Elsevier

Neuroscience

Volume 408, 1 June 2019, Pages 430-447
Neuroscience

Research Article
Inhibition of neogenin promotes neuronal survival and improved behavior recovery after spinal cord injury

https://doi.org/10.1016/j.neuroscience.2019.03.055Get rights and content

Highlights

  • Lamprey neurons express RGMa receptor Neogenin.

  • Neogenin is preferentially expressed in the low regeneration capacity neurons.

  • Downregulation of Neogenin prevents the neuronal apoptosis after SCI.

  • Neogenin inhibition improved lamprey behavior recovery after SCI.

Abstract

Following spinal cord trauma, axonal regeneration in the mammalian spinal cord does not occur and functional recovery may be further impeded by retrograde neuronal death. By contrast, lampreys recover after spinal cord injury (SCI) and axons re-connected to their targets in spinal cord. However, the identified reticulospinal (RS) neurons located in the lamprey brain differ in their regenerative capacities – some are good regenerators, and others are bad regenerators – despite the fact that they have analogous projection pathways. Previously, we reported that axonal guidance receptor Neogenin involved in regulation of axonal regeneration after SCI and downregulation of Neogenin synthesis by morpholino oligonucleotides (MO) enhanced the regeneration of RS neurons. Incidentally, the bad regenerating RS neurons often undergo a late retrograde apoptosis after SCI. Here we report that, after SCI, expression of RGMa mRNA was upregulated around the transection site, while its receptor Neogenin continued to be synthesized almost inclusively in the “bad-regenerating” RS neurons. Inhibition of Neogenin by MO prohibited activation of caspases and improved the survival of RS neurons at 10 weeks after SCI. These data provide new evidence in vivo that Neogenin is involved in retrograde neuronal death and failure of axonal regeneration after SCI.

Introduction

Spinal cord injury (SCI) in high animals (including mammals) leads to lasting loss of function since axons do not regenerate. In addition, several waves of cell death happen in the wounded spinal cord, limiting the potential for functional preservation, axon regeneration, and restorative plasticity after partial SCI. Immediate cell death due to mechanical injury is mostly necrotic. Secondary damage continues for weeks afterwards and is mostly apoptotic, affecting neurons, oligodendrocytes, microglia, and possibly astrocytes (Beattie et al., 2000, Koda et al., 2002). Neuronal death after SCI is not limited to the spinal cord, but appears in several brain structures that innervate the spinal cord in mammals (Feringa et al., 1984, Giehl and Tetzlaff, 1996, Hains et al., 2003, Lee et al., 2004) and lampreys (Shifman et al., 2008). Although the death appears to be apoptotic, little is known about its detailed mechanisms. The inductors of apoptosis after SCI are incompletely understood, but include overabundance of free radicals (e.g., NO- and reactive oxygen species (ROS)) (Hall, 2011) or absence of target-derived trophic factors. However, treatments based on neutralizing these mechanisms have had poor success in augmenting axonal regeneration, neuronal survival and functional recovery post-SCI. Thus additional mediators of delayed cell death and regenerative failure are likely to be important.

Inhibition of axon growth in the uninjured, mature CNS is adaptive because it prevents important established connections from being displaced haphazardly, but it complicates recovery from injury. During development, guidance molecules support axon growth and direct axons to their proper CNS targets. Postnatally, expression of attractive cues is downregulated, whereas repulsive cues persist, or are upregulated (Keeling et al., 1997, Manitt et al., 2004). Accumulating evidence shows that several axonal guidance molecules, including Repulsive Guidance Molecule (RGMa), continue their expression well in adulthood and are upregulated in response to CNS injury (Schwab et al., 2005a, Schwab et al., 2005b, Doya et al., 2006, Shifman et al., 2009, Chen et al., 2017). RGMa is a glycosylphosphatidylinositol-anchored (GPI-anchored) membrane-bound protein that was initially recognized as an axon growth cone repellent in the chick retinotectal system (Stahl et al., 1990, Jacobs et al., 1996, Monnier, 2002). Mouse and human genomes have three homologs of chick RGMA: RGMa, RGMb, and RGMc (Oldekamp et al., 2004, Schmidtmer and Engelkamp, 2004). RGMa is expressed primarily in the developing and adult CNS, and has the highest homology with the chick RGMA. RGMa exerts its chemorepulsive action on axons through the type-I transmembrane receptor Neogenin (Matsunaga and Chedotal, 2004, Rajagopalan et al., 2004, De Vries and Cooper, 2008), which is related to the netrin 1 receptor DCC (Deleted in Colorectal Cancer).

In the adult CNS, the expressed axon guidance molecules are predominately chemorepulsive, and Neogenin is the most abundant guidance receptor in adult neocortex and spinal cord (Manitt et al., 2004, Mueller et al., 2006, van den Heuvel et al., 2013).

RGMa and its receptor Neogenin have been involved in an assortment of CNS developmental processes, including neurogenesis, neuronal differentiation, apoptosis, neuron migration and axon guidance. Their expression is also evolutionarily conserved in the adult CNS of diverse vertebrate species (Keeling et al., 1997, Schwab et al., 2005a, Schwab et al., 2005b), including lamprey (Shifman et al., 2009). Neogenin and RGMa also appear to regulate cell death in neuronal and non-neuronal cells (Matsunaga et al., 2004, Hata et al., 2006, Bonnin et al., 2007, Liu et al., 2009) and after injury (Schwab et al., 2005a, Kyoto et al., 2007, Koeberle et al., 2010, Schnichels et al., 2011, Shabanzadeh et al., 2015).

The relevance of RGMa and Neogenin expression to retrograde cell death after SCI is not well understood. It is also very difficult to study in mammalian models of SCI because of the relative difficulty in ascertaining the quantity and specificity of neuronal death in brain regions projecting to the spinal cord. Therefore, in our research we use the unique benefits of the lamprey CNS (see below) to study the role of RGMa/Neogenin in retrograde neuronal death.

Our earlier work established that the “bad regenerating” RS neurons are likely to undertake a late retrograde neuronal death, in which TUNEL labeling was detected as early as 4 weeks post spinal cord transection, caspase activation is detectable even earlier (Hu et al., 2013), but disappearance of neurons takes 12–16 weeks (Shifman et al., 2008). Recently, we showed that in normal animals and after SCI, Neogenin was expressed preferentially in the bad-regenerating, bad-surviving RS neurons and downregulation of Neogenin expression by morpholino oligonucleotides (MO) enhanced the regeneration of RS neurons(Chen et al., 2017). The mechanism that ties the regulation of regeneration and apoptosis together is not known. This correlation cannot be a trivial consequence of dead neurons not being able to regenerate, since we observe regeneration in lampreys long before any neurons die. We hypothesize that RGMa/Neogenin signaling contributes to the retrograde neuronal death and regeneration failure of the “bad regenerating” RS neurons. To test this hypothesis, we used antisense MO to downregulate expression of Neogenin in the lamprey RS neurons, and determined the effects of this downregulation on their survival.

Most studies have employed mammalian nervous systems to study the molecular mechanisms involved in spinal cord regeneration. Most mammalian models of SCI require partial injury, making it problematic to differentiate true regeneration of injured axons from collateral sprouting of spared axons (Zheng et al., 2005, Steward et al., 2008, Blesch and Tuszynski, 2009). To overcome these limitations, we use the unique advantages of the lamprey, which recovers from complete SC transection (TX), and whose axons show robust, but incomplete regeneration. Unlike in mammals, after full spinal transection lampreys recuperate their behavior and injured spinal-projecting reticulospinal (RS) and other supraspinal axons grow selectively in their correct paths (Rovainen, 1976, Selzer, 1978, Yin et al., 1984, Mackler et al., 1986, Lurie and Selzer, 1991), making synaptic connections selectively with the types of neurons that are their normal post-synaptic targets (Mackler and Selzer, 1985, Mackler and Selzer, 1987). In limbless lower vertebrates, which lack a cortex, RS projections are the major descending motor connections that regulate trunk musculature to facilitate swimming and crawling (Tuszynski and Steward, 2012).

The anatomical projections, roles in locomotion and regenerative abilities of lamprey RS neurons have been well characterized. Eighteen pairs of them have been identified and documented by size and location in whole mount brain samples (Rovainen, 1967, Swain et al., 1993). It is possible to correlate molecular expression patterns with regenerative capabilities in individual RS neurons (Shifman and Selzer, 2000). Axons can be labeled with fluorescent dyes and regeneration of individual axons can be monitored over time. In addition, based on their regeneration abilities, the identified RS neurons have been divided into “good regenerating neurons” with probabilities of axon regeneration > 50% (including I3, I4, I5, I6, Mth’, B2, B5, B6, M1 and M4), and “bad regenerating neurons” with regeneration probabilities < 30% (including I2, Mth, B1, B3, B4, M2, M3 and I1) (Davis and McClellan, 1994, Jacobs et al., 1997). This heterogeneity has important parallels in the mammalian CNS, where some spinal-projecting neurons, e.g., CST, have been remarkably resistant to regenerative therapies, whereas other neuron types appear to be much more tractable, e.g., the median raphe serotonergic neurons (Deumens et al., 2005). The presence in lampreys of both good- and bad-regenerating neurons in the same brain location, and their projection in the same spinal cord axon tracts, suggest that variability in regenerating ability depend upon neuron-intrinsic factors, and provide an exceptional prospect to compare properties of neurons with high vs. low regenerating capacity. In mammals, comparisons of CNS neurons (low regenerating capacity) and PNS neurons (high regenerating capacity) are complicated by differences in their embryonic origin and their biochemical environment. However, new studies on mouse retinal ganglion cells begun to approach this capability (Duan et al., 2015).

Section snippets

Animals and spinal cord transection

Lampreys (Petromyzon marinus) larva (12–14 cm in length) were acquired from tributary of Lake Michigan through commercial suppliers(Northwoods Custom Products and Services, Marquette, MI or Lamprey Services, Ludington, MI), and maintained in fresh water tanks at 16 °C on a 12-h/12-h light/dark photoperiod cycle (lights on at 7:00 am) provided by ceiling-mounted fluorescent light until the day of surgery. Larval lampreys have no gender-identifying external features but presumably 50% will become

Changes in expression of RGMa mRNA after SCI

We previously reported the cellular localization and spatiotemporal expression of RGMa mRNA in normal and transected lamprey spinal cord using in situ hybridization and quantitative RT-PCR (QRT-PCR) (Shifman et al., 2009). As shown in Fig. 1A, in normal lamprey spinal cord, RGMa mRNA is abundantly expressed in several neuron types, including dorsal cells, and motor neurons. Our previous studies showed that the large RS axons retract during the first 1–2 weeks post-TX. From 2 to 4 weeks, they

Retrograde neuronal death

For reasons not completely understood, axotomized neurons often die, and thus are not available to participate in restoring connectivity. Examples of this retrograde neuronal death include apoptotic retrograde neurodegeneration after a unilateral occipital cortex ion in the dorsal lateral geniculate nucleus (Agarwala and Kalil, 1998, Al-Abdulla and Martin, 1998, Al-Abdulla et al., 1998) and the loss of retinal ganglion cells (RGCs) after transection of the optic nerve (Aguayo et al., 1991,

Acknowledgments

We thank Drs. Michael Selzer and Alan Tessler for their careful reading and valuable suggestions in writing. This work was supported by grants from Shriners Hospitals for Children (USA) grant SHC-85310 to MS and Shriners Hospitals for Children (USA) fellowship grant SHC-84297 to JC.

Author contributions

M.S. and J.C. designed the experiment; J.C. performed all parts of the experiment and carried out the RT-PCR and designed the RNA probes and Neogenin morpholino; J.C. and M.S. designed and carried out the data analysis and co-wrote the paper.

Competing financial interests

We declare that all the authors have no competing interests, or other interests that might be perceived to influence the results and discussion reported in this paper.

References (131)

  • E Demicheva et al.

    Targeting repulsive guidance molecule a to promote regeneration and neuroprotection in multiple sclerosis

    Cell Rep

    (2015)
  • R Deumens et al.

    Regeneration of descending axon tracts after spinal cord injury

    Prog Neurobiol

    (2005)
  • H Doya et al.

    Induction of repulsive guidance molecule in neurons following sciatic nerve injury

    J Chem Neuroanat

    (2006)
  • X Duan et al.

    Subtype-specific regeneration of retinal ganglion cells following axotomy: effects of Osteopontin and mTOR signaling

    Neuron

    (2015)
  • R Dubuc et al.

    The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey

    Brain Res

    (1989)
  • ER Feringa et al.

    Labeled corticospinal neurons one year after spinal cord transection

    Neurosci Lett

    (1985)
  • E Hall

    Antioxidant therapies for acute spinal cord injury

    Neurotherapeutics

    (2011)
  • J Hu et al.

    Activated caspase detection in living tissue combined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains

    J Neurosci Methods

    (2013)
  • S Kasicki et al.

    Phasic modulation of reticulospinal neurones during fictive locomotion and other types of spinal motor activity in lamprey

    Brain Res

    (1989)
  • PD Koeberle et al.

    The repulsive guidance molecule, RGMA, promotes retinal ganglion cell survival in vitro and in vivo

    Neuroscience

    (2010)
  • A Kyoto et al.

    Synapse formation of the cortico-spinal axons is enhanced by RGMA inhibition after spinal cord injury

    Brain Res

    (2007)
  • BH Lee et al.

    Injury in the spinal cord may produce cell death in the brain

    Brain Res

    (2004)
  • X Liu et al.

    Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

    Biochem Biophys Res Commun

    (2009)
  • W Liu et al.

    Dragon (repulsive guidance molecule RGMb) inhibits E-cadherin expression and induces apoptosis in renal tubular epithelial cells

    J Biol Chem

    (2013)
  • J Oldekamp et al.

    Expression pattern of the repulsive guidance molecules RGM A, B and C during mouse development

    Gene Expr Patterns

    (2004)
  • RJ Pasterkamp et al.

    Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS

    Mol Cell Neurosci

    (1999)
  • J Schmidtmer et al.

    Isolation and expression pattern of three mouse homologues of chick RGMa

    Gene Expr Patterns

    (2004)
  • S Agarwala et al.

    Axotomy-induced neuronal death and reactive astrogliosis in the lateral geniculate nucleus following a lesion of the visual cortex in the rat

    J Comp Neurol

    (1998)
  • AJ Aguayo et al.

    Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals

    Philos Trans R Soc Lond Biol

    (1991)
  • H Arakawa

    p53, apoptosis and axon-guidance molecules

    (2005)
  • A Barreiro-Iglesias et al.

    Use of fluorochrome-labeled inhibitors of caspases to detect neuronal apoptosis in the whole-mounted lamprey brain after spinal cord injury

    Enzyme Res

    (2012)
  • A Barreiro-Iglesias et al.

    Detection of activated caspase-8 in injured spinal axons by using fluorochrome-labeled inhibitors of caspases (FLICA)

  • A Barreiro-Iglesias et al.

    Complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

    JoVE

    (2014)
  • A Barreiro-Iglesias et al.

    Retrograde activation of the extrinsic apoptotic pathway in spinal-projecting neurons after a complete spinal cord injury in lampreys

    Biomed Res Int

    (2017)
  • MS Beattie et al.

    Review of current evidence for apoptosis after spinal cord injury

    J Neurotrauma

    (2000)
  • A Berg et al.

    Netrin G-2 ligand mRNA is downregulated in spinal motoneurons after sciatic nerve lesion

    Neuroreport

    (2010)
  • APS Bhalla et al.

    A forced damped oscillation framework for undulatory swimming provides new insights into how propulsion arises in active and passive swimming

    PLoS Comput Biol

    (2013)
  • A Bonnin et al.

    Serotonin modulates the response of embryonic thalamocortical axons to netrin-1

    Nat Neurosci

    (2007)
  • DE Bredesen et al.

    Apoptosis and dependence receptors: a molecular basis for cellular addiction

    Physiol Rev

    (2004)
  • A Brugeaud et al.

    Inhibition of repulsive guidance molecule, RGMA, increases afferent synapse formation with auditory hair cells

    Dev Neurobiol

    (2014)
  • JT Buchanan

    Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology

    J Neurophysiol

    (1982)
  • J Chen et al.

    Triple-labeling whole-mount in situ hybridization method for analysis of overlapping gene expression in brain tissue with high level of autofluorescence

    J Cytol Histol

    (2015)
  • RM Cooke et al.

    Locomotor recovery after spinal cord lesions in the lamprey is associated with functional and ultrastructural changes below lesion sites

    J Neurotrauma

    (2009)
  • M De Vries et al.

    Emerging roles for neogenin and its ligands in CNS development

    J Neurochem

    (2008)
  • TG Deliagina et al.

    Activity of Reticulospinal neurons during locomotion in the freely behaving lamprey

    J Neurophysiol

    (2000)
  • BW Draper et al.

    Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown

    Genesis

    (2001)
  • E Emery et al.

    Apoptosis after traumatic human spinal cord injury

    J Neurosurg

    (1998)
  • ER Feringa et al.

    Histologic evidence for death of cortical neurons after spinal cord transaction

    Neurology

    (1984)
  • Y Furuta et al.

    Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development

    Development

    (1997)
  • K Gambaro et al.

    BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors

    Cell Death Differ

    (2005)
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