Abstract
SCI is a major cause of long-term disability with no clinically accepted treatment, produced
by multi-factorial processes as a result of primary mechanical damage, secondary cell death,
reactive gliosis and a poor capacity to regenerate damaged axons.
The Wnt family of proteins has been studied intensively in recent years due to their ability to
regulate many biological events during development and adulthood in the CNS, including: cell
migration and proliferation; the maintenance of pluripotency and differentiation of neural stem
cells; oligodendrocyte precursor instruction and myelination, axonal growth and guidance,
dendritic arborization; synapse formation, function and maintenance; angiogenesis;
establishment of the blood-brain barrier (BBB); and microglia/macrophage activation. Indeed,
although a previous report described a lack of expression in the adult spinal cord of mice, strictly
induced expression of few Wnt ligands (1, 4 and 5a) and receptors (Fz1 and Ryk) has been
shown in the lesioned spinal cord [13, 14]. In this sense, it has been recently reported that the
ligands Wnt2, Wnt3a, Wnt5a and Wnt7a and the receptor Ror2 are expressed in the adult spinal
cord of mice and afferent dorsal root sensory neurons, with a role on the development of
neuropathic pain and induction of glial proliferation, in experimental models of
neurodegeneration and peripheral nerve injury [15-18]. Moreover, strategies seeking to
modulate Wnt-dependent signaling pathways, generically subdivided into canonical and noncanonical, have proved to produce benefits in different experimental models of CNS disorders
and spinal cord lesions.
Consistently, we have provided evidence that the expression of most Wnt ligands, their
inhibitors and components of their intracellular signalling pathways is prolonged in the rat and
mouse adult spinal cord, as is the activation of the canonical pathway, suggesting that the Wnt
family of proteins play a role in spinal cord function and physiology. Indeed, in a clinically
relevant rat model of SCI we demonstrated that trauma induces a dramatic and time-dependent
change in the physiological pattern of Wnt mRNA expression, as well as the activation of the
canonical pathway in cells around the wound core in a pattern suggestive for a role in the glial
scar formation.
As such, we have shown by first time that all Frizzled receptors are expressed in the adult
spinal cord of rats and that, after contusion SCI, are differentially regulated and highly
expressed by cells close to the wound core. Double immunohistochemical (IHQ)
characterization of the Frizzled-5 (Fz5) receptor, which was chosen because of its up-regulation
at all times post-injury assessed (6 hours and 1, 3, 7, and 14 days post-injury), evidenced that is
expressed by oligodendrocytes, neurons, resident microglia, NG2 precursors, astrocytes and
cells of the central canal in the non-injured spinal cord and that, after spinal cord contusion, is
up-regulated in both glial and inflammatory cells and induced in damaged axons at the injury
epicenter. Furthermore, analysis of Ryk, a well-known non-conventional Wnt receptor claimed to
be expressed by corticospinal axons to inhibit axonal regeneration after SCI, has been shown
as also prominently expressed by macrophages, fibroblasts and astrocytes with a key role in the
glial scar formation. Indeed, when inhibited by administration of a function-blocking antibody
through a subdurally implanted catheter we observed a significant reduction on the
macrophage/microglial response, which might also contribute to the sensorimotor functional
improvement also observed.
Our results provide compelling evidence that Wnts are expressed and transcriptionally
regulated by SCI with a putative role on its pathophysiology. These novel findings provide an
important foundation to further address the function of individual Wnt proteins and downstream
pathways, by loss and gain of function experiments, on the different cell populations of the
injured adult spinal cord and, eventually, allow for the development of novel therapeutic
strategies.
Brief Description of the Institution (HNP)
The Hospital Nacional de Parapléjicos (National Hospital for Paraplegics, HNP), established in
1974, is the biggest Spanish centre dedicated to the rehabilitation of patients with spinal cord
disease. It is a state owned hospital financed by public money, receiving more than two-hundred
new cases of spinal cord injured patients each year. The HNP is also a University Hospital
associated to Universidad Castilla-La Mancha (School of Medicine). In 2002, the HNP started a
department dedicated to basic, clinical and translational research: the Experimental Neurology
Department (END). The END incorporates ten research groups with more than 100 people
focused on the study of neurosciences, which have been selected on the basis of their research
excellence and interests in areas relevant to neural repair, neuroplasticity, motor and sensory
systems and pain research. The END is an example of international (more than ten different
European and non-European nationalities) and multidisciplinary approach to research. Since
2003, the non-profit foundation HNP (FUHNPAIIN) constitutes the HNP branch dedicated to
management of scientific resources.
The productivity of END is notable given it has been constituted around both experienced and
young group leaders (mean age of 42 ±8 years), many of them recognised with various awards
and prizes during their scientific careers. As of June 2012, the scientists at END had published
during their entire career more than 600 articles (with more than 10.000 citations in peer
reviewed indexed journals) and, since joining HNP, 119 articles (cited more than 600 times) and
eleven pending patents. The international competitiveness of the END is also substantiated by
the participation of its members in EU projects within the various Framework Programmes.
Moreover, the research programs of the END have been funded by National and Regional
public and private institutions, with more than fifty projects and a total amount of more than 10
millions Euros during the last six years.
Brief description of the Molecular neurology Group
Spinal Cord Injury (SCI) is a major cause of functional disability with no clinically accepted
treatment. The functional impairment following SCI is produced by multi-factorial processes as a
result of primary mechanical damage, secondary cell death and a low capacity of the CNS to
regenerate the damaged axons and replace the cells loss. The Molecular Neurology Group was
created in 2005 with the incorporation of Dr. Javier Rodríguez to the incipient END of The
National Hospital of Paraplegics, with the objective of screening for new therapeutic targets and
developing combined therapies for neuroprotection and promotion of axonal regeneration and
cell replacement. This broad experimental approach was based on a strong background on
neural repair acquired along his PhD stage at The Group of Neuroplasticity and Regeneration
from Dr. Xavier Navarro (Universitat Autonoma de Barcelona, Spain), and Postdoctoral stage at
The Molecular Neurobiology Unit from Dr. Ernest Arenas (Karolinska Institute, Sweden), with a
main focus on cell therapy, gene therapy and neural interfaces for stimulation and recording.
This broad experimental approach allows us to address the challenge of neural repair through
multidisciplinary research ranging from its very molecular aspects to the correlation in motor and
sensory functional outcome, electrophysiology and histology in clinically relevant rodent models
of CNS and PNS damage.
Major research findings include the description of a wide expression of the Wnt family of
proteins in the adult spinal cord with a key role after injury, as well as the presence of a novel
source or autologous stem cells located at the leptomeninges of the adult spinal cord with a
potential to generate new neurons and oligodendrocytes. Current research interests are
addressed to unravel the role of Wnts and Leptomeningeal Stem Cells (LeSCs) in the spinal
cord injury pathophysiology, as well as the development of new therapies based on drugs (such
as leptin and ibuprofen) and adult autologous cell transplants (such as adipose mesenchymal
cells) currently used in clinics. The final goal is to unravel the molecular and cellular underlying
mechanisms for neural damage and develop novel clinically feasible repair therapies.
The Molecular Neurology Group is currently receiving financial support from Regional,
National and International public agencies as well as funding from a private Tissue Engineering
and Cell Therapy Biotech Company. Presently, the group has one patent pending while the total
number of research articles published by its members is greater than 50.
Selected Publications
1. Decimo I, Bifari F, Rodríguez FJ et al. “Nestin- and DCX-Positive Cells Reside in Adult
Spinal Cord Meninges and Participate to Injury-Induced Parenchymal Reaction”.2011. Stem
Cells; 29(12):2062-76.
2. Fernandez-Martos CM, Gonzalez-Fernandez C, Gonzalez P, Maqueda A, Arenas A,
Rodriguez FJ. “Differential expression of Wnts after Spinal Cord Contusion Injury in adult
rats.” 2011. PLoS One 2011;6(11):e27000.
3. Fernandez-Martos CM, Gonzalez P, Rodriguez FJ. “Acute Leptin Treatment Enhances
Functional Recovery after Spinal Cord Injury.” 2012. PLoS One. 2012;7(4):e35594.
4. Gonzalez P, Fernandez-Martos CM, Gonzalez-Fernandez C, Arenas E, Rodriguez FJ.
“Spatio-Temporal Expression Pattern of Frizzled Receptors after Contusive Spinal 1 Cord
Injury in Adult Rats”. 2012. PLoS One 2012; 7(12): e50793.
5. Gonzalez P, Fernandez-Martos CM, Rodriguez FJ. “The Ryk Receptor Is Expressed in Glial
and Fibronectin-Expressing Cells after Spinal Cord Injury.” 2013. Journal of Neurotrauma.
(In Press).
6. Fernández-Martos CM, González P, Vazquez S, Fernández E, Rodríguez FJ. “Acute
ibuprofen anti-inflammatory and neuroprotective action promotes a significant functional
recovery after spinal cord injury in adult rats.” 2013. J Neurotrauma (Manuscript In
preparation).
7. González-Fernández C, Fernández-Martos CM, González P, Maqueda A, Rodríguez FJ.
“Expression analysis of the Wnt family of proteins after spinal cord injury in adult mice.”
2013. PLoS One (Manuscript in preparation).
8. Shields SD, González-Fernández C, Rodríguez FJ. “Wnt signaling is constitutively active in
adult spinal dorsal horn neurons and is altered after spinal cord injury”. 2013. PLoS One
(Manuscript In preparation).
9. Fernández-Martos CM, Maqueda A, Shields SD, Fernández-Núñez E, Rodríguez FJ.
“Combined Minocycline and Rolipram treatment improves neuroprotection and functional
recovery after spinal cord injury”. 2013. Experimental Neurology (Manuscript In preparation).
10. Maqueda A, Arenas E, Rodríguez FJ. “Wnt5a-overexpressing c17.2 Neural Precursor
transplantation increases myelin loss and glial reactivity with functional recovery impairment
in a rat model of spinal cord contusion.”. 2013. Neurobiology of Disease (Manuscript in
preparation).