Activation of neuronal Ras-related C3 botulinum toxin substrate 1 (Rac1) improves post-stroke recovery and axonal plasticity in mice

Abstract

Long-term disability after stroke is common but the mechanisms of post-stroke recovery remain unclear. Cerebral Ras-related C3 botulinum toxin substrate (Rac) 1 contributes to functional recovery after ischemic stroke in mice. As Rac1 plays divergent roles in individual cell types after central neural system injury, we herein examined the specific role of neuronal Rac1 in post-stroke recovery and axonal regeneration. Young male mice were subjected to 60-min of middle cerebral artery occlusion (MCAO). Inducible deletion of neuronal Rac1 by daily intraperitoneal injection of tamoxifen (2 mg/40 g) into Thy1-creER/Rac1-floxed mice day 7-11 after MCAO worsened cognitive (assayed by novel object recognition test) and sensorimotor (assayed by adhesive removal and pellet reaching tests) recovery day 14-28 accompanied with the reduction of neurofilament-L (NFL) and myelin basic protein (MBP) and the elevation of glial fibrillary acidic protein (GFAP) in the peri-infarct zone assessed by immunostaining. Whereas the brain tissue loss was not altered assayed by cresyl violet staining. In another approach, delayed overexpression of neuronal Rac1 by injection of lentivirus encoding Rac1 with neuronal promotor into both the cortex and striatum (total 4 μl at 1 × 10⁹ transducing units/mL) of stroke side in C57BL/6J mice day 7 promoted stroke outcome, NFL and MBP regrowth and alleviated GFAP invasion. Furthermore, neuronal Rac1 over-expression led to the activation of p21 activating kinases (PAK) 1, mitogen-activated protein kinase kinase (MEK) 1/2 and extracellular signal-regulated kinase (ERK) 1/2, and the elevation of brain-derived neurotrophic factor (BDNF) day 14 after stroke. Finally, we observed higher counts of neuronal Rac1 in the peri-infarct zone of subacute/old ischemic stroke subjects. This work identified a neuronal Rac1 signaling in improving functional recovery and axonal regeneration after stroke, suggesting a potential therapeutic target in the recovery stage of stroke.

Keywords: Rac1; functional recovery; glia scar formation; ischemic stroke; neural plasticity.

Figure 1.

Schematic representation of the study design with number of animals used for each experimental set. NORT, novel object recognition test; ART, adhesive removal test; PRT, pellet reaching test. N = number of animals.

Figure 2.

Delayed deletion of neuronal Rac1 led to poorer functional recovery after ischemic stroke in mice. Tamoxifen (2 mg/40 g/daily) was intraperitoneally injected to Thy1-creER/Rac1-floxed (T-Rac1-floxed) mice from day 7 to day 11 after stroke. Same process was performed on Rac1-floxed mice as control. (A) Immunohistochemistry assessment of cerebral Rac1 expression (red, Alexa Fluor 647) on neurons (NeuN, green, Alexa Fluor 594) 28 days after stroke (N = 4/each group). Arrows indicated the Rac1 positive neurons (yellow). Functional recovery assessed by (B) novel object recognition test, (C) adhesive removal test and (D) pellet reaching test before (Pre) up to 28 days after stroke (N = 7 in the Rac1-floxed group, N = 8 in the T-Rac1-floxed group). N = number of animals. *p < 0.05, ***p < 0.001. Mann-Whitney test was used for immune-staining. Two-way ANOVA with subsequent Bonferonni test was applied for behavioral tests.

Figure 3.

Delayed overexpression of neuronal Rac1 promoted functional recovery after ischemic stroke in mice. Highly concentrated lentivirus encoding Rac1 with neuronal promotor NSE was injected into both the cortex and striatum of WT mice at day 7 after stroke. Same process was performed using lentivirus without Rac1 sequence as controls. (A) Immunohistochemistry assessment of cerebral Rac1 expression (red, Alexa Fluor 647) on neurons (NeuN, green, Alexa Fluor 594) 28 days after stroke (N = 4/each group). Arrows indicated the Rac1 positive neurons (yellow). Functional recovery assessed by (B) novel object recognition test, (C) adhesive removal test and (D) pellet reaching test before (Pre) up to 28 days after stroke (N = 8 in the control vector group, N = 9 in the NSE-Rac1 group). N = number of animals. *p < 0.05, **p < 0.01, ***p < 0.001. Mann-Whitney test was used for immune-staining. Two-way ANOVA with subsequent Bonferonni test was applied for behavioral tests.

Figure 4.

Tamoxifen-induced deletion of neuronal Rac1 reduced axonal regeneration with no effect on tissue loss after ischemic stroke in mice. Density assessment of (A) neurofilament-L (NFL, green, Alexa Fluor 647, N = 6/each group) and (B) myelin basic protein (MBP, red, Alexa Fluor 594, N = 7/each group) in the ipsilateral (ipsi) and contralateral (contra) hemisphere 28 days after unilateral stroke. Blue indicated cell nucleus staining using DAPI. (C) Quantification of brain tissue loss 28 days after stroke was assayed by CV staining (N = 5/each group). N = number of animals. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA with subsequent Bonferonni test was applied for immune-staining. Mann-Whitney test was used for CV staining.

Figure 5.

Delayed overexpression of neuronal Rac1 increased axonal density with no effect on tissue loss 28 days after ischemic stroke in mice. Density assessment of (A) neurofilament-L (NFL, green, Alexa Fluor 647, N = 6/each group) and (B) myelin basic protein (MBP, red, Alexa Fluor 594, N = 6/each group) in the ipsilateral (ipsi) and contralateral (contra) hemisphere 28 days after unilateral stroke. Blue indicated cell nucleus staining using DAPI. (C) Quantification of brain tissue loss 28 days after stroke was assayed by CV staining (N = 7/each group). N = number of animals. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA with subsequent Bonferonni test was applied for immune-staining. Mann-Whitney test was used for CV staining.

Figure 6.

Modulation of pro-regenerative pathways by neuronal Rac1. (A) Delayed overexpression of neuronal Rac1 promoted the expression of phosphorylated (T212) PAK1 (red, Alexa Fluor 594) on neurons (NeuN, Alexa Fluor 647) at peri-infarct area 28 days after MCAO. (B) Delayed overexpression of neuronal Rac1 by cerebral injection of NSE-Rac1 vector (R) promoted phosphorylation of PAK1 (T212), MEK1/2 (S217/221), ERK1/2 (T202/Y204) and promoted BDNF production in the ipsilateral hemisphere 14 days after MCAO compared with control vector (C) group assayed by western blotting. Quantitative analysis of relative intensity of interest proteins by normalizing individuals to β-actin. N (number of animals) = 4/each group. The intensity of all bands was calculated for ERK1/2 and BDNF in individual sample. *p < 0.05, **p < 0.01, ***p < 0.001. Mann-Whitney test was used for immune-staining. Two-way ANOVA with subsequent Bonferonni test was applied for western blotting.

Figure 7.

Delayed deletion of neuronal Rac1 enhanced the glial fibrillary acidic protein (GFAP) intensity, whereas delayed overexpression of neuronal Rac1 reduced its intensity 28 days after stroke in mice. Assessment of GFAP density (red, Alexa Fluor 594) in the peri-infarct zone (A) after neuronal Rac1 gene deletion (control mice: Rac1-floxed, N = 4; Rac1 deletion mice: T-Rac1-floxed, N = 5) and (B) after neuronal Rac1 overexpression (control vector: N = 4; Rac1 overexpression: NSE-Rac1, N = 4). N = number of animals. *p < 0.05. Mann-Whitney test was used for immune-staining. Pearson correlation was used for linear correlation analyses.

Figure 8.

Protein level of Rac1 increased in the peri-infarct area of postmortem tissue of older ischemic stroke subjects. (Upper graphs in figure A and B) Representative immunohistochemical microphotographs showing Rac1 (brown) and NeuN positive cells (magenta) from age-matched control and subacute/old ischemic stroke subjects. Open arrows indicated representative Rac1⁺ cells. Arrow without bar indicated representative NeuN⁺ cells. Arrow with bar indicated representative Rac1⁺/NeuN⁺ cells. Hematoxylin (blue) was used for nucleus staining. (Lower charts in figure A and B) Quantification of Rac1⁺ and Rac1⁺/NeuN⁺ cells per mm². N (number of humans) = 10/each group. **p < 0.01, ***p < 0.001. Unpaired t-test comparisons was used for comparison between two individual groups.