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. 2024 Aug 10;25(16):8744.
doi: 10.3390/ijms25168744.

Effects of Human Neural Stem Cells Overexpressing Neuroligin and Neurexin in a Spinal Cord Injury Model

Jiwon Jeong  1 , Yunseo Choi  1 , Narae Kim  1 , Haneul Lee  1 , Eun-Jung Yoon  2 , Dongsun Park  1
Affiliations

Effects of Human Neural Stem Cells Overexpressing Neuroligin and Neurexin in a Spinal Cord Injury Model

Jiwon Jeong et al. Int J Mol Sci. .

Abstract

Recent studies have highlighted the therapeutic potential of stem cells for various diseases. However, unlike other tissues, brain tissue has a specific structure, consisting of synapses. These synapses not only transmit but also process and refine information. Therefore, synaptic regeneration plays a key role in therapy of neurodegenerative disorders. Neurexins (NRXNs) and neuroligins (NLGNs) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses, mediate trans-synaptic signaling, and shape neural network properties by specifying synaptic functions. In this study, we investigated the synaptic regeneration effect of human neural stem cells (NSCs) overexpressing NRXNs (F3.NRXN) and NLGNs (F3.NLGN) in a spinal cord injury model. Overexpression of NRXNs and NLGNs in the neural stem cells upregulated the expression of synaptophysin, PSD95, VAMP2, and synapsin, which are synaptic markers. The BMS scores indicated that the transplantation of F3.NRXN and F3.NLGN enhanced the recovery of locomotor function in adult rodents following spinal cord injury. Transplanted F3.NRXN and F3.NLGN differentiated into neurons and formed a synapse with the host cells in the spinal cord injury mouse model. In addition, F3.NRXN and F3.NLGN cells restored growth factors (GFs) and neurotrophic factors (NFs) and induced the proliferation of host cells. This study suggested that NSCs overexpressing NRXNs and NLGNs could be candidates for cell therapy in spinal cord injuries by facilitating synaptic regeneration.

Keywords: NLGN; NRXN; spinal cord injury; stem cells; synaptic markers; synaptic regeneration.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Construction of F3.NLGN and F3.NRXN cells. (A) F3.NLGN and F3.NRXN cells were constructed via the transfection of F3 human neural stem cells with a plasmid vector encoding either human NLGNs or NRXNs. (B–D) Expression of NLGNs and NRXNs in F3.NLGN and F3.NRXN cells assessed via (B) Western blotting and (C,D) immunohistochemistry. Scale bar = 30 μm
Figure 2
Figure 2
Expression of synaptic markers (synaptophysin, PSD95, VAMP2, and synapsin) in F3.NLGN and F3.NRXN cells. (A) Expression of synaptic markers assessed by means of Western blotting. (B) Band densities normalized to actin. Densitometric analysis of the Western blot was performed using ImageJ 1.54g. n = 3 per treatment group. * Significantly different from F3 cells (p < 0.05).
Figure 3
Figure 3
Improvement in locomotor recovery after F3.NLGN and F3.NRXN transplantation in SCI mice. (A) Locomotor BMS scores following T7 complete transection SCI. (B) Grid walk errors in mice 5 weeks after SCI. (C) Stride length of the hindlimbs at 5 weeks after injury in F3.NLGN and F3.NRXN groups. This value could not be measured in the vehicle and F3 groups because they crawled. (D) Representative footprints of the hindlimbs in the F3.NLGN and F3.NRXN groups. Data are expressed as mean ± standard deviation. n = 10. * Significantly different from the vehicle control (p < 0.05). nm, not measured; BMS, Basso Motor Scale; SCI, spinal cord injury.
Figure 4
Figure 4
Protein expression after F3.NLGN and F3.NRXN transplantation at the lesion site (2 mm rostral and caudal to injury center) in SCI mice. (A) Expression of synaptic markers. (B) Expression of BDNF and NGF. (C) Expression of the PI3K/PTEN/mTOR signaling pathway after F3.NLGN and F3.NRXN transplantation. Protein expression is analyzed by means of Western blotting. (D,E) Band densities normalized to actin. Densitometric analysis of the Western blot was performed using ImageJ. n = 10 per treatment group. # Significantly different from the normal control (p < 0.05). * Significantly different from the vehicle control (p < 0.05). SCI, spinal cord injury; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor.
Figure 5
Figure 5
Differentiation of transplanted F3.NLGN and F3.NRXN cells into neurons in SCI mice. hMito (red color) was used as a stem cell marker. Neurofilament (green color) was used as a neuronal marker. DAPI (blue color) was used as a counterstain for the nucleus. Scale bar = 50 μm. SCI, spinal cord injury.
Figure 6
Figure 6
Expression of VAMP2 in transplanted F3.NLGN and F3.NRXN cells in SCI mice. hMito (red color) was used as a stem cell marker. VAMP2 (green color) was used as a synaptic marker. DAPI (blue color) was used as a counterstain for the nucleus. Scale bar = 50 μm. SCI, spinal cord injury.
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