MMP-9 and MMP-2 Contribute to Neuronal Cell Death in iPSC Models of Frontotemporal Dementia with MAPT Mutations

Abstract

How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines, we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9/MMP-2 were sufficient to decrease neuronal survival. In tau-A152T neurons, inhibition of the ERK pathway decreased MMP-9 expression. Moreover, ectopic expression of 4R but not 3R tau-A152T in HEK293 cells increased MMP-9 expression and ERK phosphorylation. These findings provide insights into the molecular pathogenesis of FTD and suggest a potential therapeutic target for FTD with MAPT mutations.

Keywords: MMP-2; MMP-9; frontotemporal dementia; iPSCs; neuronal survival; neurons; tau.

Figure 1

Differentiation and Characterization of Cortical Neurons Derived from iPSC Lines from FTD Patients and Control Subjects (A) Phase-contrast images of neurospheres (upper), differentiated neurons (middle), and MAP-2 immunostaining of neurons (lower) from the iPSC lines. Scale bars, 50 μm. (B) Western blot analysis of MAP-2, PSD-95, and tau-expression levels in 4-week-old neurons differentiated from iPSC lines. Actin served as the loading control. (C) Representative action potentials elicited by current injection (0–400 pA in 100-pA steps) and blocked by tetrodotoxin (TTX) of lines 3L1 of control 1, 19L3 of patient 1, and 3L6 of patient 2. Nine to eleven cells were recorded for each group. (D) iPSC-derived neurons (n = 7 per subject) displayed synaptic responses and were sensitive to the AMPA receptor antagonist NBQX. (E) Averaged sEPSC events from neurons of different subjects. See also Figures S1 and S2.

Figure 2

MMP-9 and MMP-2 Expression Is Increased in Human Neurons with MAPT Mutations (A) Zymographic analysis of secreted MMP-9 and MMP-2 in conditioned media from tau-A152T human neurons (patient 1). Immunoblotting of cell lysates with actin served as the loading control. (B) Quantification of secreted MMP-9 and MMP-2 in (A). Values are mean ± SD of three independent experiments. p < 0.001 by Student's t test. (C) Zymographic analysis of the level of secreted MMP-9 and MMP-2 in conditioned media from MAPT IVS10+16 human neurons (patient 2). Immunoblotting of cell lysates with actin served as the loading control. (D) Quantification of secreted MMP-9 and MMP-2 in C. Values are mean ± SD of three independent experiments. p < 0.01 by Student's t test. (E) Zymographic analysis for MMP-9 and MMP-2 in conditioned media from tau-A152T human neurons (patient 3; Fong et al., 2013). Immunoblotting of cell lysates with actin served as the loading control. (F) Quantification of MMP-2 and MMP-9 levels in (E) based on three experiments. Values are mean ± SD of three experiments. ^∗∗∗p < 0.001 by Student's t test. See also Figure S3.

Figure 3

Increased MMP-9 and MMP-2 Expression Contributes to Neurodegeneration of Patient Neurons under Stress (A) TUNEL analysis of TUJ1⁺ 4-week-old cortical neurons from controls and tau patients under basal conditions. Values are mean ± SD of two independent experiments and in each experiment, 100 cells were counted for each iPSC line. (B) Percentage of human neuron survival after 48 hr of treatment with DMSO, 40 nM rapamycin, or 40 nM rapamycin and inhibitors (Inh) of MMP-2 (5 μM) and MMP-9 (200 nM). Values are mean ± SD from three independent experiments. (C) qRT-PCR analysis of MMP-2 and MMP-9 mRNA levels in human neurons after treatment with DMSO or 40 nM rapamycin (Rap). Values are mean ± SD from three independent experiments. (D) Survival of 4-week-old control human neurons 48 hr after treatment with recombinant MMP-2 (5 μg/mL) and MMP-9 (5 μg/mL). Values are mean ± SD of three independent experiments. Statistical significance is indicated by p values and was determined by Student's t test. See also Figure S3.

Figure 4

The tau-A152T Mutation Increases MMP-9 Expression through Effects on ERK (A) Western blot analysis of ERK and p-ERK in 4-week-old iPSC-derived human cortical neurons. (B and C) Neurons from line 3 of patient 1 (B) and line 6 of patient 2 (C) were treated with DMSO or the MEK inhibitor PD98059. Cell lysates were analyzed by western blot with antibodies against p-ERK, ERK, and actin, and conditioned media were analyzed by zymography for MMP-9 and MMP-2. The numbers indicate the levels of MMP-9 relative to actin. (D) Western blot analysis of tau levels in HEK293 cells transfected with human 4R WT or 4R tau-A152T. (E) Zymographic analysis of secreted MMP-9 and MMP-2 in conditioned media, and western blot analysis of actin, p-ERK, and ERK levels in respective lysates from HEK293 cells expressing 4R WT or 4R tau-A152T. (F) qRT-PCR analysis of the expression levels of MMP-2 and MMP-9 mRNA in HEK293 cells expressing 4R WT or 4R tau-A152T. Values are mean ± SD from three independent experiments. ^∗∗∗p < 0.001 by Student's t test.