Elsevier

Experimental Neurology

Volume 256, June 2014, Pages 25-38
Experimental Neurology

Regular Article
CD36 deletion improves recovery from spinal cord injury

https://doi.org/10.1016/j.expneurol.2014.03.016Get rights and content

Highlights

  • Deletion of CD36 improves locomotor recovery from contusive spinal cord injury.

  • Deletion of CD36 reduces macrophage infiltration into the contusion epicenter.

  • Deletion of CD36 improves injury vascularity and function.

  • Inhibition of CD36 reduces vascular ER stress responses in vivo.

  • Inhibition of CD36 reduces ER stress response signaling in CNS endothelial cells.

Abstract

CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36−/− mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36+/+ mice. CD36−/− mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36−/− mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.

Introduction

Contusion of the spinal cord leads to secondary complications including loss of spinal microvasculature, induction of inflammatory responses and oxidative damage. These responses exacerbate cell death, axonal degeneration, and demyelination within the contusion and injury penumbra (Alexander and Popovich, 2009, Mautes et al., 2000). Early vessel dysfunction and subsequent edema contribute to the infiltration of inflammatory cells (Mautes et al., 2000, Popovich and Longbrake, 2008). Blocking neutrophil and macrophage influx into the contusion leads to neuroprotection and improved locomotor function (Gris et al., 2004, Popovich and Jones, 2003). An initial loss of epicenter vasculature is followed by an angiogenic response 3 to 7 days following spinal cord injury (SCI), but these new vessels are immature and leaky (Benton et al., 2008, Casella et al., 2002, Loy et al., 2002, Whetstone et al., 2003). This later phase of microvascular instability has been hypothesized to contribute to chronic histopathology following SCI (Casella et al., 2002, Loy et al., 2002). Stabilization of epicenter and penumbral vasculature facilitates tissue sparing and functional recovery following SCI (Han et al., 2010).

Following SCI, cells with high protein secretory capacity initiate the unfolded protein response (UPR) to minimize toxic aggregation of misfolded proteins in the endoplasmic reticulum (ER) (Aufenberg et al., 2005, Ohri et al., 2011, Sakurai et al., 2005). Endothelial cells also initiate the UPR after ER sensors detect a high unfolded/misfolded protein to ER chaperone ratio (Marciniak et al., 2006, Yoshida, 2007). ER stress within endothelial cells contributes to many diseases, including hypertension, atherosclerosis, diabetes and bleeding disorders (Witte and Horke, 2011). Activation of the UPR leads to phosphorylation of the translation initiation protein eIF2α, which inhibits global translation in order to reduce ER-localized, KDEL-tagged proteins and minimize exacerbation of ER stress. eIF2α-independent genes such as activating transcription factor 4 (ATF4), which regulates the expression of other transcription factors, are upregulated to overcome this stress (Witte and Horke, 2011). Endothelial cells that cannot restore homeostasis induce expression of the pro-apoptotic proteins C/EBP (CCAAT enhancer binding protein) homologous protein (CHOP) and cation transport regulator-like protein 1 (CHAC-1) downstream of ATF4 (Mungrue et al., 2009). CHOP−/− mice exhibit a reduced inflammatory response, improved vascular sparing and enhanced locomotor recovery following SCI (Fassbender et al., 2012, Ohri et al., 2011). This improved vascular sparing coincides with a peak of lipid peroxidation, oxidative damage and accumulated protein adducts of 4HNE (Carrico et al., 2009), the most abundant unsaturated aldehyde generated in oxidized lipids by reactive oxygen species (Benedetti et al., 1980). Oxidized phospholipids, including 4HNE, induce ER stress in endothelial cells (Mungrue et al., 2009, Vladykovskaya et al., 2012), and these ligands trigger ERSR-induced apoptosis in macrophages through a CD36-dependent mechanism (Seimon et al., 2010).

CD36, an 88 kDa glycoprotein, is a multifunctional receptor that contributes to several pathophysiological conditions, including cerebral ischemia (Cho et al., 2005, Kunz et al., 2008), neurovascular dysfunction (Park et al., 2011) and atherosclerosis (Febbraio et al., 2000, Harb et al., 2009). High affinity CD36 agonists include lipid peroxidation products such as 4HNE (Silverstein et al., 2010) and thrombospondin-1 (TSP-1). CD36 is expressed in a variety of tissues, including macrophages and endothelial cells, and regulates inflammation (Oh et al., 2012, Seimon et al., 2010) and angiogenesis (Jimenez et al., 2000). Due to its pharmacologic accessibility and multiple pathogenic downstream pathways, CD36 has been proposed to be an ideal molecular target for developing clinically relevant therapeutic strategies for neurodegenerative diseases (Cho and Kim, 2009).

The goal of this study was to assess the role of CD36 signaling on the secondary pathophysiological effects following SCI. We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following injury. These effects were assessed in a contusive SCI model in CD36−/− mice.

Section snippets

Reagents

FITC-LEA (Lycopersicon esculentum agglutinin lectin; FL-1171) was purchased from Vector Labs (Burlingame, CA). TRITC (1:200)-, FITC (1:200)-, CY5 (1:200) or AMCA (1:100)-conjugated donkey secondary antibody F(ab′)2 fragments and normal donkey serum (017-000-121) were purchased from Jackson Immunoresearch (West Grove, PA). TRITC (1:200)-conjugated goat anti-mouse IgA (MBS674424) was purchased from MyBioSource.com. Mouse collagen IV (#354233) was purchased from BD Biosciences (San Jose, CA).

Deletion of CD36 improves functional recovery following SCI

Assessment of locomotor function after moderate contusion of the T10 spinal cord revealed that CD36−/− mice exhibited a statistically significant improvement in recovery relative to WT, beginning three weeks following injury (Fig. 1A). This improvement was sustained up to six weeks after injury (the final time point examined). The increase in motor function above a score of 5 is significant in this non-linear scale as it reflects the restoration of the capacity for plantar stepping and weight

Discussion

While uninjured CD36−/− mice appear phenotypically normal overall (Febbraio et al., 1999), these mice exhibit reduced dysfunction and disease progression to a variety of injuries including ER stress, stroke, Aβ toxicity, and oxLDL-atherosclerosis (Cho et al., 2005, Oh et al., 2012, Park et al., 2009, Rahaman et al., 2006). CD36 can reduce injury progression after stroke in neonate mice via its role in monocyte/macrophage infiltration and phagocytosis of apoptotic bodies (Woo et al., 2012).

Acknowledgments

This work was supported by NS045734 (NIH R01 NS045734), RR1556/GM103507 (NIHP30 RR1556/GM103507), the Kentucky Spinal Cord and Head Injury Research Trust, Norton Healthcare, and the Commonwealth of Kentucky Research Challenge for Excellence Trust Fund (S.R.W., T.H.). We thank Christine Yarberry for surgical assistance, Jason Beare for confocal analysis, Kim Cash for animal care, Johnny Morehouse for BMS and Treadscan analyses, and Darlene Burke for statistical analyses.

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    Current address: Department of Biomedical Sciences, East Tennessee State University, Johnson City, TN 37614, USA.

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