Methylglyoxal-derived hydroimidazolone, MG-H1, increases food intake by altering tyramine signaling via the GATA transcription factor ELT-3 in Caenorhabditis elegans.

The Maillard reaction, a chemical reaction between amino acids and sugars, is exploited to produce flavorful food ubiquitously, from the baking industry to our everyday lives. However, the Maillard reaction also occurs in all cells, from prokaryotes to eukaryotes, forming advanced glycation end-products (AGEs). AGEs are a heterogeneous group of compounds resulting from the irreversible reaction between biomolecules and α-dicarbonyls (α-DCs), including methylglyoxal (MGO), an unavoidable byproduct of anaerobic glycolysis and lipid peroxidation. We previously demonstrated that Caenorhabditis elegans mutants lacking the glod-4 glyoxalase enzyme displayed enhanced accumulation of α-DCs, reduced lifespan, increased neuronal damage, and touch hypersensitivity. Here, we demonstrate that glod-4 mutation increased food intake and identify that MGO-derived hydroimidazolone, MG-H1, is a mediator of the observed increase in food intake. RNAseq analysis in glod-4 knockdown worms identified upregulation of several neurotransmitters and feeding genes. Suppressor screening of the overfeeding phenotype identified the tdc-1-tyramine-tyra-2/ser-2 signaling as an essential pathway mediating AGE (MG-H1)-induced feeding in glod-4 mutants. We also identified the elt-3 GATA transcription factor as an essential upstream regulator for increased feeding upon accumulation of AGEs by partially controlling the expression of tdc-1 gene. Furthermore, the lack of either tdc-1 or tyra-2/ser-2 receptors suppresses the reduced lifespan and rescues neuronal damage observed in glod-4 mutants. Thus, in C. elegans, we identified an elt-3 regulated tyramine-dependent pathway mediating the toxic effects of MG-H1 AGE. Understanding this signaling pathway may help understand hedonistic overfeeding behavior observed due to modern AGE-rich diets.

Methods to Quantify and Relate Axonal Transport Defects to Changes in C. elegans Behavior.

Neuronal growth, differentiation, homeostasis, viability, and injury response heavily rely on functional axonal transport (AT). Erroneous and disturbed AT may lead to accumulation of "disease proteins" such as tau, α-synuclein, or amyloid precursor protein causing various neurological disorders. Changes in AT often lead to observable behavioral consequences in C. elegans such as impeded movements, defects in touch response, chemosensation, and even egg laying. Long C. elegans neurons with clear distinguishable axons and dendrites provide an excellent platform to analyze AT. The possibility to relate changes in AT to neuronal defects that in turn lead to quantifiable changes in worm behavior allows for the advancement of neuropathological disease models. Even more, subsequent suppressor screens may aid in identifying genes responsible for observed behavioral changes providing a target for drug development to eventually delay or cure neurological diseases. Thus, in this chapter, we summarize critical methods to identify and quantify defects in axonal transport as well as exemplified behavioral assays that may relate to these defects.

Loss of intermediate filament IFB-1 reduces mobility, density, and physiological function of mitochondria in Caenorhabditis elegans sensory neurons.

Mitochondria and intermediate filament (IF) accumulations often occur during imbalanced axonal transport leading to various types of neurological diseases. It is still poorly understood whether a link between neuronal IFs and mitochondrial mobility exist. In Caenorhabditis elegans, among the 11 cytoplasmic IF family proteins, IFB-1 is of particular interest as it is expressed in a subset of sensory neurons. Depletion of IFB-1 leads to mild dye-filling and significant chemotaxis defects as well as reduced life span. Sensory neuron development is affected and mitochondrial transport is slowed down leading to reduced densities of these organelles. Mitochondria tend to cluster in neurons of IFB-1 mutants likely independent of the fission and fusion machinery. Oxygen consumption and mitochondrial membrane potential is measurably reduced in worms carrying mutations in the ifb-1 gene. Membrane potential also seems to play a role in transport such as carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone treatment led to increased directional switching of mitochondria. Mitochondria co-localize with IFB-1 in worm neurons and appear in a complex with IFB-1 in pull-down assays. In summary, we propose a model in which neuronal IFs may serve as critical (transient) anchor points for mitochondria during their long-range transport in neurons for steady and balanced transport.

Characterization of TAG-63 and its role on axonal transport in C. elegans.

Model organisms are increasingly used to study and understand how neurofilament (NF)-based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG-63 as a NF-like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG-63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti-NEFH antibody detects a band at the predicted molecular weight of TAG-63 in Western blots of whole worm lysates and this band cannot be detected in tag-63 knockout worms. A transcriptional tag-63 reporter expresses in a broad range of neurons, and various anti-NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC-104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine-labeled in vitro reconstituted TAG-63 filaments disintegrate upon drug exposure. Speeds of UNC-104 motors are diminished in tag-63 mutant worms with visibly increased accumulations of motors along axons. UNC-104/TAG-63 and SNB-1/TAG-63 not only colocalize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals. In summary, we identified and characterized TAG-63 in C. elegans, and demonstrate that lack of this protein limits axonal transport efficiencies. Additionally, this study would aid in developing NF-related disease models in the future.

Insights on UNC-104-dynein/dynactin interactions and their implications on axonal transport in Caenorhabditis elegans.

Bidirectional cargo transport in neurons can be explained by two models: the "tug-of-war model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons.

Toxic Effects of Size-tunable Gold Nanoparticles on Caenorhabditis elegans Development and Gene Regulation.

We utilized size-tunable gold nanoparticles (Au NPs) to investigate the toxicogenomic responses of the model organism Caenorhabditis elegans. We demonstrated that the nematode C. elegans can uptake Au NPs coated with or without 11-mercaptoundecanoic acid (MUA), and Au NPs are detectable in worm intestines using X-ray microscopy and confocal optical microscopy. After Au NP exposure, C. elegans neurons grew shorter axons, which may have been related to the impeded worm locomotion behavior detected. Furthermore, we determined that MUA to Au ratios of 0.5, 1 and 3 reduced the worm population by more than 50% within 72 hours. In addition, these MUA to Au ratios reduced the worm body size, thrashing frequency (worm mobility) and brood size. MTT assays were employed to analyze the viability of cultured C. elegans primary neurons exposed to MUA-Au NPs. Increasing the MUA to Au ratios increasingly reduced neuronal survival. To understand how developmental changes (after MUA-Au NP treatment) are related to changes in gene expression, we employed DNA microarray assays and identified changes in gene expression (e.g., clec-174 (involved in cellular defense), cut-3 and fil-1 (both involved in body morphogenesis), dpy-14 (expressed in embryonic neurons), and mtl-1 (functions in metal detoxification and homeostasis)).

Cilium Length and Intraflagellar Transport Regulation by Kinases PKG-1 and GCK-2 in Caenorhabditis elegans Sensory Neurons.

To understand how ciliopathies such as polycystic kidney disease or Bardet-Biedl syndrome develop, we need to understand the basic molecular mechanisms underlying cilium development. Cilium growth depends on the presence of functional intraflagellar transport (IFT) machinery, and we hypothesized that various kinases and phosphatases might be involved in this regulatory process. A candidate screen revealed two kinases, PKG-1 (a cGMP-dependent protein kinase) and GCK-2 (a mitogen-activated protein kinase kinase kinase kinase 3 [MAP4K3] kinase involved in mTOR signaling), significantly affecting dye filling, chemotaxis, cilium morphology, and IFT component distribution. PKG-1 and GCK-2 show similar expression patterns in Caenorhabditis elegans cilia and colocalize with investigated IFT machinery components. In pkg-1 mutants, a high level of accumulation of kinesin-2 OSM-3 in distal segments was observed in conjunction with an overall reduction of anterograde and retrograde IFT particle A transport, likely as a function of reduced tubulin acetylation. In contrast, in gck-2 mutants, both kinesin-2 motility and IFT particle A motility were significantly elevated in the middle segments, in conjunction with increased tubulin acetylation, possibly the cause of longer cilium growth. Observed effects in mutants can be also seen in manipulating upstream and downstream effectors of the respective cGMP and mTOR pathways. Importantly, transmission electron microscopy (TEM) analysis revealed no structural changes in cilia of pkg-1 and gck-2 mutants.

Microfluidic Devices in Advanced Caenorhabditis elegans Research.

The study of model organisms is very important in view of their potential for application to human therapeutic uses. One such model organism is the nematode worm, Caenorhabditis elegans. As a nematode, C. elegans have ~65% similarity with human disease genes and, therefore, studies on C. elegans can be translated to human, as well as, C. elegans can be used in the study of different types of parasitic worms that infect other living organisms. In the past decade, many efforts have been undertaken to establish interdisciplinary research collaborations between biologists, physicists and engineers in order to develop microfluidic devices to study the biology of C. elegans. Microfluidic devices with the power to manipulate and detect bio-samples, regents or biomolecules in micro-scale environments can well fulfill the requirement to handle worms under proper laboratory conditions, thereby significantly increasing research productivity and knowledge. The recent development of different kinds of microfluidic devices with ultra-high throughput platforms has enabled researchers to carry out worm population studies. Microfluidic devices primarily comprises of chambers, channels and valves, wherein worms can be cultured, immobilized, imaged, etc. Microfluidic devices have been adapted to study various worm behaviors, including that deepen our understanding of neuromuscular connectivity and functions. This review will provide a clear account of the vital involvement of microfluidic devices in worm biology.

Identification and Characterization of LIN-2(CASK) as a Regulator of Kinesin-3 UNC-104(KIF1A) Motility and Clustering in Neurons.

Kinesin-3 UNC-104(KIF1A) is the major axonal transporter of synaptic vesicles. Employing yeast two-hybrid and co-immunoprecipitation (Co-IP) assays, we characterized a LIN-2(CASK) binding site overlapping with that of reported UNC-104 activator protein SYD-2(Liprin-α) on the motor's stalk domain. We identified the L27 and GUK domains of LIN-2 to be the most critical interaction domains for UNC-104. Further, we demonstrated that the L27 domain interacts with the sterile alpha motifs (SAM) domains of SYD-2, while the GUK domain is able to interact with both the coiled coils and SAM domains of SYD-2. LIN-2 and SYD-2 colocalize in Caenorhabditis elegans neurons and display interactions in bimolecular fluorescence complementation (BiFC) assays. UNC-104 motor motility and Synaptobrevin-1 (SNB-1) cargo transport are largely diminished in neurons of LIN-2 knockout worms, which cannot be compensated by overexpressing SYD-2. The absence of the motor-activating function of LIN-2 results in increased motor clustering along axons, thus retaining SNB-1 cargo in cell bodies. LIN-2 and SYD-2 both positively affect the velocity of UNC-104, however, only LIN-2 is able to efficiently elevate the motor's run lengths. From our study, we conclude that LIN-2 and SYD-2 act in a functional complex to regulate the motor with LIN-2 being the more prominent activator.