Satoshi Suo

The VNTR polymorphism of the human dopamine transporter (DAT1) gene affects gene expression

The human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) in its 3′-untranslated region (UTR). The linkage and association between the VNTR polymorphism of DAT1 and various neuropsychiatric disorders have been reported. We have determined the genomic structure of DAT1 genes containing 7-, 9-, 10-, and 11-repeat alleles and examined the effect of VNTR polymorphism in the 3′-UTR region of DAT1 on gene expression using the luciferase reporter system in COS-7 cells. Luciferase expression was significantly higher when the 3′-UTR of the DAT1 gene contained the 10-repeat allele than when it contained the 7- or 9-repeat alleles. This suggests that VNTR polymorphism affects the expression of the dopamine transporter.

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 Gαs 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 Gαq ortholog EGL-30 (egg-laying defective family member 30). The signaling cascade is only partially dependent on the phospholipase Cβ (EGL-8) and is negatively regulated by Gαo [GOA-1 (G-protein, O, α 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 Gαq.

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor.

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1‐like and D2‐like receptors. However, D2‐like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2‐like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2‐like dopamine receptors than to D1‐like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo‐lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2‐like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.

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.

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.

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.

Effect of rice-expressed amyloid β in the Tg2576 Alzheimer's disease transgenic mouse model.

One of the main hallmarks of Alzheimers disease (AD) is senile plaques composed of amyloid β (Aβ). We developed a new edible vaccine: rice expressing GFP-Aβ42. In a previous report, we described the production of anti-Aβ antibodies in B6 mice fed Aβ rice mixed with cholera toxin B subunit (CTB). In this report, we investigated whether Aβ rice had therapeutic effects in the Tg2576 AD model mice. The anti-Aβ antibody titer was increased and levels of intracerebral Aβ (soluble and insoluble) and serum Aβ decreased. Because the value of IgG1/IgG2a in the Aβ feeding group was >1, immunization via Aβ rice may induce a non-inflammatory Th2 reaction. We also found that the Aβ vaccine improved memory, as assessed in a Y-maze test. The number of arm entries in the Y-maze test was lower in the Aβ feeding group than in the control group. These results suggest that the new edible vaccine Aβ rice may have therapeutic effects in AD.

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 β-galactosidase activity. PS1 γ-secretase was ∼24-fold more active than PS2 γ-secretase in the β-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aβ generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced ∼24-fold more Aβ than PS2 γ-secretase. We found the optimal pH of Aβ 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 Aβ production.

Regulation of the alternative splicing of sarcoplasmic reticulum Ca2+-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 PKCβII 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.

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.