Extended Data Fig. 6: Myelin dysfunction alters APP processing in Cnp−/−5xFAD and Plp−/y5xFAD mice.
From: Myelin dysfunction drives amyloid-β deposition in models of Alzheimer’s disease
Continuation from Fig. 3. (a) Overview of APP-stained swellings in the fimbria of 5xFAD control and Cnp−/−5xFAD mice. Hip: Hippocampus. Fim: Fimbria. The same observations were made in 5 independent samples/mice per group. (b) Colocalisation of APP and NF200 (axonal cytoskeletal marker) in swellings confirming their axonal origin in Cnp−/−5xFAD mice. White arrowheads indicate swellings double-positive for APP and NF200. Black-contured arrows indicate swellings that are solely positive for APP. This is likely a result of cytoskeletal breakdown in axonal swellings. The same observations were made in 3 independent samples/mice per group. (c) Distinction of plaque-associated axonal swellings induced by the 5xFAD genotype and myelin-damage-associated swellings induced by the Cnp−/− genotype. Upper panels are closeups of images shown in Fig. 3c. Lower panels show a mask indicating axonal swellings. Note that plaque-associated swellings form a circular arrangement of multiple swellings (corona) around a plaque centre (asteriks). In contrast, myelin damage-associated axonal swellings occur isolated/scattered. (d) Schematic representation of plaque-associated axonal swellings and myelin damage-associated swellings, and typical stainings observed with antibodies shown in Fig. 3b. D3E10, 80C2 and 6C3 do not show cross-reactivity to full length APP and typically only very weakly stain APP-positive plaque-associated swellings, but robustly stain swellings in Cnp−/−5xFAD mice. (e) Left panels shows positive staining controls for APP processing machinery antibodies used in Fig. 3 in the 5xFAD grey matter. For APP, BACE1 and PSEN2 typical plaque-corona staining (contoured arrowheads) can be observed. For APP and BACE1 additional neuronal cell body staining (n) can be observed. Right panel shows positive staining controls for amyloid antibodies used in Fig. 3 in the 5xFAD grey matter. Contoured arrowheads indicate proper amyloid plaques, typically stained very intensely. 029-2 and 6E10 antibodies show cross-reactivity to full-length APP and also stain plaque-associated axonal swellings (white arrowheads) and neurons (n). (f) 6E10 and sAPPβswe (soluble cleavage product after β-cleavage of APP) labelling of axonal swellings in Cnp−/−5xFAD fimbria. Figure 4d continued. Quantifications are shown on the right. Bars represent means; dots represent biological replicates/mice (for sAPPβswe, n = 4 for each group; for 6E10 labelling, n = 3 for 5xFAD controls and n = 5 for Cnp−/−5xFAD). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graphs). (g) Colocalisation of BACE1 and APP/Aβ in axonal swellings in Cnp−/−5xFAD white matter/fimbria. APP/Aβ was visualised using the 6E10 antibody. White arrowheads indicate swellings that are double-positive (to various degrees) for BACE1 and 6E10. Contoured black arrowheads indicate swellings single-positive for either 6E10 or BACE1. The same observations were made in 3 independent samples/mice per group. (h) sAPPβswe staining in cortical tissue of Cnp−/−5xFAD mice and 5xFAD controls. Staining reveals abundant plaque-associated swellings arranged in a swelling corona in 5xFAD mice. In Cnp−/−5xFAD both plaque-associated swellings and numerous plaque-independent swellings (i.e. myelin-damage-associated swellings) can be found. Quantification of sAPPβswe covered area is shown on the right. Bars represent means; dots represent biological replicates/mice/n (n = 4 per group). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graph). (i) Immunostaining analysis of APP/Amyloid-β and BACE1 in axonal swellings in Plp−/y5xFAD white matter (fimbria). (j) Quantifications of immunostainings shown in (i). Bars represent means; dots represent biological replicates/mice/n (n = 3 per group). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graph). (k) Colocalisation of BACE1 and APP/Aβ in axonal swellings in Plp−/y5xFAD mice. White arrows indicate a number of swellings in which colocalisation occurs. Contoured black arrows indicate swellings without colocalisation. The same observations were made in 3 independent samples/mice per group. (l) Fluorescent immunoblot analysis of BACE1 levels in the micro-dissected white matter of Cnp−/−5xFAD and 5xFAD mice. Molecular weight marker (in kDa) is indicated on the left. Bars represent means; dots represent biological replicates/lanes/mice/n (n = 3 per group). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graph). Source data are given in Supplementary Fig. 1. (m) Immunoblot analysis of APP processing using the 6E10 antibody. Molecular weight marker (in kDa) is indicated on the left. Anti-c-terminal APP labelling was applied to identify different APP CTF fragments: (p)C99 6E10 and A8717 double-positive; C89 and C83 6E10 negative and A8717 positive. Bars represent means; dots represent biological replicates/lanes/mice/n (n = 3 per group). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graph). Source data are given in Supplementary Fig. 3. (n) Fluorescent immunoblot analysis of APP fragmentation in the membrane-bound fraction of micro-dissected white matter (corpus callosum + alveus) of Cnp−/− 5xFAD and 5xFAD control mice. Molecular weight marker (in kDa) is indicated on the left. (o) Quantification of western blot analysis shown in (n). Bars represent means; dots represent biological replicates/lanes/mice/n (n = 3 per group). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graph). Source data are given in Supplementary Fig. 1.
