vs. corresponding WT group. For interpretation of color references in figure legend, please refer to the Web version of this article.
Fig. 4
Fig. 4
Altered apoptotic responses in corticohippocampal neurons lacking RhoB. (A) TUNEL staining in primary cortico-hippocampal cultures from WT (left panels) and RhoB KO (right panels) mice, treated for 24 h with either 100 nm (upper panels) or 500 nm (lower panels) STS. Note the reduced numbers of TUNEL-positive nuclei in the neurons lacking RhoB. Scale bar = 50 µm. (B) TUNEL staining in primary cortical cultures from WT and RhoB KO mice, treated for 24 h with STS at the doses shown. Results are shown as mean ± SEM, n = 5 per group. Two-way anova: effect of STS concentration – F4,49 = 74.98, P < 0.001; effect of genotype – F1,49 = 101.10, P < 0.001; *P < 0.05, **P < 0.01; post-hoc Tukey’s test. (C) Caspase 3 activity in primary cortico-hippocampal cultures from WT and RhoB KO mice, treated for 6 h with STS at the concentrations shown; n = 4–6 per group. Note that the selective caspase 3 inhibitor Ac-DMQD-CHO completely inhibits cleavage of the fluorogenic substrate. Two-way anova: effect of STS concentration – F4,52 = 4.2, P < 0.001; effect of genotype – F1,52 = 12.87, P < 0.001. (D) Neuronal survival, in neurons from WT or RhoB KO mice, as assessed by mitochondrial XTT reduction to formazan, following exposure to 10 µm A23187 for 2.5 h, or 500 nm STS for 24 h. Neurons lacking RhoB show increased survival after exposure to STS, *P < 0.05 vs. WT (t-test).
Fig. 5
Fig. 5
Altered initiator caspase responses in corticohippocampal neurons lacking RhoB. (A) Caspase 8 activity and (B) caspase 9 activity in primary cortico-hippocampal cultures from WT and RhoB KO mice, treated for 4 h with vehicle, or either 200 or 500 nm STS. Results are shown as mean ± SEM, n = 8 per group. Two-way anova: caspase 8 activity, effect of STS concentration – F2,47 = 23.5, P < 0.001, effect of genotype – F2,47 = 0.51, P = 0.48; caspase 9 activity, effect of STS concentration – F2,47 = 162.9, P < 0.001, effect of genotype – F1,47 = 14.0, P < 0.001. ***P < 0.001 vs. corresponding WT value, post-hoc Tukey’s test.
Fig. 6
Fig. 6
Altered Bax expression in corticohippocampal neurons lacking RhoB. (A) Western blot showing expression of Bax and actin (loading control) in extracts from cortico-hippocampal cultures treated for 2 h with either vehicle or 500 nm STS. (B) Quantification of Bax immunoblotting in primary cortico-hippocampal cultures from WT and RhoB KO mice, treated for 2 h with either vehicle or 500 nm STS. Results are shown as mean ± SEM, n = 3 per group. Two-way anova: effect of STS concentration – F1,10 = 10.4, P = 0.015; effect of genotype – F1,10 = 10.0, P = 0.016; no significant interaction. *P < 0.05; **P < 0.01 vs. corresponding WT vehicle.
Fig. 7
Fig. 7
RhoB deletion modulates caspase 3 activity in adult brain. Caspase 3 activity in cortex and cerebellum samples from WT and RhoB KO (KO) mouse brain (aged 4 months). Results are shown as mean ± SEM, n = 4–5 per group. Activity in tissue from RhoB KO mice was significantly reduced relative to WT mice (two-way anova – factors: genotype and tissue; caspase 3 activity: effect of genotype F1,16 = 9.93, P = 0.008; *P < 0.05 relative to corresponding WT samples, post-hoc Tukey’s test.

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