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

Abstract

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 filopodial and growth cone responses to contact with structurally diverse aggrecan variants using the common stripe assay. Using time-lapse imaging with 15-sec 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), 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...

An in vitro assay to study induction of the regenerative state in sensory neurons

“…The fold change in neurite outgrowth was the same regardless of substrates, but the absolute growth was still not as robust as neurons replated onto control substrates. Thus, the reduced absolute growth on CSPGs is not likely a result of impaired activation or inactivation of the pro-regenerative program, but instead may be due to activation of distinct pathways to inhibit neurite outgrowth (Beller et al, 2013; Dickendesher et al, 2012; Fisher et al, 2011; Hur et al, 2011; Monnier et al, 2003; Shen et al, 2009; Sivasankaran et al, 2004). This highlights the important need to target both intrinsic and extrinsic mechanisms to achieve effective regeneration.…”

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

“…These cultures can be used for specifically analyzing growth cones in addition to analyzing the entire neuron. Previously, this procedure has been used to evaluate growth cone velocity and behaviors, such as growth cone collapse 8,9,11,13,16 . Lower plating densities on these coverslips results in dead neurons that are not adhered to the substrata.…”

“…For example, intracellular levels of cAMP and surface levels of integrins are significantly different in neurons plated on these two substrata 7,8 . Additional studies have shown that other molecules, including fibronectin and chondroitin sulfate proteoglycans, impact the expression of cell surface molecules and neuronal motility [7][8][9][10][11] . In addition, soluble molecules such as neurotrophins and neurotropins also impact cell membrane composition and neuronal motility [12][13][14][15][16] and can be easily and accurately manipulated in a cell culture model.…”

SDF-1 overexpression by mesenchymal stem cells enhances GAP-43-positive axonal growth following spinal cord injury

“…Although the precise composition of the tissue existing within the lesion environment was not identified in this study, there was a robust density of CSPGs and IBA-1 labeled cells in the tissue within the lesion site. As CSPGs have been reported to lead to the collapse of the growth cone, the fact that the transplant/lesion site was dense in CSPGs, while also containing a greater labeling of GAP-43 in SDF-1-MSC-treated animals, creates a perplexing contradiction (Beller et al, 2013;Schmalfeldt, Bandtlow, Dours-Zimmermann, Winterhalter, & Zimmermann, 2000). Such findings are suggestive that the secretome of MSCs, particularly when enhanced with SDF-1␣ expression, can help overcome the inhibitory nature that CSPGs exert on growth and regeneration.…”