Fig 1: Single synapse glutamate clearance in the striatum after expression of EAAT2-S506X. (A) Representative examples from iGluu imaging of single corticostriatal synaptic terminals in slices from WT, HET, and treated HET. Bilateral intracranial injections of HET with EAAT2-S506X. The selected images were acquired at the response peak to illustrate the extension of iGluu elevation (boxed area). Pink pixels within the black boundaries are pixels where stimulation of the bouton elicited a fluorescence increase (?F) to values larger than the resting level prior to stimulation (F). (B) Glutamate spread at the ROI peak of the glutamate elevation (see Section “Materials and Methods” for definition of the parameter “Spread”). (C) iGluu transients for the examples shown in (A). Recordings before and after electrical stimulation of the given bouton in the presence of TTX. The traces represent the mean fluorescence intensity calculated from all suprathreshold pixels in the ROI. Red curves: Monoexponential functions fitted to the iGluu traces. In black – respective time constants of decay (TauD). (D,E) Quantification of results obtained from the pixel with the highest elevation of iGluu fluorescence. (F) Cumulative probability plot to illustrate the absence of TauD values > 15 ms in WT and in EAAT2-S506X-treated HET. TauD values > 15 ms identify pathological synapses in HET. (G) Only synapses in the immediate vicinity of transduced astrocytes were included, as confirmed by the location of the tested iGluu-expressing varicosity on the territory of mRuby-positive astrocytes (arrowhead). *p < 0.05, **p < 0.05, n.s., not significant.
Fig 2: Immunohistochemical detection of EAAT2 expression in the hippocampus (scale: 20 µm). (A,D,G) show the CA1 hippocampal area; (B,E,H) show the CA3 hippocampal area; and (C,F,I) show the DG hippocampal area. Panel (J) shows the quantification of (A–I). *P < 0.05 compared with the untreated control.
Fig 3: Effects of bilateral EAAT2-S506X expression on the motor performance of Q175 mice. (A) Recordings of open field trajectories showing the mean running velocity between starts and stops (green or red circles, respectively). Gray level coding of instantaneous running velocity. (B) Motor activity at rest. Color coding of movement velocity at rest. (C,D,F–K) Quantification of results from open field testing. (E,F) Step-over latency – inverse relationship with number of starts in the open field, and sensitivity of step-over latency to mHTT and treatment. (L) Matched age-, CAG-, and body-weight composition of the test groups (only body weight illustrated). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: mHTT- and S506X-related changes in the abundance of YFP-EAAT2 binders. (A) Injected vectors. (B) Animal groups. (C) Scheme of immunoprecipitation experiment. (D) List of EAAT2 bands to be detected in the Western blots (WB) from the beads preparation (this figure) or lysates (Figure 3). (E,F) Western blot samples prepared from the beads YFP-immunoprecipitate. Note dimer bands and shift of the EAAT2-S506X band (YEX, boxed). (G) Similar amounts of mYFP immunoreactivity (YE or YEX bands) pulled down in the three animal test groups. (H) Heat-map plot illustrating the mHTT-related differences between WT:EAAT2 and HET:EAAT2. The signal from each animal was normalized to the mYFP median intensity value in a histogram constructed from all samples. The inter-group intensities were compared by a two-tailed t-test. The asterisks next to the listed genes denote the significance level of the difference. The proteins are listed with their gene names and sorted according to their abundance in WT:EAAT2. (I,J) Plot illustrating the recovery potential of significant EAAT2 binders. The LFQ intensity difference denotes the log2 difference between the compared groups. Compared were WT:EAAT2 vs. HET:EAAT2 (empty bars with black asterisks) and HET:EAAT2-S506X vs. HET:EAAT2 (filled bars with green asterisks). The dashed bars indicate the corresponding WT levels. An up-regulation is shown as upward bar, a down-regulation as downward bar. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: Neuronal cells (A–L). EAAT2 expression in the CA1, CA3, and DG hippocampal areas in rats measured 3 d after seizure onset by double-labeling immunofluorescence (yellow arrows indicate neurons; white arrows indicate astroglia-like cells) (scale: 20 µm).
Supplier Page from Abcam for Anti-EAAT2 antibody