Fig 1: Psap overexpression restores CL→mPFC functional connectivity and improves consciousness and cognitive impairments following subarachnoid haemorrhage.Schematic of the optogenetic experimental design (part a). An adeno-associated viral (AAV) vector encoding ChrimsonR was injected into the central lateral thalamus (CL), an AAV vector encoding GCaMP6s into the medial prefrontal cortex (mPFC), and a Psap-overexpressing AAV vector was injected into the corpus callosum. Representative images showing successful transfection of the light-sensitive protein ChrimsonR and the calcium indicator GCaMP6s in the CL and mPFC, respectively (part b). Post-stimulation average histogram of ΔF/F calcium signals aligned across the control (Vec) and Psap overexpression (OE) groups, with the black line indicating the mean and shaded area representing the standard error of the mean (part c; scale bar: 100 μm). Heatmap of ΔF/F calcium signals in the control (Vec) and Psap OE groups, with the colour bar indicating individual mice and groupings (part d). Quantification of normalized calcium signal area under the curve in the control (Vec) and Psap OE groups (n = 6, ****P <0.0001) (part e). Representative electroencephalogram (EEG) and electromyogram (EMG) recordings, and sleep–wake stage analysis over 24 h, recorded 48 h post-surgery in the control and Psap OE groups (part f). Proportion of time spent in each sleep–wake stage in the control (Vec) and Psap OE groups (n = 6, **P <0.01, ****P <0.0001) (part g). Representative images from the Morris water maze and probe tests in the control and Psap OE groups (parts h and k). Quantitative analysis of escape latency, swimming speed and time spent in the target quadrant during the Morris water maze and probe tests in the control and Psap OE groups (n = 12, ****P <0.0001, two-way ANOVA for part i, unpaired t-test for parts j and l) (parts i, j and l). ns, not significant.
Fig 2: SAH reduces PSAP–GPR37 interactions via selective Psap downregulation in oligodendrocytes.Bubble plot showing changes in PSAP–GPR37 interactions following subarachnoid haemorrhage (SAH) (part a). Uniform manifold approximation and projection (UMAP) plot and violin plot showing Psap gene expression in sham and SAH mice (**P<0.01) (part b). (c) UMAP plot and violin plot showing Gpr37 gene expression in sham and SAH mice (part c). Representative immunofluorescence images of GPR37 (green), PSAP (red) and CC1 (magenta) co-staining in the corpus callosum of sham and SAH mice (part d). Dashed lines outline the corpus callosum. Right panels show high-magnification images of the boxed regions. Scale bars: 50 μm. Quantification of the percentage of PSAP+CC1+ cells among total CC1+ cells in sham and SAH groups (n = 6, **P<0.01) (part e). Quantification of the percentage of GPR37+CC1+ cells among total CC1+ cells in sham and SAH groups (n = 6) (part f). Representative images of in situ proximity ligation assay (PLA) analysis showing PSAP and GPR37 interactions in the corpus callosum of brain tissue sections from sham and SAH mice (green represents interaction complexes, blue represents nuclei) (parts g and h; scale bar: 20 μm; n = 6, ****P<0.0001). ELISA-based quantification of PSAP levels in human and mouse plasma (n = 6, **P<0.01, ****P<0.0001) (parts i and j). Representative immunofluorescence images of LAMP2 (green), PSAP (red), CC1 (magenta) and 4′,6-diamidino-2-phenylindole (DAPI; blue) in the corpus callosum of sham and SAH mice (part k). Right panels show high-magnification images of the boxed regions. Yellow arrowheads indicate co-localization of PSAP with LAMP2-positive lysosomes in CC1-positive oligodendrocytes. Scale bars: 40 μm (left) and 5 μm (right). Representative immunofluorescence images of LAMP2 (green), PSAP (red), CC1 (magenta), and DAPI (blue) in brain tissue sections from human control individuals and patients with SAH (part l). Right panels show high-magnification images of the boxed regions. Yellow arrowheads indicate co-localization of PSAP with LAMP2+ lysosomes in CC1+ oligodendrocytes. Scale bars: 40 μm (left) and 10 μm (right). Quantification of PSAP+CC1+ cells per high-power field in tissue sections from human control individuals and patients with SAH (n = 6, ****P<0.0001) (part m). Quantification of GPR37+CC1+ cells per high-power field in tissue sections from human control individuals and patients with SAH (n = 6) (part n). Representative immunofluorescence images of GPR37 (green), CC1 (red), DAPI (blue) and merged channels in brain tissue sections from human control individuals and patients with SAH (part o). Right panels show high-magnification images of the boxed regions. Scale bars: 20 μm (left) and 5 μm (right). ns, not significant.
Fig 3: Oligodendrocyte-specific Psap overexpression enhances PSAP–GPR37 interactions and mitigates SAH-induced myelin and axonal damage in the CL→mPFC pathway.a Schematic representation of stereotactic injection of Psap overexpression virus into the corpus callosum. b Representative image showing Psap overexpression virus transfection, with mCherry (red) as the viral marker and 4′,6-diamidino-2-phenylindole (DAPI; blue) marking nuclei. Scale bar, 200 μm. c Representative in situ proximity ligation assay (PLA) images showing PSAP–GPR37 interactions in the corpus callosum of brain sections from subarachnoid haemorrhage (SAH) mice in the Psap overexpression (OE) and control (Psap-Vec) groups. Green signals represent interaction complexes, and blue signals represent nuclei. Scale bar, 20 μm. d The quantification of the PLA experiment representing the number of positive cells in each high-power field (n = 6, ****P<0.0001). e Representative immunofluorescence images of PSAP (magenta) and oligodendrocyte marker (CC1; green) staining in the corpus callosum of Psap OE and control (Psap-Vec) groups, with dashed lines outlining the corpus callosum. Scale bar: 20 μm. f Representative T2-weighted MRI images of the corpus callosum in Psap OE and control (Psap-Vec) groups following SAH. g Quantification of normalized T2 signal intensity in the corpus callosum of Psap OE and control (Psap-Vec) groups following SAH (n = 6, ****P<0.0001). h AAV2/9-hSyn-mCherry-labelled central lateral thalamus (CL) axons (red) in the corpus callosum co-stained for myelin basic protein (MBP) (green) and degraded MBP (dMBP; magenta). Scale bars: 50 µm. i Quantification of dMBP and MBP levels in mCherry+ regions of the corpus callosum (OE/Vec, n = 6, **P < 0.01, unpaired t-test). j Representative electron microscopy images of myelin in the corpus callosum from different groups. Scale bars: 1 μm (top panels) and 500 nm (bottom panels). k Quantification of g-ratio from myelin ultrastructure shown in part i. n >100, ***P < 0.001, ****P < 0.0001. mPFC, medial prefrontal cortex; ns, not significant.
Fig 4: HuPSAP restores CL→mPFC functional connectivity and ameliorates consciousness and cognitive impairments in SAH mice.Schematic of the optogenetic experiment (part a). An adeno-associated viral vector (AAV) encoding ChrimsonR was injected into the central lateral thalamus (CL), an AAV vector encoding GCaMP6s was injected into the medial prefrontal cortex (mPFC) and recombinant human PSAP (HuPSAP) was stereotactically delivered into the corpus callosum. Post-stimulation average histogram of ΔF/F calcium signals across HuPSAP concentration groups, with the thick black line representing the mean and the shaded area representing the standard error of the mean (part b). Quantification of normalized calcium signal area under the curve in different HuPSAP concentration groups (n = 6, ****P <0.0001, one-way ANOVA) (part c). Representative electroencephalogram (EEG) and electromyogram (EMG) recordings and sleep–wake staging over 24 h in HuPSAP and saline control groups (part d). Proportion of time spent in each sleep–wake stage in the HuPSAP and saline control groups (n = 6, ****P <0.0001) (part e). Representative images from the Morris water maze and probe tests in HuPSAP and saline control groups (parts f and h). Quantitative analysis of escape latency and time spent in the target quadrant during the Morris water maze and probe tests (n = 12, ****P <0.0001, two-way ANOVA for part g, unpaired t-test for part i) (parts g and i). Subarachnoid haemorrhage (SAH) induces consciousness impairment and downregulates Psap expression in oligodendrocytes, disrupting PSAP–GPR37 interactions that are essential for maintaining white matter integrity (part j). Targeted Psap overexpression in oligodendrocytes or administration of HuPSAP restores PSAP–GPR37 signalling, attenuates SAH-induced white matter damage, and reestablishes CL→mPFC functional connectivity, thereby improving consciousness. These findings identify PSAP as a potential therapeutic target for SAH-induced disorders of consciousness. EXN, excitatory neurons; ns, not significant; OL, oligodendrocytes.
Fig 5: SAH induces myelin and axonal damage in the CL→mPFC pathway accompanied by oligodendrocyte dysfunction and downregulation of myelination-associated genes.a Representative T2-weighted MRI images. b Quantification of T2-hyperintensity in regions of interest (n = 6, ***P <0.001). c Representative image showing AAV2/9-hSyn-mCherry transfection, with mCherry (red) as the viral marker and 4′,6-diamidino-2-phenylindole (DAPI; blue) marking nuclei. Scale bar: 200 μm. d AAV2/9-hSyn-mCherry-labelled central lateral thalamus (CL) axons (red) in the corpus callosum co-stained for myelin basic protein (MBP; green) and degraded MBP (dMBP; Magenta) at 48 h post-subarachnoid haemorrhage (SAH). Scale bars: 50 µm. e Quantification of dMBP and MBP levels in mCherry-positive regions of the corpus callosum (n = 6, ***P <0.001, unpaired t-test). f Uniform manifold approximation and projection (UMAP) visualization of oligodendrocytes, showing distinct segregation between sham and SAH groups. g Volcano plot showing differential expression of genes in oligodendrocytes following SAH. h Pathway enrichment analysis of downregulated genes in oligodendrocytes, showing significant enrichment in lipid metabolism. i Gene Ontology term analysis of five gene clusters from pseudotime analysis, identifying enriched pathways related to myelination, lipid transport and ion homeostasis. j Downregulation of key myelination-related genes in pseudotime analysis, highlighting the significant downregulation of Psap, Abca2, Mal and Mbp following SAH. mPFC, medial prefrontal cortex.
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