Sulfation patterns of chondroitin sulfates at the glial scar induce distinct reactive astrogliosis responses

Astrocytes play an essential role in regulating homeostasis and physiological functions within the central nervous system (CNS), including the homeostasis of extracellular matrix (ECM) formation and degradation. Following traumatic brain or spinal cord injury (SCI), astrocytes upregulate the expression of chondroitin sulphate proteoglycans (CSPGs), which facilitate the formation of glial scar, and inhibit axonal regeneration and neurite outgrowth. Previous studies have demonstrated that enzymatic removal of CS glycosaminoglycans (GAGs) on CSPGs using chondrotinase ABC (ChABC) enhances axonal regeneration and facilitates functional recovery. However, the specific role of CS-GAGs on astrocyte themselves remains to be elucidated. Here, we hypothesise that upregulated CS-GAGs at the glial scar contribute to the maintenance of reactive astrogliosis post-injury in the CNS.
We first assessed the presence of known CS receptors on the astrocyte surface. Both Contactin-1 (CNTN-1) and receptor tyrosine phosphatase-sigma (PTPRσ) were observed on the astrocyte, suggesting their ability to bind and potentially react to CS-GAGs. Primary astrocytes isolated from rat cortices were then treated with varying concentrations of chondroitin 4 sulphate (C4S) and chondroitin 6 sulphate (C6S), two major CS sulphation, to assess their metabolic activity and proliferation. Our results showed that incubation with C4S results in a dose-dependent decrease in astrocyte proliferation (up to 200 μg/ml), but C6S did not. Both molecules do not affect the cell viability up to 1 mg/ml and metabolic activity. Immunocytochemistry analyses revealed that both C4S and C6S treatment increases GFAP expression and induces reactive astrogliosis in 200 μg/ml and 1 mg/ml concentration. However, they induce distinct phenotypes of reactive astrogliosis. C4S induces the S100 calcium binding protein A 10 (S100A10)-positive anti-inflammatory A2 reactive astrogliosis and the complement component 3 (C3) proinflammatory reactive astrogliosis marker, while C6S induces the C3 expression only. . C4S reduces the size of astrocytes, circularity, and solidity, but C6S only reduces the circularity. Both molecules affect the CNTN1 distribution on the astrocytic surface, and CNTN1 may play a role in this process. In conditional carbohydrate sulfotransferase 11 (CHST11) knockout (KO) mice, which show reduction in C4S synthesis in the CNS, we confirmed that C4S play a vital role in A2 reactive astrogliosis and it has a crucial role in morphological changes induced by reactive astrogliosis. Our findings suggest that C4S and C6S may have a role in inducing reactive astrogliosis and maintaining glial scar after spinal cord injury, and the mechanism under this process can be an important therapeutic target to provide regeneration after spinal cord injury.