Fig 1: An enhancer at IGF2 is differentially methylated in neurons of major psychosis patients. a Hypomethylation in psychosis at the IGF2 locus. The view shows differentially methylated regions (red, comb-p) and nominal probe-level p-values (blue) in major psychosis cases (n = 55 individuals) relative to controls (n = 27 individuals), as identified by EPIC arrays. Also shown are adult frontal cortex enhancers (brown rectangles; NIH Roadmap Epigenomics Project) and region validated by targeted bisulfite sequencing (gray rectangle). b CpG probe-level methylation within differentially methylated IGF2 region, by diagnostic subgroup and sex. Boxplot center indicates median; box bounds indicate 25th and 75th percentile, and whiskers mark 1.5 times the interquartile range. c Validation of IGF2 hypomethylation using targeted bisulfite sequencing. Average % DNA methylation in a region in IGF2 that is differentially methylated in major psychosis. Box plots show the % DNA methylation averaged over the ~1.3 kb enhancer region in neuronal DNA (NeuN+; 13 cases, 13 controls) and glial DNA (NeuN-: 10 cases, 12 controls). Bases with =10× coverage are included in the analysis. P-value from ANOVA for effect of disease, after controlling for age, sex, post-mortem interval, and batch effect. Base-level methylation estimates from EPIC arrays and targeted bisulfite sequencing were strongly correlated (Pearson’s coefficient R = 0.67, p < 10-19; t-test; Supplementary Fig. 9). Boxplot elements same as in b. d Validation of neuronal IGF2 hypomethylation in cases, when samples are limited to males of European genetic ancestry (n = 25 cases, 11 controls). Nominal p-values from a nested ANOVA model for effect of diagnosis after controlling for age, post-mortem interval, and the first two genetic principal components. b–d Boxplot center indicates median; box bounds indicate 25th and 75th percentile, and whiskers mark 1.5 times the interquartile range. Boxplot elements same as in b
Fig 2: Quality assessment of DA and non-DA FACS and microarray analysis. (a) Typical FACS analysis results for sorting DA and non-DA neuron populations from VTA brain punches. DP shows the double-positive events indicating DA neurons by positive expression of TH and NeuN, while AF488 indicates the events positive only for the NeuN neuronal marker indicating the non-DA neuron population. The AF488 threshold was set to increase the specificity of non-DA neuron collection. (b) Results from principal component analysis showing clustering of the DA versus non-DA neurons as well as grouping based on saline versus nicotine treatment. Heatmaps of the microarrays showing top differentially expressed (c) miRNAs and (d) mRNAs for DA and non-DA following perinatal nicotine or saline treatment. The mRNA microarray maps also indicate a clear grouping based on neuron type.
Fig 3: Neuroinflammation and neurodegeneration analysis in the hippocampus and cortex. (A) Confocal micrographs depicting Iba1-, CD11b- and GFAP-immunolabeled cells in the cornus amonis CA1/CA3 and dentate gyrus (DG) of the hippocampus or in the cortex, and cortical immunolabeling of glutamine synthetase (GS). Typical surveillant and activated microglia (Iba1+ and CD11b+) phenotypes are indicated by the arrowheads, and expanded below the cortex micrograph. (B) HFHSD had no impact on the Iba1+ area or number of microglia cells, but increased the fraction of activated microglia, which was normalized by diet reversal. (C) Astrocytes were considered all GS+ and/or GFAP+ cells. HFHSD had no impact on the area occupied by GS+ cells or number of astrocytes. (D) Total Iba1 and GFAP levels in the hippocampus or cortex were similar across the experimental groups. (E) Expression of NF-?ß and cytokines in the hippocampus, relative to the 60S ribosomal protein L14. (F) NeuN immunolabeling of neuronal somata was used to estimate the number of mature neurons in the cortex and within the granule cell layer of CA1, CA3 and DG. (G) Doublecortin (DCX)-immunolabeling was used to count immature neurons. DCX+ cells are estimated per DG within a stained brain slice. Dashed lines in micrographs define the granule cell layer in CA1, CA3 and DG. Data is plotted as mean±SD of n=6-8 (half of either gender). Letters over data-points indicate significant differences relative to CD or as indicated (a P<0.05, b P<0.01, c P<0.001) based on Fisher’s LSD post hoc comparison following presence of significant effects of diet or interaction between diet and time in ANOVA tests.
Fig 4: Phagocyte-mediated synaptic loss and neuronal p-STAT1 signalling in Neuro-HIV. (A and B) Confocal immunofluorescence images for synaptophysin (SYP), NeuN, IBA1 and nuclei (DAPI) from brain sections of Neuro-HIV patient (A) and NND (B). Scale bar = 25 µm. (C) Quantification of synaptic densities in Neuro-HIV and NND. Symbols represent individual samples (n = 6 per group; 35 neurons evaluated per patient). (D) Correlation of synaptic density with phagocyte apposition in Neuro-HIV and NND. Symbols represent individual samples (n = 6 per group; 35 neurons evaluated per patient). (E and F) Quantification of perisomatic bouton density in Neuro-HIV (E) and NND (F) matched for brain region and age (n = 35 neurons evaluated per patient) and stratified according to the presence (+) or absence (-) of contact with CD68+ cells. Symbols represent individual samples (n = 6 per group). (G and H) 3D cell reconstruction of confocal immunostainings of a homeostatic ramified phagocyte in NND without synaptic inclusions (G) and an amoeboid phagocyte in Neuro-HIV (H) exhibiting a SYP inclusion overlapping with a CD68+ phagosome. (I) Quantification of engulfed synaptic terminals (SYP) localized in the phagosomal compartment (CD68) of CNS phagocytes (IBA1) in brain sections of Neuro-HIV patients (n = 5) and NDD (n = 5). Number of SYP+ CD68+ phagocytes per mm2 are shown. (J) Proportion of engulfed synaptic terminals out of all detected SYP punctae in brain sections of Neuro-HIV patients (n = 5) and NDD (n = 5). (C–J) Lines indicate the median. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; ns = not significant by paired Student’s t-test.
Fig 5: Astragaloside IV decreased the protein expression of CaSR in vivo and in vitro. The expression of CaSR was detected by western blotting. (A) Representative western blot images from each group in PC12 cells. (B) Relative expression levels of CaSR in PC12 cells. n=3. *P<0.05 vs. control; #P<0.05 vs. OGD/R; ^P<0.05 vs. AST-IV. (C) Representative western blot images from each group of rats. (D) Relative expression levels of CaSR in rats. n=3. *P<0.05 vs. Sham; #P<0.05 vs. MCAO/R. (E) Representative images of double immunofluorescence staining of CaSR and NeuN. Magnification, ×400. CaSR, calcium-sensing receptor; NeuN, neuronal nuclei; MCAO/R, middle cerebral artery occlusion/reperfusion; CaSR, calcium-sensing receptor; OGD/R, oxygen and glucose deprivation/reoxygenation; AST-IV, MCAO/R or OGD/R + Astragaloside IV; NPS-2143, MCAO/R or OGD/R + CaSR antagonist.
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