Fig 2: GLUD1 participates in circTICRR modulating the viability and autophagy of cervical cancer cells.A Left panel: Part of the cluster heatmap of differentially expressed mRNAs in the SiHa cells transfected with si-circTICRR#2 or negative control by RNA-seq. Each group had three repeats. Right panel: qRT-PCR analysis of 13 chosen top differentially expressed mRNAs in the SiHa and CaSki cells treated with two siRNAs targeting circTICRR or si-NC. B The relative GLUD1 mRNA (up) and protein (down) levels in the SiHa and CaSki cells transfected with two circTICRR siRNAs or negative control. Cell viability (C) was determined by CCK-8 assay, cell apoptosis (D) was measured by Flow Cytometry, cell morphology (E) was monitored by TEM, and autophagy-related proteins (F) were detected by western blot in the SiHa and CaSki cells transfected with two GLUD1 siRNAs or a negative control siRNA. G SiHa and CaSki cells transfected with mRFP-GFP-LC3B for 24 h were transfected with si-NC, si-circGLUD1#1 or #2 for another 48 h. Images were captured by confocal microscopy. Scale bar, 30 µm. SiHa and CaSki cells were transfected with si-circTICRR#1, si-circTICRR#2, si-circTICRR#1 plus GLUD1 plasmid, si-circTICRR#2 plus GLUD1 plasmid, and negative controls, respectively. CCK-8 assay (H) and Flow Cytometry (I) were performed to test cell viability. Autophagy-related proteins were detected by western blot J. Autophagy flux was monitored by confocal microscopy K. Scale bar, 30 µM. Data are representative of at least three independent experiments and presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: GLUD2 mutation aggravates the movement disorder in MPTP-treated mice.a The GLUD2 gene sequences across humans and other primates are shown. Blue coloring highlights the high homology of the T1492 site. b The experimental design of this study is shown. AAV-GLUD2, AAV-GLUD2 T1492G, or AAV-GFP was stereotaxically injected into the SNpc, and 3 weeks later, mice were intraperitoneally administered saline or MPTP for 5 weeks. One day after the last MPTP/saline injection, behavioral tests were performed, after which time the mice were sacrificed. (c, d) The expression of AAV-GLUD2 and AAV-GLUD2 T1492G in the SNpc was verified by immunostaining with GFP and GFAP antibodies. e, f Total distances traveled and numbers of entries to the center zone in the open-field are shown for AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups. g The grip strength test was used to examine the limb grip strength of mice. h The rotarod test was used to examine the motor coordination of mice. i The pole-climbing test was used to examine the bradykinesia of mice. j The grasping test was used to further examine the grip strength of mice. n = 10, 9, 12, and 9 for AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups, respectively. Results are expressed as the mean ± SEM. **p < 0.01, *p < 0.05 vs. AAV-GFP group. ##p < 0.01, #p < 0.05 vs. MPTP + AAV-GFP group. Statistical significance was determined by one-way ANOVAs and Tukey tests for post-hoc comparisons.

Fig 4: Metabolomic analysis of GLUD2 and its mutant in the SN of MPTP-treated mice.a The relative abundances of representative metabolites in each sample are shown. b Overall metabolic profiles of samples from the AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups using PCA plots. c Overview of metabolic profiles of all samples using 3-D PCA score plots. d Z-score plots showing the relative variations of each individual metabolite across all groups in the form of a heatmap. e MPEA of potential pathways involved in the differential metabolite content between the indicated groups. f The network of metabolites in the TCA cycle for MPTP + AAV-GFP versus MPTP + AAV-GLUD2 T1492G groups. Dashed boxes indicate no detection, solid boxes indicate normal expression, red boxes indicate upregulation in the MPTP + AAV-GLUD2 T1492G group compared with the MPTP + AAV-GFP group, and green boxes indicate downregulation in the MPTP + AAV-GLUD2 T1492G group compared with the MPTP + AAV-GFP group.

Fig 5: Schematic models showing the underlying mechanism by which GLUD2 T1492G variant modifies disease onset in the MPTP-induced PD animal model.In this study, the T1492G variant of GLUD2 decreases glutamate transporters expression and function, induces apoptosis by decreasing Bcl-2 and increasing Bax expression, and reduces energy metabolism by decreasing succinate dehydrogenase activity. These effects cause glutamate excitotoxicity and succinic acid accumulation. Consequently, GLUD2 variant aggravates nigral dopaminergic neuron death and exacerbates movement deficits in MPTP-induced PD mice. In the schematic model, red arrows mean downregulate and green arrows mean upregulate.