The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation.

A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.

Gaining an understanding of behavioral genetics through studies of foraging in Drosophila and learning in C. elegans.

The pursuit of understanding behavior has led to investigations of how genes, the environment, and the nervous system all work together to produce and influence behavior, giving rise to a field of research known as behavioral neurogenetics. This review focuses on the research journeys of two pioneers of aspects of behavioral neurogenetic research: Dr. Marla Sokolowski and Dr. Catharine Rankin as examples of how different approaches have been used to understand relationships between genes and behavior. Marla Sokolowski's research is centered around the discovery and analysis of foraging, a gene responsible for the natural behavioral polymorphism of Drosophila melanogaster larvae foraging behavior. Catharine Rankin's work began with demonstrating the ability to learn in Caenorhabditis elegans and then setting out to investigate the mechanisms underlying the "simplest" form of learning, habituation. Using these simple invertebrate organisms both investigators were able to perform in-depth dissections of behavior at genetic and molecular levels. By exploring their research and highlighting their findings we present ways their work has furthered our understanding of behavior and contributed to the field of behavioral neurogenetics.

A presenilin-1 mutation causes Alzheimer disease without affecting Notch signaling.

Presenilin-1 (PSEN1) is the catalytic subunit of the γ-secretase complex, and pathogenic mutations in the PSEN1 gene account for the majority cases of familial AD (FAD). FAD-associated mutant PSEN1 proteins have been shown to affect APP processing and Aß generation and inhibit Notch1 cleavage and Notch signaling. In this report, we found that a PSEN1 mutation (S169del) altered APP processing and Aß generation, and promoted neuritic plaque formation as well as learning and memory deficits in AD model mice. However, this mutation did not affect Notch1 cleavage and Notch signaling in vitro and in vivo. Taken together, we demonstrated that PSEN1S169del has distinct effects on APP processing and Notch1 cleavage, suggesting that Notch signaling may not be critical for AD pathogenesis and serine169 could be a critical site as a potential target for the development of novel γ-secretase modulators without affecting Notch1 cleavage to treat AD.

Habituation Is More Than Learning to Ignore: Multiple Mechanisms Serve to Facilitate Shifts in Behavioral Strategy.

Recent work indicates that there are distinct response habituation mechanisms that can be recruited by different stimulation rates and that can underlie different components (e.g., the duration or speed) of a single behavioral response. These findings raise the question: why is "the simplest form of learning" so complicated mechanistically? Beyond evolutionary selection for robustness of plasticity in learning to ignore, it is proposed in this article that multiple mechanisms of habituation have evolved to streamline shifts in ongoing behavioral strategy. Then, speculations are offered regarding the implications of this reconceptualization of habituation for approaching the analysis of mechanisms of more complex forms of learning and memory.

Habituation in high-throughput genetic model organisms as a tool to investigate the mechanisms of neurodevelopmental disorders.

Alterations in habituation, a highly conserved form of non-associative learning, are suspected to contribute to a range of the complex behavioural phenotypes present in multiple neurodevelopmental disorders. While progress has been made in understanding the genetics of these disorders through the application of next-generation sequencing and related technologies, the pathogenicity of genetic variants and causes of learning and memory impairments can be difficult to determine from sequencing data alone. High-throughput genetic model organisms such as the roundworm Caenorhabditis elegans, fruit fly Drosophila melanogaster, and zebrafish Danio rerio offer low-cost and efficient methods to investigate the functions of identified neurodevelopmental disorder risk genes and the functional consequences of specific disorder-associated variants. Here, we review ways assessing habituation has been used in the genotype-first approach to first validate neurodevelopmental disorder candidate genes and now to systematically characterize large candidate gene lists. We then discuss exciting ways habituation, in combination with other techniques, can be used as a tool to assess the pathogenicity of putative genes and genetic variants, uncover and confirm molecular networks, and identify potential therapeutic avenues.

But can they learn? My accidental discovery of learning and memory in C. elegans.

I did not set out to study C. elegans. My undergraduate and graduate training was in Psychology. My postdoctoral work involved studying learning and memory in 1 mm diameter juvenile Aplysia californica. As a starting Assistant Professor when I attempted to continue my studies on Aplysia I encountered barriers to carrying out that work; at about the same time I was introduced to Caenorhabditis elegans and decided to investigate whether they could learn and remember. My laboratory was the first to demonstrate conclusively that C. elegans could learn and in the years since then my lab and many others have demonstrated that C. elegans is capable of a variety of forms of learning and memory.

The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases.

Since Caenorhabditis elegans was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using C. elegans to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use C. elegans to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in C. elegans, allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution in vivo. Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease) in C. elegans and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.

Synaptic vesicle fusion is modulated through feedback inhibition by dopamine auto-receptors.

Mechanisms of synaptic vesicular fusion and neurotransmitter clearance are highly controlled processes whose finely-tuned regulation is critical for neural function. This modulation has been suggested to involve pre-synaptic auto-receptors; however, their underlying mechanisms of action remain unclear. Previous studies with the well-defined C. elegans nervous system have used functional imaging to implicate acid sensing ion channels (ASIC-1) to describe synaptic vesicle fusion dynamics within its eight dopaminergic neurons. Implementing a similar imaging approach with a pH-sensitive fluorescent reporter and fluorescence resonance after photobleaching (FRAP), we analyzed dynamic imaging data collected from individual synaptic termini in live animals. We present evidence that constitutive fusion of neurotransmitter vesicles on dopaminergic synaptic termini is modulated through DOP-2 auto-receptors via a negative feedback loop. Integrating our previous results showing the role of ASIC-1 in a positive feedback loop, we also put forth an updated model for synaptic vesicle fusion in which, along with DAT-1 and ASIC-1, the dopamine auto-receptor DOP-2 lies at a modulatory hub at dopaminergic synapses. Our findings are of potential broader significance as similar mechanisms are likely to be used by auto-receptors for other small molecule neurotransmitters across species.

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.

Accumulation of Laminin Monomers in Drosophila Glia Leads to Glial Endoplasmic Reticulum Stress and Disrupted Larval Locomotion.

The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, ß, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. We demonstrate that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the ß subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. We found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits. SIGNIFICANCE STATEMENT: This research presents a new perspective on how mutations in the extracellular matrix protein laminin cause severe consequences in glial wrapping and function. Glial-specific loss of the ß or γ laminin subunit disrupted glia morphology and led to ER expansion and stress due to retention of other subunits. The retention of the unpaired laminin subunit was key to the glial disruption as loss of Tango1 blocked secretion of the complete laminin trimer but did not lead to glial or locomotion defects. The effects were observed in the perineurial glia that envelope the peripheral and central nervous systems, providing evidence for the importance of this class of glia in supporting nervous system function.

Dopamine receptor DOP-4 modulates habituation to repetitive photoactivation of a C. elegans polymodal nociceptor.

Habituation is a highly conserved phenomenon that remains poorly understood at the molecular level. Invertebrate model systems, like Caenorhabditis elegans, can be a powerful tool for investigating this fundamental process. Here we established a high-throughput learning assay that used real-time computer vision software for behavioral tracking and optogenetics for stimulation of the C. elegans polymodal nociceptor, ASH. Photoactivation of ASH with ChR2 elicited backward locomotion and repetitive stimulation altered aspects of the response in a manner consistent with habituation. Recording photocurrents in ASH, we observed no evidence for light adaptation of ChR2. Furthermore, we ruled out fatigue by demonstrating that sensory input from the touch cells could dishabituate the ASH avoidance circuit. Food and dopamine signaling slowed habituation downstream from ASH excitation via D1-like dopamine receptor, DOP-4. This assay allows for large-scale genetic and drug screens investigating mechanisms of nociception modulation.

A review of transgenerational epigenetics for RNAi, longevity, germline maintenance and olfactory imprinting in Caenorhabditis elegans.

Inheritance of acquired characteristics without changes in DNA sequence has been called transgenerational epigenetics. This review looks at studies that used the model system Caenorhabditis elegans to uncover mechanisms of transgenerational epigenetics in studies of RNA interference, studies of longevity, studies of germline continuity and a study on olfactory imprinting. In each case, researchers have uncovered critical roles for small RNAs and for Argonaute proteins. They have revealed several different genetic pathways that mediate RNA silencing of foreign RNA for a few or for many generations, as well as identifying a related pathway responsible for recognized self-generated RNAs. Together, these studies have greatly advanced our understanding of trangenerational epigenetics.

High-throughput behavioral analysis in C. elegans.

We designed a real-time computer vision system, the Multi-Worm Tracker (MWT), which can simultaneously quantify the behavior of dozens of Caenorhabditis elegans on a Petri plate at video rates. We examined three traditional behavioral paradigms using this system: spontaneous movement on food, where the behavior changes over tens of minutes; chemotaxis, where turning events must be detected accurately to determine strategy; and habituation of response to tap, where the response is stochastic and changes over time. In each case, manual analysis or automated single-worm tracking would be tedious and time-consuming, but the MWT system allowed rapid quantification of behavior with minimal human effort. Thus, this system will enable large-scale forward and reverse genetic screens for complex behaviors.

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.

Dynamic and persistent effects of ethanol exposure on development: an in vivo analysis during and after embryonic ethanol exposure in Caenorhabditis elegans.

BACKGROUND: Defects caused by ethanol (EtOH) exposure during development can be different depending on the time of observation. To investigate this temporal component of developmental delay, we use the fast-developing nematode model Caenorhabditis elegans. We first defined the longitudinal effects of EtOH on development using age-appropriate markers and then closely followed embryonic development before, during, and after EtOH exposure. METHODS: C. elegans embryos were bathed in 0 to 20% EtOH (w/w in ddH(2)O) for 8 hours or were left untreated during embryonic development. Development was followed longitudinally and scored as embryonic stage at the end of the exposure, hatch time, hatching probability (mortality), body length, postembryonic stage, and egg-laying pattern. The rate of in vivo embryonic development was observed hourly for 24 hours covering times before, during, and after EtOH exposure. RESULTS: After exposure to 10% EtOH, embryos were at younger embryonic stages, hatched later, and had higher mortality compared to unexposed controls. Embryos exposed to 5% EtOH were at normal embryonic stages, showed no change in mortality, but hatched later than controls. Both EtOH groups showed shorter mean body lengths and slower postembryonic development; however, the 5% group recovered to control levels faster than the 10% group. The pattern of egg laying was delayed in the 10% group, but not in the 5% group. Hourly in vivo observations revealed that a developmental delay was first visible a few hours into 10% EtOH exposure and that the delay increased after the removal of EtOH exposure. CONCLUSIONS: Developmental delays occurred during and immediately after exposure and were not uniform but rather dynamic. This article highlights the importance of investigating EtOH-induced defects using different markers and at multiple time points. Attention to temporal effects during and immediately after EtOH exposure can provide understanding of these sensitive time points for observation and treatment.

Rearing C. elegans on Parafilm-wrapped NGM Plates Impacts Habituation Behavior.

Scientists use Parafilm to seal Caenorhabditis elegans cultures on Nematode Growth Media (NGM) petri plates for short-term storage to reduce the likelihood of contamination and improve moisture retention. However, we found that maintaining worms on plates wrapped with Parafilm can affect multiple behavioral metrics when assaying tap-habituation behavior using the Multi-Worm Tracker (MWT). Most notably, worms cultured on parafilm-wrapped NGM plates exhibited slower speed of initial response to tap followed by marked sensitization. These findings suggest that labs should be conscious of the possibility that Parafilm may induce behavioral changes in C. elegans when conducting experiments.

wrk-1 and rig-5 control pioneer and follower axon navigation in the ventral nerve cord of Caenorhabditis elegans in a nid-1 mutant background.

During nervous system development, neurons send out axons, which must navigate large distances to reach synaptic targets. Axons grow out sequentially. The early outgrowing axons, pioneers, must integrate information from various guidance cues in their environment to determine the correct direction of outgrowth. Later outgrowing follower axons can at least in part navigate by adhering to pioneer axons. In Caenorhabditis elegans, the right side of the largest longitudinal axon tract, the ventral nerve cord, is pioneered by the AVG axon. How the AVG axon navigates is only partially understood. In this study, we describe the role of two members of the IgCAM family, wrk-1 and rig-5, in AVG axon navigation. While wrk-1 and rig-5 single mutants do not show AVG navigation defects, both mutants have highly penetrant pioneer and follower navigation defects in a nid-1 mutant background. Both mutations increase the fraction of follower axons following the misguided pioneer axon. We found that wrk-1 and rig-5 act in different genetic pathways, suggesting that we identified two pioneer-independent guidance pathways used by follower axons. We assessed general locomotion, mechanosensory responsiveness, and habituation to determine whether axonal navigation defects impact nervous system function. In rig-5 nid-1 double mutants, we found no significant defects in free movement behavior; however, a subpopulation of animals shows minor changes in response duration habituation after mechanosensory stimulation. These results suggest that guidance defects of axons in the motor circuit do not necessarily lead to major movement or behavioral defects but impact more complex behavioral modulation.

Rapid assessment of the temporal function and phenotypic reversibility of neurodevelopmental disorder risk genes in Caenorhabditis elegans.

Recent studies have indicated that some phenotypes caused by decreased function of select neurodevelopmental disorder (NDD) risk genes can be reversed by restoring gene function in adulthood. However, few of the hundreds of risk genes have been assessed for adult phenotypic reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of NDD risk gene orthologs using a conditional protein degradation system and machine-vision phenotypic profiling in Caenorhabditis elegans. We measured how degrading and re-expressing orthologs of EBF3, BRN3A and DYNC1H1 at multiple periods throughout development affect 30 morphological, locomotor, sensory and learning phenotypes. We found that phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of rescue varied by gene and phenotype. This work highlights the critical need to assess multiple windows of degradation and re-expression and a large number of phenotypes to understand the many roles a gene can have across the lifespan. This work also demonstrates the benefits of using a high-throughput model system to prioritize NDD risk genes for re-expression studies in other organisms.