Nakano S et al. (2011) Cell "Replication-coupled chromatin assembly generates a neuronal bilateral asymmetry in C. elegans."

Horvitz HR, Nakano S, Stillman B

[Cell, 2011]

Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system.

Nakano S et al. (2010) Development "Otx-dependent expression of proneural bHLH genes establishes a neuronal bilateral asymmetry in C. elegans."

Horvitz HR, Ellis RE, Nakano S

[Development, 2010]

Bilateral asymmetry in Caenorhabditis elegans arises in part from cell lineages that differ on the left and right sides of the animal. The unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that generates the MI neuron on the right and the e3D epithelial cell on the left. We isolated mutations in three genes that caused left-right symmetry in this normally asymmetric cell lineage by transforming MI into an e3D-like cell. These genes encode the proneural bHLH proteins NGN-1 and HLH-2 and the Otx homeodomain protein CEH-36. We identified the precise precursor cells in which ceh-36 and ngn-1 act, and showed that CEH-36 protein is asymmetrically expressed and is present in an MI progenitor cell on the right but not in its bilateral counterpart. This asymmetric CEH-36 expression promotes asymmetric ngn-1 and hlh-2 expression, which in turn induces asymmetric MI neurogenesis. Our results indicate that this left-right asymmetry is specified within the two sister cells that first separate the left and right branches of the cell lineage. We conclude that the components of an evolutionarily conserved Otx/bHLH pathway act sequentially through multiple rounds of cell division on the right to relay an initial apparently cryptic asymmetry to the presumptive post-mitotic MI neuron, thereby creating an anatomical bilateral asymmetry in the C. elegans nervous system.

Nakano, S. et al. (2017) International Worm Meeting "The C. elegans MAST Kinase Acts through Stomatin and Diacylglycerol Kinase to Regulate Thermotaxis Behavior."

Kondo, R., Sano, A., Mori, I., Giles, A., Higashiyama, T., Ikeda, M., Suzuki, T., Nakano, S.

[International Worm Meeting, 2017]

C. elegans navigate themselves toward the past cultivation temperature when placed on a thermal gradient. Previous studies showed that temperature sensation and its memory in AFD thermosensory neuron is essential to achieve this thermotaxis behavior. However, how AFD exploits the temperature information to generate a proper neuronal output remains elusive. Through newly conducting a genetic screen for thermotaxis-defective mutants, we revealed here that two genes, kin-4 and mec-2, play important roles in the AFD function. A null mutation in the gene kin-4, which encodes the C. elegans homolog of MAST (Microtubule Associated Serine Threonine) kinase, caused a cryophilic phenotype, while a gain-of-function mutation in mec-2, which encodes a stomatin-like membrane-associated protein, caused a thermophilic phenotype. We also observed that this mec-2(gf) mutation suppressed the kin-4 null phenotype, suggesting that mec-2 might act downstream of kin-4. Both kin-4 and mec-2 are expressed and function in AFD. Temperature-evoked calcium responses of AFD in kin-4 and mec-2(gf) mutants were grossly normal, suggesting that these genes act downstream of the calcium influx in AFD. To identify the molecular mechanism by which KIN-4 and MEC-2 regulate thermotaxis, we screened for mutants that can suppress the thermophilic phenotype of mec-2(gf) and isolated loss-of-function mutations in dgk-1, which encodes a C. elegans homolog of diacylglycerol (DAG) kinase. We found that like kin-4 and mec-2, dgk-1 acts in AFD to regulate thermotaxis. Our results suggest a novel signal transduction pathway in which KIN-4/MAST kinase and MEC-2/Stomatin might regulate DGK-1. Since DGK-1 has been shown to be important for synaptic transmission by controlling the level of DAG, KIN-4 and MEC-2 may also regulate the DAG level in AFD to coordinate the synaptic transmission. We are currently examining the dynamics of DAG in AFD of wild-type and mutant animals, and are assessing whether KIN-4 and MEC-2 affect the subcellular localization or the activity of DGK-1. Further study will reveal a fundamental role of DAG in integrating the sensory information to neuronal output and uncover a new mechanism of the regulation of DAG, an important lipid second messenger that functions in a diverse biological processes, including synaptic vesicles cycling.

Nakano S et al. (2022) G3 (Bethesda) "Genetic screens identified dual roles of MAST kinase and CREB within a single thermosensory neuron in the regulation of C. elegans thermotaxis behavior"

Nakayama A, Nakano S, Mori I, Tsukada Y, Kuroyanagi H, Yamashiro R

[G3 (Bethesda), 2022]

Animals integrate sensory stimuli presented at the past and present, assess the changes in their surroundings and navigate themselves toward preferred environment. Identifying the neural mechanisms of such sensory integration is pivotal to understand how the nervous system generates perception and behavior. Previous studies on thermotaxis behavior of Caenorhabditis elegans suggested that a single thermosensory neuron AFD plays an important role in integrating the past and present temperature information and is essential for the neural computation that drives the animal toward the preferred temperature region. However, the molecular mechanisms by which AFD executes this neural function remained elusive. Here we report multiple forward genetic screens to identify genes required for thermotaxis. We reveal that kin-4, which encodes the C. elegans homolog of MAST kinase, plays dual roles in thermotaxis and can promote both cryophilic and thermophilic drives. We also uncover that a thermophilic defect of mutants for mec-2, which encodes a C. elegans homolog of stomatin, can be suppressed by a loss-of-function mutation in the gene crh-1, encoding a C. elegans homolog CREB transcription factor. Expression of crh-1 in AFD restored the crh-1-dependent suppression of the mec-2 thermotaxis phenotype, indicating that crh-1 can function in AFD to regulate thermotaxis. Calcium imaging analysis from freely-moving animals suggest that mec-2 and crh-1 regulate the neuronal activity of the AIY interneuron, a post-synaptic partner of the AFD neuron. Our results suggest that a stomatin family protein can control the dynamics of neural circuitry through the CREB-dependent transcriptional regulation within a sensory neuron.

Nakano, S. et al. (2019) International Worm Meeting "Presynaptic MAST Kinase Controls Bidirectional Post-Synaptic Responses to Convey Stimulus Valence during C. elegans Thermotaxis."

Ihara, K., Fei, X., Kondo, R., Ahluwalia, R., Nakano, S., Ikeda, M., Hashimoto, K., Higashiyama, T., Tsukada, Y., Mori, I., Suzuki, T., Sano, A.

[International Worm Meeting, 2019]

Presynaptic plasticity is fundamental in controlling neuronal communication and certain forms of learning. Although changes in neurotransmitter release from presynaptic neurons were shown to modulate the strength of synaptic transmission, it remains unknown whether regulation in presynaptic neurons alters the directionality -positive or negative- of the postsynaptic response. Here, we report that presynaptic regulation in C. elegans can alter excitatory or inhibitory response in the postsynaptic neuron and convey the valence information of sensory stimuli during thermotaxis behavior. During thermotaxis behavior, animals that have been cultivated at a certain temperature with food migrate toward that cultivation temperature when placed on a thermal gradient. By capturing neural activity in freely behaving animals, we showed that the AFD sensory neuron, a major thermosensory neuron known to be required for the thermotaxis behavior, stimulates a postsynaptic interneuron AIY below the cultivation temperature, while it inhibits AIY above that temperature. We identified molecular components important for this process and showed that KIN-4/MAST kinase, MEC-2/Stomatin and DGK-1/Diacylglycerol kinase function in the AFD neuron to regulate the neuronal communication. We also revealed that alteration of the AFD-AIY communication is mediated by controlling the balance of two opposing signals released from AFD, an excitatory peptidergic signaling and an inhibitory glutamatergic signaling. Further, we found that these alternative modes of transmission generate opposing bias in the curving direction and navigate the animals toward the cultivation temperature. Our results suggest that the release of multiple neurotransmitters can be independently controlled and that bidirectional responses in valence-encoding neurons are originated from its presynaptic mechanism whereby MAST kinase, Stomatin and diaclyglycerol kinase influence presynaptic outputs.

Nakano S et al. (2020) Proc Natl Acad Sci U S A "Presynaptic MAST kinase controls opposing postsynaptic responses to convey stimulus valence in Caenorhabditis elegans."

Ahluwalia R, Ihara K, Niino Y, Ikeda M, Nakano S, Suzuki T, Tsukada Y, Miyawaki A, Kondo R, Mori I, Hashimoto K, Higashiyama T, Sano A, Fei X

[Proc Natl Acad Sci U S A, 2020]

Presynaptic plasticity is known to modulate the strength of synaptic transmission. However, it remains unknown whether regulation in presynaptic neurons can evoke excitatory and inhibitory postsynaptic responses. We report here that the <i>Caenorhabditis elegans</i> homologs of MAST kinase, Stomatin, and Diacylglycerol kinase act in a thermosensory neuron to elicit in its postsynaptic neuron an excitatory or inhibitory response that correlates with the valence of thermal stimuli. By monitoring neural activity of the valence-coding interneuron in freely behaving animals, we show that the alteration between excitatory and inhibitory responses of the interneuron is mediated by controlling the balance of two opposing signals released from the presynaptic neuron. These alternative transmissions further generate opposing behavioral outputs necessary for the navigation on thermal gradients. Our findings suggest that valence-encoding interneuronal activity is determined by a presynaptic mechanism whereby MAST kinase, Stomatin, and Diacylglycerol kinase influence presynaptic outputs.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Termine D, Becuwe M, Hofbauer HF, Barrasa MI, Pincus D, Imberdis T, Selkoe D, Freyzon Y, Srinivasan S, Soldner F, Nuber S, Sandoe J, Haque A, Welte MA, Clish CB, Terry-Kantor E, Jaenisch R, Kohlwein SD, Fanning S, Dettmer U, Walther TC, Kim TE, Farese RV, Landgraf D, Baru V, Noble T, Lou Y, Lindquist S, Newby G, Ho GPH, Ramalingam N

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Ahluwalia, Rhea et al. (2021) International Worm Meeting "Discerning the Role of Neuropeptide(s) in C. elegans Thermotaxis Behavior"

Mori, Ikue, Jang, Moon Sun, Ahluwalia, Rhea, Nakano, Shunji

[International Worm Meeting, 2021]

The C. elegans genome encodes over a hundred and fifty neuropeptide genes, which consist of three classes and partake in various functions such as synaptic transmission, cellular signaling and development. Despite their ubiquitous nature, the role(s) played by neuropeptides in several behaviors in C. elegans remains elusive. One such behavioral paradigm is thermotaxis, a robust behavior wherein well-fed worms have been shown to migrate towards the region of past cultivation temperature when placed on a thermal gradient. Studies have suggested the possible involvement of neuropeptides in communication within the neural circuits required for thermotaxis, however, their exact role in thermotaxis regulation is unknown (Narayan, A., Laurent, G. and Sternberg, P. W. (2011) and Nakano, S. et al. (2020)). In order to confirm and further uncover how neuropeptide(s) might be partaking in thermotaxis, we must first identify which neuropeptide(s) are involved for this behavior. To this end, we are performing a genome-wide neuropeptide screen to look for neuropeptide candidates that may be involved in thermotaxis behavior, along with cell-specific knockout for genes crucial for neuropeptide processing in C. elegans. Thus far, we have tested over a hundred mutants, and none have shown strongly altered thermotaxis behavior. In addition, we have tested the four known pro-protein convertases in C. elegans and found that egl-3, the C. elegans ortholog of human proprotein convertase 2 (PCSK 2), displayed abnormal thermotaxis behavior. However, an AFD-specific knockout of egl-3 displayed thermotaxis behavior akin to wildtype, suggesting that egl-3 mediated regulation of thermotaxis most likely takes place through an alternative pathway, prompting further questions pertaining to site of action and the role of the neuropeptides processed by egl-3 in thermotaxis behavior. Altogether, through the genome-wide neuropeptide screen along with the study of the neuropeptide processing enzymes, we hope to gain a holistic insight into peptidergic modulation that controls thermotaxis behavior.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Coenye T, Van Nieuwerburgh F, Deforce D, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.