Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

doi: 10.1073/pnas.2017282118

Figure Lengend Snippet: Fig. 1. CNTF gene therapy, but not rCNTF, induces optic nerve regeneration. (A) Longitudinal sections through mouse optic nerve immunostained for the anterograde tracer CTB (green) to visualize regenerating axons 2 wk after NC with the indi- cated treatments. The asterisk indicates the injury site. (Scale bar, 150 μm.) (B) Whole-mounted retinas immunostained with antibody TUJ1+ (green) to visualize βIII tubulin, a marker for RGCs (treatments as in A). (Scale bar, 60 μm.) (C) Quantitation of regenerating axons 0.5 mm distal to the injury site. Whereas recombinant (rCNTF) did not promote regeneration at any concentration (Conc), CNTF gene therapy was highly effective. ***P < 0.001 (AAV2-CNTF vs. AAV2-GFP; n = 10 nerves per group). (D) Quantitation of cell survival. Whereas rCNTF did not protect RGCs, CNTF gene therapy increased cell survival by 39%. **P < 0.01 (AAV2-CNTF vs. AAV2-GFP; n = 7 retinas per group). (E and F) Expression of SOCS3. (E) Retinal cross-sections immunostained for SOCS3 (red) in TUJ1-positive RGCs (green). (Scale bar, 30 μm.) (F) CNTF gene therapy did not alter levels of SOCS3 in RGCs (P = 0.561; n = 6 to 7 retinas per group). Bars show means ± SEM. n.s., not significant.

Article Snippet: Reagents that were injected intraocularly include recombinant rat CNTF protein (0.03 to 0.5 μg/μL; 3 μL per eye; Alomone Labs); recombinant mouse CCL5 (0.1 μg/μL; 3 μL per eye; ThermoFisher Scientific); zymosan (12.5 μg/μL; sterilized before use; Sigma); recombinant rat Ocm (30 ng/μL; 3 μL per eye); the cAMP analog CPT-cAMP (50 μM; Sigma); AMD3100, a highly specific CXCR4 antagonist (100 μM; half-maximal inhibitory concentration [IC50] = 0.02 to 0.13 μM; Sigma); the Ocm peptide antagonist P1 (10) (2.3 μg/μL; 3 μL per eye); AAVs expressing green fluorescent protein (AAV2-GFP), AAV2 expressing Cas9 driven by the RGC-selective promoter γ-synuclein (Sncg) (AAV2-Sncg-Cas9), and AAV2 expressing CNTF (all from Boston Children’s Hospital Viral Vector Core); AAV2 expressing shRNA targeting CNTF receptor-α (AAV2-sh-CNTFRα; Vector Biolabs); AAV2 expressing a small guide RNA targeting CCR5 (AAV2-sgCCR5; Vigene Biosciences); and AAV2 expressing a small guide RNA targeting GPR75 (AAV2-sgGPR75; Vigene Biosciences).

Techniques: Marker, Quantitation Assay, Recombinant, Concentration Assay, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

doi: 10.1073/pnas.2017282118

Figure Lengend Snippet: Fig. 2. CNTF gene therapy does not require CNTFRα expression in RGCs. (A–D) Localization and knockdown of CNTFRα. (A) Whole- mounted retinas immunostained for CNTFRα (red), TUJ1-positive RGCs and fiber bundles (green) (top row), and GFAP-positive astro- cytes (green) (lower two rows). (Scale bar, 40 μm.) (B) In situ hy- bridization detected low levels of CNTFRα mRNA (white puncta) in the RGCs (stained with antibody to RBPMS [red] to delineate RGC cell bodies but not axon bundles). (Scale bar, 10 μm.) (C) Quantita- tion of colocalization frequency. CNTFRα colocalizes with astrocytes but not with RGCs or axon bundles. ***P < 0.001 (Mander’s value; CNTFRα with GFAP vs. CNTFRα with TUJ1; n = 4 retinas per group). CNTF gene therapy did not alter CNTFRα intensity or localization. (D) Quantitation of CNTFRα mRNA in RGCs. CNTFRα mRNA was knocked down in RGCs 2 wk after intraocular injection of AAV2 expressing an shRNA (AAV2-sh-CNTFRα). ***P < 0.001 (AAV2-sh- CNTFRα vs. AAV2-GFP; n = 4 retinas per group). (E–H) RGC- selective knockdown of CNTFRα does not diminish the effects of CNTF gene therapy. (E) Regenerating axons visualized by CTB immunostaining (green). The asterisk indicates the injury site. (Scale bar, 150 μm.) (F) Whole-mounted retinas immunostained with an- tibody TUJ1+ (green) to visualize βIII tubulin-positive RGCs. (Scale bar, 60 μm.) (G) Quantitation of axon regeneration 0.5 mm distal to the injury site 2 wk after nerve injury. RGC-selective CNTFRα knockdown did not alter the effects of CNTF gene therapy on axon regeneration (P = 0.344; n = 7 to 8 nerves per group). (H) Quantitation of RGC survival. CNTFRα knockdown in RGCs did not alter the neuro- protective effects of CNTF gene therapy (P = 0.538; n = 9 retinas per group). Bars show means ± SEM. n.s., not significant.

Article Snippet: Reagents that were injected intraocularly include recombinant rat CNTF protein (0.03 to 0.5 μg/μL; 3 μL per eye; Alomone Labs); recombinant mouse CCL5 (0.1 μg/μL; 3 μL per eye; ThermoFisher Scientific); zymosan (12.5 μg/μL; sterilized before use; Sigma); recombinant rat Ocm (30 ng/μL; 3 μL per eye); the cAMP analog CPT-cAMP (50 μM; Sigma); AMD3100, a highly specific CXCR4 antagonist (100 μM; half-maximal inhibitory concentration [IC50] = 0.02 to 0.13 μM; Sigma); the Ocm peptide antagonist P1 (10) (2.3 μg/μL; 3 μL per eye); AAVs expressing green fluorescent protein (AAV2-GFP), AAV2 expressing Cas9 driven by the RGC-selective promoter γ-synuclein (Sncg) (AAV2-Sncg-Cas9), and AAV2 expressing CNTF (all from Boston Children’s Hospital Viral Vector Core); AAV2 expressing shRNA targeting CNTF receptor-α (AAV2-sh-CNTFRα; Vector Biolabs); AAV2 expressing a small guide RNA targeting CCR5 (AAV2-sgCCR5; Vigene Biosciences); and AAV2 expressing a small guide RNA targeting GPR75 (AAV2-sgGPR75; Vigene Biosciences).

Techniques: Expressing, Knockdown, In Situ, Staining, Quantitation Assay, Injection, shRNA, Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

doi: 10.1073/pnas.2017282118

Figure Lengend Snippet: Fig. 3. CNTF gene therapy alters systemic and local inflam- mation. (A) Blood-derived immune cells stained with fluorescent antibodies to CD11b, Ly6G, and Ly6C and analyzed by flow cytometry 2 wk after intraocular injection of AAV2-CNTF or control vector. Monocytes (CD11b+Ly6GlowLy6Chigh+intermediate) and neutrophils (NEUT) (CD11b+ Ly6GhighLy6Cintermediate) were quantified as shown. (B) Quantitation of changes. CNTF gene therapy increased the ratio of monocytes to neutrophils ap- proximately sevenfold. ***P < 0.001 (AAV2-CNTF vs. AAV2-GFP; n= 3 mice per group). (C–E) Immune cells stained with F4/80 (green) (macrophages), Gr1 (red), and the nuclear marker DAPI (blue). Macrophages (Gr1lowF4/80high) (D) and neutrophils (Gr1highF4/80negative) (E) were counted manually. CNTF gene therapy enhanced macrophage numbers in peripheral blood (**P < 0.01; AAV2-CNTF vs. AAV2-GFP; n = 5 mice/group) but not neutrophil numbers. (Scale bars: C, 5 μm; C, Insets, 50 μm.) (F) CNTF gene therapy enhances neutrophil and macrophage infil- tration into the retina (both green), particularly in the optic nerve head. (Scale bar, 40 μm.) (G–J) CCR2 KO reduces the effects of CNTF gene therapy. (G) Regenerating axons visualized by CTB immunostaining (green). The asterisk indicates the injury site. (Scale bar, 150 μm.) (H) Whole-mounted retinas immunostained with antibody TUJ1+ (green) to visualize surviving RGCs 2 wk after NC. (Scale bar, 60 μm.) (I) Quantitation of regenerating axons 0.5 mm distal to the injury site. CCR2 KO reduced the effects of CNTF gene therapy on axon regeneration by 48%. ***P < 0.001 (KO vs. heterozygous KO controls; n = 9 to 10 nerves per group). (J) Quantitation. CCR2 KO diminished RGC survival by 26%. **P < 0.01 (KO vs. heterozygous KO controls; n = 11 to 14 retinas per group). Bars show means ± SEM.

Article Snippet: Reagents that were injected intraocularly include recombinant rat CNTF protein (0.03 to 0.5 μg/μL; 3 μL per eye; Alomone Labs); recombinant mouse CCL5 (0.1 μg/μL; 3 μL per eye; ThermoFisher Scientific); zymosan (12.5 μg/μL; sterilized before use; Sigma); recombinant rat Ocm (30 ng/μL; 3 μL per eye); the cAMP analog CPT-cAMP (50 μM; Sigma); AMD3100, a highly specific CXCR4 antagonist (100 μM; half-maximal inhibitory concentration [IC50] = 0.02 to 0.13 μM; Sigma); the Ocm peptide antagonist P1 (10) (2.3 μg/μL; 3 μL per eye); AAVs expressing green fluorescent protein (AAV2-GFP), AAV2 expressing Cas9 driven by the RGC-selective promoter γ-synuclein (Sncg) (AAV2-Sncg-Cas9), and AAV2 expressing CNTF (all from Boston Children’s Hospital Viral Vector Core); AAV2 expressing shRNA targeting CNTF receptor-α (AAV2-sh-CNTFRα; Vector Biolabs); AAV2 expressing a small guide RNA targeting CCR5 (AAV2-sgCCR5; Vigene Biosciences); and AAV2 expressing a small guide RNA targeting GPR75 (AAV2-sgGPR75; Vigene Biosciences).

Techniques: Derivative Assay, Staining, Flow Cytometry, Injection, Control, Plasmid Preparation, Quantitation Assay, Marker, Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

doi: 10.1073/pnas.2017282118

Figure Lengend Snippet: Fig. 4. Neutrophil depletion diminishes the effects of CNTF gene therapy. (A and B) Systemic administration of anti-Ly6G antibody eliminates neutrophils (NEUT) in peripheral blood. (A) Immune cells isolated from blood; stained with fluo- rescently conjugated antibodies to CD11b, Ly6G, and Ly6C; and analyzed by flow cytometry 2 wk after CNTF gene therapy with or without neutrophil depletion (systemic anti-Ly6G). (B) Quan- titation of blood neutrophils. **P < 0.01 (anti-Ly6G vs. IgG2a; n = 3 mice per group). (C–F) Neutrophil depletion suppresses the effects of CNTF gene therapy on optic nerve regeneration. (C) Regenerating axons visualized by CTB immunostaining (green). The asterisk indicates the injury site. (Scale bar, 150 μm.) (D) Quantitation of axon regeneration 0.5 mm past the injury site. Neutrophil depletion reduced the effects of CNTF gene therapy by 74%. ***P < 0.001 (IgG2a vs. anti-Ly6G; n = 4 to 10 nerves per group). (E) Retinal whole mounts immunostained for βIII tubulin (antibody TUJ1) (green) 2 wk after NC. (Scale bar, 60 μm.) (F) Quantitation of RGC survival. Neutrophil depletion reduced RGC survival by 21% (TUJ1+ cells) (green in E). ***P < 0.001 (IgG2a vs. anti-Ly6G; n = 8 to 10 retinas per group). Bars show means ± SEM.

Article Snippet: Reagents that were injected intraocularly include recombinant rat CNTF protein (0.03 to 0.5 μg/μL; 3 μL per eye; Alomone Labs); recombinant mouse CCL5 (0.1 μg/μL; 3 μL per eye; ThermoFisher Scientific); zymosan (12.5 μg/μL; sterilized before use; Sigma); recombinant rat Ocm (30 ng/μL; 3 μL per eye); the cAMP analog CPT-cAMP (50 μM; Sigma); AMD3100, a highly specific CXCR4 antagonist (100 μM; half-maximal inhibitory concentration [IC50] = 0.02 to 0.13 μM; Sigma); the Ocm peptide antagonist P1 (10) (2.3 μg/μL; 3 μL per eye); AAVs expressing green fluorescent protein (AAV2-GFP), AAV2 expressing Cas9 driven by the RGC-selective promoter γ-synuclein (Sncg) (AAV2-Sncg-Cas9), and AAV2 expressing CNTF (all from Boston Children’s Hospital Viral Vector Core); AAV2 expressing shRNA targeting CNTF receptor-α (AAV2-sh-CNTFRα; Vector Biolabs); AAV2 expressing a small guide RNA targeting CCR5 (AAV2-sgCCR5; Vigene Biosciences); and AAV2 expressing a small guide RNA targeting GPR75 (AAV2-sgGPR75; Vigene Biosciences).

Techniques: Isolation, Staining, Flow Cytometry, Immunostaining, Quantitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

doi: 10.1073/pnas.2017282118

Figure Lengend Snippet: Fig. 5. Inhibition of Ocm and SDF1 has minor effect on CNTF gene therapy- induced axon regeneration. (A–D) Ef- fects of blocking Ocm (Peptide P1) and SDF1 (AMD3100) on axon regeneration and RGC survival induced by zymosan (Zymo) (upper two images in A and B; positive control) and CNTF gene ther- apy (lower two images in A and B). (A) Regenerating axons visualized by CTB immunostaining (green). The asterisk in- dicates the injury site. (Scale bar, 150 μm.) (B) Whole-mounted retinas immunos- tained with antibody TUJ1+ (green) to visualize βIII tubulin-positive RGCs. (Scale bar, 60 μm.) (C) Quantitation of regen- eration. Intraocular injection of AMD3100 plus P1 eliminated zymosan-induced axon regeneration. ***P < 0.001 (zymosan + control vs. zymosan + P1/AMD3100; n = 8 nerves per group). The same inhibitors decreased CNTF gene therapy-induced axon regeneration by 19%. *P < 0.05 (CNTF gene therapy + control vs. CNTF gene therapy + P1/AMD3100; n = 8 nerves per group). (D) Quantitation of RGC survival. Antagonists to Ocm and SDF1 decreased RGC survival by 24% (**P < 0.01; zymosan + control vs. zy- mosan + P1/AMD3100; n = 8 retinas per group) but did not alter the neuro- protective effects of CNTF gene therapy (P = 0.441; n = 8 retinas/group). Bars show means ± SEM.

Article Snippet: Reagents that were injected intraocularly include recombinant rat CNTF protein (0.03 to 0.5 μg/μL; 3 μL per eye; Alomone Labs); recombinant mouse CCL5 (0.1 μg/μL; 3 μL per eye; ThermoFisher Scientific); zymosan (12.5 μg/μL; sterilized before use; Sigma); recombinant rat Ocm (30 ng/μL; 3 μL per eye); the cAMP analog CPT-cAMP (50 μM; Sigma); AMD3100, a highly specific CXCR4 antagonist (100 μM; half-maximal inhibitory concentration [IC50] = 0.02 to 0.13 μM; Sigma); the Ocm peptide antagonist P1 (10) (2.3 μg/μL; 3 μL per eye); AAVs expressing green fluorescent protein (AAV2-GFP), AAV2 expressing Cas9 driven by the RGC-selective promoter γ-synuclein (Sncg) (AAV2-Sncg-Cas9), and AAV2 expressing CNTF (all from Boston Children’s Hospital Viral Vector Core); AAV2 expressing shRNA targeting CNTF receptor-α (AAV2-sh-CNTFRα; Vector Biolabs); AAV2 expressing a small guide RNA targeting CCR5 (AAV2-sgCCR5; Vigene Biosciences); and AAV2 expressing a small guide RNA targeting GPR75 (AAV2-sgGPR75; Vigene Biosciences).

Techniques: Inhibition, Blocking Assay, Positive Control, Immunostaining, Quantitation Assay, Injection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

doi: 10.1073/pnas.2017282118

Figure Lengend Snippet: Fig. 7. CCR5 is required for the major effects of CNTF gene therapy. (A–D) CCR5 KO or the CCR5 antagonist DAPTA di- minishes most effects of CNTF gene therapy. (A) Regenerating axons visualized by CTB immunostaining (green). Treatments are as indicated. The asterisk indicates the injury site. (Scale bar, 150 μm.) (B) Whole-mounted retinas immunostained with antibody TUJ1+ (green). (Scale bar, 60 μm.) (C and D) Quanti- tation. (C) CCR5 KO reduced the effects of CNTF gene therapy on axon regeneration by 72%. ***P < 0.001 (NC + CNTF gene therapy: littermate control vs. CCR5 KO; upper pair). CCR5 antagonist DAPTA diminished the effects of CNTF gene ther- apy by 75% in wild-type 129S1 mice. ***P < 0.001 (NC + CNTF gene therapy: saline vs. DAPTA; middle set). CRISPR-mediated KO of CCR5 in RGCs diminished the effects of CNTF gene therapy on optic nerve regeneration by 64% in wild-type 129S1 mice. ***P < 0.001 (NC + CNTF gene therapy + AAV2- Sncg-Cas9: AAV2-GFP vs. AAV2-sgCCR5; n = 8 nerves per group; lower set). (D) RGC survival is decreased by 24% follow- ing CCR5 KO (**P < 0.01; NC + CNTF gene therapy: litter- mate control vs. CCR5 KO), by 20% in wild-type 129S1 mice treated with DAPTA (*P < 0.05; NC + CNTF gene therapy: saline vs. DAPTA), and by 17% in wild-type 129S1 mice with CRISPR-mediated deletion of the CCR5 gene in RGCs (*P < 0.05; NC + CNTF gene therapy + AAV2-Sncg-Cas9: AAV2- GFP vs. AAV2-sgCCR5; n = 8 retinas per group). Bars show means ± SEM.

Article Snippet: Reagents that were injected intraocularly include recombinant rat CNTF protein (0.03 to 0.5 μg/μL; 3 μL per eye; Alomone Labs); recombinant mouse CCL5 (0.1 μg/μL; 3 μL per eye; ThermoFisher Scientific); zymosan (12.5 μg/μL; sterilized before use; Sigma); recombinant rat Ocm (30 ng/μL; 3 μL per eye); the cAMP analog CPT-cAMP (50 μM; Sigma); AMD3100, a highly specific CXCR4 antagonist (100 μM; half-maximal inhibitory concentration [IC50] = 0.02 to 0.13 μM; Sigma); the Ocm peptide antagonist P1 (10) (2.3 μg/μL; 3 μL per eye); AAVs expressing green fluorescent protein (AAV2-GFP), AAV2 expressing Cas9 driven by the RGC-selective promoter γ-synuclein (Sncg) (AAV2-Sncg-Cas9), and AAV2 expressing CNTF (all from Boston Children’s Hospital Viral Vector Core); AAV2 expressing shRNA targeting CNTF receptor-α (AAV2-sh-CNTFRα; Vector Biolabs); AAV2 expressing a small guide RNA targeting CCR5 (AAV2-sgCCR5; Vigene Biosciences); and AAV2 expressing a small guide RNA targeting GPR75 (AAV2-sgGPR75; Vigene Biosciences).

Techniques: Immunostaining, Control, Saline, CRISPR