Receptor-interacting Protein Kinases Mediate Necroptosis In Neural Tissue Damage After Spinal Cord Injury

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1 Receptor-interacting Protein Kinases Mediate Necroptosis In Neural Tissue Damage After Spinal Cord Injury Haruo Kanno, M.D., Ph.D., Hiroshi Ozawa, M.D., Ph.D., Satoshi Tateda, M.D., Kenichiro Yahata, M.D., Eiji Itoi, MD, PhD. Tohoku University School of Medicine, Sendai, Japan. Disclosures: H. Kanno: None. H. Ozawa: None. S. Tateda: None. K. Yahata: None. E. Itoi: None. Introduction: Necrosis was originally thought to be a purely passive and uncontrolled type of cell death. Necrosis, which is marked by cell swelling and membrane rupture, leads to inflammation via the release of intracellular signals [1,2]. In contrast, apoptosis, which is characterized by the activation of caspases and DNA fragmentation, was once considered the sole form of programmed cell death. Chromatin condensation, nuclear shrinkage and fragmentation and membrane blebbing are the result of the proteolytic activity of caspases and define the morphological characteristics of apoptosis [1-3]. Necroptosis is a newly identified type of programmed cell death regulated by caspase-independent pathway and has the morphological features of necrosis [4]. Recent studies have shown that the receptor-interacting protein (RIP) kinase family is specifically involved in regulating necroptosis [2]. Various stimuli to cells can induce the formation of necrotic complexes that contain RIP1, TRADD, FADD and caspase-8. The interaction of RIP3 with RIP1 in the necrotic complex is an important step required for the execution of necroptosis [2]. RIP3 and RIP1 are known as a key mediator of necroptosis [5]. The amounts of RIP3 and RIP1 expressions correlate with necroptosis induction in various types of cells. Recent studies demonstrated that the increased expression of RIP3 and RIP1 in lesions and the induction of necroptosis in various disease models [6]. However, to our knowledge, there has been no study to investigate the expression of RIP proteins and the involvement of necroptosis in damaged neural tissue after spinal cord injury (SCI). In the present study, we used a spinal cord hemisection model in mice to study the alterations of RIP3 and RIP1 expressions and the involvement of necroptosis in SCI. Methods: Animals Adult female C57BL/6J mice between 8 and 10 weeks of age were used in this study. Surgical procedures The T10 vertebra was laminectomized to expose the spinal cord. With a scalpel, the cord was transected on the left side only [7]. Immunohistochemistry At different time points (4, 24 hours, 3, 7 and 21 days) after hemisection and at 24 hours after sham operation, the spinal cords at the lesion were fixed with 4% paraformaldehyde, embedded in OCT-compound and sectioned. Tissue sections were incubated with rabbit anti-rip3 antibody (1:100; Sigma-Aldrich) or mouse anti-rip1 antibody (1:100; BD Transduction Laboratories) and visualized by Alexa Fluor 594 goat anti-rabbit IgG antibody or Alexa Fluor 594 goat anti-mouse IgG antibody (1:500; Molecular Probes). Counting of RIP3- and RIP1-positive cells The numbers of RIP3- and RIP1-positive cells were counted in the injured and the contralateral sides of transverse sections at the different time points (4, 24 hours, 3, 7 and 21 days) after hemisection, and compared with those of the sham control. Western blot analysis The protein expressions of RIP3 in the injured spinal cord and the uninjured spinal cord after sham operation were determined using Western blot analysis.

2 Double staining of RIP3 and RIP1 with various cell type markers To examine the expressions of RIP3 and RIP1 in a specific population of cells, the transverse sections at 3 days after hemisection were costained for RIP3 and RIP1 with various cell type markers: NeuN for neurons, GFAP for astrocytes and Olig2 for oligodendrocytes. RIP3 staining in propidium iodide-labeled sections To investigate the RIP3 expression in dying cells, RIP3 staining was performed using propidium iodide (PI)-labeled spinal cord sections. Double staining for RIP3 and TUNEL To detect DNA fragmentation in the RIP3-positive cells, the sections were co-stained with RIP3 and TUNEL. Results: Immunohistochemical stainings of RIP3 and RIP1 Representative pictures showed the RIP3- and RIP1-expressing cells on the injured side obviously increased compared to on the contralateral side at 3 days after hemisection (Fig. 1A-D). Counting of RIP3- and RIP1-positive cells The number of RIP3-positive cells on the injured side was significantly higher than those on the contralateral side and sham control at 24 hours, 3, 7 and 21days (p < 0.05) (Fig. 1E). The number of RIP1-positive cells on the injured side was significantly higher than those on the contralateral side and sham control at 4, 24 hours, 3, 7 and 21 days (p < 0.05) (Fig. 1F). The maximum number of these cells in the injured side was observed at 3 days, and it decreased after 7 days. Western blot analysis The protein expression of RIP3 in the injured spinal cord at 3 days was significantly increased compared to the uninjured spinal cord (p < 0.05) (Fig. 1G, H). Double staining of RIP3 and RIP1 with various cell type markers Double staining with various cell type markers demonstrated the increased expression of RIP3 in neurons, astrocytes and oligodendrocytes on the injured side at 3 days after hemisection (Fig. 2A, C, E). The increased expression of RIP1 was also observed in neurons, astrocytes and oligodendrocytes on the injured side (Fig. 2B, D, F). RIP3 staining in propidium iodide-labeled sections The representative pictures show that the PI-labeled cells were frequently observed to be RIP3-positive (Fig. 3A-D). At higher magnification (Fig. 3C, D), most of the nuclei in the PI-labeled cells expressing RIP3 were round, which is normally observed in cells undergoing necrotic cell death, not fragmented or shrunken, as observed in apoptotic cells. Double staining for RIP3 and TUNEL The numbers of RIP3-expressing and TUNEL-positive cells were obviously increased in the injured spinal cord (Fig. 3E-H). At higher magnification (Fig. 3G, H), the TUNELpositive cells exhibiting shrunken or fragmented nuclei, a hallmark of apoptotic cell death, rarely expressed RIP3. Discussion: The present study demonstrated that the expressions of RIP3 and RIP1 were significantly increased in various neural cells at the lesion site after SCI. In addition, the dying cells expressing RIP3 in the injured spinal cord displayed a similar morphology to that associated with necrotic cell death but not apoptosis. These results first provided the evidence to support the involvement of necroptosis in neural tissue damage after SCI. To date, it has been considered that the secondary neural tissue damage following SCI is caused by apoptosis but not necrosis. Most previous studies related to the cell death in the injured spinal cord focused on apoptosis. In our study, the upregulation of RIP3 and RIP1 expressions was observed starting from 4-24 hours, peaked at 3 days. Importantly, the time course of RIP3 and RIP1 expressions was quite similar to that of the secondary damage following SCI [8]. These results suggest necroptosis contributes to the secondary damage in the injured spinal cord.

3 Significance: The expressions of RIP3 and RIP1 were dramatically increased at the lesion site and may contribute to induction of necroptosis as novel cell death mechanism causing secondary neural tissue damage after SCI. Therefore, necroptosis can be a novel therapeutic target to reduce neural tissue damage following SCI.

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6 ORS 2015 Annual Meeting Poster No: 0689