Familial Alzheimer's disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta

Abstract

Familial Alzheimer's disease (fAD) mutations alter amyloid precursor protein (APP) cleavage by γ-secretase, increasing the proportion of longer amyloidogenic amyloid-β (Aβ) peptides. Using five control induced pluripotent stem cell (iPSC) lines and seven iPSC lines generated from fAD patients, we investigated the effects of mutations on the Aβ secretome in human neurons generated in 2D and 3D. We also analysed matched CSF, post-mortem brain tissue, and iPSCs from the same participant with the APP V717I mutation. All fAD mutation lines demonstrated an increased Aβ42:40 ratio relative to controls, yet displayed varied signatures for Aβ43, Aβ38, and short Aβ fragments. We propose four qualitatively distinct mechanisms behind raised Aβ42:40. (1) APP V717I mutations alter γ-secretase cleavage site preference. Whereas, distinct presenilin 1 (PSEN1) mutations lead to either (2) reduced γ-secretase activity, (3) altered protein stability or (4) reduced PSEN1 maturation, all culminating in reduced γ-secretase carboxypeptidase-like activity. These data support Aβ mechanistic tenets in a human physiological model and substantiate iPSC-neurons for modelling fAD.

Fig. 1

Ratios act as internal normalisers for relative Aβ peptide abundance in conditioned media from stem cell models of AD. a The Aβ domain of APP, highlighting the canonical cleavage sites of α-, β-, and γ-secretase within the Aβ peptide sequence itself, as well as the proposed pathways of carboxypeptidase-like activity following alternative ε-cleavage. Letters coloured blue, pink, and green indicate amino acids with hydrophilic, hydrophobic, and amphipathic properties, respectively. b Western blotting of full-length APP in iPSC-derived neuronal lysates and c quantification of APP western blot band intensities. d qPCR expression analysis of APP in iPSC-derived neurons. Replicates are shown within histogram bars and error bars represent SEM. e–h Normalisation of Aβ peptides measured in cell media using ratios e Aβ42:40, f Aβ42:38, g Aβ38:40 and h Aβ43:40. e–h Display an average percentage coefficient of variance (%CV) < 4% over 6 days and < 7% over 100 days for ratios. For h Aβ43:40 average %CV was 7.1% over 6 days and 20.7% over 100 days. Data is based on multiple independent inductions per line, specifically APP V717I-1 clone 1 (n = 3), APP V717I-1 clone 3 (n = 2), PSEN1 int4del clone 4 (n = 1), PSEN1 int4del clone 6 (n = 2), Ctrl1 (n = 1), Ctrl 2 (n = 1) and Shef6 (n = 2)

Fig. 2

Aβ ratios are consistent between matched in vitro and in vivo samples from the same patient donor. a Aβ42:40 and b Aβ38:40 measured in conditioned media (n = 12), cell lysates (n = 8), lumbar CSF (n = 1) and brain tissue homogenate (n = 1) from the same individual. c Post-mortem tissue, 3D cerebral organoids and 2D iPSC-neurons from the same individual were immunostained for Aβ and MAP2

Fig. 3

fAD neurons display mutation-specific Aβ profile differences. Conditioned media was collected at 100 days post-neuronal induction for analysis. Results from the ratios a Aβ42:40, b Aβ42:38, c Aβ38:40, d Aβ43:40, e Aβ42:43, f Aβ38:43 are displayed. 2D data were generated from multiple inductions per line, specifically APP V717I-1 clone 1 (n = 7), APP V717I-1 clone 3 (n = 3), APP V717I-2 (n = 2), PSEN1 Int4del clone 4 (n = 5), PSEN1 Int4del clone 6 (n = 5), PSEN1 Y115H (n = 6), PSEN1 M139V (n = 6), PSEN1 M146I (n = 3), PSEN1 R278I (n = 6). Control data were generated from the following inductions: Ctrl 1 (n = 5), Ctrl2 (n = 6), Ctrl3 (n = 7), Ctrl4 (n = 6), and SHEF6 (n = 4). 3D data consisted of two inductions of each line, except APP V717I-1 clone 3, SHEF6, and M139V for which no data is available. Significance levels: * = < 0.05, ** = < 0.01, *** = < 0.001

Fig. 4

Mutation specific differences of Aβ secretomes from multiple proteolytic pathways. Results from mass spectrometric analysis of cell media for Aβ peptides generated by a–f BACE1 and γ-secretase activity g–i, BACE1 and BACE2 activity, and j–l BACE1 and α-secretase activity normalised as ratios. Mean data were generated at day 100 from multiple independent inductions per line, specifically non-AD (n = 10) consisting of pooled data of Ctrl1 (n = 2), Ctrl2 (n = 2), Ctrl3 (n = 2), Ctrl4 (n = 2), and Shef6 (n = 2). fAD data were generated from the following, APP V717I-1 clone 1 (n = 5), APP V717I-1 clone 3 (n = 2), PSEN1 int4del clone 4 (n = 2), PSEN1 int4del clone 6 (n = 2), PSEN1 Y115H (n = 2), PSEN1 M139V (n = 2), PSEN1 M146I (n = 2), and PSEN1 R278I (n = 2). Significance levels: * = < 0.05, ** = < 0.01, *** = < 0.001

Fig. 5

PSEN1 protein levels are variably altered in a subset of PSEN1 mutant neurons. a Representative western blot of 3 control neuron lysates and 6 fAD lysates. The asterisk depicts immature, full length PSEN1 protein at 46 kDa. b Quantification of independent neuronal lysates, replicates are depicted within histogram