Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunohistological staining for BCAN was performed on cerebellar brain sections from Pclo wt/wt and Pclo gt/gt rats. In both genotypes, BCAN is localized in GFAP-positive astrocytes, from which it is secreted. In Pclo wt/wt slices, BCAN is also found in the surrounding tissue, indicating effective secretion. In contrast, in Pclo gt/gt sections, significantly less BCAN was detected outside astrocytes, suggesting impaired secretion of BCAN from astrocytes. B , Quantification of a. BCAN intensity is slightly greater inside astrocytes in Pclo gt/gt cerebellar sections than in Pclo wt/wt cerebellar slices; however, this difference is not significant ( Pclo wt/wt : mean± SEM= 1± 0.05, n=40 astrocytes, 3 independent animals; Pclo gt/gt : mean± SEM= 1.151± 0.06, 2 independent animals; p=0.082, t test). C, Quantification of a. BCAN intensity is significantly lower outside astrocytes in Pclo gt/gt slices than in Pclo wt/wt slices ( Pclo wt/wt : mean± SEM= 1± 0.04, 3 independent animals; Pclo gt/gt : mean± SEM= 0,831± 0.06, 2 independent animals; p=0.018, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunocytochemical staining for BCAN on primary Pclo wt/wt and Pclo gt/gt cortical astrocytes. In Pclo gt/gt astrocytes, BCAN intensity accumulates around the nucleus and extends along a line from the center toward the periphery. In Pclo wt/wt astrocytes, BCAN intensity was distributed more evenly. B , Immunocytochemical staining for TNR on Pclo wt/wt and Pclo gt/gt cortical astrocytes. In Pclo gt/gt astrocytes, TNR intensity accumulates around the nucleus and extends along a line from the cell center toward the cell periphery. In contrast, in Pclo wt/wt astrocytes, the TNR intensity was distributed more evenly. C , Quantification of A. A line was drawn from the nucleus to the periphery and divided into 10 bins. BCAN intensity was subsequently measured within each bin and graphically represented. BCAN intensity is greater close to the nucleus in Pclo gt/gt primary cortical astrocytes than in Pclo wt/wt primary cortical astrocytes. D , Quantification of B. A line was drawn from the nucleus to the periphery and divided into 10 bins, subsequently TNR intensity was measured within each bin. TNR intensity is greater close to the nucleus in Pclo gt/gt cortical astrocytes than in Pclo wt/wt . E , Western blot (WB) analysis of the supernatants of Pclo wt/wt and Pclo gt/gt cortical astrocyte cultures. Significantly less BCAN was detected in Pclo gt/gt cortical supernatants ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 0.81± 0.045, n=3 independent experiments; p=0.0024, t test). F , Western blot (WB) analysis of the supernatants of Pclo wt/wt and Pclo gt/gt cortical astrocyte cultures. Significantly more TNR was detected in supernatant from Pclo gt/gt cortical astrocytes ( Pclo wt/wt : mean=1, n=3 independent experiments; Pclo gt/gt : mean± SEM= 1.653± 0.283, n=3 independent experiments; p=0.082, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunocytochemical staining BCAN, GM130, GFAP, and DAPI in Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. BCAN fluorescence intensity is diffusely distributed in Pclo wt/wt astrocytes, whereas it accumulates along a defined path in Pclo gt/gt astrocytes . B, Immunocytochemical staining of tenascin-R (TNR), GM130, GFAP, and DAPI in Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. TNR fluorescence intensity is concentrated near the nucleus and extends toward the cell periphery in Pclo gt/gt astrocytes, in contrast to a more uniform distribution in Pclo wt/wt astrocytes. C, Schematic illustration of the cargo distribution analysis method. A line from the nucleus to the cell periphery was segmented into 10 equal bins (0–9), and the fluorescence intensity of the cargo was measured in each bin and normalized to the mean intensity per bin in Pclo wt/wt astrocytes. D, Quantification of a. Normalized BCAN intensity decreases progressively from the nucleus to the periphery in Pclo wt/wt astrocytes but slightly increases in Pclo gt/gt astrocytes. E, Quantification of B. Normalized TNR intensity is greater near the nucleus in Pclo gt/gt astrocytes than in Pclo wt/wt cells. F, Western blot (WB) analysis of BCAN levels in the supernatants of Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. BCAN levels are reduced in Pclo gt/gt astrocytes ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 0.641± 0.141, n=3 independent experiments; p=0.064, t test). G, Western blot (WB) analysis of TNR levels in the supernatants of Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. TNR levels are increased in Pclo gt/gt astrocytes ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 1.246± 0.179, n=3 independent experiments; p=0.242, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining, Fluorescence, Western Blot

Journal: Scientific reports

Article Title: Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers.

doi: 10.1038/srep26882

Figure Lengend Snippet: Figure 3. Verification of differential expression of the representative proteins observed in LC-MS/MS analysis by immunohistochemistry on tissue sections. (A) shows MS/MS spectra of peptides with their reporter ion intensities for representative differentially expressed proteins - BCAN, EGFR, ENPP6 and HNRNP K. (B) immunohistochemistry (IHC) images acquired for the above proteins. IHC protocol is described under Methods and the staining and scoring details for each protein are shown in Supplementary Table S3. For BCAN, normal brain tissue shows low staining with pyramidal cells negative (a), Grade II tumor cells show strong cytoplasmic positivity (b). For EGFR, normal brain tissue shows negative staining (c) and Grade II tumor cells show medium intensity cytoplasmic staining (d). ENPP6 shows medium intensity staining of neurophils in normal brain with no staining of normal glial and neuronal cells (e), while Grade II tumor cells show low to medium intensity staining for ENPP6 in neurophil as well as in tumor cells (f). For HNRNP K, normal brain tissue scored negative (g) whereas Grade II tumor cells showed strong positivity (h).

Article Snippet: Sections were then incubated for 1 h at RT with EGFR (dilution 1:100; Cat No. HPA018530), BCAN (dilution-1:200; Cat No. HPA007865), ENPP6 (dilution-1:10; Cat No. HPA042740) and HNRNP K (dilution-1:250; Cat No. HPA007644) primary antibodies (Atlas Antibodies, Sigma) followed by peroxidase-labelled polymer conjugate to anti-rabbit or anti-mouse immunoglobulins compatible with the primary antibody, for 1 h and developed with DAB system (DAKO, Denmark).

Techniques: Quantitative Proteomics, Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Tandem Mass Spectroscopy, Staining, Negative Staining

Journal: Environmental Health

Article Title: Serum dioxin and DNA methylation in the sperm of operation ranch hand veterans exposed to Agent Orange

doi: 10.1186/s12940-019-0533-z

Figure Lengend Snippet: Top 50 EWAS loci ranked by the change in beta value comparing highest exposed with control

Article Snippet: SPAM1 , cg04471202 , 7 , 906 , 0.23 , 6.00E-04 , 0.31 , 0.17.

Techniques:

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: List of the analyzed ECM components.

Article Snippet: BCAN , BCAN-Hs00222607_m1.

Techniques:

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Invasion spectrum (the mRNA expression pattern of invasion-associated extracellular matrix components) differs in patients with ‘worse’ and ‘better’ prognoses. mRNA expression measurements were performed twice for each gene to confirm the data. A longer bar on the logarithmic scale indicates reduced expression. *P<0.05 vs. group A (Mann-Whitney U test). Group A, OS <24 months; group B, OS >24 months; BCAN, brevican; CD44, cluster of differentiation 44; CSPG5, chondroitin sulfate proteoglycan 5; EGFR, epidermal growth factor receptor; FLT4, Fms-related tyrosine kinase 4; HMMR, hyaluronan-mediated motility receptor; IDH1, isocitrate dehydrogenase 1; ITGAV, integrin-αV; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; NCAN, neurocan; PDGFA, platelet-derived growth factor α; TNC, tenascin C; VCAN, versican; OS, overall survival.

Article Snippet: BCAN , BCAN-Hs00222607_m1.

Techniques: Expressing, MANN-WHITNEY, Derivative Assay

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Mean immunohistochemistry scores of the ECM components that were determined to be significantly different in the two prognostic groups.

Article Snippet: BCAN , BCAN-Hs00222607_m1.

Techniques: Immunohistochemistry