Sexually Dimorphic Regulation of Behavioral States by Dopamine in Caenorhabditis elegans.

Sex differences in behavior allow animals to effectively mate and reproduce. However, the mechanism by which biological sex regulates behavioral states, which underlie the regulation of sex-shared behaviors, such as locomotion, is largely unknown. In this study, we studied sex differences in the behavioral states of Caenorhabditis elegans and found that males spend less time in a low locomotor activity state than hermaphrodites and that dopamine generates this sex difference. In males, dopamine reduces the low activity state by acting in the same pathway as polycystic kidney disease-related genes that function in male-specific neurons. In hermaphrodites, dopamine increases the low activity state by suppression of octopamine signaling in the sex-shared SIA neurons, which have reduced responsiveness to octopamine in males. Furthermore, dopamine promotes exploration both inside and outside of bacterial lawn (the food source) in males and suppresses it in hermaphrodites. These results demonstrate that sexually dimorphic signaling allows the same neuromodulator to promote adaptive behavior for each sex.SIGNIFICANCE STATEMENT The mechanisms that generate sex differences in sex-shared behaviors, including locomotion, are not well understood. We show that there are sex differences in the regulation of behavioral states in the model animal Caenorhabditis elegans Dopamine promotes the high locomotor activity state in males, which must search for mates to reproduce, and suppresses it in self-fertilizing hermaphrodites through distinct molecular mechanisms. This study demonstrates that sex-specific signaling generates sex differences in the regulation of behavioral states, which in turn modulates the locomotor activity to suit reproduction for each sex.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Dopamine regulates body size in Caenorhabditis elegans.

The nervous system plays a critical role in the regulation of animal body sizes. In Caenorhabditis elegans, an amine neurotransmitter, dopamine, is required for the tactile perception of food and food-dependent behavioral changes, while its role in development is unknown. In this study, we show that dopamine negatively regulates body size through a D2-like dopamine receptor, DOP-3, in C. elegans. Dopamine alters body size without affecting food intake or developmental rate. We also found that dopamine promotes egg-laying, although the regulation of body size by dopamine was not solely caused by this effect. Furthermore, dopamine negatively regulates body size through the suppression of signaling by octopamine and Gq-coupled octopamine receptors, SER-3 and SER-6. Our results demonstrate that dopamine and octopamine regulate the body size of C. elegans and suggest a potential role for perception in addition to ingestion of food for growth.

Aß induces oxidative stress in senescence-accelerated (SAMP8) mice.

According to the amyloid hypothesis, amyloid ß accumulates in brains with Alzheimer's disease (AD) and triggers cell death and memory deficit. Previously, we developed a rice Aß vaccine expressing Aß, which reduced brain Aß levels in the Tg2576 mouse model of familial AD. We used senescence-accelerated SAMP8 mice as a model of sporadic AD and investigated the relationship between Aß and oxidative stress. Insoluble Aß and 4-hydroxynonenal (4-HNE) levels tended to be reduced in SAMP8 mice-fed the rice Aß vaccine. We attempted to clarify the relationship between oxidative stress and Aß in vitro. Addition of Aß peptide to the culture medium resulted in an increase in 4-HNE levels in SH-SY5Y cells. Tg2576 mice, which express large amounts of Aß in their brain, also exhibited increased 4-HNE levels; this increase was inhibited by the Aß vaccine. These results indicate that Aß induces oxidative stress in cultured cells and in the mouse brain.

Nonredundant function of two highly homologous octopamine receptors in food-deprivation-mediated signaling in Caenorhabditis elegans.

It is common for neurotransmitters to possess multiple receptors that couple to the same intracellular signaling molecules. This study analyzes two highly homologous G-protein-coupled octopamine receptors using the model animal Caenorhabditis elegans. In C. elegans, the amine neurotransmitter octopamine induces activation of cAMP response element-binding protein (CREB) in the cholinergic SIA neurons in the absence of food through activation of the Gq-coupled octopamine receptor SER-3 in these neurons. We also analyzed another Gq-coupled octopamine receptor, SER-6, that is highly homologous to SER-3. As seen in ser-3 deletion mutants, octopamine- and food-deprivation-mediated CREB activation was decreased in ser-6 deletion mutants compared with wild-type animals, suggesting that both SER-3 and SER-6 are required for signal transduction. Cell-specific expression of SER-6 in the SIA neurons was sufficient to restore CREB activation in the ser-6 mutants, indicating that SER-6, like SER-3, functions in these neurons. Taken together, these results demonstrate that two similar G-protein-coupled receptors, SER-3 and SER-6, function in the same cells in a nonredundant manner.

Sperm function is required for suppressing locomotor activity of C. elegans hermaphrodites.

Sex differences in sex-shared behavior are common across various species. During mating, males transfer sperm and seminal fluid to females, which can affect female behavior. Sperm can be stored in the female reproductive tract for extended periods of time and used to fertilize eggs. However, the role of either sperm or embryo production in regulating female behavior is poorly understood. In the androdioecious nematode C. elegans, hermaphrodites produce both oocytes and sperm, enabling them to self-fertilize or mate with males. Hermaphrodites exhibit less locomotor activity compared to males, indicating sex difference in behavioral regulation. In this study, mutants defective in the sperm production and function were examined to investigate the role of sperm function in the regulation of locomotor behavior. Infertile hermaphrodites exhibited increased locomotor activity, which was suppressed after mating with fertile males. The results suggest that sperm, seminal fluid, or the presence of embryos are detected by hermaphrodites, leading to a reduction in locomotor activity. Additionally, females of closely related gonochoristic species, C. remanei and C. brenneri, exhibited reduced locomotor activity after mating. The regulation of locomotion by sperm function may be an adaptive mechanism that enables hermaphrodites lacking sperm or embryo to search for mates and allow females to cease their search for mates after mating.

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans.

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2-like dopamine receptors and the G protein Gi/o. The D2-like receptors work in both the octopaminergic neurons and the octopamine-responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine-mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.

Comparison of presenilin 1 and presenilin 2 γ-secretase activities using a yeast reconstitution system.

γ-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the γ-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks γ-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. γ-Secretase activity was measured based on yeast growth on selective media and ß-galactosidase activity. PS1 γ-secretase was ∼24-fold more active than PS2 γ-secretase in the ß-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aß generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced ∼24-fold more Aß than PS2 γ-secretase. We found the optimal pH of Aß production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one γ-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the γ-secretase complex were ∼28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in Aß production.

Regulation of the alternative splicing of sarcoplasmic reticulum Ca²âº-ATPase1 (SERCA1) by phorbol 12-myristate 13-acetate (PMA) via a PKC pathway.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease with no established treatment to date. Small, cell-permeable molecules hold the potential to treat DM1. In this study, we investigated the association between protein kinase C (PKC) signaling and splicing of sarcoplasmic reticulum Ca(2+)-ATPase1 (SERCA1). Our aim was to clarify the mechanisms underlying the regulation of alternative splicing, in order to explore new therapeutic strategies for DM1. By assessing the splicing pattern of the endogenous SERCA1 gene in HEK293 cells, we found that treatment with phorbol 12-myristate 13-acetate (PMA) regulated SERCA1 splicing. Interestingly, treatment with PMA for 48 h normalized SERCA1 splicing, while treatment for 1.5h promoted aberrant splicing. These two responses showed dose dependency and were completely abolished by the PKC inhibitor Ro 31-8220. Furthermore, repression of PKCßII and PKCθ by RNAi mimicked prolonged PMA treatment. These results indicate that PKC signaling is involved in the splicing of SERCA1 and provide new evidence for a link between alternative splicing and PKC signaling.

Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by γ-secretase complex.

Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.

Localization of mature neprilysin in lipid rafts.

Alzheimer's disease (AD) is characterized by senile plaques caused by amyloid-ß peptide (Aß) accumulation. It has been reported that Aß generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aß-degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aß accumulates but does not modulate the protease activity of NEP.

Alternative splicing of PDLIM3/ALP, for α-actinin-associated LIM protein 3, is aberrant in persons with myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder of muscular dystrophy characterized by muscle weakness and wasting. DM1 is caused by expansion of CTG repeats in the 3'-untranslated region (3'-UTR) of DM protein kinase (DMPK) gene. Since CUG-repeat RNA transcribed from the expansion of CTG repeats traps RNA-binding proteins that regulate alternative splicing, several abnormalities of alternative splicing are detected in DM1, and the abnormal splicing of important genes results in the appearance of symptoms. In this study, we identify two abnormal splicing events for actinin-associated LIM protein 3 (PDLIM3/ALP) and fibronectin 1 (FN1) in the skeletal muscles of DM1 patients. From the analysis of the abnormal PDLIM3 splicing, we propose that ZASP-like motif-deficient PDLIM3 causes the muscular symptoms in DM. PDLIM3 binds α-actinin 2 in the Z-discs of muscle, and the ZASP-like motif is needed for this interaction. Moreover, in adult humans, PDLIM3 expression is highest in skeletal muscles, and PDLIM3 splicing in skeletal muscles is regulated during human development.

Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans.

BACKGROUND: Parkinson's disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson's disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. METHODS: To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic and pharmacological methods to define the features of early motor dysfunction of α-synuclein-expressing C. elegans. Finally, we applied the C. elegans model to a drug repurposing screen by combining it with an artificial intelligence platform and cell culture system to identify small molecules that inhibit α-synuclein oligomers. Screen hits were validated using in vitro and in vivo mammalian models. RESULTS: We found a previously undescribed motor phenotype in transgenic α-synuclein C. elegans that correlates with mutant or wild-type α-synuclein protein levels and results from dopaminergic neuron dysfunction, but precedes neuronal loss. Together with artificial intelligence-driven in silico and in vitro screening, this C. elegans model identified five compounds that reduced motor dysfunction induced by α-synuclein. Three of these compounds also decreased α-synuclein oligomers in mammalian neurons, including rifabutin which has not been previously investigated for Parkinson's disease. We found that treatment with rifabutin reduced nigrostriatal dopaminergic neurodegeneration due to α-synuclein in a rat model. CONCLUSIONS: We identified a C. elegans locomotor abnormality due to dopaminergic neuron dysfunction that models early α-synuclein-mediated neurodegeneration. Our innovative approach applying this in vivo model to a multi-step drug repurposing screen, with artificial intelligence-driven in silico and in vitro methods, resulted in the discovery of at least one drug that may be repurposed as a disease-modifying therapy for Parkinson's disease.

Starvation induces cAMP response element-binding protein-dependent gene expression through octopamine-Gq signaling in Caenorhabditis elegans.

The nervous system plays a critical role in adaptation to a new environment. In Caenorhabditis elegans, reduced access to food requires both changes in behavior as well as metabolic adaptation for survival, which is postulated to involve the bioamine octopamine. The transcription factor cAMP response element-binding protein (CREB) is generally activated by G-protein-coupled receptors (GPCRs) that activate G alpha(s) and is known to play an important role in long-term changes, including synaptic plasticity. We show that, in C. elegans, the CREB ortholog CRH-1 (CREB homolog family member 1) activates in vivo a cAMP response element-green fluorescent protein fusion reporter in a subset of neurons during starvation. This starvation response is mediated by octopamine via the GPCR SER-3 (serotonin/octopamine receptor family member 3) and is fully dependent on the subsequent activation of the G alpha(q) ortholog EGL-30 (egg-laying defective family member 30). The signaling cascade is only partially dependent on the phospholipase C beta (EGL-8) and is negatively regulated by G alpha(o) [GOA-1 (G-protein, O, alpha subunit family member 1)] and calcium/calmodulin-dependent kinase [UNC-43 (uncoordinated family member 43)]. Nonstarved animals in a liquid environment mediate a similar response that is octopamine independent. The results show that the endogenous octopamine system in C. elegans is activated by starvation and that different environmental stimuli can activate CREB through G alpha(q).

Regulation of body size in Caenorhabditis elegans: effects of environmental factors and the nervous system.

Body size is one of the basic traits of animals and is regulated to adapt to the environment. Animals perceive environmental stimuli with sensory neurons, and signals from the nervous system alter the size of organs, thus regulating body size. The model animal Caenorhabditis elegans is particularly suited for genetic analysis of body size regulation, and has already contributed to the elucidation of various genetic pathways that regulate body size. In this review, we summarize the available literature regarding environmental factors that regulate body size and the role of the nervous system in such regulation. We discuss in detail a recent report on body size regulation by the neurotransmitter, dopamine.

Dopamine receptors in C. elegans.

Dopamine regulates various physiological functions in the central nervous system and the periphery. Dysfunction of the dopamine system is implicated in a wide variety of disorders and behaviors including schizophrenia, addiction, and attention-deficit hyperactivity disorder. Medications that modulate dopamine signaling have therapeutic efficacy on the treatment of these disorders. However, the causes of these disorders and the role of dopamine are still unclear. Studying the dopamine system in a model organism, such as Caenorhabditis elegans, allows the genetic analysis in a simple and well-described nervous system, which may provide new insight into the molecular mechanisms of dopamine signaling. In this review, we summarize recent findings on pharmacological and biochemical properties of the C. elegans dopamine receptors and their physiological role in the control of behavior.

Identification of a dopamine receptor from Caenorhabditis elegans.

The neurotransmitter dopamine regulates locomotion and egg laying in the nematode Caenorhabditis elegans. We have cloned a cDNA encoding the C. elegans G protein-coupled receptor (CeDOP1). The deduced amino acid sequence of the cloned cDNA shows high sequence similarities with D1-like dopamine receptors from other species. Three splice variants that differ in the length of the predicted third intracellular loop and C-terminal tail were identified. COS-7 cells transiently transfected with CeDOP1 showed high affinity binding to [(125)I]iodo-lysergic acid diethylamide (K(D)=3.43 +/- 0.83 nM). Dopamine showed the highest affinity (K(i)=0.186 microM) for this receptor among several vertebrate and invertebrate amine neurotransmitters tested, suggesting that the natural ligand for this receptor is dopamine.

Dysbindin-1, a schizophrenia-related protein, interacts with HDAC3.

DTNBP1 is a key candidate gene associated with schizophrenia. The expression of its protein product, dysbindin-1, is altered in the brains of schizophrenic patients; however, the physiological functions of dysbindin-1 in the central nervous system are unclear. Several studies have shown that both dysbindin-1 and histone deacetylase 3 (HDAC3) can be phosphorylated by the DNA-dependent protein kinase complex. In this study, we investigated the relationship between dysbindin-1 and HDAC3. We found that dysbindin-1 formed a protein complex with HDAC3 in human neuroblastoma cells and in mouse brain. The interaction between dysbindin-1 and HDAC3 occurred in an isoform-specific manner: HDAC3 coupled with dysbindin-1A and -1B, but not -1C. We also found that dysbindin-1B expression was increased in the nucleus in the presence of HDAC3, and, conversely, that the phosphorylation level of HDAC3 increased in the presence of dysbindin-1B. Taken together, these results identify a novel binding partner for dysbindin-1, which may potentially provide a new avenue for research into the neurological mechanisms of schizophrenia.

Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB) is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

Manumycin A corrects aberrant splicing of Clcn1 in myotonic dystrophy type 1 (DM1) mice.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.