Activating adult neural stem cells (NSCs) located within the spinal cord niche is considered a promising therapeutic approach for treating spinal cord injury (SCI). Cerebrospinal fluid (CSF)-contacting neurons expressing Pkd2l1 exhibit phenotypic and molecular traits similar to those of adult NSCs. However, the mechanism responsible for regulating the activation of Pkd2l1+ CSF-cNs still needs to be discovered. This research demonstrated that Pkd2l1+ CSF-cNs have a high concentration of vascular endothelial growth factor receptor 3 (Vegfr3) and that SCI results in elevated Vegfr3 levels. The overexpression of Vegfr3 in Pkd2l1+CSF-cNs induced potential NSC activation. Blocking Vegfr3 led to a significant reduction in the percentage of active Pkd2l1+ CSF-cNs, suggesting that Vegfr3 is involved in controlling the shift from dormancy to activation in these cells. In vivo, the downregulation of Vegfr3 by SAR131475 inhibited Pkd2l1+CSF-cN proliferation and maintained self-renewal. Injection of vascular endothelial growth factor C (Vegf-C) into the lateral ventricle of adult mice confirmed the involvement of Vegfr3 in activating Pkd2l1+ CSF-cNs. Vegf-C administration significantly increased the number of activated Pkd2l1+ CSF-cNs. Mechanistically, Vegfr3 primed quiescent Pkd2l1+ CSF-cNs for cell cycle reentry by enabling the activation of PI3K/Akt signaling. The activation of Vegfr3 may enhance SCI outcomes by promoting neuronal survival and facilitating the recovery of motor function in mice. Together, our findings highlight that Vegfr3 is a crucial functional regulator of Pkd2l1+ CSF-cNs, governing the transition from NSC quiescence to activation.

Adult neural stem cells (NSCs) offer a promising avenue for restoring spinal cord injury (SCI). However, their precise identity in the mammalian spinal cord remains unclear. Our previous research demonstrated that Pkd2l1-positive cerebrospinal fluid-contacting neurons (CSF-cNs) possess the NSC properties. Furthermore, understanding the role and molecular mechanisms of CSF-cNs as endogenous NSCs in spinal cord repair is crucial for developing effective treatments. This study utilizes a Pkd2l1-/- transgenic mouse model to investigate the role of CSF-cNs in SCI repair. We found that the CSF-cN population was almost absent in Pkd2l1-/- mice. Following SCI, these mice exhibited a significant reduction in the number of NSCs surrounding the central canal. Notably, Pkd2l1-/- mice showed impaired neuronal regeneration and compromised motor function recovery post-SCI. These findings highlight the potential importance of Pkd2l1 as a target for treating SCI by focusing on endogenous NSCs.

BACKGROUND: Recently, it was found that cerebrospinal fluid-contacting neurons near the central canal of the spinal cord have the potential of neural stem cells, but the purity of cerebrospinal fluid-contacting neurons in the in vitro research is not high, and there is no in vitro tracing technology to prove their neural stem cell characteristics. OBJECTIVE: To verify the characteristics of neural stem cells of cerebrospinal fluid-contacting neurons by multimodal imaging molecules in vitro.METHODS: According to the specific expression of the Pkd2l1 gene in cerebrospinal fluid-contacting neurons, a multimodal imaging molecular lentivirus was designed to specifically express a green fluorescent protein（GFP） in cerebrospinal fluid-contacting neurons according to the upstream promoter of Pkd2l1 gene. The primary neural stem cells were extracted from the medulla oblongata of C57BL/6 mice within 24 hours of birth, then the primary neural stem cells containing cerebrospinal fluid-contacting neurons were transfected with multimodal imaging molecular virus, and the cerebrospinal fluid-contacting neurons were screened and purified by puromycin. The screened and purified cerebrospinal fluid-contacting neurons were suspended in vitro and passaged continuously for more than four generations. Immunofluorescence was used to observe whether the third generation of cerebrospinal fluid-contacting neurons co-expressed with neural stem cell markers Nestin and Sox2. The third generation of cerebrospinal fluid-contacting neurons was induced to differentiate, and the co-expression of cerebrospinal fluid-contacting neurons with neuronal marker NeuN, astrocyte marker S100 β and oligodendrocyte marker O4 was detected by immunofluorescence. RESULTS AND CONCLUSION: The multimodal imaging molecular lentivirus was successfully constructed, and the purified cerebrospinal fluid-contacting neurons could survive, proliferate and express GFP. GFP+ cerebrospinal fluid-contacting neurons could be passaged continuously for more than four generations in vitro, and express neural stem cell markers Nestin and Sox2. After induced differentiation, GFP+ cerebrospinal fluid-contacting neurons expressed neuron marker NeuN, astrocyte marker S100β and oligodendrocyte marker O4. It is concluded that cerebrospinal fluid-contacting neurons can be specifically labeled by multimodal image molecular viruses and show the ability of self-renewal and multidirectional differentiation, which adequately proves that cerebrospinal fluid-contacting neurons have the characteristics of neural stem cells in vitro.

脊髓损伤( SCI)是一种严重的、高度致残的、致命的一种疾病,多由意外事故引起[1].在SCI中,初始机械作用引起的原发性损伤存在不可逆的神经细胞死亡,继而在炎症、局部缺血、脂质过氧化和细胞凋亡等多种因素作用下导致继发性损伤,最终造成轴突束断裂,引起运动或感觉功能障碍[2-3].

The neural stem cells (NSCs) in the ventricular-subventricular zone of the adult mammalian spinal cord may be of great benefit for repairing spinal cord injuries. However, the sources of NSCs remain unclear. Previously, we have confirmed that cerebrospinal fluid-contacting neurons (CSF-cNs) have NSC potential in vitro. In this study, we verified the NSC properties of CSF-cNs in vivo. In mouse spinal cords, Pkd2l1+ CSF-cNs localized around the central canal express NSC markers. In vitro, Pkd2l1+ CSF-cNs form a neurosphere and express NSC markers. Activation and proliferation of CSF-cNs can be induced by injection of the neurotrophic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) into the lateral ventricle. Spinal cord injury (SCI) also induces NSC activation and proliferation of CSF-cNs. Collectively, our results demonstrate that Pkd2l1+ CSF-cNs have NSC properties in vivo and may be involved in SCI recovery.