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