Journal: Brain

Article Title: Schizophrenia-associated changes in neuronal subpopulations in the human midbrain

doi: 10.1093/brain/awae321

Figure Lengend Snippet: Unsupervised clustering identifies 18 neuronal populations in the human midbrain . ( A ) Experimental design of the study . A schematic representation of the rostral midbrain at the level of the red nucleus and third cranial nerves (created with biorender.com ). The lines indicate where the superior colliculus (SC) and cerebral peduncles (CP) were removed before nuclei isolation, FANS and snRNAseq. ( B ) Unsupervised clustering of 31 669 neuronal nuclei from 14 schizophrenia (SZ) cases and 14 controls ( and ). Each dot indicates a nucleus, colours indicate clusters. ( C ) Average expression per cluster of selected neurotransmitter-, neuropeptide- or interneuron-related genes. ( D ) Bar plot depicting the total number of nuclei obtained per population, with the percentage of the total NEUN+ population enclosed in parentheses. ( E ) Violin plots depicting log-normalized expression levels of cluster markers (log 2 fold-change > 0.25, P -adjusted < 0.05) . ( F ) Violin plot depicting log-normalized expression level of DLX1 , a cluster marker for inh-CCK. ( G ) A schematic representation of the rostral midbrain indicating the periaqueductal grey and the third cranial nerve nucleus with a box and the reticular formation with a dotted box. In these regions the immunohistochemical staings, shown in H , were performed. ( H ) Representative images of immunohistochemical stainings for DLX1 (brown) and GFAP (black) in control midbrain ( n = 5). Scale bar = 50 μm. avg. = average; expr. = expression; FANS = fluorescence-activated nuclear sorting; log-norm. = log-normalized; snRNAseq = single nucleus RNA-sequencing; UMAP = Uniform Manifold Approximation and Projection.

Article Snippet: Briefly, after sucrose density centrifugation, nuclei were incubated with fluorescently-conjugated antibodies directed against neuronal marker NEUN (RBFOX3/NEUN 1B7 AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4).

Techniques: Isolation, Expressing, Marker, Immunohistochemical staining, Control, Fluorescence, RNA Sequencing

Journal: Brain

Article Title: Schizophrenia-associated changes in neuronal subpopulations in the human midbrain

doi: 10.1093/brain/awae321

Figure Lengend Snippet: Schizophrenia-associated shifts in proportions of midbrain neuronal populations and subpopulations . ( A ) Uniform Manifold Approximation and Projection (UMAP) of the clusters split by diagnosis. The colour legend indicates the number of nuclei obtained per population and the percentage of the total NEUN+ population enclosed in brackets. ( B ) Box plot depicting the percentage of exc-VGLUT1 and 2 nuclei detected in midbrain samples of schizophrenia and control cases ( n = 14 per group; ). Outlier cases are labelled with grey diamond shapes, each dot represents a case, and horizontal lines indicate the median. Outliers were defined as proportions outside of the third quartile plus 1.5 times the interquartile range. ( C ) UMAP of the subclusters split by diagnosis. The colour legend indicates the number of nuclei obtained per population and the percentage of the total NEUN+ population enclosed in brackets. ( D ) Box plots depicting the percentage of nuclei detected per case in subclusters that had significantly altered proportions in schizophrenia; and . Outlier cases are labelled with grey diamond shapes and were defined as data points outside the third quartile + 1.5 × interquartile range. ( E ) Total count of differentially expressed genes (DEGs) between schizophrenia and controls within a neuronal population. Numbers above the bar indicate the total count of increased plus decreased gene expression changes in schizophrenia. ( F ) Volcano plot depicting differentially expressed genes (absolute log 2 fold-change > 0.25, P -adjusted < 0.05) between schizophrenia and controls within the neuronal population ‘inh-CCK’. The corresponding information on DEGs is available in . ( G ) Total count of differentially expressed genes between schizophrenia and controls within a neuronal subpopulation. Numbers above the bar indicate the total count of increased plus decreased gene expression changes in schizophrenia. ( H ) Volcano plot depicting DEGs (absolute log 2 fold-change > 0.25, P -adjusted < 0.05) between schizophrenia and controls within the neuronal subpopulation ‘inh-C’. The number of gene labels was reduced to avoid overlapping text; the full information on DEGs is available in . ( I ) Heat map with the number indicating the detected schizophrenia susceptibility genes per neuronal population . The colour indicates the normalized ratio of detected susceptibility genes over the total number of expressed genes per neuronal population. High-confidence schizophrenia susceptibility genes derived from Wang et al .

Article Snippet: Briefly, after sucrose density centrifugation, nuclei were incubated with fluorescently-conjugated antibodies directed against neuronal marker NEUN (RBFOX3/NEUN 1B7 AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4).

Techniques: Biomarker Discovery, Control, Gene Expression, Derivative Assay

Journal: Acta neuropathologica communications

Article Title: Blocking peptidyl arginine deiminase 4 confers neuroprotective effect in the post-ischemic brain through both NETosis-dependent and -independent mechanisms.

doi: 10.1186/s40478-025-01951-y

Figure Lengend Snippet: Fig. 3 PAD4 expression in the different brain cell types in the post-ischemic brain (A) Schematic representation of the brain region bordering the infarct core and penumbra. (B-D1) Coronal brain sections were prepared from sham controls (B) as well as the cortical penumbra (C, C1, C2) and cortical core (D, D1) of the MCAO group at 24 h post-surgery. Triple immunofluorescence staining was performed with anti-PAD4 antibody, anti-NeuN antibody, and DAPI. Arrows indicate PAD4 immunoreactivity in neurons and arrowheads indicate PAD4 immunoreactivity in non-neuronal cells. Asterisks depict PAD4 immunoreactivity in degenerating cells or cell debris. Scale bars represent 50 μm

Article Snippet: Primary antibodies for anti-neuronal nuclei (NeuN) (MAB377; Merk Millipore), anti-ionized calcium-binding adaptor molecule-1 (Iba1) (orb18542; Biorbyt, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP) (556327; BD Biosciences, Franklin Lakes, NJ, USA), anti-CitH3 (ab281584, Abcam), anti-Myeloperoxidase (MPO) (ab90810; Abcam), anti-Lymphocyte antigen 6 complex locus G6D (Ly6g) (ab25024; Abcam), PAD4 (GTX113945, GeneTex), anti-F95 (MABN328, Merk Millipore), and anti-RECA (MCA970GA; BIO-RAD, Kidlington, UK) were all diluted 1:200.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Acta neuropathologica communications

Article Title: Blocking peptidyl arginine deiminase 4 confers neuroprotective effect in the post-ischemic brain through both NETosis-dependent and -independent mechanisms.

doi: 10.1186/s40478-025-01951-y

Figure Lengend Snippet: Fig. 5 Accumulation of deiminated proteins in neurons, especially in damaged neurons in the post-ischemic brain Coronal brain sections were prepared from sham controls (A) as well as the cortical penumbra (B-F, H, I) of the MCAO group at 12 h (B-D) or 24 h (E, F, H, I) post-MCAO. Coronal brain sec tions were further prepared from the cortical penumbra of the MCAO + BBCA group at 24 h (F, I) post-MCAO. Triple immunofluorescence staining was conducted using the anti-F95 antibody, anti-NeuN antibody, and DAPI (A-F) or with the anti-F95 antibody, TUNEL, and DAPI (H, I). (G) The ratio of F95- to NeuN-positive cells is presented (n = 12 from 3 animals). (J) The ratio of TUNEL-positive cell to F95-positive cells is presented (n = 12 from 3 animals). Arrows indicate F95 immunoreactivity localized in the neuron, while asterisks indicate localization of F95 immunoreactivity in degenerating neurons. Arrowheads highlight F95 immunoreactivity in non-neuronal cells, and double arrowheads highlight F95 immunoreactivity in vasculature. Double asterisks in H and I indicate localization of F95 immunoreactivity in TUNEL-positive cells. Scale bars represent 50 μm. **p < 0.01, ***p < 0.001 compared to the sham group and ##p < 0.01, $p < 0.05 between indicated groups

Article Snippet: Primary antibodies for anti-neuronal nuclei (NeuN) (MAB377; Merk Millipore), anti-ionized calcium-binding adaptor molecule-1 (Iba1) (orb18542; Biorbyt, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP) (556327; BD Biosciences, Franklin Lakes, NJ, USA), anti-CitH3 (ab281584, Abcam), anti-Myeloperoxidase (MPO) (ab90810; Abcam), anti-Lymphocyte antigen 6 complex locus G6D (Ly6g) (ab25024; Abcam), PAD4 (GTX113945, GeneTex), anti-F95 (MABN328, Merk Millipore), and anti-RECA (MCA970GA; BIO-RAD, Kidlington, UK) were all diluted 1:200.

Techniques: Immunofluorescence, Staining, TUNEL Assay