A novel mWGA-TYR PET reporter gene system for in vivo visualization of whole brain-level neural circuit

This study aimed to develop a novel PET reporter gene imaging system based on modified wheat germ agglutinin (mWGA) and tyrosinase (TYR) for tracing trans‑monosynaptic neural circuit in vivo.

Mouse neural stem cells NE4C were engineered via lentiviral transduction to express mWGA-TYR and GFP. TYR expression was confirmed by western blot and RT-qPCR. Melanin production was assessed using Masson-Fontana staining, gamma counting, and ¹⁸F-FPABZA PET imaging. In vitro trans-synaptic transfer was evaluated by co‑culturing transduced NE4C cells with naive cells. To validate the system in the normal brain, an rAAV vector encoding mWGA-TYR was delivered into the right sensorimotor cortex. In vivo melanin transport along neural circuits was traced by ¹⁸F-FPABZA PET imaging and confirmed by ex vivo immunofluorescent staining. Finally, the system was tested in an ischemic stroke model following NE4C transplantation.

Lentiviral transduction of NE4C cells led to successful expression of mWGA, TYR and GFP. Masson‑Fontana staining confirmed melanin production, while gamma counting and ¹⁸F‑FPABZA PET imaging showed significantly higher radiotracer uptake in TYR‑expressing cells in vitro. In co‑culture experiments, mWGA‑TYR underwent trans‑synaptic transfer from transduced to naive NE4C cells. Following rAAV injection into the right sensorimotor cortex, ¹⁸F‑FPABZA PET signals were detected in multiple brain regions, with ex vivo immunofluorescence corroborated these connectivity patterns. In the ischemic stroke model with NE4C transplantation, elevated PET signals were observed at both the transplantation site and distal sites two weeks after transplantation, which was further validated by ex vivo analyses. Furthermore, HSV‑1‑H129 tracing confirmed the anterograde trans‑monosynaptic capability of mWGA‑TYR system.

This study developed a novel mWGA-TYR PET reporter gene system for non-invasive in vivo tracing of anterograde trans-monosynaptic neural circuits. This system can be extended to monitor graft-host neural circuit connections in stem cell therapy for ischemic stroke.