Fig 2: APP and a-synuclein converge on the IP3R1-Grp75-VDAC2 axis.A The sensitivity of hippocampal mitochondria to the calcium channel antagonists 2-APB and erastin was assessed. Ten milligrams of isolated hippocampal mitochondria from 3-month-old male rats were loaded with Fluo 4-AM and then incubated with 200 µM 2-APB or 20 µM erastin. With 16 µM calcium stimulation, mitochondria in which calcium channels were inhibited showed a smaller increase in fluorescence count than untreated controls. The percentages of net fluorescence increase in the antagonist-treated mitochondria relative to its untreated controls were determined. Three independent experiments including at least five rats per genotype were performed. Data from individual rats are indicated by dots, and the mean ± SEM for each genotype is presented. T-tests were used. *p < 0.05 and ***p < 0.001 indicate significant differences, n.s. indicates no significance. B Coimmunoprecipitation of Grp78 and FL-APP in hippocampal lysates from 3-month-old WT male rats. Anti-Grp78 antibody was used for immunoprecipitation, and Grp78 protein pulled down from the lysate was used as the loading control. Anti-APP antibody was used for western blotting. The stray bands are indicated by asterisks. C Comparison of the IP3R1-Grp78 interaction in the hippocampi of 3-month-old WT and APP KO male rats. An anti-Grp78 antibody was used for immunoprecipitation, and Grp78 protein pulled down from the lysate was used as the loading control. IP3R1 levels were measured by western blotting, and the stray bands are indicated by asterisks. D Three independent experiments in (C) including three rats per genotype were used for quantification. The mean ± SEM for each genotype is presented. T-test was used for statistical analysis, *p < 0.05 indicates a significant difference. E Interactions of VDAC2 with FL-APP and a-synuclein in the hippocampi of 3-month-old WT and SNCA KO male rats. A VDAC2 antibody was used for immunoprecipitation, and VDAC2 protein pulled down from the lysate was used as the loading control. FL-APP and a-synuclein were detected through western blotting. Immunoprecipitation was performed in SNCA KO hippocampal lysates as a negative control. The stray bands are indicated by asterisks. The arrowheads indicate the IgG heavy/light chain. Three independent experiments including 3 rats per genotype were performed. F Schematic diagram of the mechanism by which APP and a-synuclein regulate calcium flow via the IP3R1-Grp75-VDAC2 axis in MAMs. FL-APP interacts with Grp78 to disrupt the binding of Grp78 to IP3R1, prohibiting calcium flow released from the ER lumen into the MAM. Both FL-APP and a-synuclein interact with VDAC2 to promote calcium flow into the matrix. At OMMcyto, only a-synuclein binds to VDAC channels to regulate calcium flux. G Representative fluorescence traces showing mitochondrial calcium uptake after a series of extra calcium stimulations ranging from 0.75 mM to 12 mM. Ten milligrams of crude hippocampal mitochondria isolated from 3-month-old male rats and loaded with Fluo4-AM were used for the assay. The fluorescence count was measured every 30 s and six times before and after each stimulation. The average fluorescence count of unstimulated mitochondria of each genotype was used as an internal reference (F0), and all fluorescence measurements were divided by F0 for normalization (F1/F0). The F1/F0 value is plotted on the y-axis, and the detection time is plotted on the x-axis. The red arrow indicates the addition of CaCl2 buffer for stimulation. H Quantification of mitochondrial calcium release after 12 mM extra calcium stimulation. Three independent experiments including at least four male rats per genotype at 3 months old were performed as described in (G). The percentage of net reduction in the average F1/F0 value after 12 mM calcium stimulation relative to the plateau level of 3–6 mM calcium stimulation was determined. The data of individual rats are indicated by dots, and the mean ± SEM for each genotype is presented. T-tests were used for statistical analysis, and differences are indicated by asterisks. ***p < 0.001 indicates significant differences.

Fig 3: Co-expression analysis of cuproptosis-related genes (CRGs) and immune checkpoint genes (ICGs). (A) Expression levels of ICGs between normal liver tissue and hepatocellular carcinoma (HCC). (B) Differences in the expression of ICGs between low-risk and high-risk groups. (C) Co-expression analysis of CRGs and ICGs. ATP7A, ATP13A2, and SNCA were significantly positively correlated with the expression of ICGs, and a negative correlation between ICGs and COX17 or F5 was observed. ns, no significance.

Fig 4: APP and SNCA deficiency lead to spatial memory impairment in young rats.A The protein levels of APP and SNCA in cortical lysates from WT, APP KO (APP-deficient), SNCA KO (SNCA-deficient) and DKO (APP- and SNCA-deficient) rats were measured by western blotting. The molecular weight of each protein is provided on the right. FL-APP was completely absent in the APP KO rat cortex, and a-synuclein was completely absent in the SNCA KO rat cortex. DKO rats lacked both APP and SNCA proteins. B–E MWM test with 3-month-old male rats. Three independent experiments including at least 5 rats per genotype were performed. The escape latency curve during the 5-day training period is shown in (B). Data from each day compared to WT rats were analyzed by one-way ANOVA. **p < 0.01 and ***p < 0.001 indicate significant differences, and n.s. indicates no significance. Representative tracks of individual rats in the probe trails are shown in (C). The percentage of the time spent in the target quadrant in the probe trial is shown in (D). The number of platform crossings in the probe trial is shown in (E). The data of individual rats are indicated by dots, and the mean ± SEM for each genotype is presented in (D) and (E). One-way ANOVA was used. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences, and n.s. indicates no significance.

Fig 5: Mitochondrial degradation and ER stress in APP- and SNCA-deficient hippocampi.A Mitochondrial content was assessed by determining the mtDNA/nDNA ratio through qPCR. Three independent experiments of three 3-month-old male rats per genotype were performed. The individual data are indicated with dots. The data for each genotype are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis, and differences were indicated by asterisks. **p < 0.01 and ***p < 0.001 indicate significant differences, and n.s. indicates no significance. B, C The expression of ER stress-related proteins in rat hippocampal lysates was measured. The stray band is indicated by an asterisk. Three independent experiments of 3-month-old male rats per genotype were performed. Each experiment detected one rat from different genotype groups at once as shown in (B). Quantification is presented as the mean ± SEM of the data from three rats per genotype in (C). T-tests were used. *p < 0.05 indicates a significant difference, and n.s. indicates no significance. D Transcription levels of ER stress-related genes were measured by RT–qPCR. RNA was isolated from hippocampal tissues of 3-month-old male rats. Three independent experiments with five rats per genotype were performed. The data are shown in the bar chart as the mean ± SEM. T-tests were performed. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences, and n.s. indicates no significance. E Subcellular fractions were probed with the indicated antibodies. A total of 15 µg protein from each fraction was loaded per lane. PM plasma membrane, Mc crude mitochondrial fraction, Mp pure mitochondrial fraction. The red box indicates the FL-APP and a-synuclein protein bands in the MAM fraction. Hippocampal tissues from six male rats of 3-month-old were mixed and used for component separation.