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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE301749 | GEO | 2026/01/28

REPOSITORIES: GEO

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Project description:The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-β1 (M(IL-10+TGF-β1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-β1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-β1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-β1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.

Project description:Spinal cord injury (SCI) often leads to persistent functional deficits due to severe neuron and glial loss, and to limited axonal regeneration after injury. Here we show that the transplantation of human dental stem cells into the completely transected adult rat spinal cord resulted in a significant recovery of hindlimb locomotor functions. These stem cells exhibited three major neuro-regenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, improving the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, by paracrine mechanisms. Third, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide novel therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuro-regenerative activities. Human dental stem cells were isolated from exfoliated deciduous teeth extracted for clinical purposes were collected at Nagoya University School of Medicine, under approved guidelines set by Nagoya University (H-73, 2003). The ethics committee of Nagoya University approved the experimental protocols (permission number 8-2). Mesenchymal stem cells of human Bone marrow line were obtained from Lonza. Total RNAs were isolated and quantified by spectrophotometer. RNA integrity was checked on 1% agarose gels. RT reactions were carried out with Superscript III reverse transcriptase (Invitrogen) using 1 μg of total RNA in a 50 μl total reaction volume. Microarray experiments were carried out using a CodeLink™ Human Whole Genome Bioarray (Applied Microarrays, Inc. Tempe, AZ) at Filgen, Inc. (Nagoya, Japan). The arrays were scanned using a GenePix4000B Array Scanner (Molecular Devices, Sunnyvale, CA), and the data was analyzed by using MicroArray Data Analysis Tool Ver3.2 (Filgen, Inc.).

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

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