Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants,in vitro

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Following spinal cord injury, a regenerating neurite encounters a glial scar enriched in chondroitin sulfate proteoglycans (CSPGs), which presents a major barrier. There are two points at which a neurite makes contact with glial scar CSPGs: initially, filopodia surrounding the growth cone extend and make contact with CSPGs, then the peripheral domain of the entire growth cone makes CSPG contact. Aggrecan is a CSPG commonly used to model the effect CSPGs have on elongating or regenerating neurites. In this study, we investigated filopodia and growth cone responses to contact with structurally diverse aggrecan variants using the common stripe assay. Using time-lapse imaging with 15-s intervals, we measured growth cone area, growth cone width, growth cone length, filopodia number, total filopodia length, and the length of the longest filopodia following contact with aggrecan. Responses were measured after both filopodia and growth cone contact with five different preparations of aggrecan: two forms of aggrecan derived from bovine articular cartilage (purified and prepared using different techniques), recombinant aggrecan lacking chondroitin sulfate side chains (produced in CHO-745 cells) and two additional recombinant aggrecan preparations with varying lengths of chondroitin sulfate side chains (produced in CHO-K1 and COS-7 cells). Responses in filopodia and growth cone behavior differed between the structurally diverse aggrecan variants. Mutant CHO-745 aggrecan (lacking chondroitin sulfate chains) permitted extensive growth across the PG stripe. Filopodia contact with the CHO-745 aggrecan caused a significant increase in growth cone width and filopodia length (112.7% ± 4.9 and 150.9% ± 7.2 respectively, p < 0.05), and subsequently upon growth cone contact, growth cone width remained elevated along with a reduction in filopodia number (121.9% ± 4.2; 72.39% ± 6.4, p < 0.05). COS-7 derived aggrecan inhibited neurite outgrowth following growth cone contact. Filopodia contact produced an increase in growth cone area and width (126.5% ± 8.1; 150.3% ± 13.31, p < 0.001), and while these parameters returned to baseline upon growth cone contact, a reduction in filopodia number and length was observed (73.94% ± 5.8, 75.3% ± 6.2, p < 0.05). CHO-K1 derived aggrecan inhibited neurite outgrowth following filopodia contact, and caused an increase in growth cone area and length (157.6% ± 6.2; 117.0% ± 2.8, p < 0.001). Interestingly, the two bovine articular cartilage aggrecan preparations differed in their effects on neurite outgrowth. The proprietary aggrecan (BA I, Sigma-Aldrich) inhibited neurites at the point of growth cone contact, while our chemically purified aggrecan (BA II) inhibited neurite outgrowth at the point of filopodia contact. BA I caused a reduction in growth cone width following filopodia contact (91.7% ± 2.5, p < 0.05). Upon growth cone contact, there was a further reduction in growth cone width and area (66.4% ± 2.2; 75.6% ± 2.9; p < 0.05), as well as reductions in filopodia number, total length, and max length (75.9% ± 5.7, p < 0.05; 68.8% ± 6.0; 69.6% ± 3.5, p < 0.001). Upon filopodia contact, BA II caused a significant increase in growth cone area, and reductions in filopodia number and total filopodia length (115.9% ± 5.4, p < 0.05; 72.5% ± 2.7; 77.7% ± 3.2, p < 0.001). In addition, filopodia contact with BA I caused a significant reduction in growth cone velocity (38.6 nm/s ± 1.3 before contact, 17.1 nm/s ± 3.6 after contact). These data showed that neuron morphology and behavior are differentially dependent upon aggrecan structure. Furthermore, the behavioral changes associated with the approaching growth cone may be predictive of inhibition or growth.

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