Fig 2: PP2A-Pep normalized receptor expression and inhibited neuronal death after MCAO-induced cerebral ischemia. Brain slices containing the somatosensory cortex were prepared from sham and MCAO operated mice and incubated for 6 h with 10 µg/ml PP2A-Pep followed by staining for GABAB receptors (red), neurons (magenta, NeuN positive cells) or PP2A-Pep (green). Representative images are shown for sections from sham operated mice treated with Ctrl-Pep (A) or PP2A-Pep (B) and for section from MCAO operated mice treated with Ctrl-Pep (C) or PP2A-Pep (D). Images on top of each panel are of low magnification (scale bar: 40 µm) and those on the bottom are of higher magnification (scale bars 10 µm). (E) Quantification of GABAB receptor expression. The data represent the mean ± SD of 21–37 frames (fields of view) derived from 4 to 5 mice per condition. Two-way ANOVA with Tukey’s multiple comparison test (ns, p > 0.05, **p < 0.005, ****p < 0.0001). (F) Quantification of neuronal loss. The data represent the mean ± SD of 22–44 frames (fields of view) derived from 4 to 6 mice per condition. Two-way ANOVA with Tukey’s multiple comparison test (ns, p > 0.05, ****p < 0.0001.

Fig 3: PP2A-Pep inhibits progressing excitotoxic neuronal death. Neurons were stressed for 60 min with 50 µM glutamate and treated with no peptide (control), PP2A-Pep (10 µg/ml) or Ctrl-Pep (10 µg/ml) 0, 3, 6, 12, 24, and 48 h after the glutamate stress. After additional 24 h of incubation, the cultures were stained with DAPI for total cells (glia plus neurons) and with an antibody directed against NeuN for neurons. (A) Schematic representation of the experimental design. (B) Representative images (scale bar: 100 µm) are shown for the untreated control and for the condition where the peptides were immediately administered after the glutamate stress (0 h), or after 6 and 48 h. (C) Quantification of neuronal loss. Number of neurons were normalized to the untreated control cultures. The data represent the mean ± SD of 35–112 frames (fields of view) per experimental condition from three independent experiments. Brown–Forsythe and Welch ANOVA with Games Howell’s multiple comparison test (ns, p > 0.05, *p < 0.05, ***p < 0.0005, ****p < 0.0001).

Fig 4: PP2A-Pep prevents constitutive internalization of GABAB receptors and normalizes glutamate-induced loss of cell surface receptors. Neurons were transfected with GABAB2 tagged with the binding site for a-bungarotoxin [GABAB2(BBS)] along with EGFP for quick identification of transfected neurons and with wild type GABAB1. After labeling cell surface receptors containing GABAB2(BBS) with AlexaFluor 555 conjugated a-bungarotoxin at 4°C, neurons were analyzed at room temperature by life cell imaging for internalization of labeled receptors at the indicated time points and treatments. (A) Representative images (scale bar: 10 µm). (B) Quantification of the immunofluorescence signal. (C) Statistical evaluation of data 10 min after initiation of internalization. The data represent the mean ± SD of 4–7 neurons derived from three independent experiments. Two-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05, ****p < 0.0001).

Fig 5: PP2A-Pep restored GABAB receptor-mediated inhibition and reduced neuronal excitability after MCAO-induced cerebral ischemia. Brain slices containing the somatosensory cortex of sham and MCAO operated mice were analyzed using whole-cell patch-clamp recordings 2–6 h after treatment with PP2A-Pep, Ctrl-Pep or saline. (A) Representative traces of baclofen-evoked currents. (A’) Quantification of baclofen-evoked current traces. The data represent the mean ± SD of 6–10 neurons. One-way ANOVA, Tukey’s multiple comparison test (ns, p > 0.05, *p < 0.05, **p < 0.01). (B–B”) Neuronal excitability was determined by injecting increasing current steps. (B) Representative current traces. (B’) Quantification of action potential firing evoked by increasing current injections. (B”) Bar graph and statistical evaluation of data depicted in the gray box in (B’). The data represent the mean ± SD of 10–16 neurons. Brown–Forsythe and Welch ANOVA with Games Howell’s multiple comparison test (ns, p > 0.05, *p < 0.05, **p < 0.01). (C) Determination of input resistance. (D) Determination of resting membrane potential. (E) Determination of action potential threshold. The data represent the mean ± SD of 10–16 neurons. One-way ANOVA with Tukey’s multiple comparison test (ns, p > 0.05, *p < 0.05, **p < 0.01).