Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology--cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.

Figure 1. The toxicity of TDP43 depends strongly on dose

(a, b) Histograms of TDP43(WT)-EGFP and TDP43(A315T)-EGFP levels in individual neurons. Increasing amounts of DNA significantly shifted the distribution of log-transformed expression levels towards higher values (p < 0.01 for all comparisons, two-sided Kolmogorov-Smirnov test). TDP43(WT)-EGFP, n=1287 neurons, with 290–342 cells per group; TDP43(A315T)-EGFP, n=1290, with 315–332 neurons per group. Values pooled from 12 wells per condition, performed in duplicate. (c) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (blue arrow) were censored. Scale bar = 25 μm. (d, e) Cumulative risk of death over time for neurons transfected with EGFP, TDP43(WT)-EGFP or TDP43(A315T)-EGFP. Supplementary Table 1 lists the number of neurons, hazard ratios, 95% CI, and p values for each condition, as determined by Cox proportional hazards analysis. Results pooled from 12 wells per condition, performed in duplicate. (f, g) Linear regression of calculated hazard ratio (± SEM) as a function of total TDP43(WT)-EGFP (R² = 0.9353) or TDP43(A315T)-EGFP (R² = 0.9574) level. The total TDP43 level was determined by quantitative immunocytochemistry using TDP43-specific antibodies (Supplementary Fig. 3) and normalized to the amount of TDP43 in non-transfected neurons. Dotted lines, reference (HR = 1).

Figure 2. TDP43 turnover in primary neurons

(a) Primary neurons were labeled with ³⁵S-labeled methionine, and endogenous TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or non-specific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R² = 0.8984). Values pooled from five independent experiments. (c) Potential confounders in half-life analyses. Ideally (top), total protein decreases over time in a predictable manner (top right panel). Toxicity (middle) reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Protein aggregation (bottom) might also shorten half-life estimations if the protein cannot be immunoprecipitated (dark arrows, bottom right panel), or lengthen it if the protein is more stable and successfully immunoprecipitated (grey arrows, bottom right panel). (d) Optical pulse labeling. Primary cortical neurons were transfected with EGFP and TDP43(WT)-Dendra2, photoconverted then imaged by AFM. Dead cells (arrow) were excluded at the time of death. (e) The half-life of TDP43(WT)-Dendra2 was determined by fitting a first-order exponential curve to the data (R² 0.9558). (f) Including cells with aggregates prolonged TDP43(WT)-Dendra2 half-life (purple line, R² = 0.9003; * p < 0.0001, F 15.34, extra sum-of-squares F-test). Excluding cells that died over the 36 h experiment also prolonged half-life (blue line, R² = 0.7171; ** p < 0.0001, extra sum-of-squares F-test), suggesting that toxicity reduces measured half-life. Error bars represent ± SEM. Values pooled from eight wells per condition, performed in quadruplicate.

Figure 3. Optical pulse labeling of TDP43

(a) Scatter plot depicting half-life measurements for individual cells expressing TDP43(WT)-Dendra2 (cyan, n=206) and TDP43(A315T)-Dendra2 (red, n=251). * p=0.0029, U = 11797, Mann-Whitney test. (b) Probability density plot for single-cell half-life measurements, demonstrating a reduced half-life of TDP43(A315T)-Dendra2 (red, n=251), compared to TDP43(WT)-Dendra2 (cyan, n=206; ** p=0.0007, two-sided Kolmogorov-Smirnov test). Values were pooled from eight wells per condition, performed in quadruplicate. Probability density plots of single-cell half-life measurements for TDP43(WT)-Dendra2 (c) and TDP43(A315T)-Dendra2 (d). At 0.5 μM, all three compounds significantly reduced TDP43(WT)-Dendra2 half-life, compared to vehicle control (DMSO, n=110; FPZ, n=96, *f p=1×10⁻⁴; MTM, n=129, *m p=2×10⁻⁷; NCP, n=111, *n p=2×10⁻⁵, two-sided Kolmogorov-Smirnov test). The compounds also reduced TDP43(A315T)-Dendra2 half-life, in comparison to vehicle control (DMSO, n=120; FPZ, n=155, *f p=2×10⁻⁹; MTM, n=152, *m p=0.004; NCP, n=111, *n p=0.005, two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons in (b), (c) and (d). Values were obtained from eight wells per condition, performed in triplicate.

Figure 4. Induction of autophagy by a family of small molecules

(a). Each compound significantly increased the LC3-II/GAPDH (b) and LC3-II/LC3-I (c) ratios at 5 μM, but not 0.5 μM [ns, p > 0.05; * 0.05 < p < 0.01, ** p < 0.01; F = 13.74 (b) and 9.74 (c), one-way ANOVA with Dunnett’s post-test]. Error bars represent ± SEM. Values pooled from five independent experiments. (d) Optical pulse-labeling of LC3-Dendra2 in rodent primary cortical neurons. Scale bar = 25 μm. (e) The change in red fluorescence intensity over time was used to calculate LC3-Dendra2 half-life, as in Fig. 2 (DMSO, R² = 0.7333; Beclin, R² = 0.6968, FPZ, R² = 0.8752; MTM, R² = 0.8447; NCP, R² = 0.8357; * p < 0.0001 for global trend, F = 12.6, extra sum-of-squares F-test). Error bars represent ± SEM. (f) The median single-cell half-life of LC3-Dendra2 was significantly reduced by beclin expression or treatment with 0.5 μM NCP, MTM, and FPZ (* p < 0.01, ** p < 0.001, Kruskal Wallis statistic = 52.56, Kruskal-Wallis with Dunn’s test). (g) Probability density plot of LC3-Dendra2 half-life measurements from individual neurons treated with vehicle control (DMSO, n=140), NCP (n=128), MTM (n=91), or FPZ (n=77), or those expressing beclin (n=101). *n, p=6×10⁻⁴; *m, p=1×10⁻¹⁰; *f, p=5×10⁻⁹; *b, p=2×10⁻³; two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons. Values pooled from eight wells per condition, performed in triplicate.

Figure 5. Autophagy induction reduces TDP43 levels, aggregation, and cytoplasmic mislocalization

(a) FPZ (n=490) and MTM (n=580), but not NCP (n=523), lowered total TDP43(WT)-EGFP levels in comparison to vehicle (DMSO, n=1007). (b) Histogram of normalized, log-transformed TDP43(WT)-EGFP levels. (c) Each compound also reduced TDP43(A315T)-EGFP levels (DMSO, n=983; FPZ, n=550; MTM, n=542; NCP, n=493). (d) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in (b) and (d) denote median values for cells exposed to vehicle (cyan), FPZ (green), MTM (blue) or NPZ (purple). For FPZ, MTM, NCP vs. DMSO in (a) and (d), p < 1×10⁻³ by two-sided Kolmogorov-Smirnov test. (e) No compound reduced TDP43(WT)-EGFP inclusions 48 h after transfection (DMSO, n=360; FPZ, n=194; MTM, n=234; NCP, n=203). (f) FPZ and MTM, but not NCP, reduced TDP43(A315T)-EGFP inclusions at 48 h (DMSO, n=327; FPZ, n=226; MTM, n=262; NCP, n=253). (g) No compound affected TDP43(WT)-EGFP localization 24 h after transfection (DMSO, n=256; FPZ, n=212; MTM, n=250; NCP, n=241). Each compound (h) prevented TDP43(A315T)-EGFP cytoplasmic mislocalization (DMSO, n=233; FPZ, n=268; MTM, n=263; NCP, n=270) and (i) reduced cytoplasmic TDP43(A315T)-EGFP levels (DMSO, n=125; FPZ, n=102; MTM, n=121; NCP, n=144. * p < 0.05; ns, p > 0.05). For (a), (c), and (e–i), * p < 0.05; ns, p > 0.05, one-way ANOVA with Dunnett’s test. Error bars represent ± SEM. Values in (a–d) pooled from 8–12 wells per condition, from six experiments; values in (e–h) pooled from eight wells per condition, in triplicate; values in (i) pooled from five wells per condition, in duplicate.

Figure 6. Autophagic stimulation improves survival in neuronal and astrocyte models of ALS

Primary neurons transfected with TDP43(WT)-EGFP (a) or TDP43(A315T)-EGFP (b) were treated with autophagy inducers and survival determined by AFM. Data obtained from eight wells per condition, performed in triplicate. See Supplementary Table 2 for the number of neurons per condition, hazard ratios, 95% CI, and p values as determined by Cox hazards analysis. (c) Autophagic induction improved survival in TDP43 M337V HB9-positive human iPSC-derived MNs. (d) FPZ and MTM also reduced the risk of death in MAP2-positive human iPSC-derived MNs Values in (c) and (d) pooled from 18 wells per condition, performed in triplicate. (e) Astrocytes differentiated from human iPSCs exhibit mutant TDP43-related toxicity that is mitigated by FPZ and MTM. Values pooled from 18 wells per condition, performed in triplicate. See Supplementary Table 3 for the number of human iPSC-derived neurons and astrocytes per condition, hazard ratios, 95% CI, and p values as determined by Cox hazards analysis.