Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression

Abstract

Background: Many neurodegenerative diseases are associated with protein misfolding/aggregation. Treatments mitigating the effects of such common pathological processes, rather than disease-specific symptoms, therefore have general therapeutic potential.

Results: Here we report that the anti-epileptic drug ethosuximide rescues the short lifespan and chemosensory defects exhibited by C. elegans null mutants of dnj-14, the worm orthologue of the DNAJC5 gene mutated in autosomal-dominant adult-onset neuronal ceroid lipofuscinosis. It also ameliorates the locomotion impairment and short lifespan of worms expressing a human Tau mutant that causes frontotemporal dementia. Transcriptomic analysis revealed a highly significant up-regulation of DAF-16/FOXO target genes in response to ethosuximide; and indeed RNAi knockdown of daf-16 abolished the therapeutic effect of ethosuximide in the worm dnj-14 model. Importantly, ethosuximide also increased the expression of classical FOXO target genes and reduced protein aggregation in mammalian neuronal cells.

Conclusions: We have revealed a conserved neuroprotective mechanism of action of ethosuximide from worms to mammalian neurons. Future experiments in mouse neurodegeneration models will be important to confirm the repurposing potential of this well-established anti-epileptic drug for treatment of human neurodegenerative diseases.

Fig. 1

Ethosuximide increases lifespan and improves sensorimotor function in C. elegans ANCL and frontotemporal dementia models. a Ethosuximide extends lifespan in dnj-14 mutants. Viability of age-synchronised dnj-14(ok237) animals grown in the presence of the indicated concentrations of ethosuximide was determined; untreated wild type control N2 worms are shown for comparison (n = 50-55 worms for each concentration). b Ethosuximide ameliorates the dnj-14 food sensing defect. The time taken to move to a bacterial food source was measured in wild type N2 and dnj-14(tm3223) strains grown until 5-6 days of age in the presence or absence of ethosuximide (n = 71-80 worms of each strain per condition). c Ethosuximide increases locomotion in Tau V337M worms, but not control worms. Thrashing in solution was measured in Tau V337M worms grown until 1 and 3 days of age and assayed in the presence of the indicated concentrations of ethosuximide (for each age group, n = 120-140 worms for 0 mg/l; n = 38-40 worms for 0.1, 0.2 and 0.5 mg/ml; n = 80-90 worms for 1 and 2 mg/ml;). Identically treated wild type control CZ1200 worms are shown for comparison (n = 20 worms per concentration). Data are shown as mean ± SEM (***p < 0.001). d Age-dependence of ethosuximide’s effect on Tau V337M locomotion. Thrashing assays were performed on age-synchronised animals grown in the presence or absence of 2 mg/ml ethosuximide (n = 30-50 worms per data point). Data are shown as mean ± SEM (***p < 0.001, *p < 0.05). e Ethosuximide increases lifespan in Tau V337M worms. Viability of age-synchronised animals grown in the presence of the indicated concentrations of ethosuximide was determined in comparison to untreated wild type control CZ1200 worms (n = 50-102 worms for each drug concentration). f Comparison of the ethosuximide concentration-dependence of mean lifespan extension in dnj-14 and Tau V337M worms

Fig. 2

Ethosuximide acts independently of T-type calcium channels and bacterial metabolism to reduce Tau aggregation. a Ethosuximide Inhibition of the T-type calcium channel, CCA-1, is not required for protection against paralysis. Tau V337M transgenic animals were crossed with loss-of-function mutations for cca-1(ad1650) to generate homozygous cross progeny. Ethosuximide supplementation increased thrashing activity of the Tau V337M transgenic strain and the double mutant Tau V337M; cca-1(ad1650) strain to similar extents. Data are shown as mean ± SEM (**p < 0.01, *p < 0.05; n = 30-50 worms per data point). b Ethosuximide extends lifespan in Tau V337M mutants in the absence of CCA-1. Lifespan assays were performed on single mutant cca-1(ad1650), transgenic (Tau V337M) and double mutant Tau V337M; cca-1(ad1650) strains grown in the presence (dashed lines) or absence (solid lines) of 1 mg/ml ethosuximide (n > 100 worms per strain/condition). c Ethosuximide extends lifespan using killed bacteria as a food source. Lifespan assays were performed on Tau V337M worms grown under control conditions or in the presence of kanamycin, or 1 mg/ml ethosuximide or both (n > 80 worms per condition). d Total Tau protein expression in Tau V337M worm lysates is not reduced by ethosuximide. The left panel shows a representative western blot; the right panel shows quantification of Tau normalised to actin and expressed as % of untreated control (mean ± SEM, n = 3; not significant). e, f Ethosuximide affects Tau proteostasis. e shows a representative western blot of the soluble and detergent-soluble (RIPA) sequentially extracted fractions in the presence or absence of ethosuximide treatment. f shows quantification of Tau fractions normalised to actin and expressed as % of the total (soluble + RIPA) protein level (data are shown as mean ± SEM (n = 3; *p < 0.05)

Fig. 3

Ethosuximide-induced genes are enriched in DAF-16-associated elements and ethosuximide-induced lifespan extension requires daf-16. a Validation of gene expression changes using qRT-PCR. Selected genes that were up-regulated by ethosuximide in microarray experiments (ugt-25, dhs-26, cyp-14A3, cyp-35B1, ttr-44, dod-6 and cyp-34A9) were confirmed to be significantly induced using qRT-PCR. No significant changes in expression of normalisation (act-1, pmp-3) or negative control (pph-6) genes was observed. Results are expressed as mean fold change ± SEM relative to the unexposed control (n = 3). b Ethosuximide-responsive genes are enriched for the DAF-16 Associated Element (DAE) motif. To identify regulatory sequences correlating with ethosuximide-responsiveness, 200-bp regions in the upstream promoter sequences of common DEGs were mined for overrepresented motifs using RSAT. c Ethosuximide increases dnj-14 lifespan in a daf-16-dependent manner. Survival curves of dnj-14(ok237) worms grown on E. coli containing empty vector (L4440), hsp-1 or daf-16 dsRNA-producing plasmids in the presence (dashed lines) or absence (solid lines) of 1 mg/ml ethosuximide. Ethosuximide treatment significantly increased the lifespan of dnj-14(ok237) worms on vector control (p < 0.001), but had no significant effect on daf-16 RNAi animals (p > 0.15) (n > 100 worms per strain/condition)

Fig. 4

Ethosuximide induces FOXO target gene expression and reduces polyglutamine protein aggregation in mammalian neurons. a mRNA levels of classical FOXO target genes were analysed by qRT-PCR in differentiated mouse N2A neuroblastoma cells treated with the indicated concentrations of ethosuximide. Data are shown as mean ± SEM (n = 3; *p ≤ 0.05, **p ≤ 0.01). (b-c) Ethosuximide reduces polyglutamine protein aggregation. b Visualisation of EGFP-tagged non-pathological (polyQ25) and pathological (polyQ97) polyglutamine tracts in N2A cells 72 h post-transfection. Phase contrast, GFP (green) and SYTOX orange staining (red, to identify dead cells) confocal images are shown to illustrate that aggregates are specific to polyQ97 and that ethosuximide redistributed polyQ97-EGFP away from aggregates into the cytoplasm and neuronal processes C) Quantification of polyQ aggregation. The number of polyQ-EGFP transfected N2A cells bearing fluorescent aggregates as a percentage of the total number of viable transfected (green) cells was counted at the indicated post-transfection times. Cell/aggregate counting was performed manually and confirmed using ImageJ software if cells were sufficiently sparse to allow this. Data are shown as mean ± SEM (n = 3, counting ~100 cells in each experiment; *p < 0.05)