Volume 17, Issue 12, Pages 2020-2030 (December 2009) Targeting a Dominant Negative Rho Kinase to Neurons Promotes Axonal Outgrowth and Partial Functional Recovery After Rat Rubrospinal Tract Lesion  Dongsheng Wu, Ping Yang, Xinyu Zhang, Juan Luo, Mohammed E Haque, John Yeh, Peter M Richardson, Yi Zhang, Xuenong Bo  Molecular Therapy  Volume 17, Issue 12, Pages 2020-2030 (December 2009) DOI: 10.1038/mt.2009.168 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Flag-tagged DNROCK blocks the formation of stress fibers in NIH 3T3 cells. NIH 3T3 cells were transfected with either (a–c) pRRL/GFP or (d–f) pRRL/DNROCK and starved of serum followed by serum challenge. F-actin was stained with rhodamine-conjugated phalloidin (red in b,c,e,f). Flag-tagged DNROCK was detected by immunostaining with an anti-Flag antibody (green in d,f). Bar = 50 µm. DNROCK, dominant negative Rho kinase; GFP, green fluorescent protein. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Expression of DNROCK in DRG neurons. Expression of Flag-tagged DNROCK was detected with anti-Flag antibody (green in a and c) in LV-transduced DRG neurons. DRG neurons were labeled with anti-PGP9.5 (red in b and c). (d) Immunoblotting using anti-Flag antibody detected the expression of Flag-tagged DNROCK (band is indicated by an arrow) in LV/DNROCK transduced DRG neurons; while the band for Flag-DNROCK was absent in LV/GFP transduced neurons. Bar = 50 µm. DNROCK, dominant negative Rho kinase; DRG, dorsal root ganglion; LV, lentiviral vector; PGP9.5, protein gene product 9.5. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 DNROCK enhances neurite outgrowth of DRG neurons on myelin substrate. DRG neurons transduced with (a) LV carrying GFP cDNA were plated on laminin (0.1 µg/well) or (b) laminin (0.1 µg/well) + myelin protein (40 µg/well) coated coverslips. DRG neurons transduced with (c) LV/DNROCK were plated on laminin or (d) laminin + myelin protein coated coverslips. Merged photomicrographs showing GFP expression (green) in LV/GFP transduced cells in a and b, and Flag-tagged DNROCK expression (green) in LV/DNROCK transduced cells in c and d. Neuronal marker PGP9.5 immunoreactivity (red) is used to define neurons and their neurites. (e) Quantification of neurite length and (f) percentage of neurite-bearing neurons in LV/GFP and LV/DNROCK groups. Values are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 [ANOVA with post hoc comparisons and Bonferroni correction on SPSS software] (Chicago, IL). Bar = 100 µm. ANOVA, analysis of variance; cDNA, complementary DNA; DNROCK, dominant negative Rho kinase; DRG, dorsal root ganglion; GFP, green fluorescent protein; LV, lentiviral vector. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 DNROCK inhibits phosphorylation of CRMP2 in the growth cones of neurons. Dorsal root ganglion neurons were infected with (a,c) LV carrying GFP cDNA or (e,g) Flag-tagged DNROCK cDNA in the (a,e) absence or (c,g) presence of myelin proteins (40 µg/well). Merged micrographs showing the expression of GFP in (a,c) LV/GFP transduced cells and phosphorylated CRMP2 immunoreactivity (pCRMP2, red), or (e,g) the expression of Flag-DNROCK (green) and pCRMP2 immunoreactivity (red). The immunoreactivity of pCRMP2 in the associated axons and growth cones (framed areas in a,c,e,g) are presented in confocal micrographs (b,d,f,h). (i) Immunofluorescence of pCRMP2 at growth cones was quantified by densitometry. Data presented are mean ± SEM of arbitrary units of densities measured with ImageJ. *P < 0.05, **P < 0.01, ***P < 0.001. Bar = 50 µm for a,c,e,g; 10 µm for b,d,f,h. cDNA, complementary DNA; DNROCK, dominant negative Rho kinase; GFP, green fluorescent protein; LV, lentiviral vector; pCRMP2, phosphor collapsin response mediator protein 2. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Neuron-specific expression of GFP and DNROCK in red nucleus and rubrospinal tract. (a) Many cells in the right red nucleus were transduced by a LV carrying GFP cDNA 18 days after injection of viruses. (b) A higher magnification of a. (c) GFP-labeled rubrospinal tract axons in left lateral funiculus were shown in a horizontal section of cervical spinal cord and some axons projected to the gray matter. (d) Transverse section of cervical spinal cord demonstrates the discrete location of GFP-labeled rubrospinal tract axons in the superficial dorsolateral quadrant of cervical spinal cord and projection to the intermediate zone of the gray matter. (e) A higher magnification of d. (f) Merged photomicrograph demonstrates that GFP+ cells (green) were colocalized (yellow) with neuronal marker NeuN (red). (g,h) Confocal photomicrographs demonstrating the co-transduction of neurons by LV/GFP (green in g) and LV/DNROCK (shown as immunoreactivity of Flag tag, red in h). (i) Merged image of g and h shows that most neurons (yellow) were co-transduced by both viral vectors. (j–l) Confocal photomicrographs showing that both GFP (green in j) and Flag-DNROCK proteins (red in k, shown as immunoreactivity of Flag tag) were transported along the same axons of rubrospinal tract after the co-transduction of neurons in red nucleus by both LV/GFP and LV/DNROCK. GFAP staining was shown as blue in l. Bar = 200 µm for a–e; 50 µm for g–i; 100 µm for f and j–l. cDNA, complementary DNA; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; LV, lentiviral vector. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 GFP-labeled rubrospinal tract axons in cervical spinal cord 10 weeks after left rubrospinal tract transection. (a) A horizontal section from an animal injected with a LV carrying GFP cDNA shows that most of GFP-labeled axons forming end bulbs stopped before they reached the injury site. Lesion border is demonstrated by the immunoreactivity of GFAP (blue). (b) An image from a LV/DNROCK-injected animal shows a panoramic view of the rostral side, the rostral border of the lesion cavity, the lesion cavity, and the caudal side. Letter-labeled white boxes indicate the areas enlarged in c–k. (c) Enlarged image from b shows GFP-labeled rubrospinal axons growing toward the rostral border and inside the lesion cavity (arrowheads). (d,e) Axons in the rostral border of the lesion cavity, with some growing beyond the border of glial scar (arrowheads). (f) Axons growing inside the lesion cavity along the glial scar. (g,h) Axons around the internal wall of the lesion cavity. (i) Axons near the caudal border of the lesion cavity. (j,k) Axons caudal to the lesion site. a, c and d–k are images from a confocal microscope. (l,m) GFP-labeled rubrospinal axons 5 mm rostral to the lesion site (l) and 5 mm caudal to the lesion centre (m) in transverse sections of spinal cord in GFP group. (n,o) GFP-labeled rubrospinal axons 5 mm rostral to the lesion site (n) and 5 mm caudal to the lesion centre (o) in transverse sections of spinal cord in DNROCK group. Arrow heads in m and o show the representative regenerating axons in left lateral funiculus of white matter. Arrow in o shows a regenerating axon growing toward its original target in the intermediate zone of gray matter. Blue in a–k represents immunoreactivity of GFAP. Bar = 100 µm for a–c; 50 µm for d–k as shown in f; 100 µm for l–o as shown in l. DNROCK, dominant negative Rho kinase; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; LV, lentiviral vector. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 DNROCK promotes axonal sprouting rostral and causal to the site of rubrospinal tract transection. (a) A drawing illustrates the injury site of the spinal cord and the regions of spinal sections used for quantification of GFP-labeled axons. (b) Projection of camera lucida drawings based on three sections from one typical animal from GFP group and (c) DNROCK group schematically show the lesion cavity and the distribution of regenerating axons 10 weeks after injury. (d) Quantification of GFP-labeled rubrospinal axon numbers at both rostral and caudal sides of the lesion cavity. The numbers of axons presented were mean ± SEM from six animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001 via one-way ANOVA for each of the distance index. ANOVA, analysis of variance; DNROCK, dominant negative Rho kinase; GFP, green fluorescent protein. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Expression of DNROCK promotes the recovery of limb functions after rubrospinal tract transection. Percentage of usage of (a) contralateral (right) forelimbs, (b) ipsilateral (left) forelimbs, and (c) both forelimbs in cylinder test. (d) Hindlimb functional recovery was assessed using error index (total slip/step ratio) of horizontal rope crossing test. Two-way ANOVA tests of all the time points after injury combined for each of the four indexes demonstrate significant difference between the DNROCK and GFP groups (P < 0.0001). Student's t-test was used to compare the difference at each time point between the two groups. *P < 0.05, **P < 0.01. Data are expressed as mean ± SEM, n = 6 for each group. ANOVA, analysis of variance; DNROCK, dominant negative Rho kinase; GFP, green fluorescent protein. Molecular Therapy 2009 17, 2020-2030DOI: (10.1038/mt.2009.168) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions