Bazopoulou, Daphne et al. (2009) International Worm Meeting "PQN-21, a prion-like protein is involved in learning and memory in C. elegans."

Tavernarakis, Nektarios, Bazopoulou, Daphne

[International Worm Meeting, 2009]

We are characterizing PQN-21, a C. elegans protein bearing a Q/N rich-''prion''domain. Prions are infectious agents implicated in a variety of neurodegenerative diseases in mammals, generally referred to as transmissible spongiform encephalopathies. The glutamine/asparagine (Q/N)-rich domain of prions appears to be responsible for their unusual capacity to fold into structurally and functionally distinct conformations, one of which is self-perpetuating. Usually the self-perpetuating-''prion'' form is organized in self-seeding polymers and can induce other proteins with similar sequences to acquire the ''prion'' state, creating a chain reaction. Prion-like proteins are widespread (found in mammals, fungi, yeast), conserved and serve diverse functions. In Aplysia, the prion-like neuronal protein CPEB functions as a positive regulator of mRNA translation in stimulated synapses and helps to maintain long-term synaptic changes associated with memory acquisition and storage. We find that pqn-21 is expressed in C. elegans neuronal and glial cells and that PQN-21 localizes in the nucleus. Deletion of the pqn-21 gene results in defective gonadal outgrowth and egg-laying. Interestingly, while PQN-21-depleted animals display normal chemotaxis to several soluble and volatile compounds, they show strongly impaired conditioning to chemicals, indicating a shortfall in associative learning and memory, as well as impaired olfactory adaptation. In addition, we find that mutants with defective glial cells show a similar phenotype. Relative to this, we showed that expression of PQN-21 in glial cells is necessary for olfactory adaptation. Based on these observations, we hypothesize that prion- like PQN-21 protein functions in glial cells to facilitate learning and memory. Given the conserved function of prion-like proteins among diverse species, C. elegans offers an attractive and versatile platform in which to dissect the relevant mechanisms. To this end, we are characterizing the function of PQN-21 and the involvement of glial cells in various paradigms of learning and memory in C. elegans.

Bazopoulou, Daphne et al. (2011) International Worm Meeting "The impact of longevity controlling genes and stress-inducing environmental factors in the functionality of the ASH chemosensory neuron in aging worms."

Chokshi, Trushal V, Bazopoulou, Daphne, Chronis, Nikos

[International Worm Meeting, 2011]

Understanding the physiology of aged neurons is a particularly challenging task, as aging studies need to be performed in a well-controllable environment and they require a large number of data in order to obtain statistically significantly trends. To address those issues, we have previously demonstrated the use of a microfluidic-based platform for calcium imaging of stimulus-evoked neural responses from single worms in an automated fashion. Extending that work, we performed ASH calcium imaging experiments on long-lived mutants in order to determine the impact of major aging-controlling pathways in the functionality of the nervous system. We found that the daf-2(e1370) mutation, which has already been implicated in lifespan extension and increased stress resistance, has a major effect in the aging of ASH neuron, by preserving the young-like functional characteristics in older ages. In addition, we quantified the effect of stressful conditions, such as heat shock, osmotic stress and hypoxia, in the functionality of the ASH neuron, during aging. Interestingly, a single treatment including a mild heat-stress early in adulthood was able to significantly increase neuronal responses in older animals. We believe that these enhanced neuronal performances are attributed to the hormetic effects of stressors that increase the resistance, improve the survival and cellular function in low doses but have inhibitory or adverse effects at high doses. Finally, we present data from a small scale compound screen that includes FDA approved drugs and dietary supplements with proposed neuroprotective function and compounds that have been reported to have beneficial effects in longevity.

Daphne Bazopoulou et al. (2007) International Worm Meeting "Characterization of PQN-21, a prion-like protein involved in learning and memory in C. elegans."

Nektarios Tavernarakis, Daphne Bazopoulou

[International Worm Meeting, 2007]

We are characterizing PQN-21, a C. elegans protein bearing a Q/N rich-‘priondomain. Prions are infectious agents implicated in a variety of neurodegenerative diseases in mammals, generally referred to as transmissible spongiform encephalopathies. The glutamine/asparagine (Q/N)-rich domain of prions appears to be responsible for their unusual capacity to fold into structurally and functionally distinct conformations, one of which is self-perpetuating. Usually the self-perpetuating-‘prion form is organized in self-seeding polymers and can induce other proteins with similar sequences to acquire the ‘prion state, creating a chain reaction. Prion-like proteins are widespread (found in mammals, fungi, yeast), conserved and serve numerous diverse functions. In Aplysia, the prion-like neuronal protein CEPB functions as a positive regulator of mRNA translation in stimulated synapses and helps to maintain long-term synaptic changes associated with memory acquisition and storage. We find that pqn-21 is expressed in C. elegans neuronal and glial cells and that PQN-21 localizes in the nucleus. Deletion of the pqn-21 gene results in defective gonadal outgrowth and egg laying. Interestingly, while PQN-21-depleted animals dispaly normal chemotaxis to several soluble and volatile compounds, they show strongly impaired conditioning to chemicals, indicating a shortfall in associative learning and memory. Based on this observation, we hypothesize that prion- like proteins roles in associative learning and memory are conserved from nematodes to mammals, thus, making C. elegans an attractive model in which to dissect the relevant mechanisms To this end, we are characterizing the function of PQN-21 and its involvement in additional phenomena of learning and memory in C. elegans.

Daphne Bazopoulou et al. (2008) European Worm Meeting "Characterization of PQN-21, a prion-like protein involved in learning and memory in C. elegans"

Daphne Bazopoulou, Nektarios Tavernarakis

[European Worm Meeting, 2008]

We are characterizing PQN-21, a C. elegans protein bearing a Q/N rich-. ''prion''domain. Prions are infectious agents implicated in a variety of. neurodegenerative diseases in mammals, generally referred to as. transmissible spongiform encephalopathies. The glutamine/asparagine (Q/N)-. rich domain of prions appears to be responsible for their unusual capacity. to fold into structurally and functionally distinct conformations, one of. which is self-perpetuating. Usually the self-perpetuating-''prion'' form is. organized in self-seeding polymers and can induce other proteins with. similar sequences to acquire the ''prion'' state, creating a chain reaction.. Prion-like proteins are widespread (found in mammals, fungi, yeast),. conserved and serve numerous diverse functions. In Aplysia, the prion-like. neuronal protein CEPB functions as a positive regulator of mRNA translation. in stimulated synapses and helps to maintain long-term synaptic changes. associated with memory acquisition and storage. We find that pqn-21 is. expressed in C. elegans neuronal and glial cells and that PQN-21 localizes. in the nucleus. Deletion of the pqn-21 gene results in defective gonadal. outgrowth and egg-laying. Interestingly, while PQN-21-depleted animals. display normal chemotaxis to several soluble and volatile compounds, they. show strongly impaired conditioning to chemicals, indicating a shortfall in. associative learning and memory. In addition, we find that mutants with. defective glial cells show a similar phenotype. Based on this observation,. we hypothesize that prion- like proteins function in glial and other cells. to facilitate associative learning and memory. Given the conserved function. of prion-like proteins among diverse species, C. elegans offers an. attractive and versatile platform in which to dissect the relevant. mechanisms. To this end, we are characterizing the function of PQN-21 and. the involvement of glial cells in various paradigms of learning and memory. in C. elegans.

Hale, Laura et al. (2015) International Worm Meeting "Developmental changes in composition of chemotaxis circuits may underlie behavioral maturation."

Chalasani, Sreekanth, Chronis, Nikos, Bazopoulou, Daphne, Lee, Eudoria, Hale, Laura

[International Worm Meeting, 2015]

How does the nervous sytem change during development to enable mature behavior? We use the development of chemotaxis behavior as model to study maturation and have shown that L3 larvae respond worse than adults in attractive diacetyl chemotaxis assays. However, L3 larvae are not generally bad at chemotaxis, since they respond similarly to adults in repulsive nonanone chemotaxis assays. To begin to identify neural mechanisms underlying maturation, we mapped both the adult and larval sensory circuits required for attractive diacetyl chemotaxis. Based on behavioral analysis of sensory neuron mutants, we found that the adult sensory circuit contains more neurons than the L3 circuit, suggesting that changing circuit composition may play a crucial role in behavioral maturation. To further characterize differences between the adult and L3 cellular circuits we examined the activity of individual sensory neurons from each circuit. While a microfluidic device for recording neuronal activity from adult worms is published, none had existed for L3 worms. In collaboration with N. Chronis and D. Bazopoulou we developed a novel microfluidic device that traps L3 animals and enables ready imaging of individual neurons. We previously reported that in both adult and L3 worms AWA sensory neurons respond to the addition of diacetyl. Interestingly, our current results show that L3 AWA responses are less reliable than adult responses at a lower diacetyl concentration. We also find, in support of our behavioral results, that different sets of sensory neurons encode diacetyl in adult and L3 animals. Moreover, the functional adult circuit is larger than the L3 one. Collectively, our results show that for adult and L3 worms different cellular circuits are required for attractive chemotaxis behavior, different sensory neurons encode diacetyl, and odors are encoded by cells which are dispensable for behavior. We will present these findings and our results analyzing differences in molecular pathways between L3 and adults.

Oleson, Bryndon et al. (2021) International Worm Meeting "H3K4me3 modifiers regulate amyloid toxicity in C. elegans"

Jakob, Ursula, Bazopoulou, Daphne, Jiang, Emily, Bhattrai, Janak, Oleson, Bryndon

[International Worm Meeting, 2021]

Neurodegenerative diseases such as Alzheimer's disease (AD) and Huntington's disease (HD) are characterized by the pathological deposition of amyloidogenic proteins which results in neurotoxicity and neurodegeneration. Aging is the greatest risk factor for these diseases, and as a result, interventions that delay aging also slow the progression of these age-associated diseases. As the precipitating events in AD and HD are thought to begin well before the onset of clinical symptoms, identifying processes early in life may be key to significantly alter the trajectory of these diseases. Our lab has recently discovered one such early-life event: naturally-occurring fluctuations in levels of reactive oxygen species (ROS) that are predictive of lifespan and stress resistance. These effects, identified in C. elegans, were found to be mediated by redox-dependent changes to the levels of trimethylated H3K4 (H3K4me3), an epigenetic mark associated with gene activation. These data demonstrate that transient changes in the levels of H3K4me3 during development can exert effects on organismal health and longevity that persist into adulthood. On this basis, we hypothesize that changes to H3K4me3 will also influence susceptibility to amyloid toxicity seen in AD and HD. In this study, we find that reduction of H3K4me3 levels by knockdown of set-2 and ash-2 (components of the H3K4me3 complex) in a C. elegans model strain of AD expressing amyloid-beta (Abeta) reduced Abeta-induced paralysis. A similar delay in paralysis was observed following transient exposure of Abeta-expressing animals to mild concentrations of the ROS-generator paraquat during development. Importantly, these protective effects of developmental ROS are lost in animals that lack set-2. In addition to models of AD, C. elegans strains expressing amyloidogenic polyglutamine repeats (Q40) characteristic of HD were also protected from paralysis by ash-2 or set-2 knockdown, indicating that disruption of H3K4me3 levels can protect against proteotoxicity from multiple types of amyloidogenic proteins. Despite having a resistance to paralysis, polyglutamine-expressing animals with lower levels of H3K4me3 appear to have an increase in aggregation of the Q40 protein in both young and aged animals. We hypothesize that disruption of H3K4me3 may heighten the capacity of these animals to mount transcriptional responses to proteotoxic stress, and we are currently investigating the mechanisms that may confer this stress resistance.

Gourgou*, Eleni et al. (2015) International Worm Meeting "Real-time behavioral study of C. elegans by dynamic in situ photopatterning of hydrogel assays."

Bazopoulou, Daphne, Chronis, Nikos, Gourgou*, Eleni, Oliver*, C. Ryan, Hart, A. John

[International Worm Meeting, 2015]

*Equal ContributionsThe use of C. elegans in neuroscience has flourished due to advances in microfluidics. However, traditional microfluidic assays require time-consuming design and fabrication and have limited flexibility during operation. We present a platform for dynamically building microfluidic assays by in situ photopatterning of a bio-compatible hydrogel on NGM plates. This method adds flexibility to the workflow, enabling the researcher to incorporate new features in the assay based on observations as the experiment proceeds. To validate the technique, we first study whether in situ photofabrication of micropillars around swimming C. elegans would influence the worms' velocity and found that going from the open frame, to the micropillar array, to the rippled microchannel, the worm's maximum speed increases 270%. Our method eliminates the need to load worms into a device one by one and sort them into individual chambers, as the chamber can be fabricated in situ around each worm. Next, we fabricate a free-floating lever around a pin anchored to the NGM plate. Worms confined within a frame surrounding this mechanism actively interact with the lever. A variety of mechanisms can be fabricated within the culture environment (one-way gates, floating microparticles, movable isolation chambers, gears) as means to investigate more complicated behaviors. In addition, the researcher can generate freeform input using a tablet, resulting in real-time modification of the assay. Using custom-built LabVIEW, the time between when the pen touches the pad and final PEG-DA photopolymerization is less than 2sec. Last, we demonstrate how worms can be tracked in custom-made mazes to determine their ability to locate food. In the absence of food, the worms choose the left or right ends with equal probability, whereas, when food is placed in one end of the maze, worms choose the leg containing the food almost twice as often. Our photopatterning technique enables rapid and flexible experimentation via micro-scale confinement of model organisms, and in the future could incorporate image analysis and machine learning techniques to acquire large datasets and accelerate breakthroughs in understanding the behavior of model organisms such as C. elegans.

Dafne Bazopoulou et al. (2006) European Worm Meeting "Characterization of PQN-21, A Prion-Like Protein Involved in Learning and Memory in C. elegans"

Nektarios Tavernarakis, Dafne Bazopoulou

[European Worm Meeting, 2006]

Dafne Bazopoulou and Nektarios Tavernarakis. Prions are infectious agents implicated in a variety of neurodegenerative diseases in mammals, generally referred to as transmissible spongiform encephalopathies. Prions contain a glutamine/asparagine (Q/N)-rich domain which appears to be responsible for the unusual capacity of these proteins to fold into structurally and functionally distinct conformations, one of which is self-perpetuating. Usually the self-perpetuating-prion form is organized in self-seeding polymers and can induce other proteins with similar sequences to acquire the prion state, creating a chain reaction. Prion-like proteins are widespread (found in mammals, fungi, yeast), conserved and serve numerous diverse functions. In Aplysia, the prion-like neuronal protein CEPB functions as a positive regulator of mRNA translation in stimulated synapses and helps to maintain long-term synaptic changes associated with memory acquisition and storage.. PQN-21 is a C. elegans protein bearing a Q/N rich-priondomain. It also contains a zinc-finger domain, which is common among DNA binding proteins. We find that the pqn-21 gene is expressed in neuronal and epithelial cells and the PQN-21 protein localizes in the nucleus. Deletion of the pqn-21 gene results in defective gonadal and axonal outgrowth. Interestingly, while PQN-21-depleted animals are normal for chemotaxis to several soluble and volatile compounds, they show strongly impaired conditioning to chemicals, indicating a shortfall in associative learning and memory. Based on this observation, we hypothesize that prion roles in associative learning and memory are conserved from nematodes to mammals, thus, making C. elegans an attractive model in which to dissect the relevant mechanisms. To this end, we are characterizing the function of PQN-21 and its involvement in additional phenomena of learning and memory in C. elegans.

Dafne Bazopoulou et al. (2006) European Worm Meeting "Towards the Development of a Minos-based Transposon Tool in C. elegans"

Dafne Bazopoulou, Nektarios Tavernarakis

[European Worm Meeting, 2006]

Dafne Bazopoulou and Nektarios Tavernarakis. We aim to develop a heterologous transposon system in C. elegans, based on the well-characterized Minos transposable element from Drosophila hydei, which has already been mobilized in several distinct species. The major advantage of Minos resides in its capacity to transpose while carrying long fragments of exogenous DNA. Therefore, Minos can be used as a vehicle to introduce foreign sequences in the genome. This feature can be used to establish enhancer-trap systems which would permit among other applications, genome-wide screens for gene regulatory regions in C. elegans. Minos could also be used for genome-wide insertional mutagenesis, as an alternative to the already established Mos trasnposon system.. We are developing a binary system for Minos transposition in the worm, similar to the one in use for the Mos element. We have generated separate lines expressing the transposase under the heat-inducible promoter and also lines carrying the Minos transposon on different extrachromosomal arrays. Furthermore, in order to test the capacity of Minos to carry exogenous DNA, we have generated lines harboring a modified Minos element, comprised of Minos ITRs along with an insert of ~2kb, encompassing a GFP reporter expressed in body wall muscles, driven by the unc-54 promoter. We obtained double-transgenic lines by crossing animals carrying the transposase source array with animals carrying either the native Minos element or the one engineered to harbor the GFP reporter gene. We confirmed proper expression of the transposase in these lines after heat shock by RT-PCR. While, we have observed mobilization of Minos in somatic cells of C. elegans, we have yet to achieve germline transposition. We are currently in the process of optimizing various parameters of our procedure in an effort to detect transposition of Minos in the germline. We are also developing alternative methods for screening such, likely rare transposition events, based on fluorescent marker expression.