Alic N (2016) Cell Metab "Of FOXes and Forgetful Worms."

Alic N

[Cell Metab, 2016]

Age-related cognitive decline is one of the most haunting aspects of human aging. In a recent publication, Coleen Murphy and colleagues (Kaletsky et al., 2016) describe the transcriptional program that maintains youthful function of aging neurons in the nematode worm.

Sohrabi, Salman et al. (2021) International Worm Meeting "CeSnAP: Machine-learning based snapshot analysis platform toward high-throughput Caenorhabditis elegans behavioral screen of Parkinson's disease"

Keyes, William, Murphy, Coleen, Sohrabi, Salman, Kaletsky, Rachel, Mor, Danielle

[International Worm Meeting, 2021]

Parkinson's disease (PD) is a movement disorder and the mechanisms that induce neurodegeneration in PD are still poorly understood. We recently found that RNAi-mediated knockdown of neuronal branched-chain amino acid transferase 1 (BCAT-1) in nematode C. elegans cause age-dependent spasm-like 'curling' phenotype mirroring PD clinical symptoms. Manual quantification of curling is labor-intensive making large-scale drug and genetic screens challenging. Here, we report the development of a machine learning-based automated workflow for C. elegans image analysis, and its application to the discovery of potential compounds that may be repurposed as late-in-life interventions for PD. This high-throughput workflow is 40X faster than the manual assay and constitutes a major advance in the efficiency and precision with which Parkinson's-like curling behavior in C. elegans can be quantified. In a screen of 50 FDA-approved drugs, we have identified four drugs (enasidenib, ethosuximide, metformin, and nitisinone) that reduced curling to <50% of vehicle-treated levels. These findings point to the utility of our high-throughput platform for screening for modifiers of the disease phenotypes, including but not limited to chemicals or genetic manipulations that may ameliorate or worsen motor function in the worms.

Moore RS et al. (2021) STAR Protoc "Protocol for transgenerational learned pathogen avoidance behavior assays in Caenorhabditis elegans."

Moore RS, Murphy CT, Kaletsky R

[STAR Protoc, 2021]

Animal experiences, including learned behaviors, can be passed down to several generations of progeny in a phenomenon known as transgenerational epigenetic inheritance. Yet, little is known regarding the molecular mechanisms regulating physiologically relevant transgenerational memories. Here, we present a method for <i>Caenorhabditis elegans</i> in which worms learn to avoid the pathogen <i>Pseudomonas aeruginosa</i> (PA14). Unlike previous protocols, this training paradigm, either using PA14 lawns or through exposure to a PA14 small RNA (P11), induces memory in four generations of progeny. For complete details on the use and execution of this protocol, please refer to Moore etal. (2019) and Kaletsky etal. (2020).

Lakhina, Vanisha et al. (2015) International Worm Meeting "Identification of a new regulator of age-dependent axon regeneration by characterizing the neuronal IIS/FOXO transcriptome from isolated adult C. elegans neurons."

Murphy, Coleen, Ashraf, Jasmine, Arey, Rachel, Lakhina, Vanisha, Williams, April, Kaletsky, Rachel, Landis, Jessica

[International Worm Meeting, 2015]

The ability of axons to regenerate depends on the age of the neuron in all model organisms tested, including humans. Young axons (including CNS axons) regenerate efficiently, whereas old axons either do not regrow at all, or make errors while navigating to their target (Verdu et al., 2000; Wu et al., 2007). Insulin/IGF-1 signaling (IIS) is a critical regulator of the most important biological decisions, from rates of growth, development, and metabolism, to reproduction and longevity. Long-lived daf-2 insulin/IGF1 receptor mutants exhibit enhanced axonal regenerative capacity in older GABAergic motor neurons and PLM mechanosensory neurons (Byrne et al., 2014, Lakhina et al., submitted). This occurs through the activity of the DAF-16/FOXO transcription factor, which regulates regeneration independent of lifespan, and in a neuron-specific manner (Byrne et al., 2014). While the global, whole-worm targets of DAF-16 were identified in C. elegans using whole-worm transcriptional analyses more than a decade ago (Murphy et al., 2003; Lee et al., 2003; McElwee et al., 2003), neuron-specific DAF-16 targets remain elusive. We have selectively isolated adult C. elegans neurons and performed transcriptional profiling. The neuron-enriched set includes synaptic machinery, channels, neurotransmitters, and signaling components, as well as >700 previously uncharacterized genes. Comparison of the embryonic and larval neuronal transcriptomes with this new adult neuronal transcriptome revealed a shift in functional categories from developmental processes to neuronal function and behavior. We next used this approach to directly identify IIS/FOXO targets from isolated daf-2 and daf-16;daf-2 neurons. IIS/FOXO neuron-specific targets are distinct from canonical IIS/FOXO-regulated longevity and metabolism targets. We also selectively sequenced six mechanosensory neurons and identified 63 mechanosensory neuron-specific DAF-16 targets. We discovered that a transcription factor that is upregulated in daf-2 neurons is almost entirely responsible for daf-2's enhanced ability to regenerate PLM mechanosensory axons with age. These studies identify a set of adult neuronal FOXO targets and potentially a new set of candidate age-dependent axonal regeneration factors.

Arey, Rachel et al. (2015) International Worm Meeting "Identification of neuronal targets of Insulin/FOXO signaling reveals novel components required for associative learning and memory formation."

Landis, Jessica, Williams, April, Stein, Geneva, Lakhina, Vanisha, Murphy, Coleen, Kaletsky, Rachel, Arey, Rachel, Kauffman, Amanda

[International Worm Meeting, 2015]

The ability to form associative memories is one of the most important functions of an organism's nervous system. However, the mechanisms underlying learning and associative memory formation have yet to be fully elucidated. Identifying molecules involved in these processes is important not only to gain an understanding of normal brain function, but can also lead to an understanding of disease states such as age-related cognitive decline. To study these processes, we developed two positive olfactory association assays to measure short-term (STAM) and long-term associative memory (LTAM). Both of these behaviors require processes that are conserved in higher organisms; STAM requires translation, while LTAM requires translation and CREB-mediated transcription. We previously found that C. elegans with a mutant form of the insulin/IGF-1 receptor daf-2, which have an extended lifespan, have threefold increased STAM, which requires the downstream FOXO transcription factor DAF-16, and two-fold increased long-term memory. Furthermore, we have characterized these behaviors with age, and find that daf-2 animals have a slower age-related decline in associative learning and STAM. We find that the extension of long-term associative memory in daf-2 mutants is likely due to increased levels and activity of the transcription factor CREB, and thus its downstream transcriptional targets, which we have identified by combining memory training with genome-wide transcriptional analysis. These targets include known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved. To identify molecular targets of DAF-2/DAF-16 that contribute to daf-2's extension of STAM, we utilized a novel technique developed in our lab that allows for transcriptional analysis of isolated neurons from adult worms. Several of these neuronal daf-2 targets, many of which had no previously defined neuronal function, were found to be required for STAM extension. Comparison of neuronal daf-2 target genes, which are potentially required for STAM, and LTAM training-induced genes reveal largely non-overlapping gene sets, suggesting that the molecular components of these two behaviors are distinct, but carry out similar functions. These results have identified novel regulators of learning and memory, and have identified potential mechanisms by which associative learning and memory can be improved and decline in these processes with age can be prevented.

Tepper, Ronald G. et al. (2013) International Worm Meeting "PQM-1: the missing "DAE Factor" and key regulator of DAF-2-mediated development, longevity, and homeostasis."

Tepper, Ronald G., Ashraf, Jasmine, Murphy, Coleen T., Bussemaker, Harmen J., Kaletsky, Rachel, Kleemann, Gunnar

[International Worm Meeting, 2013]

Reduced insulin/IGF-1-like signaling (IIS) extends C. elegans lifespan by upregulating stress response (Class I) and downregulating development (Class II) genes through a mechanism that depends on the conserved transcription factor DAF-16/FOXO. By integrating a genomewide analysis of gene expression responsiveness to DAF-16 with genomewide in vivo binding data for a compendium of transcription factors, we discovered that the transcriptional activator PQM-1 directly controls Class II genes by binding to the DAF-16 associated element (DAE). DAF-16 directly regulates Class I genes only, through the DAF-16 binding element (DBE). Loss of PQM-1 suppresses daf-2 longevity and thermotolerance and further slows development. The nuclear presence of PQM-1 and DAF-16 is controlled by IIS in opposite ways, and, surprisingly, was found to be mutually exclusive. We observe progressive loss of nuclear PQM-1 with age, explaining declining expression of PQM-1 targets. Together, our data suggest an elegant mechanism for balancing stress response and development.

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Rachel McMullan, Emma Hiley, Paul Morrison, Stephen J. Nurrish

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

Power, K. et al. (2019) International Worm Meeting "Determining the function of the kinase NEKL-4 in cilia."

Walsh, J., Barr, M., Bellotti, S., Gu, A., Zhang, W., O'Hagan, R., Power, K., Morash, M.

[International Worm Meeting, 2019]

Primary cilia are microtubule-based sensory organelles that protrude from the surface of most non-dividing cells in the human body. Disorders causing defects in the formation or function of cilia, called ciliopathies, represent major health problems, including polycystic kidney disease (PKD), situs inversus, primary ciliary dyskinesia, and retinal degeneration. C. elegans is a powerful model for human ciliopathies and to understand ciliary specialization. We previously showed that the tubulin deglutamylase CCPP-1 is essential for ciliary integrity, with defects resulting in a progressive dye filling (Dyf) phenotype (O'Hagan 2011). In humans, Ccp1 mutation causes infantile onset neurodegeneration (Shashi 2018). To identify genes that act in concert with CCPP-1 to control microtubule stability, we performed a ccpp-1 Dyf suppressor screen and identified a mutation in the NIMA-Like kinase NEKL-4. "Never-In-Mitosis A" (NIMA or NEK) kinases are important for ciliary length regulation and implicated in ciliopathies such as PKD and Short-Rib-Polydactyly Syndrome. In C. elegans, NEKL-4 is expressed in many ciliated sensory neurons (Kaletsky 2016). nekl-4(my31) and tm4910 single mutants have superficially normal cilia as judged by release of ciliary extracellular vesicles, dye filling, and ciliary glutamylation levels. We are determining the mechanism by which NEKL-4 antagonizes CCPP-1 activity. We find that NEKL-4 does not act via regulation of CCPP-1 ciliary glutamylation levels. We are currently testing the hypotheses that NEKL-4 regulates MT ultrastructure, ciliary length, and/or activity of ciliary motor-based transport. We will determine if NEKL-4 functions in the cilium or in the cytoplasm, and whether NEKL-4 acts as a kinase using CRISPR-generated kinase-active and kinase-dead alleles.

Sabatino, Brendil et al. (2021) International Worm Meeting "Investigation of the transcriptional response to starvation at the tissue level"

Taubert, Stefan, Alhusari, Deema, Sabatino, Brendil

[International Worm Meeting, 2021]

Competition for nutrients is a driving force in evolution. As such, organisms have evolved robust adaptive responses to withstand periods of starvation. In C. elegans, efforts have been made to map the acute starvation response at the transcriptome level using microarray and RNA-seq techniques. However, past studies used whole worms and thus represent an averaged response profile of all of the worms' tissues. Recently, Kaletsky and colleagues developed methods to isolate and study purified C. elegans cell populations from larvae, which allows studying tissue-specific transcriptomes. To gain a better understanding of the response to starvation and potentially reveal unique tissue-specific responses, we are adapting this technique to study how neurons, hypodermis, intestine, and body wall muscles respond to starvation. Specifically, we have used four strains that express GFP in each of these tissues of interest. We then dissociate worms into single cells and FACS-sort GFP-labeled tissues both fed and starved animals, followed by RNA isolation and RNA-sequencing. To date, we have sequenced mRNA from neuronal and body wall muscle tissues from fed animals and animals that were starved for six hours. Preliminary analysis indicates that starvation induces broader changes in neurons compared to body wall muscle. Interestingly, using a liberal significance threshold of P &lt;.05 and FDR &lt; 1, only 11% of genes are regulated by starvation in both tissues, suggesting that tissues respond in a highly specific manner to starvation. In neurons, we observed a positive enrichment for biological processes involved in guanylyl cyclase signaling and cell projection remodeling, whereas negatively enriched processes included protein translation and ribosome assembly. In muscles, we observed positive enrichment of the ER unfolded protein response and cuticle remodeling, and negative enrichment of protein synthesis. Our work demonstrates variation in responses to starvation between tissues. The methods described will allow us to assemble a profile for each tissue, which can be used to develop functional experiments to unravel the biology of the starvation response.

Yilmaz, L. Safak et al. (2019) International Worm Meeting "Tissue-level compartmentalization of metabolic network function."

Walhout, Albertha J.M., Yilmaz, L. Safak, Hang, X., Nanda, S.

[International Worm Meeting, 2019]

A major challenge is to understand how different tissues function together to control metabolism for biomass and energy generation in development and during homeostasis. We previously reconstructed the metabolic network of Caenorhabditis elegans to allow systems-level analysis of metabolic reactions in the animal as a whole (1). Here, we present our first-level analysis of tissue-level compartmentalization of metabolic network function in C. elegans. To reconstruct tissue-level metabolic network models, we integrated our previously generated model (1) with a high-quality, single-cell RNA-seq dataset obtained from L2 animals (2). Briefly, this dataset was able to reconstruct tissue-level gene expression profiles by combining relevant data from individual cells. Using this dataset, we generated tissue-level metabolic network models for seven tissues. To systematically integrate the metabolic network model with the RNA-seq data, we developed and implemented two algorithms. First, we generated an optimal flux distribution that followed highly expressed enzymes, avoided unexpressed enzymes, and had minimal flux through lowly expressed enzymes. Second, we calculated flux efficiency of each network reaction in each tissue by using relative expression levels of enzymes associated with the targeted and nearby reactions. The combination of results from the first (semi-quantitative, network level) and second (quantitative, local) integrations provided tissue-specific potentials of all possible network functions. We confirmed many previously known tissue functions in C. elegans, which validated the overall approach. We also found results that matched with mammalian tissue functions. For instance, our analysis predicts specifically that body wall muscle produces 3-aminoisobutyrate, a metabolite secreted to blood by exercising human muscle tissues. Interestingly, some hypodermis-specific pathways matched liver metabolism in mammals, consistent with the hypothesis that this tissue acts as liver in C. elegans (3). The dataset of predicted flux distributions and efficiencies will provide a resource for the understanding of tissue-relevant metabolism in C. elegans. References: 1. Yilmaz and Walhout, 2016. Cell Systems 2. Cao et al., 2017. Science 3. Kaletsky et al., 2018. PLOS Genetics