Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells

Abstract

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.

Figure 1. Generation of iPSC lines and purified neurons from APP^Dp, sAD and NDC fibroblasts

a, b, iPSC lines express NANOG and TRA1-81. c, d, iPSC-derived, FACS-purified NPCs express SOX2 and nestin. e–h, iPSC-derived, FACS-purified neurons express MAP2 and βIII-tubulin. Scale bars in a–h, 50 μm. i, Representative action potentials in response to somatic current injections. Data from iPSC line APP^Dp2.2. j, Spontaneous synaptic activity was detected (voltage clamp recording at the reversal potential of sodium (0 mV)) and reversibly blocked by GABA_A receptor antagonist SR95531 (10 μM). Each panel represents ~4 min continuous recordings separated in 25 sweeps (grey traces) and superimposed for clarity. Black traces represent a single sweep. Data from iPSC line NDC2.1. k, l, No significant difference was seen between NDCs and any patient’s cultures in the ability of iPSCs to generate NPCs at day 11 (P = 0.08, n = 9), or the ability of NPCs to form neurons at 3 weeks (P = 0.82, n = 9). Error bars indicate s.e.m.

Figure 2. Increased amyloid-β, p-tau and aGSK-3β in sAD2 and APP^Dp neuronal cultures

a, Purified neurons from sAD2, APP^Dp1 and APP^Dp2 secrete increased amyloid-β(1–40) (Aβ(1–40)) compared to NDC samples (P = 0.0012, 0.0014 and < 0.0001, respectively). b, Amyloid-β differences between patients and controls are larger in neurons versus fibroblasts. Data sets are relative to NDC mean. c, d, Neurons from sAD2, APP^Dp1 and APP^Dp2 have increased aGSK-3β (percentage non-phospho-Ser 9) and p-tau/total tau (p-tau/t-tau) compared to NDC samples (aGSK-3β, P < 0.0001, 0.0005 and 0.0001; p-tau/total tau, P < 0.0001, 0.0001 and 0.0002). In a–d, n values on graphs indicate the number of biological replicates per patient, contributed equally by three iPSC lines. e, sAD2 findings verified in two additional iPSC lines (sAD2.4 and sAD2.5). sAD2(1-3) indicates findings from initial sAD2 iPSC lines. For amyloid-β, aGSK-3β and p-tau/total tau, sAD2 remained significantly higher than controls (P < 0.0001). No significant difference was found between original and secondary sAD2 lines (P = 0.14, 0.44, 0.63). f, Strong positive correlations between amyloid-β(1–40), aGSK-3β and p-tau/total tau in purified neurons. Pearson R = 0.94, 0.91 and 0.83, respectively. g, Twenty-four hour treatment with β- and γ-secretase inhibitors reduced secreted amyloid-β(1–40) compared to control treatment. β-Secretase inhibitors partially rescued aGSK-3β and p-tau/total tau in sAD2 and APP^Dp2 neurons (P < 0.01 for aGSK-3β, P < 0.03 for p-tau). γ-Secretase inhibition did not significantly affect aGSK-3β and p-tau/total tau. In g, number of treatment sets is indicated on the graph (n), NDCs are represented by two iPSC lines each and sAD2 and APP^Dp2 are represented by three. Error bars indicate s.e.m.

Figure 3. Analysis of early endosome and synapsin levels in purified neurons co-cultured with astrocytes

a–c, Extended focus images of Rab5-stained neuronal soma from NDC1, sAD2 and APP^Dp2. Arrowhead in b marks a 1–2.1 μm³ early endosome, and the arrow marks a 2.1–7 μm³ early endosome. Scale bars, 10 μm. d, Neurons from sAD2 and APP^Dp2 have significantly increased numbers of large and very large early endosomes compared to NDC neurons (P < 0.0001, n = 40 neurons from two iPSC lines per individual). e, Representative image of synapsin I (green) on a MAP2⁺ dendrite (red). Arrowhead marks a synapsin I⁺ punctum. Scale bar, 3 μm. f, No significant difference between patients and controls in the number of synapsin⁺ structures per μm dendrite (P = 1.00, n = 40 dendrites from two iPSC lines per individual). Neurons were scored blinded to genotype. Error bars indicate s.e.m.