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. 2017 Oct 27;5(1):77.
doi: 10.1186/s40478-017-0475-z.

CRISPR/Cas9-Correctable Mutation-Related Molecular and Physiological Phenotypes in iPSC-derived Alzheimer's PSEN2 N141I Neurons

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Free PMC article

CRISPR/Cas9-Correctable Mutation-Related Molecular and Physiological Phenotypes in iPSC-derived Alzheimer's PSEN2 N141I Neurons

Maitane Ortiz-Virumbrales et al. Acta Neuropathol Commun. .
Free PMC article

Abstract

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aβ42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aβ42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aβ42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology.

Keywords: Alzheimer’s disease; BFCN; Basal forebrain; CRISPR/Cas9; Cholinergic; Electrophysiology; PSEN2; Presenilin; iPSC.

Conflict of interest statement

Ethics approval and consent to participate

Prior to their participation, all donors of skin biopsies provided their written informed consent and study approval was obtained from Western Institutional Review Board.

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All authors declare their consent for publication of this manuscript.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Overview schematic of basal cholinergic differentiation protocol. a Cells are plated and allowed to reach 100% confluency (Day 0), before the initiation of dual smad inhibition and the subsequent introduction of ventralizing agents (Day 2). At day 10 the monolayer is dissociated, sorted for p75+ cells, and kept as NEBs until day 19. Then the culture is dissociated again into a monolayer (See Methods for more details). b Left panel shows sustained EGFP expression driven by Nkx2.1 induction in NKx2.1-EGFP hESCs upon SHH plus purmorphamine or SAG plus purmorphamine treatment, maintained at Day 14, after removal of treatment at Day 8. Right panel shows Nkx2.1, Lhx8 and BF1 relative gene expression to GAPDH measured by qPCR, in NKx2.1-EGFP cell line in the presence of the indicated ventralizing agents, or unpatterned (UNP) at Day 12. n = 3, in technical triplicates. c Confocal microscope images of Nestin (green), Sox2(red) and DRAQ5 (blue) immunostaining in fControl and control lines at Day 11, showing typical neural rosettes (left panel), or Tuj1 (green), Nkx2.1 (red)right and DRAQ5(blue) in the right panel. Images representative of 3 independent experiments. d Fluorescence microscope images of immunostained NEB cryosections or dissociated NEBs into a monolayer with the BFCN markers Nkx2.1/Tuj1/p75/BF1/MAP2/ChAT. e Dissociated NEBs into a monolayer immunostained at Day 50 with MAP2(green), ChAT(red) and Hoescht (blue). Fluorescence microscope images the effect of NGF addition to SAG plus purmorphamine treatment alone. Images are representative of at least 3 independent experiments
Fig. 2
Fig. 2
Basal cholinergic markers in PSEN2 N141I neuroprecursors. a Table showing the cell lines used. Four iPS lines reprogrammed from fibroblasts were used; two controls (949 and 050643, labelled as fControl and Control, respectively) that do not carry the PSEN2 N141I mutation nor the ε4 allele; and two AD patients (948 and 950, labelled as AD1 and AD2, respectively) who carry the mutation and the ε4 allele. Three of the four iPS lines were family related (fControl, AD1, and AD2). b Representative Sanger sequencing chromatograms showing a fragment of exon 5 of PSEN2. Red arrow marks site of the missense point mutation Chr1:227,073,304 A > T. c Immunocytochemistry and RT-PCR for early neuronal and basal forebrain markers. n = 3, 3 independent experiments with technical triplicates. d RTPCR fold changes for TUJ1 and BF1. n = 3, 3 independent experiments with technical triplicates. e Representative histograms for P75 staining. n > 6. f Aβ40 and Aβ42 ELISA quantifications. n = 3, 3 independent experiments with technical triplicates. ***, p < .001. *, p < .05
Fig. 3
Fig. 3
Neuronal and basal cholinergic markers by immunocytochemistry. a Immunostaining for TrkA on DIV 21. b Immunostainings for ChAT and vAChT at different magnifications at DIV65; and Tuj1 and MAP2. Images are representative of at least 3 independent experiments
Fig. 4
Fig. 4
CRISPR/Cas9-mediated correction of PSEN2 N141I iPS lines. a Schematic showing guide RNAs used in the targeting of CRISPR/Cas9, as well as donor ssODNs utilized to introduce wild-type genotype. b Left 2 panels show GFP positive HEK293T cells indicating Cas9 system with guide RNA expression, NT refers to non-transfected; right 2 panels show sample of GFP positive iPSCs after lipofection with pCas9-gN141I-GFP vector. c Sanger sequencing results from iPSC lines, showing corrections in the N141I mutation. d Aβ 42/40 ratio detected by ELISA in 72 h conditioned media from mutant, control or Cispr-Cas9 corrected BFCNs (DIV 34). n = 4, 4 independent experiments with technical triplicates. *, p < .05; **, p < .01 Student T-test
Fig. 5
Fig. 5
BFCNs carrying various PSEN mutations are not consistently more susceptible to Aβ42 oligomer toxicity. a Sample images of BFCNs from the indicated genotypes treated with propidium iodide to visualize cell death in response to 72-h exposure to Aβ42 oligomers (5 μM). b % LDH Release recorded from media collected after 72-h exposure. n = 3, 3 independent experiments with technical triplicates. *, p < .05; **, p < .01 as detected by 2-Way ANOVA Bonferroni post hoc tests
Fig. 6
Fig. 6
NLRP2 inflammasome mRNA levels are over-expressed in some PSEN2 N141I cells, but it is not driven by mutation. RT-PCR expression of (a) NLRP2, (b) NLRP3, and (c) ASB9 in cholinergic neuroprecursors. d Western blot showing NLRP2, PSEN2 and β-Actin. RT-PCR expression of NLRP2 in Neuroprecursors (e) and BFCNs (f). n = 3, 3 independent experiments with technical triplicates, for all panels. ***, p < .001
Fig. 7
Fig. 7
Electrophysiological and morphological features of BFCN. a Top row – compound sodium and potassium currents produced by a voltage protocol shown in bottom row. Current trace produced by a voltage step to −20 mV shown in red. Inset shows first 25 ms of a current produced by a voltage step to −20 mV (scale bars 200 pA, 5 ms). b Differential interference contrast image of a patched BFCN recorded in a. Ninety-four neurons (22 wild-type control, 21 familial control, 18 AD1, 28 AD2 and 5 iAD1_control). Scale bar is 30 μm
Fig. 8
Fig. 8
Electrophysiological deficits in BFCNs from AD lines. a Co-localization of biocytin-labelled neurons (green) with cholinergic markers ChAT (red) and VAChT (blue). Arrows indicate positions of recorded neurons somas, scale bar is 50 μm. b Representative firing patterns of BFCNs produced by a 1 sec negative and positive square current injection are depicted. A grand total of 94 individual neurons were studied electrophysiologically: 22 wild-type control neurons, 21 familial control neurons, 18 AD1 neurons, 28 AD2 neurons, and 5 iAD1_ (CRISPR-corrected) neurons. The experiments on the 94 neurons required days to weeks. On each experimental day, representatives from each genotype were included, with at least three samples from each genotype studied each day. c Summary data on maximum number of action potentials that neurons are capable of sustaining (left) and height of a single action potential at rheobase (right) across all conditions. Individual data points are shown as circles, group means are shown as bars. **, p < 0.01 Tukey HSD test
Fig. 9
Fig. 9
Intrinsic electrophysiological properties of BFCNs. Summary data on all recorded BFCNs from five groups. Ninety-four neurons (22 wild-type control, 21 familial control, 18 AD1, 28 AD2 and 5 iAD1_control). Histograms show individual values from each neuron (circle) and group means (bars) for membrane resistance (a), capacitance (b), resting potential (c) and rheobase current (d). Statistical significance was tested with ANOVA and Tukey’s post hoc comparisons

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