Defective proteostasis in induced pluripotent stem cell models of frontotemporal lobar degeneration

Abstract

Impaired proteostasis is associated with normal aging and is accelerated in neurodegeneration. This impairment may lead to the accumulation of protein, which can be toxic to cells and tissue. In a subset of frontotemporal lobar degeneration with tau pathology (FTLD-tau) cases, pathogenic mutations in the microtubule-associated protein tau (MAPT) gene are sufficient to cause tau accumulation and neurodegeneration. However, the pathogenic events triggered by the expression of the mutant tau protein remain poorly understood. Here, we show that molecular networks associated with lysosomal biogenesis and autophagic function are disrupted in brains from FTLD-tau patients carrying a MAPT p.R406W mutation. We then used human induced pluripotent stem cell (iPSC)-derived neurons and 3D cerebral organoids from patients carrying the MAPT p.R406W mutation and CRISPR/Cas9, corrected controls to evaluate proteostasis. MAPT p.R406W was sufficient to induce morphological and functional deficits in the lysosomal pathway in iPSC-neurons. These phenotypes were reversed upon correction of the mutant allele with CRISPR/Cas9. Treatment with mTOR inhibitors led to tau degradation specifically in MAPT p.R406W neurons. Together, our findings suggest that MAPT p.R406W is sufficient to cause impaired lysosomal function, which may contribute to disease pathogenesis and serve as a cellular phenotype for drug screening.

Fig. 1. TFEB-regulated genes are differentially expressed in MAPT p.R406W brains.

RNAseq was performed in brains from MAPT p.R406W and neuropathology-free controls and reported in Jiang et al. [19]. A Principal component analysis reveals transcriptome-wide differences between MAPT p.R406W (n = 2) and neuropathology-free control brains (n = 16). B We tested whether genes that contain a CLEAR sequence or that are differentially expressed in the presence of TFEB in vitro were differentially expressed in human brains from MAPT p.R406W carriers compared with normal controls. Volcano plot showing log2 fold change between MAPT p.R406W and control brains and the –log₁₀ p-value for each gene. Red nodes: genes with p < 0.05.

Fig. 2. Altered proteostasis in human iPSC-neurons expressing the MAPT p.R406W mutation.

A iPSC from a MAPT mutation carrier and CRISPR/Cas9-corrected control (wild-type (WT)) were differentiated into cortical neurons and cultured for 6 weeks prior to analysis. B Representative immunostaining for Tuj1 (green) and DAPI (blue) illustrates that at 6 weeks in culture, cells are enriched for neurons. Scale bar, 10 microns. C Immunoblots of cell lysates (10 μg total protein) were probed with LAMP1, Cathepsin D and ubiquitin antibodies. D Quantification of protein analyte levels in the MAPT p.R406W neurons and isogenic controls. Graph represents mean ± SEM. Significance was determined using an unpaired, t-test. *p < 0.05; **p < 0.01. ****p < 0.0001.

Fig. 3. Human iPSC-neurons expressing the MAPT p.R406W mutation exhibit defects in lysosomal morphology.

Human iPSC-neurons neurons from a MAPT p.R406W mutation carrier and the isogenic, CRISPR/Cas9-corrected control (wild-type (WT)) were differentiated into cortical neurons and cultured for 6 weeks prior to analysis. A Representative immunostaining for LAMP1 (red) and DAPI (blue) illustrates altered LAMP1-positive vesicles in the MAPT p.R406W neurons compared with isogenic controls. Scale bar, 10 microns. White arrows indicate LAMP1-positive vesicles in the neurites of MAPT p.R406W neurons. B Diagram of quantification of LAMP1-positive vesicles represented in C–E. All quantification was performed in Tuj1-positive cells. C Distance of LAMP1-positive vesicles from the nuclear membrane is significantly greater in MAPT p.R406W neurons (n = 27 cells) compared to isogenic controls (n = 60 cells). D The size of LAMP1-positive vesicles in the soma is significantly larger in MAPT p.R406W neurons (n = 10 cells) compared with the isogenic controls (n = 12 cells). E The number of LAMP1-positive vesicles within the soma is significantly reduced in MAPT p.R406W neurons (n = 11 cells) compared to isogenic controls (n = 11 cells). Graphs represent mean ± SEM. Significance was determined using an unpaired, t-test. *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 4. Human iPSC-neurons expressing the MAPT p.R406W mutation display defects in vesicle acidity.

Human iPSC-neurons neurons from a MAPT p.R406W mutation carrier and isogenic, CRISPR/Cas9-corrected control (wild-type (WT)) were differentiated into cortical neurons and cultured for 6 weeks prior to analysis. A MAPT p.R406W neurons exhibit reduced LysoTracker staining. Live cells were incubated with Lysotracker and were imaged as described in Methods. Representative images of LysoTracker-stained neurons are shown in gray scale for clarity. Scale bar, 10 microns. Lower panel represents magnification of the cells in the black box. B Schematic of the quantification of LysoTracker staining in MAPT p.R406W neurons (n = 78 cells) and isogenic controls (n = 46 cells). C Quantification of the intensity of LysoTracker staining in individual soma. Graphs represent mean ± SEM. Significance was determined using an unpaired, t-test. ***p < 0.001.

Fig. 5. Lysosomal dysfunction in human iPSC-neurons expressing the MAPT p.R406W mutation.

Human iPSC-neurons neurons from a MAPT p.R406W mutation carrier and isogenic, CRISPR/Cas9-corrected control (wild-type (WT)) were differentiated into cortical neurons and cultured for 6 weeks prior to analysis. A MAPT p.R406W neurons exhibit increased DQ-BSA fluorescence. Live cells were incubated with DQ-BSA and were imaged as described in Methods. Representative images of DQ-BSA-stained neurons are shown in gray scale for clarity. Scale bar, 10 microns. Lower panel represents magnification of the cells in the black box. B Diagram of DQ-BSA mechanism. C Quantification of the intensity of DQ-BSA staining in soma from MAPT p.R406W neurons (n = 79 cells) and isogenic controls (n = 69 cells). D Diagram of secondary elevation of lysosomal enzymes. E Enzyme activity of β-Glucuronidase measured in cell lysates from MAPT p.R406W neurons and isogenic controls. Enzymatic activity was normalized to the total protein. Graphs represent mean ± SEM. Significance was determined using an unpaired, t-test. *p < 0.05; ****p < 0.0001.

Fig. 6. Colocalization of tau and phospho-tau with LAMP1-positive vesicles.

iPSC from a MAPT p.R406W mutation carrier and CRISPR/Cas9-corrected control (wild-type (WT)) were differentiated into cortical neurons and cultured for 6 weeks prior to analysis. A Representative confocal images showing colocalization of LAMP1 (red) and total tau (Tau5; green) or ptau (AT180; green) in the MAPT p.R406W neurons compared with isogenic controls. Scale bar, 10 microns. B Colocalization of LAMP1 and total tau in tau-positive soma (WT, n = 146 cells; p.R406W, n = 95 cells) C Colocalization of LAMP1 and ptau in ptau-positive soma (WT, n = 73 cells; p.R406W, n = 39 cells). Graphs represent mean ± SEM. Significance was determined using an unpaired, t-test. **p < 0.01; ****p < 0.0001.

Fig. 7. MAPT p.R406W organoids phenocopy lysosomal defects.

A Schematic of organoid generation. iPSC from a MAPT mutation carrier and CRISPR/Cas9-corrected control (wild-type (WT)) were differentiated into cortical organoids and cultured for 2 months. B Enzyme activity of β-Glucuronidase measured in organoid lysates (10 μg protein) from MAPT p.R406W and corrected control organoids. Enzymatic activity was normalized to total protein. Graphs represent mean ± SEM. C Immunoblots of cell lysates from organoids were probed with LAMP1 and Cathepsin D (active form shown) antibodies. D Quantification of protein analyte levels in the MAPT p.R406W organoids and isogenic controls. Graphs represent mean ± SD. Significance was determined using an unpaired, t-test. *p < 0.05.

Fig. 8. Torin-1 treatment reduces tau specifically in MAPT p.R406W neurons.

iPSC-derived neurons from MAPT p.R406W and isogenic, controls were cultured for 6 weeks prior to treatment with torin-1 or DMSO, control for 5 h. Tau was measured in cells lysates using IP/MS. Graphs represent mean ± SEM. Significance was determined using an unpaired, t-test. *p < 0.05.