EFHC1, implicated in juvenile myoclonic epilepsy, functions at the cilium and synapse to modulate dopamine signaling.

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy.

Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans.

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented-animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article, we use behavioural neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologues of Ca2+/calmodulin-dependent kinases CaMK1/4, CMK-1 and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype (i.e. larger mechanosensory responses than wild-type). Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates.

Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.

Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT).

Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.

Striated rootlet and nonfilamentous forms of rootletin maintain ciliary function.

Primary cilia are microtubule-based sensory organelles whose structures and functions must be actively maintained throughout animal lifespan to support signal transduction pathways essential for development and physiological processes such as vision and olfaction [1]. Remarkably, few cellular components aside from the intraflagellar transport (IFT) machinery are implicated in ciliary maintenance [2]. Rootletin, an evolutionarily conserved protein found as prominent striated rootlets or a nonfilamentous form, both of which are associated with cilium-anchoring basal bodies, represents a likely candidate given its well-known role in preventing ciliary photoreceptor degeneration in a mouse model [3, 4]. Whether rootletin is universally required for maintaining ciliary integrity, and if so, by what mechanism, remains unresolved. Here, we demonstrate that the gene disrupted in the previously isolated C. elegans chemosensory mutant che-10 encodes a rootletin ortholog that localizes proximally and distally to basal bodies of cilia harboring or lacking conspicuous rootlets. In vivo analyses reveal that CHE-10/rootletin maintains ciliary integrity partly by modulating the assembly, motility, and flux of IFT particles, which are critical for axoneme length control. Surprisingly, CHE-10/rootletin is also essential for stabilizing ciliary transition zones and basal bodies, roles not ascribed to IFT. Unifying these findings, we provide evidence that the underlying molecular defects in the che-10 mutant stem from disrupted organization/function of the periciliary membrane, affecting the efficient delivery of basal body-associated and ciliary components and resulting in cilium degeneration. Together, our cloning and functional analyses of C. elegans che-10 provide the first mechanistic insights into how filamentous and nonfilamentous forms of rootletin play essential roles in maintaining ciliary function in metazoans.

The FMRFamide-related neuropeptide FLP-20 is required in the mechanosensory neurons during memory for massed training in C. elegans.

Lasting memories are likely to result from a lasting change in neurotransmission. In the nematode Caenorhabditis elegans, spaced training with a tap stimulus induces habituation to the tap that lasts for >24 h and is dependent on glutamate transmission, postsynaptic AMPA receptors, and CREB. Here we describe a distinct, presynaptic mechanism for a shorter lasting memory for tap habituation induced by massed training. We report that a FMRFamide-related peptide (FMRF = Phe-Met-Arg-Phe-NH(2)), FLP-20, is critical for memory lasting 12 h following massed training, but is not required for other forms of memory. Massed training correlated with a flp-20-dependent increase in synaptobrevin tagged with green fluorescent protein in the presynaptic terminals of the PLM mechanosensory neurons that followed the timeline of the memory trace. We also demonstrated that flp-20 is required specifically in the mechanosensory neurons for memory 12 h after massed training. These findings show that within the same species and form of learning, memory is induced by distinct mechanisms to create a lasting alteration in neurotransmission that is dependent upon the temporal pattern of training: memory of spaced training results from postsynaptic changes in the interneurons of the neural circuit, whereas memory of massed training results from presynaptic changes in the mechanosensory neurons of the neural circuit.

Intensity discrimination deficits cause habituation changes in middle-aged Caenorhabditis elegans.

The ability to learn and remember is critical for all animals to survive in the ever-changing environment. As we age, many of our biological faculties decay and of these, decline in learning and memory can be the most distressing. To carefully define age-dependent changes in learning during reproductive age in the nematode Caenorhabditis elegans, we performed a parametric behavioral study of habituation to nonlocalized mechanical stimuli (petri plate taps) over a range of intensities in middle-aged worms. We found that as worms age (from the onset of reproduction to the end of egg laying), response probability habituation increases (at both 10- and 60-second interstimulus intervals) and that these age-related changes were associated with a decrease in the discrimination between stimuli of different intensities. We also used optogenetics to investigate where these age-dependent changes occur. Our data suggest that the changes occur upstream of mechanosensory neuron depolarization. These data support the idea that declines in stimulus intensity discrimination abilities during aging may be one variable underlying age-related cognitive deficits.

Tap withdrawal circuit interneurons require CREB for long-term habituation in Caenorhabditis elegans.

We investigated the role of the Caenorhabditis elegans CREB (cAMP response element binding protein) homologue, crh-1, in response to tap (nonlocalized mechanosensory stimulation) and tap habituation. Worms with a loss-of-function mutation in crh-1 performed smaller reversals in response to tap than did wild-type worms and did not show long-term memory for spaced training 24-hr posttraining; however, they did show short-term habituation to tap stimuli when stimuli were presented at both 10-s and 60-s interstimulus intervals, and showed 12-hr intermediate memory for spaced habituation training (intermediate-term memory). Expressing CRH-1 broadly throughout the nervous system and in a subset of interneurons of the tap withdrawal circuit, but not in the mechanosensory neurons, rescued the long-term memory defects observed in crh-1 mutants. Here we show for the first time that CREB is required for long-term habituation and show that the interneurons of the tap withdrawal response circuit are the locus of plasticity for long-term mechanosensory habituation in C. elegans.