Final Report Summary - AXREGEN (Axonal regeneration, plasticity & stem cells)
Executive Summary:
1.1 Executive Summary
AXREGEN is a multi-disciplinary training programme focussed on the experimental and clinical problems associated with axonal damage and repair in the central nervous system. The scientific areas of research range from studies on the molecular biology of gene expression during axonal damage and regeneration, through cellular approaches for rescuing axons, artificial systems for growing and studying axons in vitro, the use of stem cells as regenerative procedures, advanced imaging techniques for assessing axonal integrity, studies on ways to alter axonal plasticity, pharmacological approaches to preventing or restoring axonal damage, new surgical and experimental methods for studying axonal structure and function, tests of recovery (including both physiological and behavioural techniques) and the contribution of axonal malfunction to animal models of specific diseases.
The programme encompasses ten academic and two commercial organisations. Each PhD fellow based in an academic institution was enrolled in a high-quality training programme of the local doctoral (graduate) school and is further trained in a variety of theoretical and technical approaches, based on the complementary expertise of the collaborating partners. With a total of 20 ESRs and 2 ERs, the project has given a critical mass of scientists who could identify with the AXREGEN network and carried out the major part of the research program in collaboration with their host teams.
The AXREGEN project has offered a combination of local and network-wide training and capacity building to the different ESRs and ERs recruited.
Local training. The characteristics of the projects are: cutting-level research, multi-disciplinarity, and collaboration with, at least, one other ITN partner. The ESR and ER work has been directed by one principal experienced researcher (principal supervisor) from the research team in which the recruited scientists were based, but was also in contact with one external advisor from a different partner institution. The ERS students have been fully integrated to the Graduate school of the host institution and have benefited from a high quality training in Neuroscience and beyond.
Network-wide training. The AXREGEN research & training plan has been supported by a series of workshops organised by each partner in collaboration with local doctoral schools to ensure quality training. These workshops have covered the principal areas of the RTD project. In addition, the consortium is taking advantage of having two industrial partners as full partners to ensure a strong emphasis on the commercial exploitation and development of potential new therapies. This research training plan is exposing young researchers to multiple research environments to lead them to bottom-up collaborations.
Collaborations and Rotation period: In general, the ESR fellows have first been trained to achieve a certain standard of knowledge in basic science within the first 6 months of their PhD. To that end, all of the participating centres have already organized training programs like Master/PhD, MD/PhD or Graduate Programs that were joined by the new recruited researchers. This initial period was also necessary to introduce the new PhD fellows into the research topic guided by the local supervisor. After this initial training period, the researchers were sent to the collaborative laboratories.
Scientific outcome:
Axon regeneration. The role of integrins, wnt-pathway related proteins and intracellular Nogo signaling have been studied to enhance the regenerative ability of CNS neurons. Several molecules with protective features, such as the alphaB-crystallin (CRYAB) heat shock protein or neuropilin-1 bodies have been studied for their potential in reducing secondary damage after spinal cord injury (SCI). It has been demonstrated that astrocytes play an important role in maintaining the internal homeostasis and function of the CNS, including the medial prefrontal cortex (mPFC) and hippocampus.
Plasticity. The role of the extracellular matrix proteins in plasticity has been studied by several AXREGEN partners. The secretion of TIMP-1 and MMP-9 in the extracellular space seems to happen jointly in response to enhanced neuronal activity. Significant advances are being made in the study of plasticity in the cerebellum, showing, for example, that activation of integrins in Purkinje cells causes a considerable change in spine morphology. GAP-43 has been demonstrated to promote sprouting of lesioned Purkinje cells in parallel with cytoskeletal changes in the axon.
Stem cell biology. It has been demonstrated that there are two modes for oligodendrocytes to myelinate axons depending on axonal activity and glutamate release. The G-protein coupled receptor GPR17 was found to participate to the response of oligodendrocytes to injury at post-acute stages. The zinc finger transcription factor INSM has been identified as a candidate involved in regulation of neuronal progenitor cell state. Cell sorting was applied on undifferentiated and/or proliferating cells to increase the ratio of dopaminergic differentiation. An enrichment strategy for selection of neuronal progenitor cells using the MACS cell separation systems is being studied. The role of Nogo-A protein has been studied in the 6-OHDA mice model of Parkinson’s disease (DA cells destroyed by a neurotoxin). Finally, the role of calcium signals in the proliferation and differentiation of stem cells has been studied.
1.2 Project Objectives
Patients with Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and dementias, multiple sclerosis, spinal cord injury, traumatic brain injury, and glaucoma represent a large proportion of the severely disabled people in Western societies. All these conditions have damage to axons as a common feature. This training programme gains its strength by being focussed on the problem of axonal damage (axonopathy), which is central to attempts to understand how the central nervous system (CNS) can be damaged, how this damage might be prevented or limited, and how new ways of repairing the CNS might be developed. The study of axonal damage and repair can, and should, be approached in a wide variety of ways, and this is what gives the programme its multi-disciplinary scope, even though it is focussed on a common topic.
Each PhD fellow will have his/her own training schedule, encompassing a wide variety of technical and theoretical approaches, yet have a common interest in the problems of axonal degeneration and repair. In addition, there will be a strong emphasis on the commercial exploitation and development of potential new therapies. These, together with a series of workshops, seminars and online tutorials, will foster a sense of collegiality amongst the researchers themselves, as well as direct knowledge of the work going on in the laboratories of the consortium. Young researchers will also be taught the avenues available for commercial exploitation of experimental findings. Much of the wide expanse of contemporary neuroscience and associated disciplines is represented in this spectrum.
The research upon which this training programme is based falls into three, overlapping, fields: (1) axon regeneration (2) plasticity (3) stem cell biology.
1. Axon regeneration
Axon regeneration fails in the CNS due to the inhibitory environment presented by oligodendrocytes and glial scar tissue and due to the poor regeneration response of CNS axons. The partners have research programmes in these three areas, and will integrate their research through the network. Partner 1 has expertise in the role of chondroitin sulphate proteoglycans in the glial scar in blocking axon regeneration. Partner 6 and 7 have investigated the role of Nogo-A produced by oligodendrocytes. Partner 10 has shown that semaphorin3 in the lesion core of spinal cord injuries is an impediment to regeneration. New strategies based on stem cell biology and glial biology are being developed by partners 2, 5 and 9. Partner 9 has shown that treatment with different bone marrow stem cell populations has a positive effect on behavioural outcome and histopathological assessment after spinal cord injury (SCI) in rats. A clinical study suggests a possible positive effect of autologous BMC implantation in patients with SCI. Partner 5 has shown that olfactory glia can stimulate axon regeneration. The major extracellular matrix glycoprotein expressed after injury is tenascin-C. Integrins binding this molecule are not expressed in the injured CNS. A research project in collaboration between Partner 1 and 10 will examine the effect of expression of alpha9 integrin in the animal models of the partner laboratories. Some signalling pathways are activated during successful regeneration. Partner 6 has expertise in expressing such molecules following CNS injury. Following peripheral nerve injuries axons regenerate and innervate peripheral targets. However, reinnervation is inaccurate with many axons innervating the wrong targets, leading to poor functional recovery. The role of activity-dependent modulation of the specificity of target reinnervation will be investigated by partners 1, 5 and 8. To find new therapeutic targets in axon regeneration, the expertise of the industrial partner 11 in screening technologies will be used. A joint programme in microarray analysis of injured neurons and glial cells will be initiated. The goal is to identify novel regeneration-associated genes and to study the function of these genes in neurite outgrowth using high-throughput “Cellomics” screening methods followed by in vivo validation of targets using viral vector technology.
2. Plasticity
Most forms of CNS damage result in partial lesions, leaving a residual neuronal and axonal population whose effectiveness can potentially be enhanced by treatments that activate plasticity mechanisms. The partners will investigate various methods for enhancing plasticity using in vitro analysis and in vivo models. A particular focus is the extracellular matrix. Partners 1 and 4 will examine changes in the matrix during plastic changes, and the role of matrix metalloproteinase-9 and protease inhibitors in their causation. Partner 1 and 10 will collaborate on an examination of the role of perineuronal nets in plasticity, and particularly the binding of semaphorins to these structures and their role in controlling synapses. Furthermore, Partner 4 will examine role of MMP-9 (matrix metalloproteinase-9) and TIMP-1 (tissue inhibitor of matrix metallorpteinases-1) in neuronal plasticity, also in the context of perineuronal nets. Partner 8 will collaborate in this programme from a different perspective. Spinogenesis, synapse elimination and axonal remodelling in the formation of precise neuronal function and circuitry in the cerebellar Purkinje cells will be studied. Plastic events occurring spontaneously after CNS injury or induced by anti-Nogo-A antibody treatment or chondroitinase treatment are currently not well understood - both on the level of the injury site and in the sensory-motor cortex or the cerebellum. Partners 7, 6 and 1 will collaborate to analyze these anatomical and physiological changes induced after CNS injury in the rat by fMRI, tract tracing and 2-photon microscopy.
3. Stem cell biology
Production and differentiation of stem cells for CNS repair is a rapidly developing area of technology. Partner 2 will develop methods to produce differentiated dopaminergic neurons from rodent and human neural stem cells and human embryonic stem (ES) cells. Partner 9 will study mechanisms of calcium signaling cascades during the neural differentiation of hESC. Partners 3 and 11 will use large scale genomic and miRNomic approaches to study molecular regulation of the different stem cell, progenitor and neuron populations that coexist in the neurogenic regions of the adult forebrain. Partner 4 will investigate whether cyclin D2, recently discovered as playing critical role in the adult brain neurogenesis, may be considered as a target for future therapies aiming at modulation of the stem cell proliferation in the central nervous system. Integration of transplanted cells into the adult CNS is poor, except in the neurogenic hippocampus and olfactory bulb, while integration in developing CNS is impressive. Partner 6 will use transplantationto analyse the specification of cerebellar GABAergic interneuron phenotypes during development, and whether the same mechanisms are present in the adult cerebellum. In the retina, gliosis of Müller cells has been shown to prevent the integration of stem cells in the retina. These cells produce high levels of Nogo-A following retinal damage. Partners 2, 3 and 7 will investigate the role of Nogo-A in the inhibition of integration of grafted stem cells and endogenous stem cells into the damaged retina and Parkinsonian models. In the adult CNS, myelin has been shown to prevent remyelination by inhibiting oligodendrocyte precursor differentiation. Work by partners 3 and 7 will investigate whether remyelination is influenced by the inhibitory membrane proteins Nogo-A and MAG expression after experimental demyelinating lesions in the adult CNS.
The specific objectives of the project were to:
• Ensure a high quality training to 20 PhD and 2 post-doctoral fellows
• Provide multi-disciplinary / inter-laboratory knowledge based on the active participation of industrial partners and different research experts in the training plan
• Creation of a ‘community’ of open-minded and well-trained young scientists
• Promote exchange and collaborative discussions between 12 leading academic and industrial teams in Europe
• Focus on the experimental and clinical problems associated with axonal damage and repair in the CNS
• Develop new treatments that will help patients with structural damage to the CNS