Fig. 4: Interaction of SPARC on axonal membranes with extracellular collagen I is required for axonal remodeling.

A, B Mouse primary hippocampal neurons were cultured for 3 days and then treated with or without Aβ_25-35 (2.5 µM) for 3 days. Next, neurons were treated with diosgenin (0.1 or 1 µM) or vehicle solution for 4 days. The SPARC level was increased particularly on axonal shafts (yellow arrowheads) in diosgenin-treated neurons (A). SPARC expression level on axons was measured for each treatment (B). *p < 0.05, ***p < 0.001 vs Aβ_25-35 (Aβ)/Vehicle (Veh), one-way ANOVA post-hoc Bonferroni test, mean ± standard error, n = 92–427 axons. Effect size (r) = 0.248, power (1 − β) = 1. C–E Wild-type and 5XFAD mice were administered diosgenin (Dios; 0.1 µmol/kg/day) or vehicle solution (Veh) for a total of 21 days. On administration day 14, anterograde tracer BDA was injected into the hippocampal CA1 region. After 7 days, BDA-positive axons (red), SPARC expression (green), and DAPI (blue) were detected in the PFC (C). The number of BDA-positive axons (D) and percentage of SPARC-positive and BDA-positive or SPARC-negative and BDA-positive axons (E) was measured for each mouse. *p < 0.05, ***p < 0.001 vs 5XFAD/Veh, one-way ANOVA post-hoc Bonferroni test, mean ± standard deviation, n = 5 mice/group. D r = 0.835, 1 − β = 0.722, E r = 0.887, 1 − β = 0.776. F, G Mouse primary hippocampal neurons were cultured for 14 days and treated with Aβ_25-35 (2.5 µM) for 3 days, then with diosgenin (0.1 or 1 µM) or vehicle solution for 4 days. F Dot-like traces of collagen I were observed along degenerated axons in Aβ-treated neurons (blue arrowheads). G Length of axons colocalized with collagen I (pNF-H⁺, collagen I⁺) was measured in each group. ***p < 0.001 vs Aβ/Veh, one-way ANOVA post-hoc Bonferroni test, mean ± standard error, n = 12 images/group. r = 0.951, 1 − β = 0.999. H Immunohistochemistry detected pNF-H-positive axons (green) and collagen I (red) in the PFC of wild-type and 5XFAD mice. Collagen I-positive but pNF-H-negative axons was often observed in 5XFAD mice (white arrows). I Mouse primary hippocampal neurons were cultured for 14 days and treated with Aβ_25–35 (2.5 µM) for 3 days, then with diosgenin (0.1 or 1 µM) or vehicle solution for 4 days. SPARC on plasma membranes and extracellular collagen I were detected by antibodies in non-permeable immunocytochemistry. SPARC on axonal membranes and its colocalization with collagen I were observed in control and Aβ/Diosgenin groups (yellow arrowheads). Collagen I was observed along degenerated-axons in Aβ-treated neurons (blue arrowheads). J Mouse primary hippocampal neurons were cultured on PDL- or collagen I-coated dishes. AAV-Control or AAV-SPARC (5 × 10⁶ GC/µl) was treated for 7 days, and pNF-H-positive axonal lengths were quantified. *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA post-hoc Bonferroni test, mean ± standard error, n = 10–17 images/group, r = 0.740, 1 − β = 0.999. K PDL and collagen I were coated on the left and right side, respectively. AAV-Control- or AAV-SPARC (5 × 10⁶ GC/µl)-treated mouse primary hippocampal neurons were seeded on PDL-coated center part. pNF-H-positive axonal lengths were quantified after 14 days. *p < 0.05, ****p < 0.0001, one-way ANOVA; ^#p < 0.05, two-way ANOVA, post-hoc Bonferroni test, mean ± standard error, 8–13 images/group, r = 0.765, 1 − β = 0.989. A significant SPARC × collagen I interaction was found using repeated-measures two-way ANOVA [F(1, 40) = 5.81, p = 0.0206]. L–N Mouse primary hippocampal neurons were seeded on the soma space (gray) of a triple chamber neuron device and treated with 5 × 10⁷ GC/µl AAV-control for 10 days. Cerulean-labeled axons in the microgrooves were observed using live cell imaging on 10 DIV (days in vitro). After that, Aβ_25–35 (2.5 µM) was treated to soma (gray) and axonal space (pink) for 3 days. Live cell imaging in 13 DIV confirmed Cerulean-labeled axons that originally extended into the microgrooves were atrophied by Aβ_25–35. Then, triple chamber neuron devices were removed, and 5 × 10⁶ GC/µl AAV-control or AAV-SPARC were treated together with 2 µg/ml SPARC neutralizing antibody (SPARC-Ab) or control IgG (IgG). Densities of M axons pursued extracellular collagen I and N axons without pursuing extracellular collagen I were quantified at 20 DIV in each group. *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA, post-hoc Bonferroni test, mean ± standard error, 12 images/group, M r = 0.847, 1–β = 0.999, N r = 0.644, 1 − β = 0.982.