Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a For Fluorescence In Situ Hybridisation (FISH), organoid slices were processed and probed with a MetaSystems Probes for chromosome 21 (red) and chromosome 13 (green). Quantification was performed automatically using IMARIS software (nuclei were scored for 1, 2, 3 or >3 spots n>500 nuclei for each line). Scale bar: 3μm. b APP-4G8 antibody (green), BACE2 (red), MAP2 (magenta), DAPI (blue). Scale bar: 10μm. c Aβx-34 neo-epitope specific antibody (green), BACE2 (red), MAP2 (magenta), DAPI (blue). Scale bar: 10μm. d Quantification was performed blinded to the genotype, on 5 independent images representing three individual organoids per genotype, and containing 3,000-4,000 cells per image. Only images within the “cortical” part of the organoid were considered for the analysis. Graphs show total fluorescence intensity of positive signals for each wavelength for a given antibody, normalised by the total fluorescence intensity of MAP2 as a pan-neuronal marker. Additional staining (not shown) was analysed for Aβx-40 neo-epitope specific antibody. Image analysis was performed using IMARIS software. Error bars: standard error, and p-values: calculated after Holm-Bonferroni correction (α=0.05) of sequential two-tailed student t-test comparisons.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Fluorescence, In Situ, Hybridization, Software, Marker, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: The amino acid cleavage site for each secretase is noted in parentheses, and for sites in Aβ the respective peptide position is also noted. Secretases for each cleavage site are η: MT5-MMP, δ: AEP, β: BACE1, α: ADAM10, θ: BACE2, γ: gamma secretase complex. In addition to it’s role as a θ-secretase, in which C99 is cleaved to preclude Aβ formation, in this report we highlight the role of BACE2 to act on Aβ as substrate, as an Aβ-degrading protease (AβDP).

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques:

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Using Aβ IP-MS spectra from organoid (see Extended Data Figs. 1, 2, 4) conditioned media (CM), ratios were calculated of areas under the peak between the non-amyloidogenic and amyloidogenic peptides within a single mass-spectrogram. IP-MS spectra were produced for 3 timepoints (4 timepoints in exp3) for each iPSC-derived organoid line, in each of 3 independent experiments (each experiment starting at the point of undifferentiated iPSC). The team performing the IP-MS analysis was blinded to the genotypes in all experiments. BACE2-AβDP (clearance) products=[1-20&1-34], total BACE2=[1-19&1-20&1-34], amyloidogenic peptides=[1-38&1-39&1-40&1-42], α-site products=[1-16&1-17]. Exp1 and Exp2 p-values: Holm-Bonferroni sequential corrections (α=0.05) of 2-tailed student t-test comparisons. Exp3: Holm-corrected p-values after one way ANOVA. Error bars: standard error. Combined data for the isogenic iPSC lines for all 3 experiments passed the Holm-Bonferroni correction (α=0.05) of sequential 2-tailed student t-test comparisons of each peptide ratio shown in Fig1a (available on request). T21 and D21: isogenic iPCS derived from a single mosaic individual with DS published previously (Murray A et al. 2015), QM-DS1 and QM-DS2: unrelated DS iPSC, DupAPP: FEOAD iPSC. b All 3 experiments in Fig. 1a were combined to calculate the ratios of BACE2-related non-amyloidogenic peptides (1-19 or 1-34) to BACE2-unrelated non-amyloidogenic peptides (1-16 or 1-17) in organoid CM. Holm-corrected p-values after one way ANOVA are shown. Error bars: standard error. c Same ratios as in part A were calculated on IP-MS spectra obtained from cerebrospinal fluid samples of people with DS and age-matched normal controls. Data are presented as mean ± 1SD.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Protein-Protein interactions, Produced, Derivative Assay

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: Note: two new SNPs (rs746064 and rs9983496 new data in this paper) correlating (with nominal statistical significance) with the age of dementia onset found by the genetic analysis of the LonDownS cohort of people with DS (n=554) (purple font) cluster in close proximity with one previously published SNP (red font), and all 3 SNPs are contained within a 12 kb intron1 deletion (blue line) that on its own causes EOAD in a non-DS patient. Genomic position of SNPs which have been implicated previously in sporadic Alzheimer’s disease onset in Finnish population (black font). UCSC browser snapshot of human Chr21 region 41,100,000-41,345,000 (GRCh38/hg38) containing the BACE2 locus using Gencode v24 and curated RefSeq transcript annotation is given. Underneath, the layered H3K27ac track (ENCODE) indicates putative promoter and enhancer elements present in a panel human cell lines (see legend on the right). DNase clusters (ENCODE) mark accessible chromatin.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques:

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: A newly custom designed FRET reagent (spanning the Aβ34 site) was digested at pH=3.5 by the human BACE2 in presence or absence of the stated inhibitors for 2h. Enzyme activity was defined by measuring the fluorescence increase before and after the incubation. Blank-subtracted fluorescence units were normalized to the control digest and a one-way ANOVA was performed. P-values were calculated with a post-hoc Bonferroni multiple comparison (only pairs relative to the untreated control simultaneously compared). Error bars: standard error.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Activity Assay, Fluorescence, Incubation, Control, Comparison

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: Pairwise Pearson’s coefficient of colocalisation for a pair of co-stained antibodies: Aβx-34, and a specific marker for the sub-cellular vesicle compartment: lipid rafts (Flotillin1), lysosomes (LAMP1), macro-autophagosomes (LC3A), early endosomes (EEA1), macro-autophagosome-lysosome fusion/exosomes (Sortilin), late endosomes (Rab7), specific sub-sets of lysosomes (LAMP2, LAMP2A) and CMA-chaperone (HSC70). In the final two columns of the histogram, the Pearson’s colocalisation level was shown between Aβx-34 and BACE1 or BACE2, respectively (repeated in more detail in Supplementary Figure 8). Representative images of the organoid stainings from which the coefficients were calculated are shown in the panels. Last column in the bottom right panel is the zoomed-in inset from the previous column. Images were captured using Airy-Scan Zeiss confocal microscopy, and single 0.16μm slices are shown (from 20μm full z-stack analysed). Error bars: standard error, p-values: after standard one-way ANOVA using post-hoc Bonferroni multiple comparison calculation. Scale bar: 5μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Staining, Marker, Confocal Microscopy, Comparison

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Pairwise Pearson’s coefficient of colocalised volume for a pair of co-stained antibodies: Aβx-34 in all combinations, either BACE1 or BACE2 as a second antibody, and a marker of the sub-cellular vesicle compartment (shown at the bottom of each 3-columns histogram) as a third antibody. Error bars: standard error, and p-values: standard one-way ANOVA using post-hoc Bonferroni correction for multiple comparisons calculation. Representative individual z-slice images for the calculations performed in a are shown in b and c. b BACE1 (green), c BACE2 (green), with either Aβx-34, Sortilin or LAMP2 (red) and DAPI (blue). Scale bar: 10μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Immunofluorescence analysis of the brain of DS-AD-1 co-stained for Aβx-34, BACE2 and GFAP. A typical near-circular neuritic plaque is shown (in which DAPI faintly stains the fibrillar amyloid deposits). Arrows indicate 3 categories of objects in which the colocalisation of BACE2 and the AβDP product Aβx-34 is observed. White arrows: intra-neuronal fine-vesicular pattern; White arrowheads: large intra-neuronal spherical granules (lipofuscin); Black arrows with white arrowheads: amorphous extra-cellular aggregates. See Methods and FigS6 for experiments controlling the extent of lipofuscin autofluorescence effects. b DS-AD1 brain co-stained for Aβx-34 and BACE1, or Aβx-40, BACE2 and GFAP, and Pearson’s coefficient of colocalisation for proteins stained in parts A and B, with the addition of the staining for Aβx-42 neo-epitope (not shown). Error bars: SEM. c Same I.F. staining combinations as in part A (except for GFAP) were used in 3 additional brain samples: DS-AD-2, 3 and 4. d brain sample co-stained for BACE2 and Aβx-34 of a 28yrs old person with Down syndrome without dementia, and euploid non-demented (ND) controls aged 42 and 84. Scale bar: 20μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Sudan black B was used to confirm the specificity of the Aβx-34 and BACE2 antibodies in human brain sections, and to eliminate lipofuscin autofluorescence. Three different human brain samples were used: DS-AD1, DS (28 yrs) pre-AD and euploid sporadic AD (73 yrs). Both antibodies show the same pattern of expression and colocalisation after Sudan black B staining (white arrows: intraneuronal fine-vesicular pattern and black arrows with white arrowhead: amorphous extra-cellular aggregates) except for a loss of the large intraneuronal spherical granules (white arrowheads, Fig. 3), which are likely lipofuscin. Scale bar: 5μm. b and c Chromogenic, immunohistochemical analysis of the human brain sections of DS-AD1, stained using polymer-HRP/AP double-staining kit. b The primary antibody against BACE2 was labelled with DAB (brown) and primary antibody against Aβx-34 neo-epitope was labelled with GBI-permanent-red (red); b(i) is a zoomed-in inset of the rectangle in B. c same as b, but both antibodies were pre-absorbed for 12 hours, and incubated overnight, with the excess of immunogenic peptide for the BACE2 antibody; c(i) is a zoomed-in inset of the rectangle in c. d and e: BACE2 antibody specificity control for immunofluorescence on T21 organoids (100DIV). Scale bar: 10μm. d Immunofluorescent staining with Aβx-34 and BACE2, e same as b and d , but both antibodies were pre-absorbed for 12 hours, and incubated overnight, with the excess of immunogenic peptide for the BACE2 antibody. The specificity for the neo-epitope specific antibody against Aβx-34 was extensively proven in a previous report (Cabrera et al., 2018). f and g In order to distinguish the contribution of lipofuscin auto-fluorescence to the colocalised signals, specificity of primary antibodies (Aβx-34 and BACE2) has been validated using Lambda (λ) scan function on confocal microscope (see Methods). f Aβx-34 shows specific peak in different ROI and uniform pattern on the three different human brain samples: DS-AD1 (59 yrs), DS (28 yrs) pre-AD and DS (8 months). As negative control of staining, DAPI and secondary antibody alone were used. g BACE2 also shows specific peak in different ROI and uniform pattern in human brain. h secondary antibody alone control. Scale bar: 20μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Expressing, Staining, Immunohistochemical staining, Polymer, Double Staining, Incubation, Control, Immunofluorescence, Fluorescence, Microscopy, Negative Control

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a BACE2 exon3 sequence with 7bp deletion (yellow) provoked by the CRISPR/SpCas9-HF1 is shown. Red: restriction endonuclease HpyCH4IV sites (a de novo HpyCH4IV site is generated by the 7bp deletion). b agarose gel electrophoresis of the 733 bp PCR product containing the targeted site before (uncut) and after digestion with HpyCH4IV(cut), for the initial clone 2.5, and its colony-purified sub-clone 2.3.5 (renamed further below as “Δ7”). The 294bp fragment in 2.3.5 is reduced to 65% of the wt value (normalized to the 439bp band), and a de novo 255 bp fragment appears in CRSPR targeted line (red asterisk). c Western blot stained with anti-BACE2 antibody of the lysates of the iPSC line Δ7 compared to the wt T21C5 iPSC line. Quantification of the total actin-normalised BACE2 signal showed a significant reduction in Δ7 compared to T21 unedited line. Error bars: standard error, p-value: student’s t-test. d BACE2 AβDP/amyloidogenic peptides ratio after IP-MS analysis of CM produced by the 48DIV organoids derived from the iPSC line Δ7 compared to the T21C5wt control were significantly decreased. Error bars: standard error, p-values: two-tailed t-test comparison.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Sequencing, CRISPR, Generated, Agarose Gel Electrophoresis, Purification, Western Blot, Staining, Protein-Protein interactions, Produced, Derivative Assay, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Following CRISPR editing, C5Δ7 iPSC line was assessed by SNP array and no genomic alternations were detected compared to the parental C5 iPSC line. B-allele frequency and LogR ratio plots comparing these two lines are shown for chromosome 21. Data for the whole genome available on request. b To further confirm retention of trisomy after BACE2 CRISPR editing, selected other genes on chromosome 21 were validated as trisomic using quantitative paralogous amplification/pyrosequencing method. GABPA and ITSN allele number were quantified relative to paralogous sequence mismatches on other chromosomes. GABPA and ITSN both show approximate 60:40 ratios for trisomic cells, and 50:50 ratios for disomic cells as expected. Quantified nucleotides are shown on the pyrogram in shaded grey boxes and the relative values for the corresponding peaks are shown.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: CRISPR, Amplification, Sequencing

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: Western blot stained with anti-BACE2 antibody or anti-APP antibody of the lysates of the iPSC line Δ7 compared to the wt T21C5, and D21C3 iPSC lines. Quantification of the total actin-normalised BACE2 signal showed a 27% reduction in Δ7 compared to T21 unedited line, and no significant difference compared to D21 control. Quantification of the total actin-normalised APP signal showed no significant difference between Δ7 and unedited T21 line, whereas they both had significantly higher APP protein levels compared to the disomic control line. Error bars: standard error, p-values after standard one way ANOVA and Tukey’s multiple comparisons test.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Western Blot, Staining, Control