The HECT Family Ubiquitin Ligase EEL-1 Regulates Neuronal Function and Development

Abstract

Genetic changes in the HECT ubiquitin ligase HUWE1 are associated with intellectual disability, but it remains unknown whether HUWE1 functions in post-mitotic neurons to affect circuit function. Using genetics, pharmacology, and electrophysiology, we show that EEL-1, the HUWE1 ortholog in C. elegans, preferentially regulates GABAergic presynaptic transmission. Decreasing or increasing EEL-1 function alters GABAergic transmission and the excitatory/inhibitory (E/I) balance in the worm motor circuit, which leads to impaired locomotion and increased sensitivity to electroshock. Furthermore, multiple mutations associated with intellectual disability impair EEL-1 function. Although synaptic transmission defects did not result from abnormal synapse formation, sensitizing genetic backgrounds revealed that EEL-1 functions in the same pathway as the RING family ubiquitin ligase RPM-1 to regulate synapse formation and axon termination. These findings from a simple model circuit provide insight into the molecular mechanisms required to obtain E/I balance and could have implications for the link between HUWE1 and intellectual disability.

Keywords: C. elegans; EEL-1; GABA; HUWE1; acetylcholine; intellectual disability; motor neuron; seizure; synaptic transmission; RPM-1.

Figure 1. eel-1 RNAi and eel-1 (lf) mutations cause hypersensitivity to aldicarb

(A) Schematic of human HUWE1, Drosophila Huwel and C. elegans EEL-1 protein sequence. Conserved mutations associated with intellectual disability (R2981H and R4187C) are highlighted. Deletions generated by ok1575 and zu462 are shown below. Conserved protein domains are annotated as follows: DUF (domain of unknown function, annotated in NCBI conserved domain database), CD (conserved domain of unknown function, annotated here), UBA (ubiquitin associated domain), WWE (WWE domain), CAD (conserved acidic domain), and HECT (homologous to E6AP c-terminus domain). (B) Aldicarb time course for eel-1 RNAi using eri-1; lin-15B animals. (C) Aldicarb paralysis at the 90-minute time point for eel-1 RNAi using eri-1; lin-15B. (D) Aldicarb time course for eel-1 RNAi using uIs57; lin-15B animals. (E) Aldicarb paralysis at the 60-minute time point for eel-1 RNAi using uIs57; lin-15B animals. (F) Aldicarb time course for eel-1 mutants, zu462 and ok1575. (G) Aldicarb paralysis at the 60-minute time point for eel-1 mutants. goa-1 and ric-3 mutants were included as positive controls for Hic and Ric phenotypes. Significance was determined using an unpaired Student's t test, and error bars represent standard error of the mean. *P<0.05, **P<0.01, ***P<0.001

Figure 2. EEL-1 functions in GABAergic motor neurons to regulate aldicarb hypersensitivity

(A) Aldicarb time course showing that transgenic expression of EEL-1 rescues aldicarb hypersensitivity of eel-1 mutants. (B) Aldicarb paralysis at the 150- minute time point for EEL-1 transgenic rescues. (C) Aldicarb time course showing that transgenic expression of EEL-1 in GABAergic motor neurons (PGABA∷EEL-1) rescues aldicarb hypersensitivity of eel-1 mutants. (D) Aldicarb paralysis at the 150-minute time point for EEL-1 rescue in GABAergic motor neurons. (E) Aldicarb time course showing that transgenic overexpression of EEL-1 in wild-type animals using a pan-neuronal promoter (Pneuro∷EEL-1), or a promoter specific for the GABAergic motor neurons (PGABA∷EEL-1) results in resistance to aldicarb. (F) Aldicarb paralysis at the 180- minute time point for transgenic overexpression of EEL-1. Significance was determined using an unpaired Student's t test, and error bars represent standard error of the mean. ***P<0.001 and ns = not significant

Figure 3. EEL-1 is required for GABAergic transmission at the NMJ

(A) GABAergic mIPSC frequency is reduced in eel-1 (zu462) mutants. (B) Representative GABAergic traces for the indicated genotypes. (C) The integrated transgenic line bggIs16 (Peel-1∷EEL-1) rescues GABAergic mIPSC frequency defects in eel-1 mutants. (D) Representative GABAergic traces for the indicated genotypes. (E) Cholinergic mEPSC frequency and amplitude is unchanged in eel-1 mutants (F) Representative cholinergic traces for the indicated genotypes. *P<0.05, **P<0.01, *** P<0.001, ns = not significant

Figure 4. EEL-1 is expressed in the nervous system and present at GABAergic presynaptic terminals

Transgenic animals expressing Peel-1∷GFP were analyzed using epifluorescent microscopy. Shown are representative images of (A) neurons in the head, the nerve ring, and the ventral nerve cord. (B) Cholinergic motor neurons (arrows) coexpressing Peel-1∷GFP and Punc-129∷ mCherry (cholinergic marker). (C) GABAergic motor neurons (arrows) coexpressing Peel-1∷GFP and Punc-25∷mCherry (GABAergic marker). (D) ALM mechanosensory neuron (arrow) coexpressing Peel-1∷GFP and P mec-7∷mCherry (ALM marker). (E) PLM mechanosensory neuron (arrow) coexpressing Peel-1∷GFP and Pmec-7∷mCherry (PLM marker). (F) GABAergic presynaptic terminals in the dorsal cord coexpressing SNB-1∷GFP (juIs1, green) and mCherry∷EEL-1 (magenta). Left is a merged confocal image of a multi-slice z-projection, and right is a higher magnification single confocal slice. Scale bar 10pm.

Figure 5. Locomotion and electroshock response are impaired in eel-1 mutants

(A) Quantitation of reverse locomotion following harsh head touch for the indicated genotypes. (B and C) MWT analysis of forward locomotion for the indicated genotypes. (D) Quantitation of time to recovery following electroshock induced paralysis. Recovery from electroshock is impaired in eel-1 mutants, and is rescued by the transgene bggIs16 (Peel-1∷EEL-1). (E) Treatment of eel-1 mutants with RTG suppresses defects in recovery from electroshock. Significance determined using unpaired Student's t test; error bars represent standard error of mean. ** P<0.01, ***P<0.001

Figure 6. Motor neuron synapse formation is largely normal in eel-1 mutants, but enhanced in eel-1; fsn-1 double mutants

(A) Schematic of a cholinergic motor neuron (purple) innervating dorsal muscles; presynaptic terminals in green. Blue box highlights the region of dorsal cord that was visualized using the transgene nuIs152 (Punc-129∷SNB-1∷GFP) for the indicated genotypes. Highlighted are regions lacking SNB-1 puncta (arrows), and abnormal SNB-1 aggregation (arrowheads). (B) Quantitation of SNB-1 puncta in cholinergic motor neurons for the indicated genotypes. (C) Schematic of a GABAergic motor neuron (purple) innervating dorsal muscles; presynaptic terminals in green. Blue box highlights the region of dorsal cord visualized using the transgene juIs1 (Punc-25∷SNB-1∷GFP) for the indicated genotypes. (D) Quantitation of SNB-1 puncta in GABAergic motor neurons for the indicated genotypes. Significance determined using unpaired Student's t test; error bars represent standard error of the mean. ***P< 0.001, ns = not significant. Scale bar 10μm.

Figure 7. Conserved, disorder-associated mutations impair EEL-1

Wild-type EEL-1, or EEL-1 carrying point mutations associated with intellectual disability (EEL-1 R2991H or R3990C) tested for transgenic rescue of aldicarb hypersensitivity in eel-1 mutants. (A) Aldicarb time course for the indicated genotypes. (B) Aldicarb paralysis at the 150- minute time point for the indicated genotypes. ***P < 0.001, ns = not significant.