Erucin, a Natural Isothiocyanate, Prevents Polyglutamine-Induced Toxicity in Caenorhabditis elegans via aak-2/AMPK and daf-16/FOXO Signaling

Abstract

Several neurodegenerative diseases (NDDs), such as Huntington's disease, six of the spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy, and spinobulbar muscular atrophy, are caused by abnormally long polyglutamine (polyQ) tracts. Natural compounds capable of alleviating polyQ-induced toxicity are currently of great interest. In this work, we investigated the modulatory effect against polyQ neurotoxic aggregates exerted by erucin (ERN), an isothiocyanate naturally present in its precursor glucoerucin in rocket salad leaves and in its oxidized form, sulforaphane (SFN), in broccoli. Using C. elegans models expressing polyQ in different tissues, we demonstrated that ERN protects against polyQ-induced toxicity and that its action depends on the catalytic subunit of AMP-activated protein kinase (aak-2/AMPKα2) and, downstream in this pathway, on the daf-16/FOXO transcription factor, since nematodes deficient in aak-2/AMPKα2 and daf-16 did not respond to the treatment, respectively. Although triggered by a different source of neurotoxicity than polyQ diseases, i.e., by α-synuclein (α-syn) aggregates, Parkinson's disease (PD) was also considered in our study. Our results showed that ERN reduces α-syn aggregates and slightly improves the motility of worms. Therefore, further preclinical studies in mouse models of protein aggregation are justified and could provide insights into testing whether ERN could be a potential neuroprotective compound in humans.

Keywords: AMPK; Caenorhabditis elegans; daf-16/FOXO; erucin; neuroprotection; polyQ toxicity.

Figure 1

Pharmacokinetics and bioavailability of ERN. The isothiocyanate ERN is a reduced analog of the isothiocyanate SFN. It is released both from the enzymatic hydrolysis of glucoerucin, a GL found in rocket salad leaves (Eruca sativa Mill., Diplotaxis tenuifolia L.) and from the in vivo reduction of SFN, derived from the conversion of glucoraphanin, the GL typical of broccoli (Brassica oleracea L. ssp italica). Myrosinase is the enzyme responsible for the hydrolysis of GLs.

Figure 2

Erucin restores neuronal function and reduces polyglutamine-induced muscular toxicity. (A) The expression of 112Q:TdTom in mechanosensory neurons induces an impairment of neuronal function compared to animals that do not have the transgene. Treatment in N2 wild-type worms does not affect the rate of response to touch for each concentration of ERN tested compared to untreated animals. Treatment with 100 μM and 200 μM ERN rescues neuronal function in 112Q: TdTom young adult animals compared to untreated nematodes. (B) Erucin treatment reduces polyQ-induced muscular aggregation in 40Q young adult animals significantly. *** p < 0.001, **** p < 0.0001 and ns, not significant.

Figure 3

Erucin reduces polyglutamine-induced neuronal toxicity. (A) Erucin treatment reduces polyQ-induced aggregate formation in the ventral nerve cord in 40Q young adult animals. (B) Representative images from fluorescence microscopy showing five neuronal aggregates of polyQs (white arrows) in the ventral nerve cord of F25B3.3p::40Q::YFP young adult animals at 20× magnification. ** p < 0.01, *** p < 0.001.

Figure 4

Reduction in polyQ aggregation induced by erucin requires AMPK catalytic function and daf-16/FOXO transcription factor. (A) The treatment with 100 µM and 200 µM ERN of 40Q::YFP worms defective of the catalytic subunit of AMPK (aak-2/AMPKα2) does not produce any significant difference in polyQ-induced muscular aggregate formation in comparison to untreated worms. In parallel, ERN treatment reduces polyQ-induced muscular aggregation in 40Q young adult animals of the control strain significantly. (B) The treatment with 100 µM and 200 µM ERN of 40Q::YFP worms defective of the daf-16 gene does not produce any significant difference in polyQ-induced neuronal aggregate formation in comparison to untreated worms. In parallel, ERN treatment significantly reduces polyQ-induced muscular aggregation in 40Q young adult animals of the control strain. (C) Representative images from fluorescence microscopy showing neuronal aggregates of polyQs (white arrows) in the ventral nerve cord of F25B3.3p::40Q::YFP and F25B3.3p::40Q::YFP; daf-16(mu86) young adult animals at 20× magnification. In each fluorescent image, two neuronal aggregates are counted and indicated by white arrows, showing no difference between the control strain (F25B3.3p::40Q::YFP) and the mutated one (F25B3.3p::40Q::YFP; daf-16(mu86)). ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns, not significant.

Figure 5

Treatment with erucin reduces α-synuclein aggregation and slightly restores motor capacity. (A) Treated animals with 100 µM and 200 µM ERN show a reduction in α-syn aggregates compared to untreated α-syn::YFP 2-day-old adult animals. (B) α-syn aggregates induce an impairment of motility compared with wild-type animals. Treated wild-type worms with both concentrations of ERN tested did not show motility impairment. Treated α-syn::YFP nematodes with 100 µM and 200 µM ERN rescue motility capacity of 2-day-old adult animals significantly after treatment only with the higher dose of ERN tested. * p < 0.05, **** p < 0.0001 and ns, not significant.