Genetic inactivation of RIP1 kinase activity in rats protects against ischemic brain injury

Abstract

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.

Fig. 1. RIP1 KD rats and cells are resistant to necroptotic cell death and TNF driven hypothermia.

A Schematic depiction of D138N kinase-inactivating mutation in rat RIP1. B Lymphocyte subsets from spleen (Spl) and lymph nodes (LN) of RIP1 KD and WT rats. Data from individual rats of each genotype (n = 5) are plotted, with the mean value for each group indicated by a horizontal line. There were no significant differences between two genotypes as evaluated by unpaired t-test (p > 0.1). C, D BMDMs derived from WT and RIP1 KD rats were treated with TNF, BV6, and zVAD (TBZ) (C) or LPS and zVAD (LZ) (D) with or without Nec1 overnight. Cell viability was assessed by Cell Titer-Glo assay. Numbers in parentheses indicate that cells were derived from different animals. E BMDMs derived from WT and RIP1 KD rats were treated with TBZ for indicated periods of time. Cellular lysates were immunoblotted with the indicated antibodies. Numbers in parentheses indicate that cells were derived from different animals. F, G WT and KD RIPK1 rats were treated with TNF (300 µg/kg) and zVAD 10 mg/kg or PBS (unstimulated) for 4 h. Survival (F) and body temperatures (G) were evaluated 2 and 4 h after treatment. *p < 0.05 and **p < 0.01 between WT and RIP1 KD by Mantel–Cox test. # in (G) indicates that no animals from this group survived to this time point. Data are represented as mean ± S.E.M.

Fig. 2. Genetic inactivation of RIP1 kinase activity ameliorates neurological function following ischemic brain injury.

A Experimental timeline and endpoints in the tMCAO model. B Body weights of WT and RIP1 KD rats before and after sham or tMCAO surgery. C–F Behavioral scoring of WT and RIP1 KD rats before and after sham or tMCAO surgery. 20-point neuroscore (C), limb placing (D), and beam walking (forepaw (E) and hindpaw (F)) tests were performed. n = 8–10 rats/group. At the indicated time points, **p < 0.01 between WT-tMCAO and WT-Sham (in gray), and *p < 0.05 and **p < 0.01 between RIP1 KD-tMCAO and WT-tMCAO (in black) by Three-Way ANOVA (Holm-Sidak). Data are represented as mean ± S.E.M.

Fig. 3. Genetic inactivation of RIP1 kinase activity preserves tissue integrity following ischemic brain injury.

A T2-MRI imaging in WT and RIP1 KD rats following tMCAO surgery. Representative images at days 2-, 15-, and 30 post-tMCAO are shown. In groups where there are two clusters with respect to lesion sizes (as in RIP1 KD-tMCAO 15 and 30 days post-tMCAO, B), representative images from each cluster are separately shown. B Quantification of lesion size based on blinded volumetric analysis of T2-relaxation time, from (A) (longer time correlates with severity of the lesion). The closed symbols correspond to the animals with T2-MRI images shown in (A). n = 8–10 rats/group. *p < 0.05 and **p < 0.01 by Two-way ANOVA (Holm-Sidak). C Quantification of tissue percent area with edema, from (A). n = 8–10 rats/group. At the indicated time points, **p < 0.01 between WT-tMCAO and WT-Sham (in gray), and between RIP1 KD-tMCAO and WT-tMCAO (in black) by Three-way ANOVA (Holm-Sidak). D Correlation between 20-point neuroscore and lesion volume at 2–3 days (open circles) and 14–15 days (closed circles) post-tMCAO, from (B) and Fig. 2C. **p < 0.01 by Spearman test. E Immunohistochemistry by a myelin stain in WT and RIP1 KD rats at 30-days post-tMCAO. Representative images across the brain are shown. Darker color highlights the intact tissue. Scale bar, 5 mm. F Quantification of the ratio of ipsilateral to contralateral myelin stain intensity, from (E). n = 8–10 rats/group. **p < 0.01 by Two-way ANOVA (Holm-Sidak). Data are represented as mean ± S.E.M.

Fig. 4. Genetic inactivation of RIP1 kinase activity reduces inflammation following ischemic brain injury.

A Immunohistochemistry for Iba1, CD68, and GFAP in WT and RIP1 KD rats at 30-days post-tMCAO. Representative images across the brain are shown. Scale bar, 5 mm. B–D Quantification of percent tissue area positive for high-intensity Iba1 (B), CD68 (C) and GFAP (D) in the ipsilateral side, from (A). n = 8–10 rats/group. **p < 0.01 by Two-way ANOVA (Holm-Sidak). Data are represented as mean ± S.E.M.

Fig. 5. Genetic inactivation of RIP1 kinase activity reduces plasma NfL and additional markers of injury following ischemic brain injury.

A Plasma NfL levels in WT and RIP1 KD rats before and 2-, 15-, and 30-days after sham or tMCAO surgery. n = 8–10 rats/group. At the indicated time points, **p < 0.01 between WT-tMCAO and WT-Sham (in gray), and *p < 0.05 and p = 0.07 between RIP1 KD-tMCAO and WT-tMCAO (in black) by Two-way ANOVA (Holm-Sidak). Data are represented as mean ± S.E.M. B Correlation between plasma NfL levels at 2 days versus 20-point neuroscore at 3 (open circles) and 28 days (closed circles) post-tMCAO, from (A) and Fig. 2C. *p < 0.05 by Spearman test. C Plasma protein profiling by mass spectrometry in WT and RIP1 KD rats before and 2-, 15-, and 30-days after sham or tMCAO surgery. n = 10 rats/group. Each graph represents log2 fold-change in plasma protein abundance at the indicated time points, post-surgery compared to pre-surgery, in WT (x-axes) and RIP1 KD (y-axes) rats. Proteins that show significant difference in abundance upon sham surgery in WT animals (>1.2 fold-change, adjusted p-value < 0.1, open symbols in Supplementary Fig. 3B) are excluded from the graphs. Symbol colors represent changes in plasma protein abundance according to the genotypes such that blue symbols represent significant change in both WT and RIP1 KD plasma, red symbols represent significant change only in WT plasma, and gray symbols represent no significant change in WT plasma. D Graphs representing the log2 fold-change in plasma protein abundance of the indicated proteins compared to baseline levels in WT and RIP1 KD rats. Symbol colors represent WT or RIP1 KD rats. Symbol types, closed or open, represent statistical significance as indicated. Error bars represent standard error of the log2 fold change.