TTBK2-Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration after Spinal Cord Injury

Primary cilia (PCs) are microtubule-based organelles that function as critical signaling hubs in both embryonic neurodevelopment and mature neurons. Tau tubulin kinase 2 (TTBK2) is a key initiator of ciliogenesis, but its role in neuronal regeneration following spinal cord injury (SCI) remains unclear. Here, we investigated whether TTBK2 regulates axonal regeneration and circuit remodeling after SCI via a cilia-dependent mechanism. Using both in vitro spinal neuron cultures and an in vivo conditional knockout (TTBK2^fl/fl-Cre ERT^+/-) mouse model of T10 spinal hemisection, we demonstrate that TTBK2 deletion impairs PC formation, downregulates Sonic Hedgehog (SHH) signaling, reduces MAP2 expression, and disrupts axonal elongation. Overexpression of TTBK2 rescued these deficits, while loss of ciliary function via KIF3A knockdown replicated the phenotypes. Transcriptomic and proteomic profiling revealed that TTBK2 deficiency suppresses axonogenesis-related pathways while enhancing synaptic excitability markers. Notably, activation of SHH signaling by Smoothened agonist partially restored MAP2 expression and axonal growth in TTBK2-deficient neurons. In vivo, TTBK2 knockout mice displayed impaired motor recovery, increased glial scarring, disrupted axonal projection, and elevated expression of axon growth inhibitors (NogoA/NogoR). Confocal imaging and electron microscopy confirmed the loss of PCs and morphological signs of demyelination and aberrant synaptogenesis. Furthermore, endogenous neural stem cells in TTBK2-deficient mice failed to differentiate into mature neurons, correlating with reduced SHH activity. Collectively, our findings identify a TTBK2–PC–SHH–MAP2 regulatory axis that governs axonal regeneration and circuit reassembly after SCI. This study expands the functional relevance of primary cilia beyond neurodevelopment and highlights TTBK2 as a potential therapeutic target for promoting neural repair in the adult CNS. Overall design: Neuronal cells were isolated from the spinal cord of fetal mice at 15 days of gestation. Cells were cultured at a density of 1.5*10^6/well (six-well plate) for immunofluorescence experiments Neuronal cells were grown in u-Plate 24 Well Black (ibidi, Germany). Neuronal growing media included DMEM (Gibco, USA) and FBS (Gibco, USA), and neuronal media included Neurobasal medium (Gibco, USA), 2% B27neural supplement (Gibco-Invitrogen, USA), 1 mM L- glutamine solution (Sigma-Aldrich, G7513, USA) and 100 ng/ml penicillin-streptomycin (Gibco-Invitrogen, USA). Neuronal cells were changed every 2 days and a half, and subsequent experiments were performed on day 5 to identify neuronal cells by immunofluorescence assay. The experiment was divided into five groups, including: NC, shKIF3A, shTTBK2, OETTBK2, shKIF3A+OETTBK2. Cells were seeded on day 5 of the plate and infected using adenovirus, using the 1/2-volume infection method, half of the medium in the original well plate was removed, and the corresponding viral solution was added according to the MOI=100. Adv5-MIR30 (shTTBK2, NM_001024856), Adv5-MIR30(shKIF3A, NM_001290805), pDC316-mCMV(mTTBK2 , NM_001024856) The sequences of the target genes are shown in Table S1, and silencing of KIF3A, TTBK2, overexpression of TTBK2 and silencing of KIF3A+ overexpression of TTBK2, and negative plasmids were added to the control group. After 8h of infection, the virus-shouting medium was discarded and replaced with neuronal medium for further cultivation, and the infection efficiency was identified by qRT-PCR. National Natural Science Foundation of China, NO.82171388