A molecular switch between scarring and central nervous system regeneration

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By Sandy Vascotto and Arthur Brown
Salamanders and other evolutionarily primitive animals are capable of regenerating complex structures that serve as inspiration to modern medicine. Even such sensititve systems as severed spinal cords are regrown to full functionality in short order. Humans are not nearly so adept at regeneration. A simple event like an uninformed dive into a swimming pool, automobile accident, slip on a set of stairs, or mere progression of the normal process of aging can have a profound impact upon an individual’s capacity to maintain an independent existence.

While modern medicine has achieved great success at saving the lives of spinal cord-injured patients there is not a single therapeutic treatment designed to improve nerve growth or function after spinal cord injury. Why are salamanders successful where we are not? The answer, in part, lies in the absence of a scar and a series of molecular switches that both recognize the status of the damaged tissues and implement a program of repair and regeneration. Scientific discoveries have suggested that scarring and regeneration are largely incompatible and are a conserved source of challenge across the full spectrum of central nervous system disorders.

Scarring in the CNS following trauma and stroke affects over 15 Million people in the US alone
There are over 12,000 new spinal cord injuries per year in the U.S. and Canada. New patients are the target of most experimental therapies – generally designed to mitigate further damage during the acute phase of injury (ie. limiting immune response, inflammation, cytotoxic cascade, apoptosis). The economic burden of increased survival following severe trauma is extensive particularly considering the limited capacity to regenerate to normal function. The average cost in the first year following spinal cord injury ranges between $220,000 and $740,000 depending upon the severity of injury. Following the first year, the average yearly burden per patient ranges between $15,000 and $130,000 with severity and averages at $22,000/person/year.

Beyond new patients, there are approximately 275,000 people in North America who have survived the acute phase and live with associated disability. These individuals, and the estimated other three million people worldwide, represent a market for therapeutic strategies that are not dependent upon treatment immediately following injury and constitute a health care cost of approximately $10 billion/year for spinal cord injury alone.

While a therapy that “cures” spinal cord injury represents a huge opportunity, one that is able to increase the level of function of the severely disabled to a level requiring less intensive care could reduce the annual patient cost 10-fold and bring the patients a step closer to independent function.

The potential broader application of scar modulating strategies for CNS repair includes booming age-related applications such as stroke and Alzheimer’s; as well as traumatic brain injury. A therapy that can limit damage following stroke can impact the 700,000 new patients per year in the U.S. or 20 million people worldwide. In the U.S. alone, there are approximately six million individuals living with the effects of a previous stroke that would benefit from a regenerative strategy that remodels the associated scar to establish new neural connections. Combined with the approximately 1.4 million new traumatic brain injuries per year and the standing 5.3 million individuals in the U.S. recovering from traumatic brain injury, the population of patients that could be impacted by a regenerative medicine strategy that augments both the deposition of rate limiting scar proteins versus pro-regenerative factors is staggering.

Setting the stage for a molecular switch for CNS regeneration
There are a number of stages associated with natural repair and regeneration following nervous system damage. After trauma, there is an acute phase wherein the body attempts to limit the extent of degradation through wound stabilization.

This is followed by an active stage of inflammation and further damage produced by factors secreted through the immune response and degeneration of the wound site. This process is generally stabilized by the formation of a scar. The scar, which is generated by astrocytes, and infiltrating leukocytes consists of extracellular matrix molecules and serves as a physical and chemical barrier to new nerve growth. However the same cells that produce key nerve-repelling elements of the scar can also, in some contexts, deposit factors that tell nerves to extend and establish new connections. Thus neurological recovery, when seen, is slow because the balance of protein expression by astrocytes favours growth-inhibiting molecules over growth-promoting molecules.

One may consider the astrocytes as the switch box for extracellular matrix composition and regeneration.

The negative effects of the scar on recovery from spinal cord injury have been well documented. The most infamous of the scar proteins belong to the chondroitin sulfate proteoglycan (CSPG) family. CSPGs have been shown to be potent inhibitors of nerve growth. In culture CSPGs will stunt the growth of extensions from plated neurons and in experimental models of spinal cord injury digestion of CSPGs at the scar improve regeneration. Nerve-repelling CSPGs are produced by astrocytes, support cells in the central nervous system after injury. Could removing astrocytes from the injured spinal cord be therapeutic? The answer is clearly no. Animals’ recovery from spinal cord injury is worsened by astrocyte ablation. Part of the reason for this is that astrocytes carry out many functions in the injured spinal cord aside from the production of the nerve-repelling CSPGs.

One of these functions is the production of growth promoting proteins such as laminin. Thus, as is often the case, nature strikes a balance. Unfortunately for spinal cord-injured patients the production of nerve repelling CSPGs seems to outweigh the production of growth promoting proteins after injury. Changing the balance of power to favour the expression of growth promoting proteins such as laminin over growth inhibiting proteins such as CSPGs would be predicted to improve recovery after spinal cord injury and potentially other neurological diseases in which scar prevents regeneration.

The SOX9 transcription factor is a molecular switch between scar formation and regeneration
Regenerative medicine is the new buzz word for considering therapeutic approaches to repairing damaged tissues and systems.

Instead of attempting to impose one’s will upon a biological system, the regenerative medicine approach seeks to harness and augment the normal processes of repair and restoration. Traditional approaches to wound repair typically deal with the elements of the wound healing cascade individually or in limited combination through a series of technological advances. This would be analogous to attempting to turn on or off a light in a room by physically placing and removing a shroud over the lightbulb depending upon the lighting interests of an individual. A regenerative medicine approach would be to identify and modulate the switch on the wall to toggle between the preferred lighting.

The transcription factor, SOX9, is a molecular switch that tells the astrocytes to either deposit components of the scar, or turn off the scar and produce factors that promote nerve repair. SOX9 is largely expressed as an instructive messenger during the early development of a fetus, with limited role in adulthood. In the CNS, evidence indicates that SOX9, increases the expression of enzymes that synthesize the nerve-repelling CSPGs within the scar. Since a large number of genes encode the core proteins of all the different CSPGs investigative work has focused on identifying proteins that regulate the genes that put the inhibitory side chains on the core proteins. Using software to compare the human mouse and rat DNA sequences in the promoter regions of these genes identified SOX9 as a key candidate that binds and regulates these genes. In a manner analogous to a switch, SOX9 has also been demonstrated to limit the expression of inhibitory scar proteins and to increase the expression of growth promoting scar proteins.

An examination of the presence of SOX9 in relevant models of disease supports its role as a candidate molecular switch in CNS repair. Reactive astrocytes within animal models of spinal cord injury demonstrate elevated SOX9 expression that correlates with CSPG production. An examination of human tissues demonstrates SOX9 expression in damaged spinal cord, the scar around an eruptive stroke, and associated with the degenerative plaques in Alzheimer’s patients. This expression data suggests that SOX9 serves a role in pathologies associated with human scarring of the CNS.

Functional testing of the role of SOX9 in reactive fibroblasts provides strong insight into the mechanism of action. In vitro evidence demonstrates that SOX9 expression directly increases in the expression of CSPG modifying proteins and the production of CSPGs. Knockdown of SOX9, both at the level of expression or by the chemical modulation of its activity, decreases the expression of these genes as well as CSPG production. Importantly, the pharmaceutical inhibition of SOX9 both downregulates CSPGs and increases the expression of such nerve promoting factors as laminin.

The relevance of SOX9 as a molecular switch to scar composition in vivo is promising. Gene expression patterns in spinal cord-injured rats that have received an anti-inflammatory treatment show that treated rats have greatly reduced levels of SOX9 in their spinal lesions. This is accompanied by a reduction in CSPGs and an increase in laminin in the lesions of treated spinal cord injured rats. These findings are consistant with the observation that certain cytokines increase the expression of SOX9 in astrocytes. Finally, preliminary evidence of the impact of pharmaceutical compounds directly upon the activity of SOX9 in rodent models serve as excellent leads as future therapeutic modulators of CNS repair and regeneration.

Molecular switches to regeneration constitute a high impact therapeutic opportunity

Typical strategic approaches to treating spinal cord injury and other neural tissue repair are oftentimes unidimensional, ie. limit the immune response, limit the apoptosis and cytotoxic cascade associated with the degeneration of the nerves, or promote nerve repair afterwards. Such approaches do not take into account the network of cascades that ultimately direct how a cell will interact with its microenvironment and are often limited to a window of time wherein treatment must occur. Scar production is often a secondary effect and they do not address the rate limiting step of the natural remodeling of the scar.

The prime advantage of SOX9 as a molecular switch is that it targets the natural process of neural regeneration that is normally occurring at a prohibitively slow rate. SOX9 has a limited role outside of early human development and its inhibition is predicted to replace nerve repelling extracellular matrix molecules with ones that promote nerve growth. Furthermore, since scar tissue is constantly being remodelled and replaced the strategy of SOX9 inhibition could be applied any time after injury including immediately following damage and after subsequent stabilization. This provides therapeutic opportunity to both new patients and to those currently living with the consequences of CNS damage. Modulation of the molecular switch to CNS regeneration has the potential to affect the quality of life and cost of care for a growing population of patients, families, and of the health care system as a whole.

Arthur Brown is a Scientist with the Biotherapeutics Group at the Robarts Research Institute and Associate Professor in at the University of Western Ontario, London, Ontario. Arthur Brown’s research focus is on understanding of the molecular and cellular basis of CNS damage and on developing strategies to enhance repair and regeneration.

Sandy Vascotto is a Business Development Manager with the Robarts branch of WORLDiscoveries™. WORLDiscoveries™ is the integrated business development and technology commercialization arm of the University of Western Ontario, Robarts Research Institute, and Lawson.