Journal: Acta Neuropathologica Communications
Article Title: Systemic delivery of a specific antibody targeting the pathological N-terminal truncated tau peptide reduces retinal degeneration in a mouse model of Alzheimer’s Disease
Figure Lengend Snippet: Pathological N-terminal tau truncation occurs in eyes of symptomatic Tg2576 mice and is successfully immunodepleted by 12A12mAb systemic delivery. a Study design. 6-month-old Tg2576 Alzheimer’s disease (AD) mice were intravenously (i.v.) injected with 12A12mAb or mouse IgG (isotype control). On day 15, mice were sacrificed and eyes were used for biochemical (Western blotting) and morphological (immunofluorescence) evaluations. Wild type (WT) mice immunized with vehicle (saline) or mouse IgG under the same experimental conditions (antibody dosage, time of treatment, administration route) were used as controls. Picture was assembled by means of Biorender online software ( https://biorender.com ). Western blotting analyses ( b ) and semi-quantitative densitometric analysis (n = 6) ( c ) carried out on soluble extracts from three experimental groups (wild-type, Tg2576 and Tg2576 + mAb) showing the presence of the NH 2 htau peptide in retina and vitreous body of Tg2576 mice and its 12A12mAb-mediated neutralization following i.v. administration. Filters were probed with three different tau antibodies reacting against different epitopes located around the extremity and middle N-terminal end of protein, including caspase-cleaved protein (CCP)-NH 2 tau (26-36aa) [ 51 , 64 ], BT2 (194-198aa) and DC39N1 (45-73aa). β-actin was used as loading control. Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test for multiple comparison among more than two groups. p
Article Snippet: The following antibodies were used: Caspase-cleaved protein (CCP) NH2 -tau antibody rabbit (D25-(QGGYTMHQDQ) epitope, phosphorylation-independent state) [ , , ]; Tau Antibody (BT2) mouse MN1010 ThermoFisher Scientific; anti-N-tau (45-73aa) DC39N1 mouse T8451 Sigma-Aldrich; Phospho-PHF-tau pSer202+ Thr205 mouse MN1020 ThermoFisher Scientific; PC1C6 Tau1 Ser-195/Ser-198− epitopes mouse MAB3420 Merck Millipore; anti-pan tau protein HT7 (1-150aa of N-terminus) rabbit sc-5587 Santa Cruz Biotechnology; anti-Aβ/APP protein 6E10 (4-9aa) mouse MAB1560 Chemicon; GFAP antibody (2E1) mouse sc-33673 Santa Cruz; anti-GFAP (clone GA5) mouse MAB360 Millipore; Iba1 antibody (1022-5) mouse sc-32725 Santa Cruz; anti-Iba1 rabbit 019-19741 Wako; NMDAζ1 antibody (C-20) goat sc-1467 Santa Cruz; anti-synapsin I antibody rabbit AB1543P Millipore; anti-synaptophysin antibody (D-4) mouse sc-17750 Santa Cruz; anti-syntaxin 1 mouse S1172 Sigma-Aldrich; anti-SNAP25 antibody (clone SMI 81) mouse 836301 BioLegend; anti-α synuclein antibody (clone 42) mouse 610786 BD Transduction Laboratories; cleaved caspase-6 (Asp162) antibody rabbit 9761 Cell Signaling; anti-choactase antibody (H-95) rabbit sc-20672 Santa Cruz; anti-mAChR M1 antibody (H120) rabbit sc-9106 Santa Cruz; anti-vGLUT1 antibody rabbit 135 302 Synaptic System; anti-vGAT antibody rabbit 131 003 Synaptic System; anti-VDAC/Porin antibody rabbit ab34726 Abcam; Tomm20 antibody (FL-145) rabbit sc-11415 Santa Cruz; cytochrome C (136F3) rabbit 4280 Cell Signaling Technology; acetylated α Tubulin (6-11B-1) mouse sc-23950 Santa Cruz; rat anti tubulin alpha mouse MCA77G Bio-Rad; OPA1 antibody mouse 612606 BD Transduction Laboratories; SOD II (MnSOD, mitochondrial superoxide dismutase) rabbit SOD-110D Stressgen Biotechnologies; anti-β-actin antibody mouse S3062 Sigma-Aldrich; anti-mouse IgG (whole molecule)-Peroxidase antibody A4416 Sigma-Aldrich; anti-rabbit IgG (whole molecule)-Peroxidase antibody A9169 Sigma-Aldrich; donkey anti-goat IgG-HRP antibody sc2056 Santa Cruz.
Techniques: Mouse Assay, Injection, Western Blot, Immunofluorescence, Software, Neutralization, Molecular Weight, Migration