Investigating the impact of nervous system maturation on transmembrane receptor localisation and transport within CNS axons

In the UK alone, 40,000 people live with paralysis, with 1,200 people becoming injured each year. Throughout the world, approximately 2.5 million people are living with paralysis. To date, there is no cure for paralysis and spinal cord injury, only rehabilitative therapy regimes. Once severed, the best instances of nerve fibre (axon) regrowth is limited to local sprouting at the site of injury, with few prospects for reconnecting damaged fibres or regaining lost function.

Further understanding about how the environment of the adult brain and spinal cord interacts with nerve cells is required in order to develop successful tools to treat neurological conditions. Evidence for failed central nervous system (CNS) regrowth implicates both the hostile injury environment and an inherent inability of nerve cells to repair themselves. This latter fact is due largely to a developmental reduction of growth-promoting molecules in the adult CNS. Studies reintroducing growth-promoting molecules in cultured neurons (nerve cells) show enhanced growth but, upon translation into an animal model, the resulting repair is very modest. A primary example is the re-expression of growth-inducing integrin receptors, which are critical for central nerve development and promote significant neurite growth in cultured neurons. In a model of CNS injury, however, the regrowth is not as robust but we went on to show that re-expressed integrins are only present in the nerve cell body and not transported within axons. Interestingly however, this is not the case in early postnatal neurons where integrins are present in perinatal brain and spinal cord axons. At this age, specialised structures such as myelin (the insulating cover of nerve fibres that increase the speed of nerve signals) as well as the extracellular matrix (structural support) are not fully developed. Likewise, in certain adult neuronal subtypes (neurons of the eye and peripheral nerve) this transport defect is also not present. These neurons do not contain specialised extracellular matrix (known as 'perineuronal nets' or PNNs) which cover many adult neurons. Without understanding how the transport of these molecules is affected by the mature nervous system, the success of integrin-based and similar treatments in future clinical translation is severely limited.

Thus, this project will determine what factors in the CNS inhibit axonal transport/expression of integrin molecules, and also use modifications previously shown to target molecules to axons to target integrins. More specifically, our initial aim will use two different experimental models: the first will utilise a transgenic mouse in which we can eliminate myelin-producing cells specifically in the area where neurons have been modified to express integrins; and the second will utilise an enzyme (chondroitinase ABC) to breakdown PNNs in the area where neurons have been modified to express integrins. The second aim will identify molecular strategies that target integrins into axons by modifying the integrin structure, thus overriding any external influence that the CNS may have on expression.

Results from all of the above experiments will involve advanced microscopic analysis of brain and spinal cord tissue in order to determine integrin expression within each experimental group. Fluorescently-labelled integrins will be used to visualise the expression within axons at an early (3 wks) and late (8 wks) time point, the former to allow expression of the integrins, and the latter to assess transport in conjunction with a spinal injury. These data will contribute greatly to neurological repair by determining factors both extracellular and intracellular which can affect expression of potential therapies involving re-expression of growth-promoting neuronal receptors. Additionally, these experiments will develop advanced techniques for bio-imaging and gene therapy that will be of benefit to the scientific community.

Extracellular factors within the central nervous system (CNS) as well as intrinsic neuronal factors contribute to the inability of axons to regenerate. Certain treatments have demonstrated enhanced neurite outgrowth in cell culture models but fail to produce robust growth when translated into an in vivo paradigm. A prime example is the reintroduction of growth-promoting alpha9 integrin which results in a strong regenerative response in cultured neurons, with much less growth observed in an in vivo model injury.
Experiments have demonstrated an axonal localisation deficit of integrins and other transmembrane receptors which may contribute to reduced in vivo repair. It is unclear however what CNS factors modulate this localisation except that integrins localise within perinatal axons, prior to myelination or establishment of the mature CNS extracellular matrix (ECM), and also localise within adult axons of neurons devoid of ECM-rich perineuronal nets (dorsal root ganglia, retinal ganglion cells). This proposal will determine how the mature CNS environment affects axonal localisation of integrin receptors and will identify molecular modifications which target integrins into axons in vivo. We will use an in vivo approach to assess axonal localisation of integrins in adult rodent cortical neurons following either modification to the CNS environment or molecular modification of integrin receptors. CNS modifications will include demyelination using a targeted genetic approach to ablate oligodendrocytes with viral caspase 3 in a PLP-Cre mouse or enzymatic removal of ECM proteoglycans using chondroitinase ABC. Molecular modifications of integrin receptors will utilise successful strategies for axonal targeting in cultured neurons. Results which will be demonstrated using advanced bio-imaging techniques (confocal and light sheet microscopy) will identify in situ factors which preclude axonal localisation of integrins, directly impacting future therapeutic treatments.

Who will benefit from this research?
The impact of this research will benefit both the scientific community as well as those concerned with future therapies for neurological conditions such as clinicians, patients, and the pharmaceutical industry.

Academic Research Community
The primary beneficiaries of the research outcomes in this proposal will be academic researchers interested in understanding how development and aging of the nervous system influences axonal function, expression and localisation of proteins, and potentially experimental clinical treatments. Researchers interested in targeting axons for different therapeutics will benefit greatly from the findings in this project. Furthermore, from a technique perspective, we will be developing tools throughout this project that will have far-reaching effects in the scientific community specifically with: 1) a novel focal demyelination mouse model that will be developed and used in our first Aim; 2) molecular strategies for driving axonal transport in vivo used in our second Aim; and 3) the potential advancement of bio-imaging techniques through Light Sheet microscopy for tissue imaging.
The results of this proposal will be disseminated through scientific publications in high impact peer-reviewed journals and through presentations at national and international conferences. There will be direct discussion of these results in internal seminars and group meetings in the University of St Andrews, fostering further collaborations locally and beyond.

Biotechnology and Pharmaceutical Industry
In the current climate of failed clinical trials, an increased understanding of how the body interacts with potential therapeutics is required to optimise treatments for human conditions. Those in biotechnology and pharmaceutical industries looking to develop novel and improved approaches for gene therapy in the nervous system and across disciplines will greatly benefit from the outcomes of this study.

Healthcare sector
Although not imminently translatable, patients and clinicians working in the area of neural injury and degenerative conditions may be particularly interested in these results which could directly contribute to optimised therapeutics in the future.

Education
The training of a postdoctoral researcher provided by this proposal will be a valuable contribution to the scientific community. Additionally, the research outcomes of this project will impact our current neuroscience knowledge and understanding of how neuronal function and intra-neuronal protein expression and transport can be affected by the external CNS environment. Furthermore, our results will contribute to the understanding of how the nervous system environment may control the intrinsic neuronal environment, and how these controls can be overcome through molecular modification.

General Public
Data and results generated in this project will be disseminated to the general public through outreach programs such as the University of St Andrews annual Science Discovery Day, as well as publicised through the University of St Andrews press office and appropriate social media. Both PI and the postdoctoral researcher will engage in these activities to provide and promote discussion of our work with the greater community.

How will they benefit from this research?
The education and awareness of the public on the research being performed within this project will stimulate interest into how basic research can impact on the treatment of human health conditions. This may also perk the interest of young biomedical scientists in their future careers. Additionally, the University of St Andrews has recently appointed a dedicated Public Engagement Officer for the Biomedical Sciences Research Complex who will assist in identifying activities and events for promoting our research.