AAV-mediated glia-to-neuron conversion in mice with oral squamous cell carcinoma
Introduction
Recently, an in vivo cell conversion technique has been used to regenerate functional new neurons in rodent adult brain by directly converting glial cells to neurons. In the present study, we examine whether AAV-mediated ectopic expression of NeuroD1 and Dlx2 transcription factors can produce in vivo direct glia-to-neuron conversion. Our preliminary data have shown microglia activation in the spinal trigeminal nucleus caudalis (Sp5C, the primary trigeminal pain center) of mice with chronic oral cancer pain. It is well known that microglia activation contributes to the central mechanisms of chronic oral cancer pain. On the other hand, disinhibition due to reduced release of ʏ-aminobutyric acid (GABA) is critical for the development of oral cancer pain. Therefore, direct conversion of pro-nociceptive microglia into GABA-releasing inhibitory GABAergic neurons in pain-related brain regions can decrease activated microglia and reverse GABA-lacking caused disinhibition, thereby inhibiting central sensitization and treating chronic oral cancer pain.
Methods
In this study, chronic oral cancer pain in C57BL/6J WT mice (8-10 weeks) was induced by intra-tongue injection of squamous cell carcinoma (SCC) cells (3x105, 30 µl) under isoflurane anesthesia, and we performed AAV-mediated co-expression of NeuroD1 and Dlx2 for specifically converting microglia into GABAergic neurons in the Sp5C. We constructed AAV5-CD68-NeuroD1-2A-Dlx2-2A-eGFP-WPRE and its control AAV5-CD68-eGFP-WPRE. The promoter CD68 can guide specific expression of NeuroD1 and Dlx2 in microglia of mice. Two weeks following the injection of SCC cells, we injected AAV5-CD68-eGFP-WPRE (control) or AAV5-CD68-NeuroD1-2A-Dlx2-2A-eGFP-WPRE (1x1012 vg/ml, 0.3 μl) into Sp5C. Three weeks later, we harvested brainstem tissues containing Sp5C and conducted immunofluorescence double staining with GFP and GABA/Iba1 antibodies.
Results
By co-expressing NeuroD1 and Dlx2 in microglia under chronic oral cancer pain condition, we successfully converted microglia into GABAergic neurons in the Sp5C. We observed that in the mice injected with the control AAV, all GFP-positive cells in the Sp5C were co-labeled with Iba1, and in the mice injected with the AAV carrying NeuroD1 & Dlx2, GFP-positive cells in the Sp5C were not co-labeled with Iba1 and most GFP-positive cells were co-labeled with GABA.
Conclusion
These results indicate that AAV-mediated co-expression of NeuroD1 and Dlx2 can convert microglia to GABAergic neurons in the Sp5C.