Elsevier

Brain, Behavior, and Immunity

Volume 76, February 2019, Pages 258-267
Brain, Behavior, and Immunity

Lysophosphatidic acid receptor type 2 activation contributes to secondary damage after spinal cord injury in mice

https://doi.org/10.1016/j.bbi.2018.12.007Get rights and content

Highlights

  • LPA2 is upregulated in the spinal cord parenchyma after traumatic injury.

  • LPA2 signaling leads to demyelination.

  • Microglia express LPA2 in the adult CNS, but also in cell culture conditions.

  • LPA2 signaling in microglia triggers oligodendrocyte cell death.

  • Microglia LPA2 cytotoxicity is mediated by activation of P2X7 on oligodendrocytes.

Abstract

Lysophosphatidic acid (LPA) is an extracellular lipid mediator involved in many physiological functions by signaling through six known G-protein-coupled receptors (LPA1-LPA6). In the central nervous system (CNS), LPA mediates a wide range of effects, including neural progenitor cell physiology, astrocyte and microglia activation, neuronal cell death, axonal retraction, and contributions to pain, schizophrenia and hydrocephalus. We recently reported that LPA-LPA1 signaling mediates functional deficits and myelin loss after spinal cord injury (SCI). Here, we provide clear evidence on the deleterious contribution of another LPA receptor, LPA2, to myelin loss after SCI. We found that LPA2 is constitutively expressed in the spinal cord parenchyma and its transcripts were up-regulated after contusion injury, in part, by microglial cells. We also found that the demyelinating lesion triggered by intraspinal injection of LPA into the undamaged spinal cord was markedly reduced in the lack of LPA2. Similarly, LPA2 deficient mice showed enhanced motor skills and myelin sparing after SCI. To gain insights into the detrimental actions of LPA2 in spinal cord we performed cell culture studies. These experiments revealed that, similar to LPA1, activation of microglia LPA2 led to oligodendrocyte cell death. Moreover, we also found that the cytotoxic effects underlaying microglial LPA-LPA2 axis were mediated by the release of purines by microglia and the activation of P2X7 receptor on oligodendrocytes. Overall, this study provides new mechanistic insights into how LPA contributes to SCI physiopathology, and suggest that targeting LPA2 could be a novel therapeutic approach for the treatment of acute SCI.

Introduction

Traumatic spinal cord injury (SCI) is a major cause of disability that results in a functional decline due to disruption of axonal pathways and death of neurons and oligodendrocytes (David et al., 2012, Hilton et al., 2017). There are two waves of tissue degeneration after SCI which are known as a primary and secondary injury. The first is caused by the mechanical damage to the spinal cord parenchyma followed by the activation of several cellular and molecular events that take place in the spinal cord tissue from hours to weeks after trauma (David et al., 2012, Hilton et al., 2017). Primary injury cannot be avoided, however, the mechanisms participating in secondary injury can be substantially abrogated or even blocked, providing some options to minimize secondary degeneration and functional loss.

LPA is an extracellular lipid mediator with a wide range of biological functions. LPA receptors are expressed in almost all cells of the nervous system (Choi and Chun, 2013, Choi et al., 2010, Yung et al., 2015). The different responses of LPA in neural cells include neuronal cell death (Holtsberg et al., 1998), axonal retraction (Tigyi et al., 1996), inhibition of oligodendrocyte maturation (Dawson et al., 2003), and proliferation of astrocytes and mouse microglial cells (Shano et al., 2008, Sorensen et al., 2003). Moreover, LPA has been related with some nervous system pathologies such as fetal hydrocephalus (Yung et al., 2011, Yung et al., 2014), psychiatric diseases (Harrison et al., 2003, Roberts et al., 2005), neuropathic pain (Halder et al., 2013, Inoue et al., 2004, Ma et al., 2009), and tissue damage after trauma to the spinal cord (Goldshmit et al., 2012, Santos-Nogueira et al., 2015) and brain (Crack et al., 2014). We recently reported that LPA triggers demyelination after SCI by signaling, in part, via microglia LPA1 (Santos-Nogueira et al., 2015). However, considering the pleiotropic effects of LPA and the variety of receptors with which it interacts, it is possible that LPA could exert beneficial or detrimental actions in SCI depending on the receptor it signals through, as previously observed with other lipid mediators, such as prostaglandins (Kawano et al., 2006, Kerr et al., 2008, Liang et al., 2011, Redensek et al., 2011).

In the present work we provide evidence for the first time that the LPA-LPA2 axis contributes to the physiopathology of SCI. In particular, we found that mice lacking LPA2 are protected against demyelination triggered by intraspinal injection of LPA and by SCI. Our in vitro work also reveals that LPA2 stimulation in oligodendrocytes does not cause cell death. However, activation of microglia LPA2 leads to release of purines that mediate cytotoxic effects on oligodendrocyte via P2X7.

Section snippets

Animal genotyping

PCR analysis was used to detect the presence of WT and lpar2 mutated allele. For tissue sampling, the tip of the tail was cut at the time of weaning the mice. Genomic DNA extraction was carried out by using the ArchivePure DNA Purification System (5PRIME), as described by the manufacturer. Allele amplification was performed by PCR reaction, using the Taq DNA Polymerase kit (Invitrogen), and PCR products were analyzed by standard electrophoresis in 1% agarose gels. Primers sequences were the

Expression of LPA2 in the injured and uninjured mouse spinal cord

We first assessed the changes in mRNA levels of LPA2 in the spinal cord parenchyma after contusion injury by using real time PCR. We found that LPA2 was constitutively expressed in the spinal cord and its levels were notably up-regulated for the first 3 days following injury, reaching the peak expression at day 3 (one-way ANOVA p = 0.022 at day 1 and p < 0.001 at day 3 vs naive; n = 3 per group) (Fig. 1A).

We previously reported that increasing the local levels of LPA in the intact spinal cord

Discussion

In the present work, we studied whether LPA2 signaling contributes to the physiopathology of SCI. We show that mRNA levels of LPA2 are increased in the spinal cord after contusion injury and that LPA2 activation leads to demyelination and functional deficits after SCI. We demonstrate, by doing cell culture work, that LPA2 signaling does not mediate direct harmful effects on oligodendrocytes, but evokes microglial cells to release purines, which in turn, mediates cytotoxic cell death of

Acknowledgments

This work has been supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2010-17851; SAF2013-48431-R), Marie-Curie International Reintegration Program Grant (MC IRG 249274), Wings for Life Foundation International Foundation for Research in Paraplegia and by funds from the Fondo de Investigación Sanitaria of Spain (TERCEL and CIBERNED) to R.L-V. and NIH NS084398 to JC.

References (50)

  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method

    Methods (San Diego, Calif)

    (2001)
  • C. Lopane et al.

    Implications of the lysophosphatidic acid signaling axis in liver cancer

    Biochim. Biophys. Acta

    (2017)
  • M. Mekhail et al.

    Oligodendrocyte-protection and remyelination post-spinal cord injuries: a review

    Prog. Neurobiol.

    (2012)
  • J. Park et al.

    LPA-induced migration of ovarian cancer cells requires activation of ERM proteins via LPA1 and LPA2

    Cell. Signalling

    (2018)
  • S. Shano et al.

    Lysophosphatidic acid stimulates astrocyte proliferation through LPA1

    Neurochem. Int.

    (2008)
  • Y.C. Yung et al.

    Thematic review series: lysophospholipids and their receptors LPA receptor signaling: pharmacology, physiology, and pathophysiology

    J. Lipid Res.

    (2014)
  • Y.C. Yung et al.

    Lysophosphatidic Acid signaling in the nervous system

    Neuron

    (2015)
  • Y. Zhang et al.

    Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse

    Neuron

    (2016)
  • J. Amo-Aparicio et al.

    Neuroinflammation quantification for spinal cord injury

    Curr. Protoc. Immunol.

    (2018)
  • D.M. Basso et al.

    Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains

    J. Neurotrauma

    (2006)
  • J.W. Choi et al.

    LPA receptors: subtypes and biological actions

    Annu. Rev. Pharmacol. Toxicol.

    (2010)
  • M. Coll-Miro et al.

    Beneficial effects of IL-37 after spinal cord injury in mice

    Proc. Natl. Acad. Sci. U.S.A.

    (2016)
  • P.J. Crack et al.

    Anti-lysophosphatidic acid antibodies improve traumatic brain injury outcomes

    J. Neuroinflammation

    (2014)
  • J. Dawson et al.

    Lysophosphatidic acid induces process retraction in CG-4 line oligodendrocytes and oligodendrocyte precursor cells but not in differentiated oligodendrocytes

    J. Neurochem.

    (2003)
  • R.J. Dumont et al.

    Acute spinal cord injury, part I: pathophysiologic mechanisms

    Clin. Neuropharmacol.

    (2001)
  • Cited by (27)

    • Bioactive Lipid Mediators in the Initiation and Resolution of Inflammation after Spinal Cord Injury

      2021, Neuroscience
      Citation Excerpt :

      LPA2 in the CNS is expressed mainly in oligodendrocytes, neurons, and microglia. The suppression of LPA-LPA2 signaling reduces demyelination after intraspinal injection of LPA and after SCI, indicating a key deleterious role of LPA2 in SCI pathology (Lopez-Serrano et al., 2019). Like LPA1, the demyelinating actions of LPA2 after neurotrauma are likely to be mediated by microglial cells.

    • TMEM16F inhibition limits pain-associated behavior and improves motor function by promoting microglia M2 polarization in mice

      2019, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Presently, no pharmacological therapy could effectively treat this disease [2]. The pathogenesis of SCI begins with mechanically inflicted trauma, which incites primary and secondary injury phases [3]. Accumulating studies have indicated that the secondary injury phase in SCI functions as a crucial therapeutic window, during which neuroprotective treatment could be implemented in efforts to improve functional recovery after SCI [4].

    View all citing articles on Scopus
    1

    Both authors contributed equally to this work.

    View full text