Caenorhabditis elegans dnj-14, the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform for neuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol

Adult onset neuronal lipofuscinosis (ANCL) is a human neurodegenerative disorder characterized by progressive neuronal dysfunction and premature death. Recently, the mutations that cause ANCL were mapped to the DNAJC5 gene, which encodes cysteine string protein alpha. We show here that mutating dnj-14, the Caenorhabditis elegans orthologue of DNAJC5, results in shortened lifespan and a small impairment of locomotion and neurotransmission. Mutant dnj-14 worms also exhibited age-dependent neurodegeneration of sensory neurons, which was preceded by severe progressive chemosensory defects. A focussed chemical screen revealed that resveratrol could ameliorate dnj-14 mutant phenotypes, an effect mimicked by the cAMP phosphodiesterase inhibitor, rolipram. In contrast to other worm neurodegeneration models, activation of the Sirtuin, SIR-2.1, was not required, as sir-2.1; dnj-14 double mutants showed full lifespan rescue by resveratrol. The Sirtuin-independent neuroprotective action of resveratrol revealed here suggests potential therapeutic applications for ANCL and possibly other human neurodegenerative diseases.

(A) Sequence alignment of the predicted products of the human DNAJC5 and C. elegans dnj-14a genes (CSPα and DNJ-14 proteins, respectively).
(B) Exon structure of the dnj-14 gene and location of the ok237 and tm3223 alleles.
(C) Confirmation of the ok327 deletion. Genomic DNA from wild type N2 and dnj-14(ok237) worms was amplified using primers flanking the deletion (left panel) or with one flanking primer and a primer within exon 2 of dnj-14 (right panel).
(D) Characterisation of the tm3223 insertion/deletion. Genomic DNA from wild type N2 and dnj-14(tm3223) worms was amplified using primers flanking the mutation site. Wild type N2 and dnj-14(ok237) worms were synchronised and grown on NGM plates for at least 9 days. Animals were then immobilised for GFP imaging. Typical images are shown for various animals. These illustrate the loss of neuronal cell bodies, reduction in the number of visible neurites, or the presence of contorted neuronal processes in the head of the worms that was frequently seen in dnj-14 mutants. In contrast, age-matched wild type N2 animals generally exhibited obvious neuronal cell bodies and had clearly labelled multiple neurites that extended straight to the end of the worm's head without twisting.

FIG. S4: Mechanosensation is unaffected in aged dnj-14 mutants.
Wild type N2, dnj-14(tm3223) and dnj-14(ok237) worms were synchronised and grown on NGM plates until 6 days of age. After transfer to unseeded plates, mechanosensation was assessed by gently touching an eyelash to the side of the worm's head at a perpendicular angle. Each worm was assayed ten times and the number of times the worm either stopped or reversed its direction of movement was recorded. No significant differences between wild type and dnj-14 mutants were seen (n=15 animals per strain).

FIG. S5: Pharyngeal pumping is unaffected in aged dnj-14 mutants.
Wild type N2, dnj-14(tm3223) and dnj-14(ok237) worms were synchronised and grown on NGM plates until 6 days of age. After transfer to plates freshly seeded with OP50 bacteria, the number of contraction/relaxation cycles of the pharynx per thirty seconds was for each worm was recorded. No significant differences between wild type and dnj-14 mutants were seen (n=15 animals per strain).

FIG. S6: Measurement of cAMP levels in worms treated with resveratrol and rolipram.
Wild type N2 worms were age-synchronised and grown on 60-mm NGM plates seeded with JB1669 adenylyl cyclase deficient bacteria. Approximately fifteen such plates of day 2 worms were washed in M9 buffer and then treated in M9 buffer containing vehicle control (ethanol), 100 µM resveratrol or 100 µM rolipram for 120 mins. The worms were then lysed, centrifuged to pellet any debris, and the supernatant used immediately for assay. Endogenous cyclic AMP levels were measured by ELISA and normalised to total protein concentration.
Data shown are mean + SEM (n = 4 biological replicates). No significant differences between treatments were seen.

MOVIE S1: Superficially normal locomotion in dnj-14(ok237) mutants
The movement of worms on NGM agar plates was recorded. Wild type N2 worms are shown first, followed by dnj-14(ok237) mutants.