TDP-43 promotes tau accumulation and selective neurotoxicity in bigenic Caenorhabditis elegans

Abstract

Although amyloid β (Aβ) and tau aggregates define the neuropathology of Alzheimer's disease (AD), TDP-43 has recently emerged as a co-morbid pathology in more than half of patients with AD. Individuals with concomitant Aβ, tau and TDP-43 pathology experience accelerated cognitive decline and worsened brain atrophy, but the molecular mechanisms of TDP-43 neurotoxicity in AD are unknown. Synergistic interactions among Aβ, tau and TDP-43 may be responsible for worsened disease outcomes. To study the biology underlying this process, we have developed new models of protein co-morbidity using the simple animal Caenorhabditis elegans. We demonstrate that TDP-43 specifically enhances tau but not Aβ neurotoxicity, resulting in neuronal dysfunction, pathological tau accumulation and selective neurodegeneration. Furthermore, we find that synergism between tau and TDP-43 is rescued by loss-of-function of the robust tau modifier sut-2. Our results implicate enhanced tau neurotoxicity as the primary driver underlying worsened clinical and neuropathological phenotypes in AD with TDP-43 pathology, and identify cell-type specific sensitivities to co-morbid tau and TDP-43. Determining the relationship between co-morbid TDP-43 and tau is crucial to understand, and ultimately treat, mixed pathology AD.

Keywords: Caenorhabditis elegans; Alzheimer's disease; Amyloid β (Aβ); Proteotoxicity; TDP-43; Tau.

Fig. 1.

Co-expression of TDP-43 with tau but not Aβ leads to enhancement of motility deficits. (A) tau+TDP Tg-low animals have significantly decreased activity. Unstimulated activity on a seeded agar plate was detected using unbiased computer-assisted video tracking and analysis. Movement (velocity) was recorded (µm/sec). n=115 (non-TG), 66 (TDP Tg-low), 89 (tau Tg), 93 (tau+TDP Tg-low), from three independent replicates. (B) tau+TDP Tg-low animals showed significantly decreased thrashing in liquid. Rates of thrashing were measured using unbiased computer-assisted tracking and analysis. The number of turns (thrashes) per minute were recorded (turns/min). n=73 (non-TG), 89 (TDP Tg-low), 81 (tau Tg), 87 (tau+TDP Tg-low), from three independent replicates. (C) Unstimulated activity on a seeded agar plate was detected using unbiased computer-assisted video tracking and analysis. Movement (velocity) was recorded (µm/sec). n=147 (non-TG), 119 (TDP Tg-low), 163 (Aβ Tg), 182 (Aβ+TDP Tg-low), from three independent replicates. (D) Rates of thrashing in liquid were measured using unbiased computer-assisted tracking and analysis. The number of turns (thrashes) per minute were recorded (turns/min). n=85 (non-TG), 105 (TDP Tg-low), 101 (Aβ Tg), 119 (Aβ+TDP Tg-low), from three independent replicates. One-way ANOVA with Tukey's multiple-comparison test. **P<0.01, ****P<0.0001. ns, not significant.

Fig. 2.

tau and TDP-43 do not synergize with poly-glutamine. (A-C) tau does not synergize with poly-glutamine. (A) Unstimulated activity on a seeded agar plate is detected using unbiased computer-assisted video tracking and analysis. Movement (velocity) was recorded (µm/sec). n=140 (non-TG), 179 (tau Tg), 138 (polyQ Tg), 110 (polyQ+tau Tg), from three independent replicates. (B) Rates of thrashing in liquid were measured using unbiased computer-assisted tracking and analysis. The number of turns (thrashes) per minute were recorded (turns/min). n=149 (non-TG), 204 (tau Tg), 167 (polyQ Tg), 132 (polyQ+tau Tg), from three independent replicates. (C) Data from B with decreased y-axis to allow visual comparisons between polyQ Tg and tau+polyQ Tg strains. (D-F) TDP-43 does not synergize with poly-glutamine. (D) Unstimulated activity on a seeded agar plate is detected using unbiased computer-assisted video tracking and analysis. Movement (velocity) was recorded (µm/sec). n=123 (non-TG), 99 (TDP Tg-low), 116 (polyQ Tg), 87 (polyQ+TDP Tg-low), from three independent replicates. (E) Rates of thrashing in liquid were measured using unbiased computer-assisted tracking and analysis. The number of turns (thrashes) per minute were recorded (turns/min). n=82 (non-TG), 94 (TDP Tg-low), 116 (polyQ Tg), 83 (polyQ+TDP Tg-low), from three independent replicates. (F) Data from E with decreased y-axis to allow visual comparisons between polyQ Tg and TDP+polyQ Tg strains. One-way ANOVA with Tukey's multiple-comparison test. *P<0.05, ****P<0.0001. ns, not significant.

Fig. 3.

tau+TDP Tg-low exhibit mechanosensory defects but intact pharyngeal pumping. (A) tau+TDP Tg-low animals have significantly worse mechanosensation. Mechanosensation was assayed by responsiveness to a light touch and scored as: 2, normal response; 1, abnormal response; 0, no response. n=45 (all strains), from three independent replicates. (B-D) C. elegans pharyngeal pumping was evaluated by recording pharyngeal muscle and neuron action potentials to generate an electropharyngeogram (EPG). No significant differences were detected among strains. (B) Representative EPG traces of pharyngeal action potentials showing both positive (excitatory) and negative (relaxation) spikes. (C) Average pump frequency and (D) average pump duration over a 2 min recording. n=39 (non-TG), 36 (TDP Tg-low), 32 (tau Tg), 35 (tau+TDP Tg-low), from three independent replicates. One-way ANOVA with Tukey's multiple-comparison test. ****P<0.0001. ns, not significant. Data are mean±s.e.m.

Fig. 4.

tau and TDP-43 co-expression promotes accumulation and phosphorylation of tau. (A-E) Developmentally synchronized day 1 adult C. elegans were harvested and tested by immunoblot for total tau, phosphorylated tau (AT8), total TDP-43, phosphorylated TDP-43 (phospho-S409/410) and tubulin (load control). (A) Immunoblot shown is representative of three independent replicate experiments. (B-E) Quantification of protein levels normalized to tubulin. Total tau (B) and phosphorylated tau (C) are elevated in tau+TDP-low Tg animals. Total TDP-43 (D) and phosphorylated TDP-43 (E) are not elevated in tau+TDP-low Tg animals. One-way ANOVA with Tukey's multiple-comparison test. *P<0.05. ns, not significant. Data are mean±s.e.m.

Fig. 5.

Co-expression of tau and TDP-43 leads to selective glutamatergic and serotonergic neurodegeneration. (A-C) Assessment of glutamatergic neurons in tau+TDP Tg-low animals. Depiction of neurons scored (green) (A). Quantification of neurons lost in developmentally synchronized day 1 adult (B) and day 4 adult (C) C. elegans. n>40 for all strains and time points scored, from at least three independent replicates. (D-F) Assessment of serotonergic neurons in tau+TDP Tg-low animals. Depiction of neurons scored (green) (D). Quantification of neurons lost in developmentally synchronized day 1 adult (E) and day 4 adult (F) C. elegans. n>45 for all strains and time points scored, from at least three independent replicates. One-way ANOVA with Tukey's multiple-comparison test. *P<0.05, ****P<0.0001. ns, not significant. Data are mean±s.e.m.

Fig. 6.

Co-expression of tau and TDP-43 leads to selective cholinergic and GABA-ergic neurodegeneration. (A-C) Assessment of cholinergic neurons in tau+TDP Tg-low animals. Depiction of neurons scored (green) (A). Quantification of neurons lost in developmentally synchronized day 1 adult (B) and day 4 adult (C) C. elegans. n>45 for all strains and time points scored, from at least three independent replicates. (D-F) Assessment of GABA-ergic neurons in tau+TDP Tg-low animals. Depiction of neurons scored (green) (D). Quantification of neurons lost in developmentally synchronized day 1 adult (E) and day 4 adult (F) C. elegans. n>45 for all strains and time points scored, from at least three independent replicates. One-way ANOVA with Tukey's multiple-comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns, not significant. Data are mean±s.e.m.

Fig. 7.

Loss of sut-2 expression prevents motility deficits and neurodegeneration in a model of combined tau and TDP-43 expression. (A) tau+TDP Tg-low animals have significantly decreased thrashing in liquid, which is rescued by sut-2(−). Rates of thrashing were measured using unbiased computer-assisted tracking and analysis. The number of turns (thrashes) per minute were recorded (turns/min). ***P<0.0008, ****P<0.0001. TDP-43 Tg-low versus sut-2(−); TDP Tg-low: P=0.9833; tau Tg versus sut-2(−); tau Tg: P<0.0001; tau+TDP Tg-low versus sut-2(−); tau+TDP Tg-low: P<0.0001. n=149 (non-TG), 134 (TDP Tg-low), 167 (tau Tg), 185 (tau+TDP Tg-low), 145 (sut-2(−); TDP Tg-low), 121 (sut-2(−); tau Tg), 131 (sut-2(−); tau+TDP Tg-low) from three independent replicates. (B,C) Quantification of neurons lost in developmentally synchronized day 1 adult (B) and day 4 adult (C) C. elegans show that sut-2(−) prevents neuron loss in tau+TDP Tg-low animals. *P<0.05; ***P<0.001; ****P<0.0001. (B) TDP-43 Tg-low versus sut-2(−); TDP Tg-low: P=0.9504; tau Tg versus sut-2(−); tau Tg: P=0.0002; tau+TDP Tg-low versus sut-2(−); tau+TDP Tg-low: P<0.0001. (C) TDP-43 Tg-low versus sut-2(−); TDP Tg-low: P>0.9999; tau Tg versus sut-2(−); tau Tg: P<0.0001; tau+TDP Tg-low versus sut-2(−); tau+TDP Tg-low: P<0.0001. n>45 for all strains and time points scored, from at least three independent replicates. One-way ANOVA with Tukey's multiple-comparison test. ns, not significant. Data are mean±s.e.m.

Fig. 8.

Loss of sut-2 expression prevents tau protein accumulation in a model of combined tau and TDP-43 expression. (A-E) Developmentally synchronized day 1 adult C. elegans were harvested and tested by immunoblot for total tau, phosphorylated tau (AT8), total TDP-43, phosphorylated TDP-43 (phospho-S409/410) and tubulin (load control). Immunoblot shown (A) is representative of three independent replicate experiments. (B-E) Quantification of protein levels performed by ImageJ software analysis of scanned film images. Total tau (B) and phosphorylated tau (C) are reduced in sut-2(−); tau+TDP-low Tg animals. Total TDP-43 (P=0.0917) (D) and phosphorylated TDP-43 (E) are not significantly reduced in sut-2(−); tau+TDP-low Tg animals. One-way ANOVA with Tukey's multiple-comparison test. ****P<0.0001. ns, not significant. Data are mean±s.e.m.