Fig 2: Representative fluorescent images of the cerebellar cortex showing the correlation of the PC or BG transduction efficiency with microglial density.Mice received AAV9 vectors expressing GFP under the control of the NSE promoter. The cerebellar slices that were produced 1 week after the injection were double immunostained for GFP and Iba1, a marker of microglia. Left and middle panels show images that were immunolabelled for GFP and Iba1, respectively. Right panels are merged images of GFP and Iba1. Each image with blue, yellow or red circle corresponds to dots of the same colour as in Fig. 3E,F. Scale bar, 100 µm.

Fig 3: Schematic depicting a possible mechanism regulating the epigenetic switch of the NSE promoter in BG and PCs.In normal conditions, PCs use glucose as a primary energy resource, which is metabolized to pyruvate via the glycolysis pathway, which involves the glycolytic enzyme NSE (NSE promoter is ON). The pyruvate is oxidized and enters the tricarboxylic (TCA) cycle in the mitochondria to generate ATP. In the traumatic brain injury, high amounts of glutamate are released from injured cells, which in turn excites neurons to further release the glutamate. The excess amount of extracellular glutamate is taken up by BG that were activated by cytokines released from activated microglia. In the activated BG, the NSE promoter is activated to produce the NSE, which converts glucose into lactate, with the subsequent transfer of lactate to neurons (astrocyte-neuron lactate shuttle). The lactate inhibits glycolysis in the PCs through hydroxycarboxylic acid receptor 1 (HCA1), leading to suppression of the NSE promoter activity in PCs and facilitating lactate metabolization into pyruvate.

Fig 4: Regulation of the endogenous NSE promoter activity in parallel with that of virally introduced rat NSE promoter.The cerebellar slices treated with AAV vectors expressing GFP under the control of the NSE promoter were immunolabelled for GFP, NSE and S100. (A–C) Immunofluorescent images from region 1 (square in an upper left drawing). NSE immunoreactivity was present in the cell bodies of GFP-expressing PCs (insets in A,B), whereas it was absent in the BG processes (arrows in B,C). (D–F) Immunofluorescent images from region 2 (square in a lower left drawing). The NSE immunoreactivity was absent in the cytoplasm (insets in D,E) and dendrites (arrows in E) of GFP-negative PCs. Scale bar, 50 µm.

Fig 5: Transduction switch of the NSE promoter from BG to PC, triggered by the disappearance of local inflammation.Mice that received an injection of AAV9 vectors expressing GFP under the control of the NSE promoter to the cerebellum and the sagittal sections were examined via triple immunolabelling for GFP, Iba1 and S100 or parvalbumin 1, 2 and 3 weeks after injection. In addition, non-treated mice were used as controls. (A) Schema showing a position that was used for the analysis (an orange square in the lobule 6). (B–D) Cerebellar cortical images that were immunostained for GFP (green) and Iba1 (magenta) 1 (B), 2 (C) and 3 (D) weeks after viral injection. (E) A cerebellar image that was immunostained for parvalbumin (Parv, green) and Iba1 (magenta) from an age-matched naïve mouse. Scale bar, 100 µm for (B–E). (F) The number of microglia per µm3 × 10-6 (microglia density) in the granule cell layer 1, 2 and 3 weeks after viral injection and that from naïve control mice (Co). (G) The number of GFP-expressing PCs per 100 µm PC layer 1, 2 and 3 weeks after viral injection. (H) The number of GFP-expressing BGs per 100 µm PC layer 1, 2 and 3 weeks after viral injection. (I,J) Graphs showing the correlation of transduced PC density (I) or transduced BG density (J) with the density of microglia (Spearman’s rank correlation coefficient, (I) r = -0.7172, p = 0.0008, (J) r = 0.711, p = 0.0009). The numbers of GFP-expressing PCs and BGs per 100 µm PC layer were plotted against the microglia density in the contiguous granule cell layer (µm3 × 10-6). (K) A graph showing the correlation of transduced PC density with transduced BG density (Spearman’s rank correlation coefficient, r = -0.9546, p < 0.0001). The circles and diamonds are results that were obtained from the mice at 1 (circles) and 3 (diamonds) weeks after viral injection. Three sections per mouse from 3 mice (total 9 sections) were analysed each week. Asterisks and daggers indicate statistically significant differences compared to the mice at 1 week or 2 weeks post injection, respectively, as determined via a one-way ANOVA followed by Tukey post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, †p < 0.05, ††p < 0.01.