Takeshi Ishihara

An Expanded Palette of Genetically Encoded Ca2+ Indicators

Directed protein evolution provides a series of fluorescent protein-based indicators for multicolor Ca2+ imaging. Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP–yellow FP–based indicator, Ca2+ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.

Identification and characterization of the high-affinity choline transporter

In cholinergic neurons, high-affinity choline uptake in presynaptic terminals is the rate-limiting step in acetylcholine synthesis. Using information provided by the Caenorhabditis elegans Genome Project, we cloned a cDNA encoding the high-affinity choline transporter from C. elegans (cho-1). We subsequently used this clone to isolate the corresponding cDNA from rat (CHT1). CHT1 is not homologous to neurotransmitter transporters, but is homologous to members of the Na+-dependent glucose transporter family. Expression of CHT1 mRNA is restricted to cholinergic neurons. The characteristics of CHT1-mediated choline uptake essentially match those of high-affinity choline uptake in rat brain synaptosomes.

Retarded Axonal Transport of R406W Mutant Tau in Transgenic Mice with a Neurodegenerative Tauopathy

Intracellular accumulations of filamentous tau inclusions are neuropathological hallmarks of neurodegenerative diseases known as tauopathies. The discovery of multiple pathogenic tau gene mutations in many kindreds with familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) unequivocally confirmed the central role of tau abnormalities in the etiology of neurodegenerative disorders. To examine the effects of tau gene mutations and the role of tau abnormalities in neurodegenerative tauopathies, transgenic (Tg) mice were engineered to express the longest human tau isoform (T40) with or without the R406W mutation (RW and hWT Tg mice, respectively) that is pathogenic for FTDP-17 in several kindreds. RW but not hWT tau Tg mice developed an age-dependent accumulation of insoluble filamentous tau aggregates in neuronal perikarya of the cerebral cortex, hippocampus, cerebellum, and spinal cord. Significantly, CNS axons in RW mice contained reduced levels of tau when compared with hWT mice, and this was linked to retarded axonal transport and increased accumulation of an insoluble pool of RW but not hWT tau. Furthermore, RW but not hWT mice demonstrated neurodegeneration and a reduced lifespan. These data indicate that the R406W mutation causes reduced binding of this mutant tau to microtubules, resulting in slower axonal transport. This altered tau function caused by the RW mutation leads to increased accumulation and reduced solubility of RW tau in an age-dependent manner, culminating in the formation of filamentous intraneuronal tau aggregates similar to that observed in tauopathy patients.

HEN-1, a Secretory Protein with an LDL Receptor Motif, Regulates Sensory Integration and Learning in Caenorhabditis elegans

Animals sense many environmental stimuli simultaneously and integrate various sensory signals within the nervous system both to generate proper behavioral responses and also to form relevant memories. HEN-1, a secretory protein with an LDL receptor motif, regulates such processes in Caenorhabditis elegans. The hen-1 mutants show defects in the integration of two sensory signals and in behavioral plasticity by paired stimuli, although their sensation capability seems to be identical to that of the wild-type. The HEN-1 protein is expressed in two pairs of neurons, but expression in other neurons is sufficient for wild-type behavior. In addition, expression of HEN-1 at the adult stage is sufficient. Thus, HEN-1 regulates sensory processing non-cell-autonomously in the mature neuronal circuit.

The conserved nuclear receptor Ftz-F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans

Nuclear receptors are essential players in the development of all metazoans. The nematode Caenorhabditis elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz‐F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila.

Olfactory plasticity is regulated by pheromonal signaling in Caenorhabditis elegans

Too Close for Comfort Pheromones are often used for sexual communications in animals, but they can also serve as a measure of population density. Now, Yamada et al. (p. 1647) have found that population density in the nematode worm Caenorhabditis elegans regulates plasticity of olfactory behavior, in which attraction to an odorant decreases after prolonged exposure. Using two rounds of genetic screens, a peptide named SNET-1 and a homolog of a mammalian transmembrane peptidase neprilysin were found to mediate pheromonal regulation. This regulation of olfactory behavior may serve to coordinate the behavior of individual animals in relation to the status of the whole population. A nematode odor response is regulated by population density through dauer pheromone, a neuropeptide, and neprilysin peptidase. Population density–dependent dispersal is a well-characterized strategy of animal behavior in which dispersal rate increases when population density is higher. Caenorhabditis elegans shows positive chemotaxis to a set of odorants, but the chemotaxis switches from attraction to dispersal after prolonged exposure to the odorants. We show here that this plasticity of olfactory behavior is dependent on population density and that this regulation is mediated by pheromonal signaling. We show that a peptide, suppressor of NEP-2 (SNET-1), negatively regulates olfactory plasticity and that its expression is down-regulated by the pheromone. NEP-2, a homolog of the extracellular peptidase neprilysin, antagonizes SNET-1, and this function is essential for olfactory plasticity. These results suggest that population density information is transmitted through the external pheromone and endogenous peptide signaling to modulate chemotactic behavior.

Behavioral choice between conflicting alternatives is regulated by a receptor guanylyl cyclase, GCY-28, and a receptor tyrosine kinase, SCD-2, in AIA interneurons of Caenorhabditis elegans.

Animals facing conflicting sensory cues make a behavioral choice between competing alternatives through integration of the sensory cues. Here, we performed a genetic screen to identify genes important for the sensory integration of two conflicting cues, the attractive odorant diacetyl and the aversive stimulus Cu2+, and found that the membrane-bound guanylyl cyclase GCY-28 and the receptor tyrosine kinase SCD-2 regulate the behavioral choice between these alternatives in Caenorhabditis elegans. The gcy-28 mutants and scd-2 mutants show an abnormal bias in the behavioral choice between the cues, although their responses to each individual cue are similar to those in wild-type animals. Mutants in a gene encoding a cyclic nucleotide gated ion channel, cng-1, also exhibit the defect in sensory integration. Molecular genetic analyses suggested that GCY-28 and SCD-2 regulate sensory integration in AIA interneurons, where the conflicting sensory cues may converge. Genetic ablation or hyperpolarization of AIA interneurons showed nearly the same phenotype as gcy-28 or scd-2 mutants in the sensory integration, although this did not affect the sensory response to each individual cue. In gcy-28 or scd-2 mutants, activation of AIA interneurons is sufficient to restore normal sensory integration. These results suggest that the activity of AIA interneurons regulates the behavioral choice between the alternatives. We propose that GCY-28 and SCD-2 regulate sensory integration by modulating the activity of AIA interneurons.

Caenorhabditis elegans Integrates the Signals of Butanone and Food to Enhance Chemotaxis to Butanone

Behavioral plasticity induced by the integration of two sensory signals, such as associative learning, is an important issue in neuroscience, but its evolutionary origin and diversity have not been explored sufficiently. We report here a new type of such behavioral plasticity, which we call butanone enhancement, in Caenorhabditis elegans adult hermaphrodites: C. elegans specifically enhances chemotaxis to butanone by preexposure to butanone and food. Mutant analysis revealed that this plasticity requires the AWCON olfactory neuron, whose fate is known to be determined by the NSY-1/ASK1 MAPKKK (mitogen-activated protein kinase kinase kinase) cascade as well as the DAF-11 and ODR-1 guanylyl cyclases. These proteins also control many aspects of olfactory sensation/plasticity in AWC neurons and seem to provide appropriate cellular conditions for butanone enhancement in the AWCON neuron. Butanone enhancement also required the functions of Bardet-Biedl syndrome genes in the AWCON neuron but not other genes that control ciliary transport. Furthermore, preexposure to butanone and the odor of food was enough for the enhancement of butanone chemotaxis. These results suggest that the AWCON olfactory neuron may conduct a behavioral plasticity resembling associative learning and that the functions of Bardet-Biedl syndrome genes in sensory cilia may play an important role in this plasticity.

hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1.

Proteins of the tolloid/bone morphogenetic protein (BMP)‐1 family play important roles in the differentiation of cell fates. Among those proteins are BMP‐1, which plays a role in cartilage and bone formation in mammals, the TOLLOID protein, which is required for the establishment of the dorsoventral axis of Drosophila embryos and BP10/SpAN, which are thought to act in the morphogenesis of sea urchins. These proteins have some properties in common. First, they contain the astacin metalloprotease domain, the CUB domain and the epidermal growth factor‐like domain. Second, they are expressed in embryos at stages expected for their role in cell differentiation. Third, at least BMP‐1 and TOLLOID are thought to interact with proteins of the transforming growth factor‐beta family. We report that the hch‐1 gene of the nematode Caenorhabditis elegans encodes a tolloid/BMP‐1 family protein. The protein has the characteristic domains common to the tolloid/ BMP‐1 family. Like other members of the family, it is expressed in embryos. However, the phenotype of hch‐1 mutants shows that it is required for normal hatching and normal migration of a post‐embryonic neuroblast. Furthermore, in spite of its expression in embryogenesis, it is not required for the viability of embryos. These results show new functions of the tolloid/BMP‐1 family proteins and give insight into their evolution.

Left-right olfactory asymmetry results from antagonistic functions of voltage-activated calcium channels and the Raw repeat protein OLRN-1 in C. elegans

BackgroundThe left and right AWC olfactory neurons in Caenorhabditis elegans differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWCOFF, and the contralateral AWC becomes AWCON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWCON.ResultsWe show here that the asymmetry gene olrn-1 acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWCON. OLRN-1, a protein with potential membrane-association domains, is related to the Drosophila Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. olrn-1 opposes the action of two voltage-activated calcium channel homologs, unc-2 (CaV2) and egl-19 (CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWCOFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from olrn-1.Conclusionolrn-1 and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.