Targeted downregulation of N‐acetylgalactosamine 4‐sulfate 6‐O‐sulfotransferase significantly mitigates chondroitin sulfate proteoglycan‐mediated inhibition

Abstract

Chondroitin Sulfate-4,6 (CS-E) glycosaminoglycan (GAG) upregulation in astroglial scars is a major contributor to CS proteoglycan (CSPG) mediated inhibition (Gilbert et al. 2005). However, the role of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S6ST) catalyzed sulfation of CS-E, and its contribution to CSPG mediated inhibition of CNS regeneration remains to be fully elucidated. Here, we used in situ hybridization to show localized upregulation of GalNAc4S6ST mRNA after CNS injury. Using in vitro spot assays with immobilized CS-E, we demonstrate dose dependent inhibition of rat embryonic day 18 (E18) cortical neurons. To determine whether selective downregulation of CS-E affected the overall inhibitory character of extracellular matrix produced by reactive astrocytes, single [against (chondroitin 4) sulfotransferase 11 (C4ST1) or GalNAc4S6ST mRNA) or double (against C4ST1 and GalNAc4S6ST mRNA] siRNA treatments were conducted and assayed using quantitative real-time PCR (qRT-PCR) and high performance liquid chromatography (HPLC) to confirm the specific downregulation of CS-4S GAG (CS-A) and CS-E. Spot and Bonhoeffer stripe assays using astrocyte conditioned media (ACM) from siRNA treated rat astrocytes showed a significant decrease in inhibition of neuronal attachment and neurite extensions when compared to untreated and TGFα treated astrocytes. These findings reveal that selective attenuation of CS-E via siRNA targeting of GalNAc4S6ST significantly mitigates CSPG mediated inhibition of neurons, potentially offering a novel intervention strategy for CNS injury.

Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation

“…Although much evidence has accumulated suggesting that it is the GAG chain moieties that are recognized by nerve growth cones, fewer studies have specifically addressed the differential effects that individual GAG moities have on a particular nerve cell. 13,20,22,46,47,100,101 Here, we used a modified spot assay designed to mimic an in vivo environment whereby GAGs are immobilized and presented solely to nerve growth cones, but not to their neuronal cell bodies, similar to how GAGs would be encountered by growth cones in a developing embryo. Our results show that each GAG tested in the study displayed its own unique potential for inhibiting trigeminal nerve growth cone migration in vitro.…”

“…In contrast to our studies using sensory neurons of the PNS, rat CNS embryonic cortical neurons did not display increased fasciculation when growth cones were presented with the same purified, commercial preparation of CSA as was used in our study. 13 Moreover, immobilized, purified CSA and KS were shown to significantly increase CNS dopaminergic (DA) neurite branching on poly-L-ornithine. 46 Collectively, data from these studies and ours indicate that the differential effects on neurite behavior when encountering immobilized GAGs, likely depends on the source of the neuron (PNS versus CNS), as well as on the actual charge density/GAG density on the surface.…”

“…8,13,38 Such differences may be attributed to a difference in PG or GAG conformation, in turn altering their ability to interact with neurite growth cone receptors and may shed insight into the mechanism by which GAGs regulate neurite growth cone behavior in vivo, since growth cones may encounter PGs or GAGs in varying forms in vivo, such as bound to a cell surface or to ECM (immobilized) or as soluble molecules diffusing through the ECM.…”

“…3,4 Chondroitin sulfate proteoglycans (CSPGs) influence many biological functions, including cell adhesion, cell migration, axonal guidance and pathfinding, and axonal fasciculation/ defasciculation behavior. [5][6][7] With respect to axonal growth cone movement, CSPGs in the ECM have been shown to be inhibitory both in vitro [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] and in vivo. [23][24][25][26][27][28][29] Interestingly, other studies have reported positive effects of CSPGs on neurite outgrowth.…”

Pathophysiology of the brain extracellular matrix: a new target for remyelination

“…The disaccharide with sulphates at both the C4 and C6 positions of GalNAc has been shown to be particularly responsible for the inhibitory properties of GAGs [17][18][19] , probably by binding to a newly described CSPG receptor, protein tyrosine phosphatase-σ (PTPσ) 19,20 . Intriguingly, it has been suggested that CSPGs with C6-sulphated GalNAc may actually be permissive for axonal regrowth 21 .…”