Increased Expression of Transcription Factor SRY-box-Containing Gene 11 (Sox11) Enhances Neurite Growth by Regulating Neurotrophic Factor Responsiveness

Abstract

The peripherally projecting axons of dorsal root ganglion (DRG) neurons readily regenerate after damage while their centrally projecting branches do not regenerate to the same degree after injury. One important reason for this inconsistency is the lack of pro-regeneration gene expression that occurs in DRG neurons after central injury relative to peripheral damage. The transcription factor SRY-box-containing gene 11 (Sox11) may be a crucial player in the regenerative capacity of axons as previous evidence has shown that it is highly upregulated after peripheral axon damage but not after central injury. Studies have also shown that overexpression or inhibition of Sox11 after peripheral nerve damage can promote or block axon regeneration, respectively. To further understand the mechanisms of how Sox11 regulates axon growth, we artificially overexpressed Sox11 in DRG neurons in vitro to determine if increased levels of this transcription factor could enhance neurite growth. We found that Sox11 overexpression significantly enhanced neurite branching in vitro, and specifically induced the expression of glial cell line-derived neurotrophic factor (GDNF) family receptors, GFRα1 and GFRα3. The upregulation of these receptors by Sox11 overproduction altered the neurite growth patterns of DRG neurons alone and in response to growth factors GDNF and artemin; ligands for GFRα1 and GFRα3, respectively. These data support the role of Sox11 to promote neurite growth by altering responsiveness of neurotrophic factors and may provide mechanistic insight as to why peripheral axons of sensory neurons readily regenerate after injury, but the central projections do not have an extensive regenerative capacity.

Keywords: dorsal root ganglia; molecular biology; neurite growth; neurotrophic factor; regeneration; transcription factor.

Figure 1. Penetratin-1 enhances the transfection efficiency of primary DRG neurons and promotes increased protein expression in vitro

A: Few primary DRG neurons express a GFP reporter when transfected with naked CMV-Sox-IRES-eGFP plasmids (arrow). B: In contrast, binding plasmids with Penetratin-1 greatly enhances the transfection of primary DRG neurons (arrows). C: Sox11 mRNA is substantially enhanced in DRG cultures compared to IRES control at both two and three days in vitro. Sox11 protein expression is also significantly increased compared to Untreated and IRES controls. *p<0.001; n=3 mice per condition. D: Co-expression of eGFP and NeuN is minimal (arrows) in CMV-Sox-IRES-eGFP treated cultures (n=3 mice). Most NeuN positive cells do not contain eGFP (dashed arrows) E: Extensive co-labeling of NeuN and eGFP (arrows) is detected in Penetratin bound Sox11 containing plasmids (n=3 mice). Scale Bar, 100μm (all panels).

Figure 2. Overexpression of Sox11 enhances neurite branching but not maximum neurite length in DRG neurons in vitro

A: Vector control (PenIRES) transfected neurons immunoreactive for PGP9.5 exhibit some branching and neurite elongation after three days in vitro. B: Sox11 transfected neurons (PenSOX) immunoreactive for PGP9.5 display significant branching at three days. Scale Bar (A and B), 200μm. Higher magnification images of neurons in cultures treated with PenIRES (C) and PenSOX (D). Scale Bar (C and D), 100μm. E: The number of primary neurites three days in vitro in Sox11 transfected neurons (PenSOX) was greater relative to vector control transfected neurons and untreated neurons (**p<0.05). Number of primary neurites in the PenIRES condition was also significantly increased from untreated (*p<0.05). F: The number of secondary neurites three days in vitro in Sox11 transfected neurons (PenSOX) was also greater relative to vector control transfected neurons (PenIRES) and untreated neurons (**p<0.05). G: Maximum neurite length three days in vitro in Sox11 transfected neurons (PenSOX) however, was not significantly different relative to vector control transfected neurons (PenIRES) or untreated neurons. n=4 mice per condition.

Figure 3. Sox11 enhances responsiveness to the neurotrophic factor artemin

A: Primary DRG neurons immunopositive for PGP9.5 transfected with vector control plasmids and treated with artemin (PenIRES+ART) exhibit extensive branching. B: Primary DRG neurons transfected with Sox11 containing plasmids and treated with artemin (PenSOX+ART) display less higher order branching, but have longer neurites. Scale Bar (A and B), 200μm. Higher magnification images of neurons in cultures treated with PenIRES+ART (C) and PenSOX+ART (D). Scale Bar (C and D), 40μm. E: Quantification of higher order branching in primary DRG neurons treated with Sox11 containing plasmids and artemin (PenSOX+ART) are significantly reduced relative to control plasmid transfected cells treated with artemin (PenIRES+ART). F: Maximum neurite length is substantially increased with overexpression of Sox11 in the presence of artemin (PenSOX+ART) in primary DRG neurons relative to control conditions (PenIRES+ART). Data from PenSOX treated cultures alone is provided for reference. *p<0.05; n=6 mice per condition.

Figure 4. Sox11 alters responsiveness to the neurotrophic factor GDNF

A: Primary DRG neurons immunopositive for PGP9.5 transfected with vector control plasmids and treated with GDNF (PenIRES+GDNF) exhibit some branching and a few long neurites. B: Primary DRG neurons transfected with Sox11 containing plasmids and treated with GDNF (PenSOX+GDNF) however, display more branching, but shorter neurites. Scale Bar (A and B), 200μm. Higher magnification images of neurons in cultures treated with PenIRES+GDNF (C) and PenSOX+GDNF (D). Scale Bar (C and D), 40μm. E: Quantification of higher order branching in primary DRG neurons treated with Sox11 containing plasmids and GDNF (PenSOX+GDNF) are significantly increased relative to control plasmid transfected cells treated with GDNF (PenIRES+GDNF). F: Maximum neurite length is substantially decreased with overexpression of Sox11 in the presence of GDNF (PenSOX+GDNF) in primary DRG neurons relative to control conditions (PenIRES+GDNF). Data from PenSOX treated cultures alone is provided for reference. *p<0.05; n=6 mice per condition.

Figure 5. Transfection of Sox11 plasmid treated DRG cultures with GFRα3 targeting siRNAs significantly inhibits the expression of GFRα3 protein

A: Examples of western blot images for Sox11, GFRα3 and GAPDH obtained from cultures treated with Penetrain-bound Sox11 containing plasmids (PenSOX) and either Penetratin-linked control (PenCON) or GFRα3 targeting siRNAs (Penα3). Untreated and PenIRES transfected control cultures are provided for reference. B: Increased expression of Sox11 is verified at the protein level under both conditions. C: Sox11-induced increase of GFRα3 protein is significantly reduced with Penα3 siRNAs. F_{2, 6}= 9.7, p<0.02; n=3–4 mice per condition.

Figure 6. siRNA-mediated inhibition of the GDNF family receptors GFRα3 and GFRα1 blocks the effects of overexpressing Sox11 in vitro

A: Cells treated with vector control plasmids (PenIRES) and non-targeting control siRNAs (PenCON) display moderate branching in vitro. B: PenCON transfected cultures with Sox11 overexpression (PenSOX) display increased neurite branching. C: Knockdown of GFRα3 (Pena3) in vector control treated cultures does not significantly alter neurite growth. D: Inhibition of GFRα3 (Pena3) in cells co-transfected with Sox11 containing plasmids (PenSOX) show reduced branching. E: Similar to GFRα3 knockdown, inhibition of GFRα1 (Penα1) in vector control treated cultures does not significantly alter neurite growth. F: However, knockdown of GFRα1 in cells with enhaced Sox11 also reduces neurite branching. Scale Bars, 200μm. G–I: Higher order branching in vector control treated cells that are also treated with PenCON siRNAs is not altered by knockdown of either GFRα3 or GFRα1. However, the Sox11 induced increased in branching in PenCON treated conditions is blocked by GFRα1 and GFRα1 treatment. J–L: Similar effects of GFRα3 and GFRα1 targeting siRNAs are found in regard to primary neurite numbers whereby the Sox11 induced increase in primary neurites observed in control conditions of blocked by knockdown of GFRα3 and GFRα1. *p<0.05 vs. controls; n=3 mice per condition.

Figure 7. Inhibition of the Sox11-induced expression of GDNF family receptors GFRα3 and GFRα1 blocks the effects of artemin and GDNF on neurite growth, respectively

A: Higher order branching in primary DRG neurons transfected with Sox11 containing plasmids and treated with artemin display more branching with knockdown of GFRα3 (PenSOX+Penα3+ART) compared to control siRNA transfected cells (PenSOX+PenCON+ART). *p value < 0.05. B: Maximum neurite length is substantially decreased when cells are transfected with Sox11 containing plasmids and treated with artemin and GFRα3 siRNA (PenSOX+Penα3+ART) compared to treatment with control siRNA (PenSOX+PenCON+ART). ^#p<0.08; *p<0.05. C: Higher order branching in primary DRG neurons transfected with Sox11 containing plasmids and treated with GDNF and GFRα1 siRNA (PenSOX+Penα1+GDNF) exhibit more branching compared to control siRNA transfected cells (PenSOX+PenCON+GDNF), although not statistically significant. D: Maximum neurite length is substantially increased when transfected with Sox11 containing plasmids and treated with GDNF and GFRα1 siRNA (PenSOX+Penα1+GDNF) compared to controls (PenSOX+PenCON+GDNF). *p value < 0.002; n=6 mice per condition (artemin treated) and n=5 mice per condition (GDNF treated).