rabbit polyclonal synapsin 1 and 2  (Synaptic Systems)

Journal: Cells

Article Title: Hypothermia Shifts Neurodegeneration Phenotype in Neonatal Human Hypoxic–Ischemic Encephalopathy but Not in Related Piglet Models: Possible Relationship to Toxic Conformer and Intrinsically Disordered Prion-like Protein Accumulation

doi: 10.3390/cells14080586

Figure Lengend Snippet: Antibodies Used for Cell Type and Synapse Identification in Combination with Antibodies to Toxic Conformer Proteins.

Article Snippet: Synapsin 1 and 2 , Presynaptic terminals , Rabbit polyclonal , Synaptic Systems, 106002.

Techniques: Drug discovery, Ubiquitin Proteomics, Transduction

Journal: Cells

Article Title: Hypothermia Shifts Neurodegeneration Phenotype in Neonatal Human Hypoxic–Ischemic Encephalopathy but Not in Related Piglet Models: Possible Relationship to Toxic Conformer and Intrinsically Disordered Prion-like Protein Accumulation

doi: 10.3390/cells14080586

Figure Lengend Snippet: Synucleinopathy occurs in three different piglet models of HI brain damage as demonstrated by the accumulation of aggregated α-Syn. ( A ) Piglets were exposed to hypoxia–ischemia (HI) and treatment with hypothermia (HT) for 20 h followed by rewarming at 0.5 °C/hours or left at normothermia (NT) or treated with the sham procedure (with and without HT) and survived for 29 h. Western blotting of forebrain extracts show that aggregated α-Syn (oligomer-specific antibody Syn33) accumulated subacutely after HI. Ponceau S staining shows protein loading. ( B ) Graph of Western blot densitometry quantification of aggregated α-Syn in forebrain of sham-NT ( n = 4), sham-HT ( n = 4), HI-NT ( n = 4), and HI-HT ( n = 4) piglets. Box plot showing mean values (with IQR and 5–95 percentile whiskers). ( C ) Piglets were exposed to HI, but no hypothermia treatment, or were exposed to sham procedure and survived for 4 days. Western blotting of somatosensory cortex extracts shows that aggregated α-Syn (oligomer-specific antibody Syn33) accumulated days after HI. Brain sample from human mutant α-Syn-A53T transgenic mouse was the + control. Ponceau staining shows protein loading. ( D ) Graph of Western blot densitometry quantification for aggregated α-Syn at 4 days after HI (sham n = 4, HI n = 4). Box plot showing mean values (with IQR and 5–95 percentile whiskers). ( E ). Immunohistochemical localization of aggregated α-Syn in HI-QA piglets and 14 days after injury. Aggregated α-Syn was found enriched in attritional neurons in striatum (black arrow) while other neurons were lesser-enriched (gray arrow), and other neurons were negative (white arrow). Presynaptic bouton-like structures in the neuropil (thin arrows) were also positive. Scale bar = 10 µm (same for F ). ( F ) Sham piglet neurons were largely negative for aggregated α-Syn suggesting that the antibody is detecting a pathological form of α-Syn. ( G ) Counts of positive aggregated α-Syn boutons in striatum of HI-QA and sham piglets at 15 days after injury (sham piglets n = 5, HI-QA piglets n = 6; 3 sections/piglet). Box plots show mean values (with IQR and 5–95 percentile whiskers). Statistically significant p values are indicated. ( H ) Immunofluorescence demonstrated that some aggregated α-Syn immunoreactivity (green) localizes to presynaptic terminals (white arrows, yellow) identified by SV2 (red) in the neuropil and in perineuronal/peridendritic regions of the HI piglet neocortex. Scale bar = 3 µm. ( I ) Immunofluorescence shows that aggregated α-Syn (green) colocalizes (white arrows, yellow) with synaptophysin (red) in the striatum of HI-QA piglets. Scale bar = 5 µm. ( J ) In a favorably preserved sample of the human HIE neocortex, immunofluorescence also showed that aggregated α-Syn (green) colocalized with SV2 (red) seen as yellow (white arrows). Some presynaptic terminals appeared swollen (left most white arrow). Scale bar = 6 µm.

Article Snippet: Synapsin 1 and 2 , Presynaptic terminals , Rabbit polyclonal , Synaptic Systems, 106002.

Techniques: Western Blot, Staining, Mutagenesis, Transgenic Assay, Control, Immunohistochemical staining, Immunofluorescence

Journal: bioRxiv

Article Title: CONDENSATES OF SYNAPTIC VESICLES AND SYNAPSIN ARE MOLECULAR BEACONS FOR ACTIN SEQUESTERING AND POLYMERIZATION

doi: 10.1101/2024.07.19.604346

Figure Lengend Snippet: A. Domain organization of Synapsin 1. B. SDS-PAGE gel of the proteins – EGFP-Syn1-FL (102.235 kDa), EGFP-Syn1-IDR (57.621 kDa) and EGFP-Syn1-domain C (63.116 kDa) employed for in-vitro reconstitutions in the present study. C. Schematic illustration of Syn1-actin reconstitution assay. Actin polymerization from Syn1 phases was examined by first forming 6 µM EGFP-Syn1-FL condensates with 3% (w/v) PEG 8000 on a glass bottom dish. After 5 minutes, when EGFP-Syn1-FL condensates became 3-4 µm in size, ATTO647 labelled G-actin monomers were added from top into these pre-formed EGFP-Syn1-FL condensates such that the final concentration of actin and EGFP-Syn1-FL in the final reaction mix was 4 and 4 µM respectively.

Article Snippet: Synapsin condensates were co-labeled using a rabbit polyclonal antibody made against the IDR of synapsin 1a/b (Synaptic Systems 106 008; 1:100), which was characterized in Supplemental figure 8.

Techniques: SDS Page, In Vitro, Reconstitution Assay, Concentration Assay

Journal: bioRxiv

Article Title: CONDENSATES OF SYNAPTIC VESICLES AND SYNAPSIN ARE MOLECULAR BEACONS FOR ACTIN SEQUESTERING AND POLYMERIZATION

doi: 10.1101/2024.07.19.604346

Figure Lengend Snippet: Representative confocal images of the reconstituted EGFP-Syn1-IDR [4 µM, 3% (w/v) PEG 8000] and SVs (3 nM, labelled with FM4-64, 1.65 µM) condensates after adding ATTO-647 labelled G-actin monomers (4 µM) at t = 0 (top) and 90 minutes (bottom). Excited wavelengths were 488 nm for EGFP-Synapsin 1 IDR, 560 nm for SVs labelled with FM4-64 and 647 for ATTO647 G-actin. Scale bar: 5 µm.

Article Snippet: Synapsin condensates were co-labeled using a rabbit polyclonal antibody made against the IDR of synapsin 1a/b (Synaptic Systems 106 008; 1:100), which was characterized in Supplemental figure 8.

Techniques:

Journal: bioRxiv

Article Title: CONDENSATES OF SYNAPTIC VESICLES AND SYNAPSIN ARE MOLECULAR BEACONS FOR ACTIN SEQUESTERING AND POLYMERIZATION

doi: 10.1101/2024.07.19.604346

Figure Lengend Snippet: A, B. Two-color super-resolution STED images of hippocampal neurons (14 DIV) from (a) wild-type and (b) synapsin triple knockout neurons stained with StarRed-phalloidin and anti-synaptophysin (Star580). Scale bar, 1 µm. C. Magnified region from (a) showing a cohort of synaptic vesicles (anti-synaptophysin) colocalizing with actin (StarRed-phalloidin). D. Magnified region from (b) indicating a more dispersed signal of SVs (anti-synaptophysin) lacking the colocalization with actin (StarRed-phalloidin). Scale bar, 0.5 µm. E, F. Representative images of synapsin triple knockout neurons rescued with (e) full-length EGFP-synapsin 1 or (f) EGFP-synapsin 1 IDR and stained with StarRed-phalloidin and anti-synaptophysin (Star580). Scale bar, 1 µm. G, H. Magnified regions from (e) and (f), respectively, showing colocalization of SVs with actin. Scale bar, 0.5 µm. I. Autocorrelation of the actin signal along the axons (StarRed-phalloidin; see dotted/while lines in the images in a and b ) indicates that actin rings remain unaltered in the absence of synapsins. J. Actin enrichment in synaptic vesicle cohorts defined as an intensity signal of actin channel within regions positive for synaptophysin (wild-type and synapsin triple knockout) or both synaptophysin and EGFP (in rescue experiments).

Article Snippet: Synapsin condensates were co-labeled using a rabbit polyclonal antibody made against the IDR of synapsin 1a/b (Synaptic Systems 106 008; 1:100), which was characterized in Supplemental figure 8.

Techniques: Triple Knockout, Staining