Cytokinin response factor 6 (CRF6) is an Arabidopsis AP2/ERF transcription factor which is transcriptionally induced by cytokinin. Cytokinin is known to delay leaf senescence in wild-type (WT) plants, for example in dark-incubated detached leaves. This response is mediated by the cytokinin receptor Arabidopsis histidine kinase receptor 3 (AHK3). Similar to ahk3 mutants, crf6 leaves show decreased sensitivity to this cytokinin effect. Leaves overexpressing CRF6 retain more Chl than those of the WT under these conditions without exogenous cytokinin. It therefore appears that an increase in expression of CRF6 downstream of the perception of cytokinin by AHK3 is involved in the delay of leaf senescence. Intact crf6 plants also begin to undergo monocarpic senescence sooner than WT plants. Interestingly, plants overexpressing CRF6 display a more extreme acceleration of development than crf6 mutants, suggesting that a specific expression level or localization of CRF6 is necessary to prevent premature senescence. Expression analyses indicate that CRF6 is highly expressed in the veins of mature leaves and that this expression decreases with age. CRF6 expression is shown to be induced by abiotic stress, in addition to increased cytokinin. Together, these findings suggest that CRF6 functions to regulate developmental senescence negatively and may have a similar role in response to stress. CRF6 may therefore be involved in fine-tuning the timing of developmental and stress-induced senescence. CRF6 functioning in negative regulation of senescence is significant in that it is the first process known to be regulated by cytokinin, in which a CRF can be placed specifically downstream of the cytokinin signaling pathway.

Summary 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 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. While 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.

The microscopic nematode Caenorhabditis elegans has emerged as a valuable model for understanding the molecular and cellular basis of neurological disorders. The worm offers important physiological similarities to mammalian models such as conserved neuron morphology, ion channels, and neurotransmitters. While a wide-array of behavioral assays are available in C. elegans, an assay for electroshock/electroconvulsion remains absent. Here, we have developed a quantitative behavioral method to assess the locomotor response following electric shock in C. elegans. Electric shock impairs normal locomotion, and induces paralysis and muscle twitching; after a brief recovery period, shocked animals resume normal locomotion. We tested electric shock responses in loss-of-function mutants for unc-25, which encodes the GABA biosynthetic enzyme GAD, and unc-49, which encodes the GABAA receptor. unc-25 and unc-49 mutants have decreased inhibitory GABAergic transmission to muscles, and take significantly more time to recover normal locomotion following electric shock compared to wild-type. Importantly, increased sensitivity of unc-25 and unc-49 mutants to electric shock is rescued by treatment with antiepileptic drugs, such as retigabine. Additionally, we show that pentylenetetrazol (PTZ), a GABAA receptor antagonist and proconvulsant in mammalian and C. elegans seizure models, increases susceptibility of worms to electric shock.

The nematode Caenorhabditis elegans is a useful model organism for dissecting molecular mechanisms of neurological diseases. While hermaphrodite C. elegans contains only 302 neurons, the conserved homologous neurotransmitters, simpler neuronal circuitry, and fully mapped connectome make it an appealing model system for neurological research. Here we developed an assay to induce an electroconvulsive seizure in C. elegans which can be used as a behavioral method of analyzing potential anti-epileptic therapeutics and novel genes involved in seizure susceptibility. In this assay, worms are suspended in an aqueous solution as current is passed through the liquid. At the onset of the shock, worms will briefly paralyze and twitch, and shortly after regain normal sinusoidal locomotion.The time to locomotor recovery is used as a metric of recovery from a seizure which can be reduced or extended by incorporating drugs that alter neuronal and muscular excitability.