MST1 activation is associated with disease progression in 5xFAD mice

Labeling amyloid content and distribution in the brain parenchyma is a crucial indicator for evaluating the pathological status of AD. In 5xFAD mice, the presence of Aβ1-42 emerges at an early age and progressively increases thereafter, facilitating the early formation of amyloid deposition plaques [32]. Therefore, amyloid plaque deposition was assessed in the brains of 5xFAD mice at 1, 3, 6, and 9 months old using thioflavin staining. Mature amyloid plaques were present in the hippocampus and cortex beginning at 3 months, with deposition gradually intensifying as the mice aged (Fig. 1A, B).

Fig. 1

Activation of MST1 is involved in the pathological process of 5xFAD mice. A Representative thioflavin S staining images for detection content in the hippocampus of 1-, 3-, 6-, 9-month-old WT and 5xFAD mice. B Quantitative analysis of the fluorescence intensity of amyloid plaque in the hippocampus of 1-, 3-, 6-, 9-month-old WT and 5xFAD mice (n = 3). C Hippocampus MST1, p-MST1, Bax, and Bcl-2 protein levels in 3-, 6-, 9-month-old WT and 5xFAD mice as showed using immunoblotting analysis. D Quantitative analysis of p-MST1/MST1 (n = 4). E Quantitative analysis of Bax/Bcl-2 (n = 4). F–I Representative immunohistochemical images and relative expression of p-MST1 in the hippocampus (F, G) and cortex (H, I) of 6-month-old WT and 5xFAD mice (n = 3, Scale bar is 200 µm and enlarged images scale bar is 20 µm). Nuclei were stained in blue (DAPI). All data represent means ± SEMs. *, 5xFAD vs. WT group at different months of age. *p < 0.05; **p < 0.01 and ***p < 0.001. #, Comparison between 5xFAD at different months of age, #p < 0.05; ##p < 0.01

To explore the activation of MST1 in 5xFAD, its activated form p-MST1 (Thr183) was assessed. The WB results showed a gradual increase in the p-MST1 to MST1 ratio as the age of 5xFAD mice increased, aligning with the observed changes in hippocampal neuronal apoptotic protein Bax and amyloid plaque deposition. However, these results did not differ significantly in age-matched WT mice. Therefore, the activation of MST1 to p-MST1 in 5xFAD mice may correlate with the pathological advancement of the disease (Fig. 1C–E). To further validate p-MST1 activity and distribution, IHC assays were conducted, revealing a significant increase in p-MST1 levels within the hippocampus (Fig. 1F, G) and cortex (Fig. 1H, I) of 6-month-old 5xFAD mice compared with the WT group, consistent with the WB results.

MST1 promotes cognitive deficits and neuronal damage in 5-month-old 5xFAD mice

To assess the effect of MST1 on cognitive and memory impairment in AD mice, 4-month-old C57 mice and 5xFAD mice were randomly assigned to four groups: C57 + AAV vehicle, C57 + AAV MST1, 5xFAD + AAV vehicle, and 5xFAD + AAV MST1, with 20 animals in each group. AAV-GFP vehicle and the AAV-GFP MST1 were administered into the DG region of mice through hippocampal stereotaxic injection to upregulate MST1 (Fig. 2A). Four weeks post-injection, spontaneous GFP fluorescence from the virus was observed in the hippocampus, indicating successful injection (Fig. 2B). The expression efficiency of MST1 was also evaluated via WB and RT-qPCR. The results showed significantly higher levels of MST1 expression at the protein and mRNA levels than in the control group (Fig. 2C–E).

Fig. 2

MST1 Overexpression aggravates cognitive impairment in 5-month 5xFAD mice. A Schematic diagram of experimental arrangement. AAV-GFP-vehicle and AAV-GFP-MST1 was injected into the hippocampus of 4-month C57 mice and 5xFAD mice, and then the mice were divided into four groups: C57 + AAV vehicle, C57 + AAV MST1, 5xFAD + AAV vehicle, and 5xFAD + AAV MST1 for experiments. One month later, MWM test was performed, and finally all mice were euthanized for other experiments. B Representative spontaneous fluorescence images of the AAVs-infected slices (n = 4, Scale bar is 2.5 µm). C Representative immunoblotting bands of hippocampus MST1 and p-MST1 after AAVs injection. D Quantitative analysis of MST1 and p-MST1 in hippocampus (n = 4). E MST1 mRNA levels in the hippocampus after AAVs injection were detected using RT-qPCR (n = 3). F-J MWM test results of four groups of mice, including swimming speed (F), escape latency (G), total time spent in the target quadrant (H), number of platform crossings (I), representative traces on fifth day the MWM training period (J). (n = 12). All data represent means ± SEMs. *, Comparison between C57 + AAV-vehicle and 5xFAD + AAV-vehicle group, *p < 0.05; #, Comparison between 5xFAD + AAV-vehicle and 5xFAD + AAV-MST1 group, #p < 0.05; ##p < 0.01; ###p < 0.001

Four weeks post-injection, the MWM test was performed to assess the spatial learning and memory abilities of the mice. During the training period, the mice did not exhibit significant differences in swimming speed (Fig. 2F). Compared with the C57 + AAV-vehicle group, the 5xFAD + AAV-vehicle group exhibited a significantly prolonged escape latency (time to find the hidden platform) beginning from the third day of training. Moreover, the escape latency of the 5xFAD + AAV-MST1 group during the training was significantly longer than that of the 5xFAD + AAV-vehicle group (Fig. 2G, J). The number of platform crossings and total time spent in the target quadrant were reduced considerably in the 5xFAD + AAV-MST1 group compared with the 5xFAD + AAV-vehicle group. Similar reductions were observed in the 5xFAD + AAV-vehicle group compared with the C57 + AAV-vehicle group (Fig. 2H, I). These MWM results suggested that MST1 overexpression exacerbated spatial learning and memory impairment in 5xFAD mice.

Due to the pivotal role of neuronal loss in cognitive decline, we examined the effect of MST1 expression on neurons in the CA1, CA3, and DG regions of the hippocampus using Nissl and HE staining, and then assessed the cellular morphology changes and the level of cellular death. Nissl staining showed neurons have normal morphology, clear nuclei, and dense arrangement in the C57 + AAV-vehicle group. However, hippocampal neurons in the 5xFAD + AAV vehicle group were mostly damaged (vacuolated, shrunk, and loosely arranged), having a dramatic reduction in the live: dead neuronal ratio. Furthermore, the degree of neuronal damage in the 5xFAD + AAV-MST1group was more severe, and the live: dead neuronal ratio was further reduced compared to the 5xFAD + AAV vehicle group (Fig. 3A, B).

Fig. 3

MST1 Overexpression aggravates neuronal damage in 5-month 5xFAD mice. A Representative Nissl staining images of hippocampal three subregions (DG, CA1 and CA3) in different group (Scale bar is 200 µm and enlarged images scale bar is 50 µm). red arrows indicate dead neuronal cells. B The ratio of live and dead neuronal cells of the hippocampus in different groups (n = 4). C HE staining images of hippocampal three subregions (DG, CA1 and CA3) in different group (n = 3, Scale bar is 200 µm and enlarged images scale bar is 50 µm). D Representative immunoblotting bands of hippocampus Bax, Bcl-2, Cleaved Caspase 9, Cleaved Caspase 3, and Cyt-C after AAVs injection E Quantitative analysis of Bax/Bcl-2, Cleaved Caspase 9, Cleaved Caspase 3, and Cyt-C in hippocampus (n = 4). F–H Representative immunofluorescence images and quantitative analysis of PSD95 and SYP in the hippocampus DG region of different group (n = 4, Scale bar is 50 µm). Nuclei were stained in blue (DAPI). I The levels of Aβ1-42 in the hippocampus of different group were measured using ELISA (n = 4). All data represent means ± SEMs. *, Comparison between C57 + AAV-vehicle and 5xFAD + AAV-vehicle group, *p < 0.05; **p < 0.01; ***p < 0.001; #, Comparison between 5xFAD + AAV-vehicle and 5xFAD + AAV-MST1 group, #p < 0.05; ##p < 0.01. ns, no significance

HE staining revealed a compact arrangement and normal morphology of neuronal cells in the C57 + AAV vehicle group. In contrast, the 5xFAD + AAV vehicle group exhibited sparse neuron arrangement with some pyknotic nuclei and increased chromatin. MST1 overexpression further aggravated the increase in the number of abnormal neurons (Fig. 3C).

Additionally, the expression of apoptosis-related proteins (Bax, Bcl-2, Cleaved Caspase 9, Cleaved Caspase 3, and Cyt-C) was assessed through immunoblotting. The Bax/Bcl-2 ratio and expressions of Cleaved Caspase 9, Cleaved Caspase 3, and Cyt-C in the 5xFAD + AAV vehicle group were significantly higher than in the C57 + AAV vehicle group. In the 5xFAD + AAV-MST1 group, the expression levels of apoptosis-related proteins were higher than in the 5xFAD + AAV vehicle group (Fig. 3D, E). These data suggest that MST1 promoted neuronal apoptosis.

Finally, WB revealed reduced expression levels of the synaptic marker proteins PSD95 and SYP in the 5xFAD + AAV-MST1 group compared with the 5xFAD group (Fig. S1A, B). IF of PSD95 and SYP validation yielded similar results (Fig. 3F–H), suggesting that MST1 overexpression also impaired the synaptic structure of neurons. ELISA results further revealed that Aβ deposition did not differ significantly between the 5xFAD + ad-MST1 and 5xFAD + ad-vehicle groups, implying that overexpression of MST1 did not significantly increase Aβ1-42 content (Fig. 3I).

Collectively, these findings suggest that MST1 promotes neuronal apoptosis and exacerbates the pathological process in 5xFAD mice through a mechanism that does not involve increased Aβ deposition.

MST1 exacerbates mitochondrial dysfunction and oxidative stress levels in 5-month-old 5xFAD mice

Mitochondria play an important role in satisfying the high energy metabolism of neurons, contributing more than 90% of the energy required by synapses. Hence, mitochondrial dysfunction causes significant damage to neurons. Mitochondrial dysfunction can manifest as changes in mitochondrial ultrastructure, increased mitochondrial fission, decreased fusion and biogenesis, increased ROS, and reduced ATP production. The mitochondrial perimeter is indicative of morphological alterations [33]. Hence, TEM was employed to monitor mitochondrial ultrastructural changes in mouse hippocampal neurons. The mitochondrial morphology (round or oval) appeared normal, exhibiting clear and intact outer membranes and cristae in the C57 + AAV vehicle group. Moreover, the mitochondrial perimeter was relatively longer in the C57 + AAV vehicle group. Although the mitochondria exhibited abnormal morphology and smaller perimeters in the C57 + AAV-MST1 group, there was no statistically significant difference when compared to the C57 + AAV vehicle group (Fig. 4A, B). However, in the 5xFAD + AAV-vehicle and 5xFAD + AAV-MST1 groups, some of the mitochondria exhibited significant shrinkage, swelling, and incomplete cristae, accompanied by decreased matrix density and shortened perimeters. The mitochondrial morphology in the 5xFAD + AAV-MST1 group deteriorated further than in the 5xFAD + AAV-vehicle group (Fig. 4A, B). This suggests that MST1 overexpression in 5xFAD mice exacerbates mitochondrial morphological and ultrastructural damage.

Fig. 4

MST1 Overexpression aggravates mitochondrial damage and oxidative stress levels in 5-month 5xFAD mice. A Morphological change of mitochondria in hippocampal neurons was measured by transmission electron microscopy after AAVs injection (Scale bar is 500 nm). red arrows indicate mitochondria. B Quantitative analysis of single mitochondrial perimeter in hippocampal neurons after AAVs injection (n = 20 mitochondrial/group). C, D Representative immunoblotting bands (C) and relative expression (D) of mitochondrial dynamics (OPA1, MFN2, Drp1, Fis1) and mitochondrial biogenesis (PGC1α, Nrf1) related proteins in hippocampus after AAVs injection(n = 4). E Representative images of mitochondrial ROS in the hippocampal DG region measured by MitoSOX Red staining (Scale bar is 20 µm). Nuclei were stained in blue (DAPI). F Quantitative analysis of MitoSOX Red staining (n = 4). G Levels of ATP in each group after AAVs injection (n = 3). H–J Levels of oxidative stress related indicators (SOD, GSH, MDA) (n = 4). All data represent means ± SEMs. *, Comparison between C57 + AAV-vehicle and 5xFAD + AAV-vehicle group, *p < 0.05; **p < 0.01; ***p < 0.001. #, Comparison between 5xFAD + AAV-vehicle and 5xFAD + AAV-MST1 group, #p < 0.05; ##p < 0.01; ###p < 0.001

The WB results showed significant variations in mitochondrial dynamic-related proteins (OPA1, MFN2, Drp1, and Fis1) and mitochondrial biogenic proteins (PGC1α and Nrf1) between the C57 + AAV-vehicle and 5xFAD + AAV-vehicle groups, as well as between the 5xFAD + AAV-vehicle and 5xFAD + AAV-MST1 groups (Fig. 4C, D). Hence, compared with the C57 + AAV-vehicle group, the mitochondrial fission in the 5xFAD group increased, while the mitochondrial fusion and biogenesis decreased. Notably, MST1 overexpression in 5xFAD mice further increased mitochondrial fission and inhibited mitochondrial fusion and biosynthesis compared with the 5xFAD + AAV-vehicle group.

The impact of MST1 overexpression on 5xFAD mice mitochondrial oxidative stress was also evaluated via MitoSOX red fluorescence staining. Mitochondrial ROS in the C57 + AAV-MST1 group increased, with no significant differences compared with the C57 + AAV-vehicle group. In contrast, the MitoSOX red fluorescence staining in the 5xFAD + AAV-vehicle group tended to increase compared to the C57 + AAV-vehicle group. Meanwhile, significantly more mitochondrial ROS was detected in the 5xFAD + AAV-MST1 group compared with the 5xFAD + AAV-vehicle group (Fig. 4E, F and Fig. S1C, D). These results confirmed that MST1 could enhance the mitochondrial ROS level in the hippocampus of 5xFAD mice, further aggravating mitochondrial damage.

ATP levels were reduced in the 5xFAD + AAV-vehicle group compared with the C57 + AAV-vehicle group. Importantly, the reduction in ATP levels was more pronounced in 5xFAD mice overexpressing MST1 (Fig. 4G). These data suggest that overexpression of MST1 interferes with energy metabolic processes in 5xFAD mice.

The activities of SOD, GSH, and MDA serve as indicators of cellular oxidative stress levels. In the 5xFAD + AAV-vehicle group, the levels of SOD and GSH decreased (Fig. 4H, I), while MDA increased (Fig. 4J) compared with the control group. Meanwhile, the 5xFAD + AAV-MST1 group exhibited significantly reduced SOD and GSH levels, along with a significant increase in MDA activity compared with the 5xFAD + AAV-vehicle group (Fig. 4H–J). Hence, MST1 promoted oxidative stress in 5xFAD mice.

Taken together, these findings imply that overexpression of MST1 in the hippocampus of 5xFAD mice impairs mitochondrial morphology and function, causing oxidative stress and imbalanced energy metabolism, which leads to hippocampal damage and exacerbation of cognitive deficits in mice.

Down-regulation of MST1 alleviates cognitive impairment and mitochondrial dysfunction in 8-month-old mice

To verify the effect of MST1 downregulation on cognition, mitochondrial function, and neurons in AD mice, AAVs were administered into the DG region of 7-month-old C57 and 5xFAD mice (Fig. 5A). Four weeks later, a significant decrease in MST1 protein and mRNA expression levels was observed compared with the control group (Fig. 5B–D). Additionally, the abundance of p-MST1 protein was significantly lower than in the control group (Fig. 5B, C).

Fig. 5

MST1 Knockdown alleviates cognitive impairment and mitochondrial damage in 8-month 5xFAD mice. A Schematic of experimental proposition in vivo. AAV-GFP-vehicle and AAV-GFP-shMST1 were injected into the hippocampus of 7-month-old C57 mice and 5xFAD mice, and then the mice were divided into four groups: C57 + AAV-vehicle, C57 + AAV-shMST1, 5xFAD + AAV-vehicle, and 5xFAD + AAV-shMST1 for experiments. One month later, MWM test was performed, and finally all mice were euthanized for other experiments. B Western blotting was used to analyze the levels of hippocampus MST1 and p-MST1 after AAVs injection. C Quantitative analysis of MST1 and p-MST1 in hippocampus (n = 3). D MST1 mRNA levels in the hippocampus after AAVs injection were detected using RT-qPCR (n = 3). E–I MWM test results of four groups of mice, including swimming speed (E), escape latency (F), total time spent in the target quadrant (G), number of platform crossings (H), representative traces on fifth day the MWM training period (I). (n = 12/group). J-K Representative immunoblotting bands (J) and relative expression (K) of mitochondrial dynamics (OPA1, MFN2, Drp1, Fis1) and mitochondrial biogenesis (PGC1α, Nrf1) related proteins in hippocampus after AAVs injection. L Representative images of mitochondrial ROS in the hippocampal DG region measured by MitoSOX Red staining (Scale bar is 20 µm). Nuclei were stained in blue (DAPI). M Quantitative analysis of MitoSOX Red staining (n = 4). All data represent means ± SEMs. *, Comparison between C57 + AAV-vehicle and 5xFAD + AAV-vehicle group, *p < 0.05, **p < 0.01, ***p < 0.001. #, Comparison between 5xFAD + AAV-vehicle and 5xFAD + AAV-shMST1 group, #p < 0.05; ##p < 0.01, ###p < 0.001

Subsequently, the MWM was employed to assess the spatial learning and memory of each mouse group. the mice did not exhibit significant differences in swimming speed during the training period (Fig. 5E). The 5xFAD + AAV-vehicle group exhibited a significantly prolonged escape latency (time to find the hidden platform) starting from the third day of training compared with the C57 + AAV-vehicle group. Meanwhile, the escape latency was significantly shorter in the 5xFAD + AAV-shMST1 group during the training compared to the 5xFAD + AAV-vehicle group (Fig. 5F, I). The number of platform crossings and total time spent in the target quadrant were reduced considerably in the 5xFAD + AAV-vehicle compared with the C57 + AAV-vehicle group. These factors were significantly increased in the 5xFAD + AAV-shMST1 mice compared with the 5xFAD + AAV-vehicle group (Fig. 5G, H). These MWM results showed that MST1 knockdown improved the ability to locate the hidden platforms and enhanced the cognitive function of 8-month-old 5xFAD mice.

To observe the effect of knocking down MST1 on neuronal synapses, immunofluorescence staining of synaptic-related markers (PSD95 and SYP) in the DG region of the hippocampus was performed. A significant reduction in synapse-related proteins was observed in 8-month-old 5xFAD mice, whereas MST1 knockdown significantly increased the abundance of these proteins in 5xFAD mice (Fig. S2A–C). These results indicate that 8-month-old 5xFAD mice experience significant synaptic damage, which can be prevented by MST1 knockdown.

Compared with the 5xFAD + AAV-vehicle group, The 5xFAD + AAV-shMST1 mice exhibited significantly increased abundances of mitochondrial fusion (OPA1 and MFN2) and biogenic (PGC1α and Nrf1) proteins and a significant decrease in mitochondrial fission proteins (Drp1 and Fis1) (Fig. 5J, K). MitoSOX red staining results showed a reduction in mitochondrial ROS levels following MST1 downregulation (Fig. 5L, M and Fig. S2D, E).

Transcriptomic analysis of the regulatory effects of MST1 in 8-month-old 5xFAD mice

To elucidate the molecular mechanism underlying MST1’s effect on AD, RNA-seq technology was employed. Transcriptome sequencing was conducted on hippocampal tissues from mice in the 5xFAD + AAV-vehicle and 5xFAD + AAV-shMST1 groups. The volcano plot displays the upregulated and downregulated genes within the differentially expressed genes (DEGs) (Fig. S3A). Overall, 359 DEGs (p ≤ 0.01) were assessed, with 162 downregulated (log2FC ≤  − 0.5) and 197 upregulated (log2FC ≥ 1.0). The heatmap presents the DEG clustering analysis results, revealing the expression of an identical gene across various samples and confirming biological replicate consistency (Fig. 6A). The Venn diagram illustrates the gene counts detected in each group, while overlapping areas indicate co-expressed genes between the two groups (Fig. 6B). To elucidate the functional roles of the identified DEGs, Gene Ontology (GO) enrichment analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. GO enrichment of DEGs was primarily associated with cellular metabolism (NAD(P) + nucleosidase activity, NAD + nucleotidase, cyclic ADP-ribose generation, NAD + nucleosidase activity) and immune inflammation regulation (positive regulation of immune effector processes, negative regulation of immune system processes, interleukin-6 production, and regulation of immune effector processes) (Fig. S3B). KEGG analysis revealed a significant enrichment of unigenes encoding enzymes in the oxidative phosphorylation pathway, with the PI3K-Akt signaling pathway the most significantly enriched (Fig. 6C). Enrichment was also observed in antioxidant resistance and chemical carcinogenic ROS pathways. Representative GSEA results aligned with the KEGG pathway findings (Fig. 6D, E). Significant upregulation of mitochondrial respiratory chain-related genes was observed following MST1 knockdown, including NADH dehydrogenase subunit 4 (MT-ND4L), ATP synthase F0 subunit 6 (MT-ATP6), cytochrome b oxidase (MT-CO2), ATP synthase F0 subunit 8 (MT-ATP8), Gm28439, and BC002163 (Fig. S3C).

Fig. 6

Exploring the Mechanisms of MST1 in Alzheimer's Disease through RNA-seq. The analysis of differentially expressed genes (DEGs) between 5xFAD + AAV-vehicle group and 5xFAD + AAV-shMST1 group. A Hierarchical clustering heatmap analysis of DEGs in each sample. The intensity of color represents the level of differentially expressed genes. Red indicates relatively high expression and blue represents relatively low expression. C1, C2, and C3 represent three samples of the 5xFAD + AAV-vehicle, M1, M2, and M3 represent three samples of the 5xFAD + AAV-shMST1. B Venn diagram of DEGs for two datasets. A total of 23776 co-expression genes were obtained. C Sankey diagram showing KEGG pathways enriched by DEGs. D GSEA plot of the Oxidative phosphorylation pathway. p < 0.05, p-adjust < 0.05. E GSEA plot of the PI3K-Akt pathway p < 0.05, p-adjust < 0.05

MST1 effects on SH-SY5Y cell model induced by β-amyloid in vitro

To investigate the effects and potential mechanisms of MST1 on cell models, SH-SY5Y cells were induced by Aβ1-42 as the AD in vitro model. Following treatment of SH-SY5Y cells with various concentrations of Aβ1-42 (0, 5, 10, 20, 40 μM) for 24 h, WB results revealed that the level of MST1 activation (p-MST1) and the ratio of phosphorylated MST1 to total MST1 (p-MST1/MST1) gradually increased in a concentration-dependent manner. Given that statistical significance was achieved following 20 μM Aβ1-42 exposure (Fig. 7A, B), this concentration was selected for subsequent experiments to establish the cell model. MST1 activation was assessed in the model and control groups using IF. Aβ caused a significant increase in the fluorescence intensity of p-MST1, consistent with the WB results (Fig. 7C, D).

Fig. 7

MST1 effects upon cell viability and mitochondria in vitro. A, B Western blotting measured the expression of MST1 and p-MST1 in SH-SY5Y cells cultured in 0, 5, 10, 20 and 40 μM Aβ1-42 for 24 h. B Quantitative analysis of p-MST1/ MST1 (n = 3). C, D Representative immunofluorescence images (C) and quantitative analysis (D) of p-MST1 in control and Aβ-treated groups (Scale bar is 20 μm). Nuclei were stained in blue (DAPI). (n = 3). E CCK8 assay was used to detect the cell viability of SH-SY5Y cells after treatment with Aβ1-42 or MST1 overexpressed plasmid. (n = 3) F CCK8 assay was used to detect the cell viability of SH-SY5Y cells after treatment with Aβ1-42 or MST1 specific siRNA (n = 3). G, H Flow cytometry analysis cells apoptosis of SH-SY5Y cells in different groups (Control, Aβ, ad-MST1 + Aβ, si-MST1 + Aβ). I Representative images of TMRM staining in SH-SY5Y cells in the Control, Aβ, ad-MST1 + Aβ, si-MST1 + Aβ groups (Scale bar is 20 μm). Nuclei were stained in blue (DAPI). J Quantitative analysis of TMRM. (n = 3). K Representative images of mitochondrial ROS in SH-SY5Y cells in the Control, Aβ, ad-MST1 + Aβ, si-MST1 + Aβ groups measured by MitoSOX Red staining (Scale bar is 20 μm). Nuclei were stained in blue (DAPI). L Quantitative analysis of MitoSOX Red staining (n = 3). All data represent means ± SEMs. *, Compared with the control group, *p < 0.05, **p < 0.01 and ***p < 0.001. #, Comparison between intervention groups, #p < 0.05; ##p < 0.01

Additionally, an MST1 overexpression plasmid and MST1-specific small interfering RNA (siRNA) were constructed, along with their respective vehicles, for cell transfection. WB and RT-qPCR results showed successful upregulation (Fig. S4A–C) or downregulation (Fig. S4D–F) of MST1, respectively. Additionally, CCK8 assay results revealed a decrease in SH-SY5Y cell viability upon treatment with Aβ1-42. Moreover, MST1 overexpression further reduced cell viability (Fig. 7E). However, loss of MST1 expression promoted cell survival (Fig. 7F). Additionally, the abundance of mitochondria-dependent apoptosis-related proteins (Bax, Cleaved Caspase 3, and Cyt-C) was increased in the AD cell model, with a more pronounced increase following transfection with the MST1 overexpression plasmid (Fig. S4G, H). However, transfection with siRNA reversed this effect (Fig. S4I, J). The trend in the antiapoptotic protein Bcl-2 abundance was opposite to that of the apoptotic proteins in each group (Fig. S4G–J). To further quantify the effect of MST1 on apoptosis, a flow cytometric analysis of apoptotic cells was performed. Increased apoptosis was observed in the Aβ and ad-MST1 + Aβ groups compared with the control group. The apoptosis rate of the ad-MST1 + Aβ group was significantly higher than in the Aβ group. Meanwhile, MST1 knockdown significantly decreased the apoptosis rate (Fig. 7G, H). These results indicate that MST1 was activated in Aβ-treated cells, promoting SH-SY5Y cell apoptosis.

The effects of MST1 activation on mitochondrial morphology and function was also evaluated in SH-SY5Y cells. The MitoTracker staining data showed more mitochondrial fragmentation in the Aβ and ad-MST1 + Aβ groups (Fig. S4K). Moreover, based on the TMRM and Mitosox Red staining results, the mitochondrial membrane potential decreased and mitochondrial ROS content increased in the ad-MST1 + Aβ group compared with the control and Aβ groups, whereas MST1 knockdown reversed these manifestations (Fig. 7I–L). These findings support the potential of MST1 to regulate mitochondrial functions.

MST1 regulates the transcription of mitochondrial genes and affects mitochondrial oxidative phosphorylation by binding PGC1α

Following RNA-seq analysis, KEGG enrichment analysis was performed, identifying enrichment of the oxidative phosphorylation pathway. Among the DEGs, MT-ND4L, MT-ATP6, and MT-CO2 differed significantly, all of which are associated with the oxidative phosphorylation pathway. To verify whether MST1 regulates mitochondrial gene transcription, the expression of these candidate genes was validated via RT-qPCR. The results showed a significant decrease in the mRNA expression of MT-ND4L, MT-ATP6, and MT-CO2 in the ad-MST1 + Aβ group and a significant increase in the si-MST1 + Aβ group, compared with the Aβ group (Fig. 8A, B). Additionally, the abundance of MT-ND4L, MT-ATP6 and MT-CO2 proteins was significantly decreased within the Aβ and si-Ctrl + Aβ groups, whereas knocking down MST1 effectively reversed this effect (Fig. S5A, B). Furthermore, upon overexpression of MST1, the abundance of these proteins significantly decreased (Fig. S5C, D). These results suggest that MST1 modulates mitochondrial DNA transcription and the expression of ECT proteins in an AD cell model.

Fig. 8

MST1 regulates the mitochondrial genes expression by binding to PGC1α in SH-SY5Y cell. According to the results of RNA-seq detection, three genes (MT-ND4L, MT-ATP6, MT-CO2) in the top 10 were selected for verification in Aβ-treated SH-SY5Y cells. A, B Relative expression MT-ND4L mRNA, MT-ATP6 mRNA, and MT-CO2 mRNA after overexpression or knockdown of MST1 in AD cell models (n = 3). C Representative images showing co-localization of p-MST1 and MitoTracker (Scale bar is 10 µm). (n = 3). Magnified image showing details of co-localisation (Scale bar is 5 μm). D Co-localization analysis of p-MST1 and MitoTracker (n = 3). E Co-IP followed by western blot analyses confirmed the binding between MST1 and PGC1α in the AD cell model. F–H Mitochondrial stress test analysis of oxygen consumption rate (OCR) in SH-SY5Y cells (n = 5) (F). Analysis of oxygen consumption rate (OCR), including basal respiration (G), proton leak (G), maximal respiration (H), ATP production (H). I, J Relative levels of MT-ND4L, MT-ATP6, and MT-CO2 protein in control, Aβ, ad-MST1 + Aβ, ad-MST1 + Aβ + ad-PGC1α group (n = 3). K Relative complex I-V enzyme activity in SH-SY5Y cells in the Control, Aβ, ad-MST1 + Aβ, ad-MST1 + Aβ + ad-PGC1α groups (n = 3). All data represent means ± SEMs. *, Compared with the control group, **p < 0.01. #, Comparison between intervention groups, #p < 0.05; ##p < 0.01 and ###p < 0.001

The subcellular localization of the activated form of p-MST1 at baseline was primarily concentrated in the cytoplasm and nucleus. However, in the AD cell model, the co-localization of p-MST1 and MitoTracker increased in the Aβ group (Fig. 8C, D). This suggests that Aβ treatment of SH-SY5Y cells promotes the activation of MST1 to the p-MST1 form and causes p-MST1 to accumulate on mitochondria.

To further explore the downstream molecular mechanism of MST1, Co-IP analyses were performed. PGC1α was found to potentially bind to MST1 (Fig. 8E). PGC1α is a key transcriptional co-activator that induces gene expression under physiological and pathological stress conditions. Moreover, a key function of PGC1a is the activation of mitochondrial biosynthesis and oxidative phosphorylation [34].Accordingly, we hypothesized that the effect of MST1 on mitochondrial oxidative phosphorylation-related genes involves PGC1a.

WB and PCR experiments showed that the reduction in MT-ND4L, MT-ATP6, and MT-CO2 proteins and mRNA expression after MST1 overexpression was partially reversed by PGC1a overexpression (Fig. 8F, G and Fig. S5E). Furthermore, MST1 overexpression reduced the maximum respiratory capacity and ATP production in cells while also increasing proton leakage within the ETC. However, overexpression of PGC1α reversed the OCR impairment caused by MST1 (Fig. 8H–J). Additionally, the activity of mitochondrial respiratory chain complexes I–V was assessed. Overexpression of MST1 worsened the decline in complex enzyme activity induced by Aβ; PGC1α overexpression mitigated this effect (Fig. 8K).

Taken together, these data support the notion that PGC1α is an important downstream effector molecule of MST1, impacting mitochondrial oxidative phosphorylation in Aβ-induced SH-SY5Y cells.

MST1 regulates oxidative stress through PI3K-Akt signaling in SH-SY5Y cells

Analysis of the RNA-seq results revealed enrichment of the PI3K-Akt pathway, suggesting its potential involvement in the effect of MST1 on AD regulation. To investigate the potential mechanism, changes in PI3K, Akt, and p-Akt protein expression was assessed upon upregulation or downregulation of MST1. WB revealed that p-Akt abundance was reduced in the Aβ group and further decreased in the group overexpressing MST1 (Fig. 9A, B). However, the inhibitory effect observed in the Aβ group was reversed when MST1 was knocked down (Fig. 9C, D). Total PI3K and Akt expression remained unchanged in all groups. To further evaluate the influence of the PI3K-Akt pathway on oxidative stress and mitochondrial function, MST1-overexpressing cells were treated with 740Y-P (25 μM) for 24 h to activate the PI3K-Akt pathway. The expression of mitochondrial apoptosis proteins (Bax, Cleaved Caspase-3, and Cyt-C), initially induced by MST1 overexpression, was significantly reduced by 740Y-P. Hence, MST1-induced apoptosis was reduced by 740Y-P (Fig. 9A, B). Treatment of MST1-overexpressing cells with 740Y-P significantly suppressed ROS levels (Fig. 9E, F), facilitating MMP restoration (Fig. 9I, J).

Fig. 9

MST1 affects oxidative stress through PI3K-Akt pathway, thereby affecting mitochondrial function. SH-SY5Y cells were incubated with 25 μM of PI3K-Akt activator (740 Y-P) for 1 h before treatment with Aβ after transfection with MST1 overexpression plasmid. SH-SY5Y cells were incubated with 20 μM of PI3K-Akt Inhibitor (LY294002) for 1 h before treatment with Aβ after transfection with MST1 specific siRNA. A, B Relative levels of PI3K, Akt, p-Akt, Bax, Bcl-2, Cleaved-Caspase 3, and Cyt-C protein in control, Aβ, ad-MST1 + Aβ, ad-MST1 + Aβ + 740Y-P group (n = 3). C, D Relative levels of PI3K, Akt, p-Akt, Bax, Bcl-2, Cleaved-Caspase 3, and Cyt-C protein in control, Aβ, si-MST1 + Aβ, si-MST1 + Aβ + LY294002 group (n = 3). E–H Relative intensity of ROS by Flow cytometry analysis in different groups. I, J Representative images (I) and quantitative analysis (J) of TMRM staining in SH-SY5Y cells in the Control, Aβ, ad-MST1 + Aβ, ad-MST1 + Aβ + 740Y-P groups (Scale bar is 20 µm). Nuclei were stained in blue (DAPI). (n = 3). K, L Representative images (K) and quantitative analysis (L) of TMRM staining in SH-SY5Y cells in the Control, Aβ, ad-MST1 + Aβ, si-MST1 + Aβ + LY294002 groups (Scale bar is 20 µm). Nuclei were stained in blue (DAPI). (n = 3). All data are repeated at least three times. All data represent means ± SEMs. *, Compared with the control group, *p < 0.05; **p < 0.01 and ***p < 0.001. #, Comparison between intervention groups, #p < 0.05; ##p < 0.01 and ###p < 0.001

Subsequently, MST1-knockdown cells were treated with LY294002 (20 μM), a PI3K-Akt pathway inhibitor; the abundance of mitochondrial apoptosis-related proteins, namely Bax, Caspase-3, and Cyt-C, increased. In contrast, the antiapoptotic protein Bcl-2 was decreased in the si-MST1 + Aβ + LY294002 group compared with the si-MST1 + Aβ group (Fig. 9C, D). This indicates that the administration of LY294002 counteracted the protective effect of MST1 knockdown. Moreover, MST1 knockdown significantly alleviated oxidative stress and improved MMP compared with the Aβ group, while LY294002 treatment exacerbated oxidative stress (Fig. 9G, H) and reduced MMP (Fig. 9K, L). These findings suggest that LY294002 reverses the inhibitory effects on oxidative stress and enhanced mitochondrial function induced by MST1 knockdown. Thus, as expected, MST1 might serve as a major regulator of the PI3K-Akt-ROS signaling pathway in Aβ-induced SH-SY5Y cells.

XMU-MP-1 relieves AD symptoms by inhibiting MST1 activity

XMU-MP-1—a novel Hippo kinase inhibitor—can effectively suppress MST1 expression [26]. Thus, 7-month-old WT and 5xFAD mice were intraperitoneally injected with XMU-MP-1 or DMSO for one month (Fig. 10A). The MWM results revealed significantly impaired spatial cognitive and memory abilities in the 8-month-old 5xFAD mice. However, following one month of treatment with XMU-MP-1, the 5xFAD mice exhibited shortened search times for hidden platforms, prolonged time spent in the target quadrant, and increased platform crossings (Fig. 10B–E). These results indicated that XMU-MP-1 rescued cognitive function impairments in 8-month-old 5xFAD mice.

Fig. 10

XMU-MP-1 improves cognitive and mitochondrial function in 8-month-old 5xFAD mice. A Schematic of experimental proposition in vivo after treatment XMU-MP-1. B–E MWM test results of four groups (C57 + DMSO, C57 + XMU-MP-1, 5xFAD + DMSO, 5xFAD + XMU-MP-1) of mice, including swimming speed (B), escape latency (C), total time spent in the target quadrant (D), Number of platform crossings (E). (n = 10/group). F, G Relative levels of MST1, p-MST1 protein in the hippocampus (n = 4). H, I Relative levels of PSD95, and SYP protein in the hippocampus (n = 4). J, K Relative levels of Bax, Bcl-2, Cleaved Caspase 3, Cleaved Caspase 9 protein in the hippocampus (n = 4). L, M Relative levels of Mitochondrial biogenic protein (PGC1α, Nrf1) in the hippocampus (n = 4). N, O Relative levels of MT-ND4L, MT-ATP6, and MT-CO2 in the hippocampus (n = 4). P, Q Relative levels of PI3K, Akt, and p-Akt protein in the hippocampus (n = 4). All data represent means ± SEMs. *, Comparison between C57 + DMSO and 5xFAD + DMSO group, *p < 0.05; **p < 0.01 and ***p < 0.001. #, Comparison between 5xFAD + DMSO and 5xFAD + XMU-MP-1 group, #p < 0.05; ###p < 0.001

Following one month of treatment, the abundance of certain proteins was evaluated via WB. The expression of p-MST1 was significantly suppressed, and p-MST1/MST1 levels decreased in the 5xFAD + XMU-MP-1 group (Fig. 10F, G). Moreover, synaptic-related proteins (PSD95 and SYP) were increased in the 5xFAD + XMU-MP-1 group compared with the 5xFAD + DMSO group (Fig.