Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: TTBK2 regulates primary cilium formation and axonal growth in spinal neurons. (A) Schematic diagram of the primary cilium. Kinesin‐2 comprises KIF3A; TTBK2 is related to the formation of basal bodies. (B, C) Quantitative RT‐qPCR analysis showing efficient knockdown or overexpression of KIF3A and TTBK2 in spinal neurons via adenoviral infection ( n = 6 from 3 independent experiments). (D, E) Representative immunofluorescence images of spinal neurons labeled with MAP2 (green), ACIII (red), and DAPI (blue) in five experimental groups: NC, shKIF3A, shTTBK2, TTBK2‐OE, and shKIF3A + TTBK2‐OE. White arrows indicate PCs. Compared with that in NC, the cilium length was significantly reduced in the shKIF3A, shTTBK2, and shKIF3A + TTBK2‐OE groups, while TTBK2‐OE overexpression led to elongated cilia ( n = 6 from 3 independent experiments). Scale bars, 20 μm. (F, G) Representative images showing immunolabeling of TUJ1 (green, axons), MAP2 (orange, dendrites), and DAPI (blue, nuclei). Axonal morphology and length were assessed across five groups. KIF3A or TTBK2 knockdown significantly reduced axon length, while TTBK2 overexpression enhanced elongation. shKIF3A + TTBK2‐OE partially rescued axon length compared with that under shKIF3A alone ( n = 5 from 3 independent experiments). Scale bars, 10 μm. Data are presented as mean ± SEM. One‐way ANOVA was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Infection, Immunofluorescence, Labeling, Immunolabeling

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: TTBK2 regulates axonal regeneration via the primary cilium–SHH pathway. (A–C) Volcano plots showing differentially expressed proteins between shTTBK2 vs. NC, shKIF3A vs. NC, and shKIF3A + TTBK2‐OE vs. shKIF3A. The x ‐axis represents log2 (fold change), and the y ‐axis represents −log10( p ‐value). Gray dots indicate proteins that did not meet significance thresholds ( p > 0.05). Blue and red dots indicate downregulated and upregulated proteins, respectively. (D) The heatmap displays differentially expressed proteins identified in each of the four experimental groups relative to the NC control group, with color intensity representing expression levels. (E–H) Western blotting analysis demonstrating significant reductions in MAP2, Gli1, and Smo protein expression in the shTTBK2 group ( n = 3 from 3 independent experiments). (I, J) Treatment with the SHH pathway agonist SAG restored MAP2 expression in shTTBK2 neurons ( n = 3, from 3 independent experiments * p < 0.05). (K, L) Representative immunofluorescence images of spinal neurons stained for TUJ1 (green), PSD95 (red), and DAPI (blue). TTBK2‐OE group showed markedly increased PSD95 expression compared to other groups ( n = 6 from 3 independent experiments). Scale bar, 5 μm. Data are presented as mean ± SEM. One‐way ANOVA was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Control, Expressing, Western Blot, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: The TTBK2–SHH–MAP2 axis regulates endogenous neuronal repair following SCI. (A) Representative immunofluorescence images of frozen spinal cord sections stained with GFAP (green), MAP2 (orange), TUJ1 (red), and DAPI (blue). Scale bars: Left, 200 μm; right, 50 μm ( n = 6 from six mice in each group). (B–D) The quantification of immunostaining showed increased GFAP in all injured groups. MAP2 levels in WT‐SCI were comparable to those in uninjured controls, while Ttbk2 fl/fl ‐SCI showed a marked reduction. TUJ1 staining indicated significantly higher immature neuron proportion in WT‐SCI than in other groups ( n = 6 from six mice in each group). (E, F) Co‐staining of NF200 (green) and Nestin (red) revealed elevated neural progenitor marker Nestin in injured groups. Ttbk2 fl/fl ‐SCI mice exhibited a higher Nestin/NF200 ratio than did WT‐SCI mice ( n = 6 from six mice in each group). (G–J) Western blotting results confirmed that MAP2, Smo, and Gli1 protein levels were significantly reduced in Ttbk2 fl/fl ‐SCI mice, indicating SHH pathway suppression ( n = 3 from three mice in each group). Bars and errors represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (one‐way ANOVA).

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Immunofluorescence, Staining, Immunostaining, Marker, Western Blot

Journal: bioRxiv

Article Title: Targeting NGF but not VEGF or BDNF signaling reduces endometriosis-associated pain in mice

doi: 10.1101/2023.12.05.570181

Figure Lengend Snippet: (A) scheme of experimental procedures. (B) representative image from endometriotic lesions stained for NGF and beta-III tubulin. (C) quantification of TrkA + and pNF-κB + TrkA + neurons in dorsal root ganglia (DRG) of sham and endometriosis lesion-bearing mice. Lesions and DRG were dissected at 56 dpi. Results are presented as mean ± SEM of the percentage of positive neurons. n = 6 or 7 mice per group. (*P < 0.05 vs. sham). (D) Representative images of DRG neurons stained for TrkA (red) and p-NF-kB (green) by confocal microscopy.

Article Snippet: Sections were hydrated with PBS, blocked in 5% BSA in PBS 0.5% triton-X 100 (m/v/v) for 1h at RT, following overnight incubation at 4°C with primary antibodies: mouse anti-mouse phosphorylated-NF-κB (pNF-κB, Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc-136548, 1:200 [RRID:AB_10610391]), rabbit anti-mouse TrkA (Invitrogen, Waltham MA, USA, cat. # MA5-32123, 1:100 [RRID:AB_2809414]), rabbit anti-mouse NGF (Abcam, Boston, MA, USA, cat. # AB52918, 1:300 [RRID:AB_881254]), and β-III tubulin (Novus Biologicals, Centennial, CO, USA, cat. # NB120-11314, 1:200 [RRID:AB_792496]).

Techniques: Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: Intranasal delivery of nanomicelle curcumin promotes corneal epithelial wound healing in streptozotocin-induced diabetic mice

doi: 10.1038/srep29753

Figure Lengend Snippet: The effect of 7-day treatment with topical and/or intranasal nanomicelle curcumin in promoting diabetic corneal nerve regeneration after corneal epithelium abrasion was investigated. Corneas were harvested, flat-mounted, and immunostained with β-III tubulin antibody. ( A ) Images of central and peripheral corneal nerve were taken and combined as a whole flat-mounted corneal nerve. ( B ) Central and peripheral corneal nerve densities were calculated and are expressed as a percentage of that in the NC group (* P < 0.05 when compared with the DC group; ** P < 0.01, with the DC group; # P < 0.05, with the OT group; n = 3).

Article Snippet: Corneal nerve staining was performed, as described previously, with an anti-neuron-specific β-III tubulin antibody (NL1195R; R&D System Inc. Minneapolis, USA).

Techniques:

Journal: ACS nano

Article Title: Combination Nanomedicine Strategy for Preventing High-Risk Corneal Transplantation Rejection.

doi: 10.1021/acsnano.4c06595

Figure Lengend Snippet: Figure 7. Corneal nerve regeneration after HR PKP surgery. (A) Immunostaining images of corneal nerve marker β-tubulin III, scale bar 200 μm. (B) Corneal nerve regeneration was quantified as the percentage of threshold area positive for β-tubulin III staining in the center part of cornea with the diameter of 3.5 mm. Statistical analysis for B: one-way ANOVA with a Tukey post hoc test for multiple comparison. (*p ≤0.05; **p ≤0.01; and ***p ≤0.001). N = 3 for each group. All data are plotted from mean ± SEM.

Article Snippet: After that, cornea samples were washed with PBS with 0.3% Triton X-100 (Sigma-Aldrich) for 4 × 10 min followed by blocking with 2% BSA-PBS-Triton X-100 solution at room temperature for another 2 h. The cornea was incubated at 4 °C overnight in fluorescein-labeled antirat β-tubulin III antibody (R&D systems, Cat No: NL1195R).

Techniques: Immunostaining, Marker, Staining, Comparison