Induced pluripotent stem cell models of progranulin-deficient frontotemporal dementia uncover specific reversible neuronal defects

Abstract

The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell lines from a control subject, a patient with sporadic FTD, and an FTD patient with a novel heterozygous GRN mutation (progranulin [PGRN] S116X). In neurons and microglia differentiated from PGRN S116X induced pluripotent stem cells, the levels of intracellular and secreted PGRN were reduced, establishing patient-specific cellular models of PGRN haploinsufficiency. Through a systematic screen of inducers of cellular stress, we found that PGRN S116X neurons, but not sporadic FTD neurons, exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover, the serine/threonine kinase S6K2, a component of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by PGRN expression. Our findings identify cell-autonomous, reversible defects in patient neurons with PGRN deficiency, and provide a compelling model for studying PGRN-dependent pathogenic mechanisms and testing potential therapies.

Figure 1. Generation and Characterization of FTD Patient-Specific iPSCs

(A-C) Total and endogenous (Endo) levels of the reprogramming factors in control, sporadic, and PGRN S116X iPSC lines relative to the values in H9, as assessed by qRT-PCR. Values are mean ± SEM. (D) Genomic DNA sequencing of the heterozygous PGRN S116X mutation g.4627C>A (p.S116X: nonsense mutation) in PGRN S116X iPSCs. (E) Methylation status of the OCT4 promoter for control iPSC line 20, sporadic iPSC line 9, and PGRN S116X iPSC line 26. Open circles, unmethylated CpG dinucleotides. Filled circles, methylated CpG dinucleotides. (F) Immunofluorescence analysis of pluripotency markers in control iPSC line 20, sporadic iPSC line 9, and PGRN S116X iPSC line 26, and their respective normal karyotypes. Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (G) In vitro (EB formation) and in vivo (teratoma formation) differentiation of control iPSC line 20, sporadic iPSC line 9, and PGRN S116X iPSC line 26 into cells of all three germ layers. For in vitro studies, cells were immunostained with α-fetoprotein (AFP, endoderm), desmin (mesoderm), βIII-tubulin (ectoderm), and DAPI (nuclei). Hematoxylin/eosin staining of teratoma sections showed neuron/rosette structures (ectoderm), smooth muscle (mesoderm), and ducts (endoderm). Scale bar, 50 μm. See also Figure S1.

Figure 2. Differentiation and Characterization of Human Postmitotic Neurons Derived from FTD and Control iPSCs

(A-E) Phase-contrast images of the cells at different stages of neuronal differentiation. Scale bar, 100 μm. (F) Representative images of 2-week-old cells stained with MAP2. Nuclear staining is shown in blue. (G, K) Cells positive for MAP2 and GFAP as a percentage of all cells in 2-week-old cultures. (H-J) Cells positive for VGLUT1, GABA, and TH (glutamatergic, GABAergic and dopaminergic markers, respectively) as a percentage of MAP⁺ cells. On average, 200 cells were analyzed per experiment in panels G-K (n = 3 independent cultures). Values are mean ± SEM. (L) Electrophysiological properties of control neurons and PGRN S116X neurons. Representative action potentials responding to step depolarization by current injection from 0 pA to 400 pA (100 pA step) that could be blocked by tetrodotoxin. n = 24 for each line. (M) Sample traces of mEPSCs from control (blue) and PGRN S116X neurons (black). All neurons (n = 10 for each line) displayed synaptic responses and were abolished by AMPA receptor antagonist NBQX. (N) Averaged mEPSC traces from control (blue) and PGRN S116X neurons (black). See also Figure S2.

Figure 3. An FTD Patient-Specific Neuronal Model of Progranulin Haploinsufficiency

(A) GRN mRNA expression levels in fibroblasts. (B, D) GRN mRNA expression levels in iPSC lines derived from control, sporadic, and PGRN S116X subjects (B) and neurons differentiated from iPSCs (D). The values from control (fibroblasts) or control line 20 were set to 1 (n = 3-4 independent cultures). (C, E) Amount of progranulin secreted into the medium over 24 h by iPSCs (C) and iPSC-derived neurons (E). Values of control line 20 were set to 100% (n = 3-4 independent cultures). (F) Intracellular progranulin levels in iPSC-derived neurons after medium collection. Values of control line 20 were set to 100% (n = 3-4 independent cultures). In panels (A-F), Values are mean ± SEM. **p < 0.01; ***p < 0.001, patient cells were compared with control cells. See also Figure S3.

Figure 4. Novel Cellular and Molecular Defects of PGRN S116X Neurons Can be Rescued by Progranulin Expression

(A-C) Effects of the stress inducers on human neurons. Values are expressed as a percentage of the cells exposed to DMSO (control) (n = 3-4 independent cultures). (D) Caspase-3-like activity after exposure to 10 nM staurosporine, 0.5 μM tunicamycin, or DMSO for 24 h. (E and F) Measurement of cell viability (E) and caspase-3 activation (F) after rescue with progranulin expression. n=5-6 independent cultures. (G) Heatmap depicting fold changes of gene expression in 2-3 neuron cultures differentiated from each one of the four iPSC lines of sporadic FTD case (blue) or PGRN S116X patient (fucsia) compared to control neurons. (H) Gene expression changes on the array for GRN and RPS6KB2. The log fold change is relative to control neurons. (I and J) Progranulin expression restores S6K2 protein levels in PGRN S116X neurons. Representative western blotting image for S6K2 (control line 17 and PGRN S116X line 1) (I) and quantification of S6K2 relative to GAPDH for three experiments performed on lines 17 and 20 (control) and, 1 and 26 (PGRN S116X) (J). In all panels, values are mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001. See also Figure S4.