W39 - Therapeutic Targeting of Notch1-Expressing Cells with Gsx1 Gene Therapy in Acute SCI

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Andie McCullough

Undergraduate Researcher
Bucknell University
Ebensburg, Pennsylvania, United States

Co-Author(s)

Andie McCullough

Undergraduate Researcher
Bucknell University
Ebensburg, Pennsylvania, United States
TC

Tanner Clifford

Graduate Student
Rutgers University, United States

Primary Investigator(s)

LC

Li Cai

Professor
Rutgers University, United States

Introduction: : Spinal cord injury (SCI) is a complex tissue injury that causes damage to the central nervous system (CNS), leading to permanent loss of motor and sensory function and impacting quality of life. Recent studies have found that globally, 20.6 million people are affected by SCIs, with 250,000 to 500,000 new cases occurring each year [1]. Immediately following injury, glial and immune cell types travel to the injury site, forming a scar around its border. Many of these cell types also contribute to the formation of an inhibitory microenvironment that is unfavorable for cellular regeneration and spared tissue function [1]. Neural stem/progenitor cells (NSPCs) are quiescent in the adult spinal cord and serve as a potential target cell population for regenerative cell reprogramming therapies due to their pluripotency. The Cai lab has identified cis-regulatory element Notch1CR2 (N1CR2) as an enhancer of Notch1, a key regulator of stem cell activation [2]. Preliminary studies show that lentivirus-mediated (LV) genomic screened homeobox 1 (Gsx1) administration activates N1CR2 in NSPCs and promotes neuronal regeneration following SCI, resulting in increased locomotor function in contusion mouse models [2]. Benefits to locomotor recovery are seen as early as 7 days post-injury (dpi) despite the generation of new neurons and their contribution to recovery being unlikely within this time, necessitating an assessment of glial and immune cell function in the spinal cord at the acute phase. This work aims to assess Notch1 signaling and immune cell function three days post-injury and Gsx1 gene therapy administration.

Materials and

Methods: : Adult C57BL/6 mice with contusion SCI received local injections of the control (lentivirus encoding reporter GFP) or Gsx1 treatment (lentivirus overexpressing Gsx1) at the lesion site. Spinal cord tissues were harvested 3 dpi and cryosectioned. These frozen tissues were removed from −80 °C and placed at room temperature for 30 minutes to thaw. Samples were then processed following an established lab standard immunohistochemistry protocol.

Images were captured at the same exposure, threshold, and intensity per condition using Zeiss AxioVision imager A1 and Echo Revolve at wavelengths 488, 549, 647 nm. Five to six sections from each animal were analyzed at 20x magnification. Co-labeled cells with viral reporter GFP and specific markers were manually counted in separate RGB channels and merged images in a 438 μm by 328 μm region within the lesion site. GraphPad Prism 6 was used for all statistical analyses. Comparisons between treatment and control groups were analyzed using a two-tailed T-test (α = 0.05).

Results, Conclusions, and Discussions:: Notch1 signaling pathways exist in various CNS cell types, resulting in a range of effects like cell activation and axogenesis [2]. To evaluate Notch1 upregulation via Gsx1 gene therapy, we screened relevant cell types for enhanced Notch1 signaling to elucidate its effects on secondary injury. Immunostaining was performed to quantify Notch1 signaling in combination with cell-specific markers to determine if Notch1 was upregulated in astrocytes, neurons, homeostatic microglia, NG2 glial cells, or oligodendrocytes, using markers of GFAP, MAP2, TMEM119, NG2, and OLIG1, respectively. Following immunofluorescent staining, samples were imaged and expression was quantified via manual cell counting. Cell count analyses found that LV-mediated N1CR2-driven Gsx1 gene therapy administration significantly increased (p < 0.05) Notch1 activation in NG2 glial cells as compared to controls (Figure 2A). NG2-expressing glial cells, primarily composed of oligodendrocyte precursor cells (OPCs) and astrocytes, have stem-like potential, making their involvement in injury response reasonable.
In contrast, Gsx1 had no significant effect on Notch1 expression in homeostatic microglia or astrocytes (Figure 2B, 2C). Given Notch1’s well-characterized activation pathways in astrocytes and microglia, two critically important glial populations in scar formation and secondary injury, we assessed the downstream markers of Notch1-driven activation in these cells. Iba1 and Cd68, as well as TNFa and GFAP, were used to determine these proinflammatory activation states in microglia and astrocytes, respectively. Cell count analysis found N1CR2-targeted Gsx1 did not affect the number of activated astrocytes post-injury (Figure 1B) but significantly increased (p < 0.001) reactive microglia in acute SCI (Figure 1D).

While Gsx1 treatment does not alter Notch1 activation in homeostatic microglia or astrocytes, it significantly increases the presence of reactive microglia and enhances Notch1 activation in NG2 glial cells. These findings suggest that N1CR2-driven Gsx1 promotes activation of NG2+ glial cells and microglia around the injury, highlighting these cell types as possible mediators for early injury response and locomotor improvement in acute SCI. These results provide insight into immune and glial cell involvement in inflammation and scar formation, furthering our understanding of gene therapy as a potential treatment for SCI.

Acknowledgements and/or References (Optional): :

References:
[1] Clifford T, Finkel Z, Rodriguez B, Joseph A, Cai L. Current Advancements in Spinal Cord Injury Research-Glial Scar Formation and Neural Regeneration. Cells. 2023 Mar 9;12(6):853. doi: 10.3390/cells12060853. PMID: 36980193; PMCID: PMC10046908.
[2] Li, Y., Tzatzalos, E., Kwan, K. et al. Transcriptional Regulation of Notch1 Expression by Nkx6.1 in Neural Stem/Progenitor Cells during Ventral Spinal Cord Development. Sci Rep 6, 38665 (2016). https://doi.org/10.1038/srep38665
[3] Finkel Z, Esteban F, Rodriguez B, Clifford T, Joseph A, Alostaz H, Dalmia M, Gutierrez J, Tamasi MJ, Zhang SM, Simone J, Petekci H, Nath S, Escott M, Garg SK, Gormley AJ, Kumar S, Gulati S, Cai L. AAV6 mediated Gsx1 expression in neural stem progenitor cells promotes neurogenesis and restores locomotor function after contusion spinal cord injury. Neurotherapeutics. 2024 Jul;21(4):e00362. doi: 10.1016/j.neurot.2024.e00362. Epub 2024 Apr 24. PMID: 38664194; PMCID: PMC11452562.