cGMP is a ubiquitous second messenger implicated in a multitude of neurobiological processes, including sensory transduction, learning and memory. FRET-based and GFP-based cGMP reporters have been developed to complement the genetic and biochemical tools used to probe the role of cGMP in these processes. While FRET-based cGMP sensors have been expressed in C. elegans to explore the spatiotemporal regulation of cGMP in sensory neurons in response to stimuli, their use requires a dual emission system, which limits their ability to be coexpressed with other fluorophores, such as red calcium sensors that can indirectly read out neural activity. This work demonstrates that WincG2, a GFP-based cGMP sensor codon-optimized for use in C. elegans, can report changes in cGMP levels in living, behaving C. elegans. We demonstrate that coexpression of WincG2 and light-activatable guanylyl cyclases in body wall muscle cells results in an increase in WincG2 fluorescence upon light exposure that corresponds with the rate of cGMP production. Furthermore, WincG2 fluorescence changes in the cell bodies of the gustatory neuron ASER and the phasmid neuron PHB in response to [NaCl] step changes and sodium dodecyl sulfate, respectively. This provides the first visual evidence that suggests GMP levels change in these neurons in response to stimuli. Intriguingly, preliminary data suggest that in ASER, cGMP levels decrease linearly in the cell body while increasing transiently in the cilia in response to a [NaCl] downstep, which could have implications for cGMP’s potential role in both sensation and memory in ASER. Finally, we demonstrate that cGMP could act as a neuromodulator in a nociceptive neural circuit. WincG2 fluorescence increases in the nociceptive neuron ASH - which is not known to express guanylyl cyclases - in the absence of food while remaining relatively constant in the presence of LB. These results suggest that cGMP could be flowing from other neurons into ASH to signal food status, resulting in the modulation of ASH activity. Taken together, this work demonstrates that WincG2 could be used the uncover cGMP’s role in diverse neurobiological processes in living, behaving C. elegans.

Progranulin is an evolutionarily conserved protein. Insufficient levels have been implicated in frontotemporal lobar degeneration with TDP-43 aggregates. Human progranulin is comprised of multiple cysteine-rich, biologically active granulin peptides. Granulin peptides accumulate with age and stress, however their functional contributions relative to full-length progranulin remain unclear. To address this, we generated C. elegans strains that produce measurable levels of progranulin and granulins. Using these strains, we demonstrate that an increase in granulin to progranulin-1 (PGRN-1) ratio suppresses the activity of the lysosomal aspartyl protease activity. This increased granulin to PGRN-1 ratio also compromises animal fitness as measured by progress through development and stress response. Further, this increased ratio impairs the degradation of human TDP43, corroborating the impaired lysosomal function. In summary, these studies suggest that the balance between full length PGRN-1 and its cleavage products is important in regulating the lysosomal biology. Given their importance in neurodegenerative disease, this suggests that the processing of progranulin into granulins may be a site of intervention in therapeutic development.

Learning and memory are essential for survival—organisms must respond to their environment and update their knowledge accordingly. Memories undergo multiple stages before being stored long term: acquisition, consolidation, and maintenance. Various factors contribute to long-term memory formation, including sleep, synaptic plasticity, and molecular mechanisms. Sleep is thought to drive memory consolidation by promoting synaptic plasticity. However, the exact molecular, cellular, and synaptic mechanisms occurring during sleep to drive memory maintenance are not well known. To achieve single-cell and synaptic resolution of memory in a genetically-amenable organism, I utilized C. elegans and an odor spaced-training long-term memory paradigm. I show that sleep post training is required to consolidate the odor memory. I found that one specific interneuron (AIY) is required for long-term odor memory maintenance, and that synapses between that interneuron and one olfactory sensory neuron (AWC) are decreased after the long-term memory is formed, only in animals that slept. In addition, I found that the human transient receptor potential vanilloid (TRPV) channel osm-9, an ortholog of human TRPV5 and TRPV6, is required specifically for memory consolidation in order to establish this long-term olfactory memory, independent of sleep. Understanding the mechanisms underlying how learning and memory are mediated is important to understand how organisms learn from their environment to ensure survival, as well as what happens when these processes go awry, such as in the disease context. Expanded CGG repeat RNAs from the fragile X mental retardation 1 (FMR1) gene are present in the brain of patients with fragile X and fragile-X tremor/ataxia syndrome (FXTAS). We show that when human FMR1 with these expanded RNA repeats is expressed in C. elegans olfactory sensory neurons, that it blocks short-term odor memory. Behavioral plasticity perturbations are also observed in FXTAS patients, such as altered prepulse inhibition. We find that this aberrant plasticity requires the miRNA-Argonaute pathway. Thus, these studies may help to understand the molecular and cellular mechanisms and dynamics of how neural plasticity drives learning and memory, and what happens molecularly in diseases that limit plasticity.

Early mammalian development is driven by precise molecular changes that enable cells to establish, maintain, and exit from the pluripotent state, which is characterized by ability to generate all of the cells that comprise the adult mammal. Embryonic stem cells (ESCs) can be used to model the developmental progression through which pluripotent cells become differentiated somatic cells, and remarkably, ESC-like cells called induced pluripotent stem cells (iPSCs) can be generated from somatic cells like fibroblasts through a process called reprogramming or de-differentiation. Differentiation and de-differentiation are both highly regulated by post-transcriptional mechanisms mediated in part by non-coding RNAs and RNA binding proteins (RBPs). Here, we investigated the roles of microRNAs (miRNAs) and RBPs in the regulation of pluripotency.

Using a fluorescent reporter system, we found that the miR-290 and miR-302 miRNA families are expressed in that order during embryonic development and ESC differentiation but that the loci are transcriptionally activated in a stochastic manner during reprogramming. Addition of the reprogramming enhancer Sall4, however, promoted the transcription of the miR-302 locus before that of miR-290, suggesting that reprogramming does not necessarily represent development in reverse and that the sequence of regulatory changes that occur during the process depends on the specific cocktail of reprogramming factors used. Surprisingly, we found that, despite their expression in pluripotent cells, miR-290 and miR-302 are also both dispensable for the generation of iPSCs.

In a separate set of studies investigating the role of RBPs in pluripotency, we performed an RNAi screen in an ESC differentiation system that allowed us to uncover NF45 and NF90/NF110 as factors important for maintaining pluripotency. Our molecular, cellular, and genomic analyses of NF45 and NF90/NF110 knockdown and knockout ESCs suggested that NF45 and NF90/NF110 regulate the expression of one another and function both independently and in complexes to promote ESC proliferation and proper differentiation.

The studies described here demonstrate the importance and complexity of miRNA and RBP control over the pluripotent state. Developing a more nuanced understanding of these post-transcriptional regulatory processes will enable the more effective and informed use of pluripotent cells to address clinical problems in the future.

Many mechanisms govern both how animals respond to stimuli and how these responses inform physical state and future behavior. In C. elegans, these mechanisms include the generation and spread of various species of small RNAs. Emerging as a potent regulator of gene expression in both a single animal and its progeny, small RNAs are reshaping the fields of behavior and transgenerational inheritance. The rising prominence of small RNA within the context of these fields highlights gaps in the knowledge of essential processes, such as the precise mechanisms of small RNA export. RNA import, thanks to earlier efforts characterizing systemic RNA interference (RNAi), are relatively well understood. However, much less is known about how RNAi and other small RNA exit cells. This manuscript details tools and approaches for identifying mobile small RNAs, members of RNA export pathways, and points of regulation in the export process.