Trisomy of Hsa21-homologous genes including or excluding <i>App</i> does not affect APP half-life in MEFs.

A) Degradation curve of APP in Dp1Tyb, Dp1Tyb/App+/- and WT MEFs. B) Half-life of APP is not significantly different in Dp1Tyb, Dp1Tyb/App+/- and WT MEFs (One-way ANOVA, p = 0.48, N = 5/6). Average APP half-life in minutes: Dp1Tyb = 84 ± 9; Dp1Tyb/App+/- = 97 ± 17; WT = 77 ± 6). C) APP abundance is not significantly different in Dp1Tyb, Dp1Tyb/App+/- and WT MEFs (One-way ANOVA, p = 0.77, N = 6). Average APP/β-actin: Dp1Tyb = 0.93 ± 0.07; Dp1Tyb/App+/- = 0.83 ± 0.1; WT = 0.93 ± 0.15). Each dot corresponds to a biological repeat (i.e. an independent MEF line used). For each biological repeat, three technical repeats (i.e. western blot) were performed. Error bars = SEM. All full uncropped western blots are available at Figshare 10.6084/m9.figshare.17316434.</p

Custom ImageJ Macro.

Macro designed by Dr Dale Moulding to smooth the cell surface and clear its outside in 3D, enabling accurate quantification of the volume of the cell and of the number and volume of endosomes. (PDF)</p

Trisomy of Hsa21-homologous genes, including or excluding <i>App</i>, does not affect Aβ40/Aβ42 ratio.

In WT, Dp1Tyb and Dp1Tyb/App+/- MEFs overexpressing APP-β-CTF led to no difference in A) Aβ40 abundance (Dp1Tyb = 134.9 ± 42.5; Dp1Tyb/App+/- = 138.2 ± 47.75; WT = 154.1 ± 76.59. One-way ANOVA, p = 0.97, N = 3); B) Aβ42 abundance (Dp1Tyb = 7.78 ± 2.5; Dp1Tyb/App+/- = 7.56 ± 2.88; WT = 9.02 ± 4.61 in pg/ml. One-way ANOVA, p = 0.95, N = 3) or C) Aβ40/Aβ42 ratio (Dp1Tyb = 17.51 ± 0.24; Dp1Tyb/App+/- = 19.29 ± 1.37; WT = 17.38 ± 0.31. One-way ANOVA, p = 0.26, N = 3). Each dot corresponds to a biological repeat using an independent MEF lines. Error bars = SEM.</p

Process of quantification of RAB5⁺ endosomal staining.

A) WT MEF stained for Integrinβ (cell membrane, green) and RAB5 (endosomes, red). B, D) Endosomal staining after deconvolution and background clearance C, E) 3D reconstruction of endosomal staining. Deconvolution and 3D reconstruction to accurately quantify the volume of endosomes. Z-stacks of each cell were taken with 150 nm interval between slices and fixed voxel volume (x = 50 nm, y = 50 nm, z = 150 nm) on confocal microscope LSM880. Each stack was deconvolved using Huygens software to improve image signal to noise and resolution. ImageJ software was used to remove the background with a macro written by Dr Dale Moulding. Imaris software was used to reconstruct the deconvolved staining in 3D. The area of Integrinβ was used to create a mask to define cellular volume. (TIF)</p

Endosomal structure and APP biology are not altered in a preclinical mouse cellular model of Down syndrome

Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer’s disease, characterised by the accumulation in the brain of amyloid-β plaques. Amyloid-β is a product of the processing of the amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-β accumulation is within endosomes, and changes to endosome biology occur early in Alzheimer’s disease. Here, we determine if primary mouse embryonic fibroblasts isolated from a mouse model of Down syndrome can be used to study endosome and APP cell biology. We report that in this cellular model, endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in the model, despite three copies of App

Number of endosomes per cell, endosomal volume distribution and mean volume of the largest endosomes are not different in WT and Dp1Tyb MEFs.

A) Schematic of gene content of Dp1Tyb mouse model. B) No difference was found in the number of RAB5+ endosomes (normalised to cell volume) in WT and Dp1Tyb MEFs (Nested t-test p = 0.83, N = 5 biological repeats, N = 3–5 technical repeats). C-D) Endosomes were binned in three size categories: small (0-50th percentile of WT MEFs), medium (50-90th percentile of WT MEFs) and large (90-100th percentile of WT MEFs). The categories were determined using the endosomes in WT MEFs. C) No difference in RAB5+ endosome volume distribution was observed in WT and Dp1Tyb MEFs (Mann-Whitney U test). N = 5 biological repeats, N = 3–5 technical repeats. D) Volume of the RAB5+ endosomes classified as ‘large’ is not different in WT and Dp1Tyb MEFs (Nested t-test, p = 0.21). N = 5 of biological repeats (independent MEF lines), N = 3–5 technical repeats. Error bars = SEM.</p

Distribution and quantification of endosomes in MEFs transfected with PBS and RAB5CA.

A) The normal distribution of endosomal size in WT MEFs transfected with PBS was determined to define the parameters for classification of “large” endosomes (small: endosomes in the 0–50th percentile, medium: endosomes in the 50–90th percentile, large: endosomes in the >90thpercentile). B) A nested t-test showed that ‘large’ endosomes in cells transfected with RAB5CA had a significantly higher volume than the endosomes in cells transfected with PBS (p = 0.007, N = 3 of biological repeats). The dots indicate the average volume of the ‘large’ endosomes in one cell imaged (technical repeat). Error bars = SEM. C, D) Representative images of WT MEFs transfected with PBS (C) or RAB5CA (D), endosomes labelled with RAB5 antibody (red), the RAB5CA plasmid is GFP-tagged (green); enlarged endosome indicated with (white arrow). (TIF)</p

Detail of the pCI-neo βCTF-3xFLAG plasmid map.

The APP signalling sequence was inserted in a pCI-neo plasmid followed by the β-CTF fragment of APP and by a 3xFLAG sequence. The primers used for sequencing the insert (sequencing forward and reverse) are also shown. (TIF)</p

Additional file 1 of Cathepsin B abundance, activity and microglial localisation in Alzheimer’s disease-Down syndrome and early onset Alzheimer’s disease; the role of elevated cystatin B

Additional file 1. Supplementary Figure 1: changes to cathepsin B and cathepsin D activity in a sub analysis of EOAD and AD-DS Braak VI cases

Downregulated Wnt/β-catenin signalling in the Down syndrome hippocampus.

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/β-catenin pathway in the hippocampus of adult DS individuals with Alzheimer's disease and the 'Tc1' DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/β-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/β-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer's disease treatment