Ab64581

Anesthetized rats (4 /group) were perfused with 300 ml 0.9% NaCl and 4% paraformaldehyde in phosphate-buffered saline (PBS), and the extracted brains were stored with 4% paraformaldehyde and then 30% sucrose for 48 hours before being frozen in an embedding compound. The frozen whole brains were cut to a thickness of 30 μm with a cryostat (HM525, Thermo Scientific). The section slides were incubated overnight at 4 °C with primary antibodies against GFAP (1:1000; catalog ab7260, Abcam), NeuN (1:1000; catalog MAB377, Millipore), MAP2 (1:1000; catalog ab11267, Abcam), PSD95 (1:500; catalog ab18258, Abcam), synapsin (1:500; catalog ab64581; Abcam), and CD68 (1:1000; cat MAB1435; Millipore), washed with PBS, and incubated for 2 hours at room temperature with Alexa Fluor 488 and Cy^tm 3-conjugated AffiniPure F(ab’)₂ Fragment Donkey Anti-Mouse IgG secondary antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA, USA). DAPI was used for counterstaining. Immunofluorescent sections were imaged by LSM700 confocal microscope (Zeiss, Oberkochen, Germany) using 10× and 40× PlanApo oil-immersion lens. Briefly, 12 μm-thick confocal Z-stacks of the synaptic zone in ZI were captured. Three image stacks per rat (4 /group) were used for analyses. The number of cells with colocalized GFAP and mGluR1 was quantified. Three images per rat (4 /group) were used for analyses.

Three selected proteins, sortilin, synapsin I, and TMCC2 were analysed by Western blot in the hippocampus using 15 μg of total protein per lane in a 4–20% Criterion TGX Precast Gel (Bio-Rad, Madrid, Spain). Membranes were incubated overnight with rabbit polyclonal anti-Sortilin (catalogue number: S0697; Sigma-Aldrich, Madrid, Spain), anti-Synapsin I (ab64581; Abcam, Madrid, Spain), or anti-TMCC2 (25042–1-AP; Proteintech, AntibodyBcn, Barcelona, Spain) antibody. Antibodies were diluted 1:400, 1:1000, and 1:1000, respectively, in 0.02 M Tris-buffered saline and 0.1% Tween-20. Beta actin antibody (37,005, Cell Signalling, Werfen, Barcelona, Spain) was used as transfer and loading control. Infrared-dyed secondary anti-IgG antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used, membranes were scanned in Odyssey Imager (LI-COR Biosciences).

Immunoblotting was performed as previously described [28 (link)]. Antibodies and dilutions used in this study include: anti-TrkB (1:1000; # ab18987, Abcam), anti-phospho-TrkB (phTrkB Y817; 1:1000; # ab81288, Abcam), anti-synapsin I (1:1000; # ab64581, Abcam), anti-PSD-95 (1:1000; # ab12093, Abcam), anti-tau (1:1000; # ab75714, Abcam), anti-phospho-tau (phospho T181; 1:1000; # ab75679, Abcam), anti-phospho-tau (phospho S262; 1:1000; # ab131354, Abcam), anti-phospho-tau (phospho S396; 1:1000; # ab109390, Abcam), and anti-β-actin (1:1000; # ab1801, Abcam). Quantitative densitometric analyses were performed on digitized images of immunoblots in the ImageJ software (NIH, USA).

All immunoprecipitations were performed on fresh half brains of 3-month-old wt male mice. Brains were dissected and lysed in 1.2 ml RIPA lysis buffer (Santa-Cruz Biotechnology, France) using Precellys^® homogenizer tubes. After centrifugation at 2800 g for 2×15 s, 1 ml brain extract was incubated with 2 µg of antibody of interest at 4°C for 1 h under gentle rotation. An aliquot of the remaining supernatant was kept for further immunoblotting as homogenate control. Then, 20 µl protein G agarose beads, previously washed three times with bead buffer, were added to the mix and gently rotated at 4°C for 30 min. After a 1 min spin at 10,000 g and removal of the supernatant, the pelleted immune complexes were washed three times with bead buffer before WB analysis with appropriate antibodies directed against DYRK1A (H00001859 M01, Interchim; 1:1000), PSD95 (ab18258, Abcam, France; 1:1000), SYN1 (ab64581, Abcam; 1:1000), CAMK2A (PA5-14315, Thermo Fisher Scientific; 1:1000) and GAPDH (MA5-15738, Thermo Fisher Scientific; 1:3000). Immunoblots were revealed with Clarity Western ECL Substrate (Bio-Rad).

Other commercially available antibodies used were: goat anti-human IgG Fcγ (31125, Thermo Fisher), AF488 goat anti-rabbit IgG (H + L; A-11008, Thermo Fisher), mouse anti-MAP2 (M9942, Sigma-Aldrich), AF647 goat anti-mouse IgG (H + L; A-21235, Thermo Fisher), HRP goat anti-mouse IgG (115-035-003, Jackson), HRP goat anti-rabbit IgG (P0448, Dako), rabbit anti-ADAM22 (PA5-65610, Thermo Fisher), rabbit anti-ADAM23 (C680120, LSBio), rabbit anti-synapsin-1 (ab64581, Abcam), mouse anti-PSD-95 (75-028, NeuroMab), mouse anti-Kv1.1α (75-105, NeuroMab), mouse anti-β-actin (A2228, Sigma-Aldrich). GraphPad Prism_v8, R version 3.5.1 (R Foundation for Statistical Computing, Vienna, Austria) and ggplot2 (Wickham, 2009 ) were used for statistical analyses and data presentation. Relevant animal procedures were carried out with UK Home Approval under licence P996B4A4E. The study was approved by the Research Ethics Committee (REC16/YH/0013) and all participants gave written consent.