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Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains

  1. Diane M Bushman
  2. Gwendolyn E Kaeser
  3. Benjamin Siddoway
  4. Jurgen W Westra
  5. Richard R Rivera
  6. Stevens K Rehen
  7. Yun C Yung
  8. Jerold Chun  Is a corresponding author
  1. Dorris Neuroscience Center, The Scripps Research Institute, United States
  2. University of California, San Diego, United States
Research Article
Cite this article as: eLife 2015;4:e05116 doi: 10.7554/eLife.05116
7 figures, 6 videos and 5 tables

Figures

Methodologies used in assessing genomically mosaic AD.

(A) Neuronal nuclei were isolated from the prefrontal cortex and cerebellum of postmortem human brain (see ‘Materials and methods’ for samples used) as described (Westra et al., 2008). (B) Nuclear DNA was stained with propidium iodide (PI) and DNA content was quantified using flow cytometric analysis. (C) APP copy number variations were analyzed in small populations of nuclei (∼75 genomes) using custom primers for exon 14 of APP. (D) Single-cell qPCR assessed APP copy number variations in individual neuronal nuclei via TaqMan probes and a modified Biomark integrated fluidic chip system (Fluidigm Corporation, South San Francisco). (E) FISH paints against the whole q arm of chromosome 21 and a point probe against a region on the q arm of 21 (21q22.13-q22.2) were used to double-label and call aneusomies in AD samples. (F) Peptide nucleic acid (PNA) FISH was combined with super resolution microscopy for threshold detection of APP copy number above ∼2 occurring at a single locus.

https://doi.org/10.7554/eLife.05116.003
Figure 2 with 2 supplements
AD cortical nuclei show increased DNA content variation (DCV) by flow cytometry.

(A) Histogram displaying gating parameters used in sorting ‘high’ and ‘low’ DNA content populations for validation of DNA content. (B) Validation of DNA content analyses using semi-quantitative MDA whole-genome amplification (WGA) on ‘high’ and ‘low’ DNA content populations of 1000, 500, and 100 nuclei. (C and D) Representative DNA content histograms for lymphocytes (LYM), AD cerebellum (CBL), and AD prefrontal cortex (CTX). Each colored histogram represents a separate sample in each set; CTX and CBL samples are from paired brains. Chicken erythrocyte nuclei (CEN) were used as internal calibration controls. (E) Representative orthogonal view of DNA content vs forward scatter width (FSC-W). For each brain sample, the area to the right of the vertical line indicates a DNA content increase of the population of nuclei. AD-6 CTX is a representative right-hand peak shift and AD-7 is a representative right-hand shoulder (see Figure 3A for more examples). (F) DNA content changes for all human LYM, ND, and AD brain samples examined (AD CTX N = 32, AD CBL N = 16, LYM N = 15 [20 meta analysis], ND CTX = 21 [36 meta analysis], ND CBL = 11 [12 meta analysis]). Red bars denote average for each group relative to lymphocytes. Averages are as follows (including metadata from Westra et al. (2010)): AD CTX 8.219%; AD CBL −0.1104%; LYM −0.2915%; ND CTX 2.239%; ND CBL −3.358%. (G) DNA content changes of the current study (AVOVA p < 0.0001). (H) DNA content changes of the current study combined with metadata from Westra et al. (2010) (ANOVA p < 0.0001). (I) Comparison of mean coefficient of variation (CV statistic from FlowJo of the population, included metadata from Westra et al., 2010) demonstrates that there is an average increase in the variation of AD samples (ANOVA p < 0.0001). *p = 0.05, **p = 0.01, ***p = 0.001, ****p < 0.0001, See Figure 2—source data 1 for exact p values. See Figure 2—figure supplement 1 for age, PMI and Braak score correlations. See Figure 2—figure supplement 2 for control of nuclear size analysis.

https://doi.org/10.7554/eLife.05116.004
Figure 2—source data 1

DNA Index (DI) and percent change values and statistics.

https://doi.org/10.7554/eLife.05116.005
Figure 2—figure supplement 1
DNA content shows no correlation with age or post-mortem index (PMI).

(A) Comparison of mean skew values for each sample group, skew determined as: (Mean − Mode/Standard Deviation of the diploid DNA content peak). (B) No correlation was observed between DNA content and Braak score. (CE) No correlation was observed between DNA content and age across all brains analyzed. (FH) No correlation was observed between DNA content and post-mortem index across all brains analyzed.

https://doi.org/10.7554/eLife.05116.006
Figure 2—figure supplement 2
Analysis of nuclear size and DNA content.

(AC) Representative flow cytometry scatter plots of nuclei. (A) Lymphocytes (LYM), (B) CTX nuclei, (C) CBL nuclei. (D) Overlay of red boxes shown in (AC), demonstrating that cortical nuclei similar in size to LYM and CBL consistently display a DNA content shift.

https://doi.org/10.7554/eLife.05116.007
Pairwise DNA content analyses in AD cortical nuclei vs AD cerebellum.

(A) Pairwise analysis of overlaid DNA content histograms (CTX = solid red, CBL = black dashed lines) in the same AD individual (each graph represents a unique AD individual). (B) Pairwise analysis of overlaid DNA content histograms (CTX = solid blue, CBL = black dashed lines) in the same ND individual.

https://doi.org/10.7554/eLife.05116.008
DNA content increases observed in AD cortical nuclei are attributable to neurons.

(AC) The gating procedure used for NeuN-positive flow cytometry analysis. (A) DNA content peak for identified nuclei. (B) A sample of unlabeled neuronal nuclei that display no NeuN-positive signal. (C) Selection for NeuN-positive nuclei for downstream analysis. (D) DNA content histograms of four AD samples displaying NeuN-positive nuclei (solid purple) vs NeuN-negative nuclei (black dashed line). NeuN-positive populations display distinct cortical histograms with prominent right-shifted peaks (arrows). (E) Comparison of DNA index (DI) increases from NeuN-positive nuclei (solid purple) vs NeuN-negative nuclei (black dashes) from AD CTX samples. NeuN-positive nuclei (DI = 1.10) showed an average gain of 9% over NeuN-negative nuclei (DI = 1.01), **p = 0.0011. (F) Comparison of DNA content in NeuN-positive nuclei from AD CTX (DI = 1.10) (red) vs AD CBL (DI = 0.94) (pink) from the same individual; CTX nuclei displayed a 15.6% gain over CBL nuclei, *p = 0.0335. Statistics are paired two-tailed t-test. Bars indicate ± SEM.

https://doi.org/10.7554/eLife.05116.009
Figure 5 with 1 supplement
Mosaic amplification of the APP locus in small cohorts of AD cortical neurons unrelated to trisomy 21.

(A) Comparison of relative copy number of APP in CBL and CTX fractions from six AD brains. APP locus-specific amplification was determined relative to reference gene SEMA4A; paired CBL nuclei were used as a calibrator sample for each brain, normalized to 2.00 for a diploid cell. Differences in ΔΔCt ± SEM of APP in the cortex vs cerebellum were assessed in each individual using an unpaired, two-tailed t-test (****p = 0.0001, *p = 0.0165, *p = 0.0489) (B) Comparison of relative copy number of APP in CBL and CTX fractions from 4 non-diseased brains. (C) Average relative copy number in non-diseased vs AD brains. Control genes and DS individuals were also examined (Figure 5—figure supplement 1). (DJ) FISH strategy of chromosome 21 counting through simultaneous labeling using chr 21 q arm ‘whole’ chromosome paint (WCP, green) and chr 21 regional FISH probe for 21q22.13-q22.2 (red) (see Figure 5—source data 1 for raw counts). (D and E) The ability to detect aneuploidy was validated using interphase nuclei from a human trisomy 21 brain, where three regional spots (red, encompassing the APP gene) were seen, despite WCP spatial variation (see also Rehen et al. (2005)). (FI) Chromosome 21 aneusomy was examined in prefrontal cortical nuclei. Examples of chr 21 (F) monosomy, (G) disomy, (H) trisomy, and (I) tetrasomy (please note tetrasomy is not an example of aneuploidy). (J) Quantification of individual FISH signals showed no significant differences in monosomy, disomy, trisomy, or tetrasomy. 5 control brains and 9 AD brains were used. At least 450 nuclei were quantified per brain sample. Scale bar = 10 um. 4974 total nuclei examined.

https://doi.org/10.7554/eLife.05116.010
Figure 5—source data 1

Raw dual point-paint probe FISH counts.

https://doi.org/10.7554/eLife.05116.011
Figure 5—figure supplement 1
Controls for small population qPCR.

(A) Reference genes validated in small population qPCR via examination of APP exon 14 in Down Syndrome (DS) nuclei as a positive control. (B) Representative males (AD-1 and AD-6) displayed reduced copy number of PCDH11X, a gene located on the X chromosome, while a representative female demonstrates two copies of PCDH11X and CCL18, a second single copy control gene.

https://doi.org/10.7554/eLife.05116.012
Figure 6 with 1 supplement
Mosaic APP locus amplification in single neurons from AD brains.

(A) Single nuclei relative copy numbers for exon 3 of APP from non-diseased (ND) CBL, ND CTX, AD CBL, and AD CTX; each black diamond represents one neuron. For each group, the mean is displayed in red and bars represent 95% confidence intervals. AD CTX showed a mean APP copy number of 3.80; this is significantly higher than AD CBL (2.23), ND CTX (1.60), and ND CBL (2.28). *p = 0.0147, **p = 0.0015, **p < 0.0012, ANOVA p < 0.0001 (see Figure 6—source data 1 for raw numbers and statistics). (B) Single nuclei relative copy numbers for exon 14 of APP, similar to (A). The two exons showed a high concordance (Figure 6—figure supplement 1) where the AD CTX showed a mean APP copy number of 3.40 while the AD CBL (2.34), ND CTX (1.44), and ND CBL (1.92) remained closer to 2 copies. *p = 0.0163, **p = 0.0016, ****p < 0.0001, ANOVA p < 0.0001. (CF) Distribution of copy number calls for exon 3 (C and D) and exon 14 (E and F) binned by relative copy number. The AD CTX for both exons displayed unique distributions, with more nuclei falling into the high copy number bins. (G and I) Distribution of nuclei with copy numbers less than, equal to, and greater than two copies. (H and J) Average copy number increases in nuclei binned with greater than two copies (gold columns in G) (AD CTX: Exon 3 = 5.01, Exon 14 = 4.96, *p = 0.0361). All statistics represent an ANOVA with a Tukey's multiple comparison test. Bars indicate ± SEM.

https://doi.org/10.7554/eLife.05116.013
Figure 6—source data 1

Single Cell qPCR Data and Statistics.

https://doi.org/10.7554/eLife.05116.014
Figure 6—figure supplement 1
Concordance of APP exon 3 and 14 from single cell qPCR.

(A) Relative copy numbers (RCN) for APP exon 3 and APP exon 14 displaying concordance between exons. 100 of 115 nuclei examined for both exons display copy numbers within one copy number call. 10 of the 15 remaining nuclei, while more than one copy number apart, were both called as gains. Bars represent RCN Min and RCN Max. (B) Scatter plot of average relative copy numbers. The data remain consistent with those displayed for individual exons (Figure 6A,B). Statistics represent an ANOVA with a Tukey's multiple comparison test. **p < 0.01, ***p < 0.001.

https://doi.org/10.7554/eLife.05116.015
Figure 7 with 1 supplement
Visualization of APP copy number increases in neuronal nuclei from AD brain samples.

(A) Peptide nucleic acid probes (PNA) were developed against nine separate sites on APP (4 sites within exon 3 and 5 sites within exon 14). Each PNA probe consists of a peptide backbone conjugated to a single fluorophore, with separately conjugated nucleotides, substantially increasing specificity (Lansdorp et al., 1996). Single copies of APP are not detectable because of fluorophore detection limits. Detection of increased copy number by PNA probes can be visualized as copies of APP increase (Figure 7—figure supplement 1B,C). Positive internal controls using PNA probes directed against telomere sequences were simultaneously hybridized. (B) Visualization of copy number increases in neuronal nuclei. Green puncta (arrow 1, insets) indicate visualized APP increases. Telomere labeling (red puncta) was present in all nuclei, demonstrating probe accessibility and template fidelity. Lipofuscin (arrow 2, orange puncta) was detected in nuclei, visualized by extensive fluorescence signal in all channels, but was eliminated from quantifications. Limited nuclei displayed two green puncta (arrow 3). V1-6 Refers to the supplemental videos where 3-D projections can be visualized. (C) Graphic representation of non-diseased (blue) and DS (grey) brains displayed limited numbers of threshold-detected increases in APP (Figure 7—source data 1). AD (red) brains displayed significant and consistent threshold-detected increases in APP. (D) Individual threshold-detected APP increases were quantified and plotted on a relative intensity scale (blue diamonds: non-diseased, red diamonds: AD). Dotted line represents the threshold below which APP copy number was undetectable, only limited puncta were identified in non-diseased nuclei. Bars indicate ± SEM, *p < 0.05.

https://doi.org/10.7554/eLife.05116.016
Figure 7—source data 1

Data and statistics for PNA-FISH counts.

https://doi.org/10.7554/eLife.05116.017
Figure 7—figure supplement 1
PNA FISH controls.

(A) PNA probe specificity was verified via dot blots of APP sequence followed by PNA probe hybridization and immunoblotting against the Alexa-488 fluorophor. Probes designed against exon 3 exhibited specific binding to the 5′ region of APP, and probes designed against exon 14 exhibited specific binding to the 3′ region of APP, while probes did not display significant binding to non-specific sequences. (B) Plasmids containing all 9 APP PNA binding sites were blotted at 1× (1.8 µg), 2× (3.6 µg), and 3× (5.4 µg) DNA concentration, and PNA probes were hybridized and an empty plasmid at 1.8 μg was used for a negative control in lane 1. Fluorescent output demonstrated a linear increase with increasing DNA concentration. (C) 10 μg of plasmids containing 0, 3, 6, and 9 copies of the APP PNA binding sites was blotted onto membrane and probes were hybridized. Fluorescent output showed an expected linear increase with the number of PNA probe binding sites. (D) Quantification of the variable APP signal increases observed across four brains. (E) Representative APP signals visualized and verified using super-resolution 3D projections displayed a range of variable intensities.

https://doi.org/10.7554/eLife.05116.018

Videos

Video 1
PNA-Fish analysis of APP in nuclei from non-diseased cortical neurons.

Video of 3-D projection from Figure 7B, V1. Green puncta indicating APP increases were infrequently visualized in non-diseased brains. Red puncta indicate telomere labeling with separate telomere-specific PNA probes and were visualized as a positive control for PNA hybridization.

https://doi.org/10.7554/eLife.05116.019
Video 2
PNA-Fish analysis of APP in nuclei from non-diseased cortical neurons with lipofuscin.

Video of 3-D projection from Figure 7B, V2. Green puncta indicating APP increases were infrequently visualized in non-diseased brains. Lipofuscin (orange puncta), visualized by extensive fluorescence signal in all channels, was detected in some nuclei, but was excluded from analysis. Red puncta indicate telomere labeling with separate telomere-specific PNA probes and were visualized as a positive control for PNA hybridization.

https://doi.org/10.7554/eLife.05116.020
Video 3
PNA-Fish analysis of APP in nuclei from non-diseased cortical neurons.

Video of 3-D projection from Figure 7B, V3. Green puncta indicating APP increases were infrequently visualized in non-diseased brains. Red puncta indicate telomere labeling with separate telomere-specific PNA probes and were visualized as a positive control for PNA hybridization.

https://doi.org/10.7554/eLife.05116.021
Video 4
PNA-Fish analysis of APP in nuclei from AD cortical neurons.

Video of 3-D projection from Figure 7B, V4. Green puncta indicate visualized APP increases. Red puncta indicate telomere labeling with separate telomere-specific PNA probes and were visualized as a positive control for PNA hybridization.

https://doi.org/10.7554/eLife.05116.022
Video 5
PNA-Fish analysis of APP in nuclei from AD cortical neurons with lipofuscin.

Video of 3-D projection from Figure 7B, V5. Green puncta indicate visualized APP increases. Lipofuscin (orange puncta), visualized by extensive fluorescence signal in all channels, was detected in some nuclei, but was excluded from analysis. Red puncta indicate telomere labeling with separate telomere-specific PNA probes and were visualized as a positive control for PNA hybridization.

https://doi.org/10.7554/eLife.05116.023
Video 6
PNA-Fish analysis of APP in nuclei from AD cortical neurons.

Video of 3-D projection from Figure 7B, V6. Green puncta indicate visualized APP increases. Limited nuclei in AD displayed two green puncta. Red puncta indicate telomere labeling with separate telomere-specific PNA probes and were visualized as a positive control for PNA hybridization.

https://doi.org/10.7554/eLife.05116.024

Tables

Table 1

Human samples used in each experiment

https://doi.org/10.7554/eLife.05116.025
SexAgePaper codeSampleExperimentsPost mortem intervalBraak score
Alzheimer's disease prefrontal cortex, N = 32F811521D23VI
F831562D6U
F74AD-131866DTUU
F79AD-51868DSTUU
F82AD-101875DSTUU
F831893D9U
F871899D5VI
F62AD-91912DSTUU
F80AD-81913DTUU
F54AD-41916DTBUU
F72AD-21921DTBUU
F772400D3.7V
F80AD-112500DP2.3VI
F10150341D19V
F9161788D11V
F9862405D11V
F8962439D11V
F7762509D18VI
M88AD-1102DSBP3IV
M90268D91V
M83736D27U
M821211D18U
M79AD-151252D9U
M921748D5.5V
M85AD-121861DTUU
M85AD-141870DTUU
M82AD-32401DSBP3VI
M84AD-62499DS3.4VI
M63AD-74199DP3VI
M8013173D22IV
M9430022D20V
M9160987D22.5V
Mean82.1
Alzheimer's disease cerebellum, N = 15F74AD-131866DUU
F79AD-51868DSUU
F82AD-101875DSUU
F80AD-81913DUU
F62AD-91912DSUU
F54AD-41916DBUU
F72AD-21921DBUU
M88AD-1102DSB3IV
M79AD-151252D9U
M701625D1U
M85AD-121861DUU
M85AD-141870DUU
M82AD-32401DSB3VI
M84AD-62499DS3.4VI
M63AD-74199D3VI
Mean75.9
Non-diseased prefrontal cortex, N = 40F74ND-21901DSBP2.3II
F74ND-8299D2.8II
F84ND-3703S5.8III
F53ND-111379D15III
F73713*TUU
F9560831D9II
F511568P22U
F171230P16U
F87ND-101502D5II
F8060728D13II
M79ND-9827*DTUU
M96ND-11102DSB3.4II
M83ND-52501DB1.7II
M95ND-41301DS3.5I
M871471*TUU
F711571*TUU
M531344*DTUU
F93318D2.3VI
F92955D20.5III
F564238D12
M704534D28
F9111488D16II
F7913188D12.5
F9013204D9.5II
F10360329D5III
F8560428D8.5III
F9960524D15II
F9562043D20.5
M71389M15
F83719M17III
M69946M12
M872039M6.3III
F864546M22
M9160772M16II
F8061218M5.5
M8761334M8II
M88PDC2MU
M80PDC5MU
M75PDC8MU
Mean79.54
Non-diseased cebellum, N = 15F74ND-21901DSB2.3II
F74ND-8299D2.8II
F84ND-3703S5.8III
F771569D8III
F83719DUU
F531379D15III
F711571DTUU
F87ND-101502D5II
M531344DTUU
M96ND-11102DSB3.4II
M83ND-52501DB1.7II
M95ND-41301DS3.5I
M79ND-9827DUU
M871471DUU
F864546M22U
Mean78.8
DS/AD, N = 3F51DS-1M1864B19U
F47DS-2M3233S24U
F44DS-31258S13U
Mean47.3
LYM, N = 21F405162DN/AN/A
F403963DN/AN/A
F634984DN/AN/A
F604519DN/AN/A
M55Lym 1DN/AN/A
M354651DN/AN/A
29DN/AN/A
187DN/AN/A
4781DN/AN/A
4801DN/AN/A
4903DN/AN/A
M285259DN/AN/A
M5683M
F521344M
F564603M
M544609M
M58Lym 2M
F51Lym 3M
M52Lym 4M
Mean50.0M
  1. Samples in bold are paired CBL and CTX.

  2. *

    Denotes mid frontal gyrus (MFG), D = DNA content analyses, S= Small population qPCR, T = FISH Analysis, B = single cell qPCR on Biomark HD, P=PNA FISH, M = Westra et al. DNA content metadata.

Table 2

Primers used for small population and single nuclei qPCR

https://doi.org/10.7554/eLife.05116.026
GeneProteinLocusAssay typePrimer sequenceProbeProduct lengthEfficiency
APP, Exon 14Amyloid precursor protein21q21.3SYBR GreenF-TGCACGTGAAAGCAGTTGAAG, R-AAAGATGGCATGAGAGCATCGN/A2140.973
SEMA4ASemaphorin 4A1q22SYBR GreenF- ATGCCCAGGGTCAGATACTAT, R-TTCTCCGAGATCCTCTGTTTCN/A1770.997
CCL18Chemokine (C–C motif) ligand 1817q11.2SYBR GreenF-TTCCTGACTCTCAAGGAAAGG, R-CTGGCACTTACATGACACCTGN/A2091.006
PCDH11XProtocadherin 11 X-linkedXq21.3SYBR GreenF-TCTTTTGGTCAGTGTTGTGCG, R-CAACAAGTCGCCTATCAGGACN/A1880.993
APP, Exon 14Amyloid precursor protein21q21.3TaqManCGGTCAAAGATGGCATGAGAGCATC*, Assay Hs01255859_cnFAM-MGB911.040
APP, Exon 3Amyloid precursor protein21q21.3TaqManF-GCACTTCTGGTCCCAAGCAT, R-CCAGTTCTGGATGGTCACTGROX-IB1400.992
SEMA4ASemaphorin 4A1q22TaqManGTTCAAGGGTATGTGAGGTGAGATG*, Assay Hs00329046_cn_VICVIC-MGB901.016
  1. *

    Denotes probe sequence provided by Life Technologies.

Table 3

Quality control between 48.48 Dynamic Array runs

https://doi.org/10.7554/eLife.05116.027
Cell 1Cell 2Cell 3Cell 4Cell 5Cell 6Cell 7Cell 8
APP 14Run 121.3718.7718.0218.3417.1119.3320.1218.02
Run 220.6018.7718.1418.4117.2519.5020.6018.26
APP3Run 122.7920.3518.5319.11
Run 221.9719.8418.6519.28
SEMA4ARun 125.5424.2922.4424.2322.8125.7424.4624.67
Run 225.2624.5323.9224.3923.0325.4224.1124.30
Table 4

Confidence Intervals for (CI) calling Copy Number (CN)

https://doi.org/10.7554/eLife.05116.028
CNCIRCN, value
1Lower0.92156
Upper1.08512
2Lower1.84312
Upper2.17023
3Lower2.76468
Upper3.25535
4Lower3.68624
Upper4.34047
5Lower4.60780
Upper5.42559
6Lower5.52936
Upper6.51070
Table 5

Peptide nucleic acid (PNA) probe sequences

https://doi.org/10.7554/eLife.05116.029
GeneProteinLocusAssaySequence
APP Exon 3Amyloid precursor protein21q21.3PNA FISHA488-GATGGGTCTTGCACTG, A488-CCCCGCTTGCACCAGTT, A488-GGTTGGCTTCTACCACA, A488-CAGTTCAGGGTAGAC
APP Exon 14Amyloid precursor protein21q21.3PNA FISHA488-CTCCATTCACGG, A488-GTGGTTTTCGTTTCGGT, A488-ACTGATCCTTGGTTCAC, A488-ACTGATCCTTGGTTCAC, A488-ACGTCATCTGAATAGTT
TelomereN/ATelomeresPNA FISHTelC-Cy3 (F1002, PNA BIO)

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