Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

WHY I CHOSE THIS PREPRINT

Researchers working in the field of regenerative biology are chasing the molecules and mechanisms that positively regulate tissue repair and regeneration. As such, a common research approach is the characterization of the injury response in highly regenerative species like zebrafish, and its subsequent comparison to the response occurring in poorly regenerative animals, like mammals. The question behind these studies is “What are the molecules and mechanisms that enable regeneration in zebrafish, and how can we reactivate them in, or translate them to, mammals?” In this preprint, Kolb and colleagues turn the question the other way around: “Are there any molecules that prevent– that is, negatively regulate- regeneration in mammals? What happens when we force the expression of such molecules in the highly regenerative zebrafish?” This cascade of questions intrigued me enough to read the whole manuscript as if I was reading a mystery novel. Who does prevent successful regeneration in injured mammals? Who is the culprit?

BACKGROUND

In the lesioned spinal cord of mammals, the formation of a fibrous scar in the tissue inhibits regeneration of neuronal connections and functional recovery. Specifically, excessive deposition of extracellular matrix (ECM) molecules upon lesion creates an inhibitory environment to axon growth. By contrast, the ECM composition of the lesioned spinal cord in zebrafish is permissive for regeneration. In fact, zebrafish larvae regrow neuronal connections and recover their swimming function within days after the lesion, whereas adult fish take 6-8 weeks to achieve functional spinal regeneration. In this preprint, Kolb and co-authors surveyed and compared the ECM composition of the lesioned spinal cord of rats and zebrafish, looking for the proteins that explain the difference between poorly regenerating species (mammals) and highly regenerative ones (zebrafish).

RESULTS

1. A mass spectrometry survey uncovers the absence of small leucine-rich proteoglycans in the regeneration-permissive zebrafish ECM

First, the authors used mass spectrometry to investigate the ECM protein composition in the lesioned spinal cord of larval zebrafish. Then, they compared the resulting ECM dataset to a formerly published dataset that had analysed the ECM composition in the lesioned rat spinal cord. They observed that seven members of the small leucine-rich proteoglycans (SLRPs) family were highly enriched in the injury ECM of rats, but showed low abundance after spinal cord injury in zebrafish. Specifically, they focused their investigation on a subset of four SLRPs, namely chondroadherin (Chad), fibromodulin (Fmod), lumican (Lum) and prolargin (Prelp). SLRPs are part of the building blocks of the ECM, and consist of a protein core bound to short chains of sugar. Since SLRPs had previously not been associated with regeneration failure after spinal cord lesion, the authors moved on to study the role of SLRPs as potential inhibitors of mammalian axonal regeneration in the central nervous system.

2. SLRPs are highly abundant in the lesioned human brain and spinal cord

Second, the authors wondered whether SLRP enrichment is a general characteristic of the ECM in the lesioned central nervous tissues of poorly regenerating species. They used immunofluorescence to survey the brain as well as the spinal cord of human patients who had undergone traumatic brain injury, brain surgery or spinal cord injury. They found prominent immunoreactivity for anti-CHAD, anti-FMOD, anti-LUM and anti-PRELP in the lesioned area of the human brain, and increased, local immunoreactivity of anti-LUM, anti-PRELP, anti-FMOD, and, less frequently, of anti-CHAD, in the lesioned human spinal cord. The authors concluded that SLRP enrichment in the lesion area is a feature of the lesioned central nervous system in mammals, and in particular humans.

3. SLRP protein enrichment decreases axonal regrowth as well as swimming recovery upon zebrafish spinal cord lesion

Next, the authors investigated what happens when SLRP protein abundance increases in the spinal cord ECM of highly regenerative zebrafish upon injury. Specifically, they engineered fibroblast cells, which are responsible for ECM production, to increase extracellular deposition of SLRPs. Enrichment of SLRPs in the lesioned zebrafish spinal cord decreased the thickness of the regenerated axonal bridge that reconnects the severed spinal cord ends, as compared to control spinal lesioned zebrafish lacking SLRPs in the injury ECM. The thickness of the axonal bridge is a proxy to measure successful spinal cord regeneration, and correlates well with recovery of swimming function in zebrafish. By increasing the deposition of SLRPs in the ECM of the lesioned fish spinal cord, the researchers also observed a reduced swimming distance as compared to the lesioned fish without SLRP enrichment. The swimming distance is a proxy of recovery of the locomotor function in zebrafish after spinal cord injury. In summary, high abundance of SLRPs in the ECM of the injury area correlates with poor axonal regrowth and poor functional recovery.

4. SLRPs do not act directly on neurons, the immune system or fibroblasts to inhibit axon regeneration in the lesioned zebrafish spinal cord

Finally, the authors investigated how SLRPs inhibit axon regeneration in the zebrafish spinal cord. Like a private investigator, who proceeds by excluding the suspects of a murder one by one, Kolb and colleagues excluded possible mechanisms of SLRPs-mediated inhibition one by one. In fact, SLRPs might inhibit spinal cord regeneration at multiple levels. For instance, they might act directly on neurons to impair axonal extension. However, inducing SLRPs specifically in neurons in the zebrafish spinal cord did not affect the outcome of regeneration. Both lesioned animals with or without neuron-specific SLRP secretion exhibited the same degree of axonal regrowth. It is important to note here that SLRP induction in neurons does not result in their enrichment in the ECM of the lesion area, in contrast to induction of SLRPs specifically in fibroblasts. Second, SLRPs might act at the level of the inflammatory response, for instance by modifying the number or the action of immune cells that are recruited to the lesion area to resolve inflammation. However, the inflammatory response was resolved both in SLRP-enriched and in non-SLRP-enriched lesioned spinal cords. Finally, SLRPs might affect fibroblast-mediated deposition as well as composition of molecules in the ECM of the lesioned fish spinal cord. However, there was no difference between the ECM molecular composition of the SLRP-enriched and of non-SLRP-enriched, spinal lesioned zebrafish.

5. SLRPs change the structure and mechanics of the ECM in the lesioned zebrafish spinal cord

Tissues have specific molecular and structural characteristics that correlate with specific mechanical properties, which, in turn, can be affected by ECM composition. Accumulating evidence shows that tissue mechanical properties can influence several cellular functions, including axonal growth. Moreover, there is emerging evidence that tissue scarring in the lesioned spinal cord is a barrier to axonal outgrowth not only because of the altered biochemical composition of the tissue, but also because of changes in the elastic stiffness of the local microenvironment at the lesion site. However, correlation between biochemical composition and mechanical properties of a tissue in vivo is currently missing. There are different techniques that measure specific parameters to describe tissue mechanics in vivo. For example, mechanics can be described in terms of tissue elasticity, such as tissue compressibility. The authors used optical techniques to measure the structure and the mechanical properties of the ECM in the lesioned spinal cord of fish enriched with SLRPs in vivo. Intriguingly, increased abundance of Fmoda, Lum and Prelp altered the structural properties of the ECM in the lesioned zebrafish spinal cord, as compared to the non-SLRP-enriched ECM lesioned controls. Moreover, enrichment of Lum and Prelp (but not of Chad or Fmoda) decreased a measured parameter called longitudinal modulus. Such decrease in the longitudinal modulus may be interpreted as an increase in the tissue compressibility as compared to non-SLRP-enriched, lesioned controls. Hence, the authors suggest that SLRPs inhibit axonal re-growth and spinal regeneration by altering the structural and mechanical properties of the ECM in the lesioned spinal cord of the zebrafish. In particular, they put forward Lum and Prelp as the causal factors that make the difference between the poorly regenerative mammalian spinal cord, which highly expresses these two specific SLRPs, and the highly regenerative fish spinal cord, in which Lum and Prelp abundance does not increase upon injury.

Tags: extracellular matrix, fish, movement, regeneration

doi: https://doi.org/10.1242/prelights.33855

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