Fig 2: Analysis of selected proteins following IA learning.Dorsal hippocampal protein expression levels were measured using extracts from animals sacrificed 24 hours following IA training (A) or 6 hours following IA retention testing (B). Protein expression level of MCT1, Na/K alpha2, Glut1, MCT4 and Pygb were assessed by the western blotting. Results are expressed as percentage of control value and are means ± SEM of OD units from each group normalized to ß-actin, except for Glut1 which was normalized to ß-tubulin. Asterisks indicate a significant difference (Student’s t-test; * P < 0.05, ** P < 0.01, *** P < 0.001 vs CS group, n = 7 per group).

Fig 3: miR-338-3p targets PYGB to inhibit glycogenolysis in Neuro2a cells. (A) Sequence analysis of miR-338-3p mature miRNA binding with the 3'-UTR of PYGB. (B) Luciferase reporter assay showing reduction of luciferase activity in N2a cells compared with wild-type PYGB 3'UTR (luc-PYGB-WT) plasmids, but not in the seed region mutant PYGB 3'UTR (luc-PYGB-MT) plasmids. Data are shown as the mean ± SEM values and are the result of three independent experiments. Statistical analysis was performed by using two-tailed unpaired t-test. N.S., no statistical significance, and ***P < 0.001. (C,D) Western blots and quantifications showing the expression of PYGB in Neuro2a cells transfected with miR-338-3p mimics, inhibitors or scramble sequence (100 nM, respectively). Data are shown as the mean ± SEM values and are the result of four independent experiments. Statistical analysis was performed by using one-way ANOVA test followed by a Turkey post hoc test. *P < 0.5 and **P < 0.01. miR-NC: miRNA negative control (scramble sequence). (E) Relative glycogen levels in Neuro2a cells transfected with miR-338-3p mimics, inhibitors or scramble sequence (100 nM, respectively). Data are shown as the mean ± SEM values and are the result of four independent experiments. Statistical analysis was performed by using one-way ANOVA test followed by a Turkey post hoc test. *P < 0.5. (F) A schematic model to show that regional glycogen accumulation in the lumbar spinal cord of ALS mice, which is caused by increased miR-338-3p and decreased PYGB.

Fig 4: Screening of miRNAs targeting PYGB. (A) Overlap examinations performed on four different miRNA prediction databases by VENNY. Eight miRNAs were found, namely miR-338-3p, miR-183-5p, miR-491-5p, miR-19b-5p, miR-451b-5p, miR-2110-5p , miR-149-3p, and miR-23a-5p. (B) Luciferase reporter assay showing miR-338-3p and miR-149-3p may directly bind to the 3′UTR of PYGB. Data are shown as the mean ± SEM values and are the result of three independent experiments. Statistical analysis was performed by using one-way ANOVA test followed by a Turkey post hoc test. **P < 0.01 and ***P < 0.001. (C–F) RT-qPCR results showing the changes of miR-338-3p and miR-149–3p in the lumbar spinal cord and motor cortex. Data are shown as the mean ± SEM values and are the result of six independent experiments. Statistical analysis was performed by using two-tailed unpaired t-test. N.S., no statistical significance, and **P < 0.01.