Journal: bioRxiv

Article Title: Ageing-associated myelin dysfunction drives amyloid deposition in mouse models of Alzheimer’s disease

doi: 10.1101/2021.07.31.454562

Figure Lengend Snippet: (a) Schematic representation of binding sites and specificity of antibodies to APP and amyloid-β used in the following panels and typical staining observed (plaque-associated axonal swellings vs Aβ plaque staining). Plaque-associated axonal swellings typically form a corona consisting of multiple swellings around amyloid plaques. (b) High pressure freezing EM of optic nerves from 6-month-old wildtype and CNP -/- optic nerves. Examples of (plaque-independent) axonal swellings in CNP-deficient animals show abundant accumulation of endosomal/lysosomal structures and multivesicular bodies. (c) Fluorescent and chromogen immunostainings against APP and APP cleavage enzymes BACE1 (β-secretase) and Psen2 (as part of the γ-secretase complex) showing accumulation of APP, BACE1 and PSEN2 in plaque-independent axonal swellings in CNP -/- 5×FAD. Contoured arrows mark plaque-associated axonal swellings typically forming a corona as abundantly found in the cortex of 5×FAD mice. Non-contoured arrows indicate plaque-independent axonal swellings as observed in CNP -/- mice. (d) Fluorescent and chromogenic immunostainings against different species of Aβ-peptides in CNP -/- 5×FAD vs 5×FAD in the white matter. As additional control, typical plaque staining in 5×FAD cortex are shown. Contoured arrows indicate proper amyloid plaques, typically stained very intensely. Non-contoured arrows indicate swellings stained positive by the respective β-amyloid antibody, typically less intensely stained and of round structure. D3E10, 80C2 and 6C3 do not show cross-reactivity to full length APP and typically do not stain plaque-associated swellings, but stain swellings in CNP -/- 5×FAD mice. 029–2 and 6E10 antibody show certain cross-reactivity to full-length APP and also stain axonal swellings. (e) Fluorescent immunoblot analysis of BACE1 levels in micro-dissected cortex and white matter of CNP -/- 5×FAD and 5×FAD mice. P-value of unpaired, two-sided Student’s t-test is indicated in the respective quantification bar plots. Dots represent lanes/single animals. n=3 for each group. (f) Fluorescent immunoblot analysis of APP fragmentation in the membrane-bound fraction of micro-dissected cortical tissue of CNP -/- 5×FAD and 5×FAD control mice. Quantification of band intensity is given on the right. fAPP levels were normalised against total protein fastgreen staining. CTF levels were normalised to fAPP levels. fAPP: full length APP. CTFs: c-terminal fragments. P-values of unpaired, two tailed Student’s t-test is indicated in the respective quantification bar plots. Dots represent lanes/single animals. n=3 for each group.

Article Snippet: Antibodies used in this study were: anti-Iba1 (rabbit, Wako; 1:1000); anti-Aβ-6E10 (mouse, Biolegend; 1:1000), anti-CNP (mouse, AMAb91072, Atlas; 1:1000), anti-PLP-clone aa3 (rat, culture supernatant; 1:200), anti-BACE1 (rabbit, ab183612, Abcam; 1:100), anti-MBP (rabbit, serum, custom Nave Lab; 1:1000), anti-GFAP (mouse, GA5, Leica, 1:200), anti-n-terminal APP (22c11, Merck; 1:1000), anti-c-terminal APP (rabbit, 127–003, Synaptic Systems; 1:1000), anti-Aβx-42-D3E10 (rabbit, Cell Signalling Technology; 1:1000), anti-Psen2 (rabbit, Abcam, 1:100), anti-ApoE D7I9N (rabbit, Cell Signalling Technology; 1:500), anti-Aβ 1-x (mouse, 80C2, Synaptic Signalling, 1:200), anti-Aβ 1-x (mouse, Moab2-6C3 , Abcam, 1:200), anti-Aβ 4-x (guinea pig, 029–2 , Oliver Wirths, custom, 1:200).

Techniques: Binding Assay, Staining, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Development and Technical Validation of an Immunoassay for the Detection of APP 669–711 (Aβ −3–40 ) in Biological Samples

doi: 10.3390/ijms21186564

Figure Lengend Snippet: Immunohistochemical staining of Alzheimer’s disease (AD) transgenic mouse models. ( a ) Staining using an antibody directed against the carboxyterminus of APP (APP C-term) showed abundant cellular and neuritic immunoreactivity in 5XFAD and APP Ld mice (left). In contrast, 101-1-1 showed an abundant staining only in APP Ld mice harboring APP with an aminoterminal wildtype (K670–M671) sequence, but a complete lack of immunoreactivity in 5XFAD mice overexpressing APP with the Swedish mutation (N670–L671) (right). ( b ) Double-staining using an Aβ 42 -specific antibody (D3E10, green) and 101-1-1 (red) revealed abundant Aβ immunoreactivity in the central amyloid plaque cores, while the 101-1-1 signal was restricted to surrounding dystrophic neurites. Scale bar: 100 µm.

Article Snippet: The rabbit monoclonal anti Aβ 42 antibody mAb D3E10 was purchased from Cell Signaling Technology (Danvers, MA, USA), while the rabbit polyclonal anti C-terminus APP A8717 was purchased from Sigma-Aldrich (Munich, Germany).

Techniques: Immunohistochemical staining, Staining, Transgenic Assay, Sequencing, Mutagenesis, Double Staining

Journal: International Journal of Molecular Sciences

Article Title: Development and Technical Validation of an Immunoassay for the Detection of APP 669–711 (Aβ −3–40 ) in Biological Samples

doi: 10.3390/ijms21186564

Figure Lengend Snippet: Receiver operating characteristic (ROC) analysis of Aβ 42 , the Aβ 42 /Aβ 40 ratio, and the Aβ 42 /Aβ −3–40 ratio as discriminators of patients with other dementias (OD) and Alzheimer’s type dementia (AD-D). The ROC curves of the indicated biomarker candidates in blood plasma are shown in discriminating the diagnostic groups OD and AD-D. The areas under the curves (AUCs) were 0.760 (Aβ 42 ), 0.790 (Aβ 42 /Aβ −3–40 ratio), and 0.854 (Aβ 42 /Aβ 40 ratio), respectively.

Article Snippet: The rabbit monoclonal anti Aβ 42 antibody mAb D3E10 was purchased from Cell Signaling Technology (Danvers, MA, USA), while the rabbit polyclonal anti C-terminus APP A8717 was purchased from Sigma-Aldrich (Munich, Germany).

Techniques: Biomarker Assay, Diagnostic Assay