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

abstract Article history:Received 19 December 2012Revised 6 February 2013Accepted 18 February 2013Available online 1 March 2013Keywords:Neuronal growth conesDorsal root ganglia neuronsVelocityExtracellular matrix (ECM)RegenerationCell culture Following spinal cord injury, a regenerating neurite encoun ters a glial scar enriched in chondroitin sulfate proteo-glycans (CSPGs), which presents a major barrier. There are two points at which a neurite makes contact withglialscarCSPGs:initially, filopodiasurroundingthegrowthconeextendandmakecontactwithCSPGs,thenthepe-ripheral domain of the entire growth cone makes CSPG contact. Aggrecan is a CSPG commonly used to model theeffect CSPGs have on elongating or regenerating neurites. In this study, we investigated filopodia and growthcone responses to contact with structurally diverse a ggrecan variants using the common stripe assay. Usingtime-lapse imaging with 15-s intervals, we measured g rowth 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 ofaggrecan: two forms of aggrecan derived from bovine articular cartilage (puri fied and prepared using differenttechniques),recombinantaggrecanlac kingchondroitinsulfatesidechains(p roducedinCHO-745cells)andtwoad-ditional recombinant aggrecan preparations with varying l engths of chondroitin sulfate side chains (produced inCHO-K1 and COS-7 cells). Responses in filopodia and growth cone behavior differed between the structurally di-verseaggrecanvariants.MutantCHO-745aggrecan(lacking chondroitinsulfatechains)permittedextensivegrowthacross the PG stripe. Filopodia contact with the CHO-745 aggrecan caused a signi ficant increase in growth conewidthand filopodialength(112.7% ± 4.9and150.9% ± 7.2respectively,p b 0.05),andsubsequentlyupongrowthcone contact, growth cone width remained elevated along with a reduction in filopodia number (121.9% ± 4.2;72.39% ± 6.4, p b 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 b 0.001), and while these parameters returned to baseline upon growth cone contact, a reduction in filopodianumber and length was observed (73.94% ± 5.8, 75.3% ± 6.2, p b 0.05). CHO-K1 derived aggrecan inhibitedneurite outgrowth following filopodia contact, and caused an increase in growth cone area and length(157.6% ± 6.2;117.0% ± 2.8,p b 0.001).Interestingly,thetwobovinearticu larcartilageaggrecanpreparationsdif-feredin theireffectson neurite outgrowth. The proprieta ryaggrecan (BA I, Sigma-Aldri ch)inhibitedneurites at thepoint of growth cone contact, while our chemically puri fied aggrecan (BA II) inhibited neurite outgrowth at thepoint of filopodia contact. BA I caused a reduction in growth cone width following filopodia contact (91.7% ± 2.5,p b 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 b 0.05), as well as reductions in filopodia number, total length, and max length (75.9% ± 5.7,p b 0.05;68.8% ± 6.0;69.6% ± 3.5,p b 0.001).Upon filopodiacontact,BAIIcausedasigni ficantincreaseingrowthcone area, and reductions in filopodia number and total filopodia length (115.9% ± 5.4, p b 0.05; 72.5% ± 2.7;77.7% ± 3.2,p b 0.001).Inaddition, filopodiacontactwithBAIcausedasigni ficantreductioningrowthconeveloc-ity (38.6 nm/s ± 1.3 before contact, 17.1 nm/s ± 3.6 after contact). These data showed that neuron morphologyandbehavioraredifferentiallydependentuponaggrecans tructure.Furthermore,thebehavioralchangesassociatedwith the approaching growth cone may be predictive of inhibition or growth.© 2013 Elsevier Inc. All rights reserved.