Ultrastructural localization of calcium homeostasis modulator 1 in mouse taste buds.

Taste receptor cells are morphologically classified as types II and III. Type II cells form a unique type of synapses referred to as channel synapses where calcium homeostasis modulator 1 (CALHM1) together with CALHM3 forms voltage-gated channels that release the neurotransmitter, adenosine triphosphate (ATP). To validate the proposed structural model of channel synapses, the ultrastructural localization of CALHM1 in type II cells of both fungiform and circumvallate taste buds was examined. A monoclonal antibody against CALHM1 was developed and its localization was evaluated via immunofluorescence and immunoelectron microscopy using the immunogold-silver labeling technique. CALHM1 was detected as puncta using immunofluorescence and along the presynaptic membrane of channel synapses facing atypical mitochondria, which provide ATP, by immunoelectron microscopy. In addition, it was detected along the plasma membrane lined by subsurface cisternae at sites apposed to afferent nerve fibers. Our results support the validity of a previously proposed structural model for channel synapses and provide insights into the function of subsurface cisternae whose function in taste receptor cells is unknown. We also examined the localization of CALHM1 in hybrid synapses of type III cells, which are conventional chemical synapses accompanied by mitochondria similar to atypical mitochondria of channel synapses. CALHM1 was not detected in the six hybrid synapses examined using immunoelectron microscopy. We further performed double immunolabeling for CALHM1 and Bassoon, which is detected as puncta corresponding to conventional vesicular synapses in type III cells. Our observations suggest that at least some, and probably most, hybrid synapses are not accompanied by CALHM1.

A light-controlled phospholipase C for imaging of lipid dynamics and controlling neural plasticity.

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCß (opto-PLCß). Opto-PLCß uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCß triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCß can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCß offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

All-optical presynaptic plasticity induction by photoactivated adenylyl cyclase targeted to axon terminals.

Intracellular signaling plays essential roles in various cell types. In the central nervous system, signaling cascades are strictly regulated in a spatiotemporally specific manner to govern brain function; for example, presynaptic cyclic adenosine monophosphate (cAMP) can enhance the probability of neurotransmitter release. In the last decade, channelrhodopsin-2 has been engineered for subcellular targeting using localization tags, but optogenetic tools for intracellular signaling are not well developed. Therefore, we engineered a selective presynaptic fusion tag for photoactivated adenylyl cyclase (bPAC-Syn1a) and found its high localization at presynaptic terminals. Furthermore, an all-optical electrophysiological method revealed rapid and robust short-term potentiation by bPAC-Syn1a at brain stem-amygdala synapses in acute brain slices. Additionally, bPAC-Syn1a modulated mouse immobility behavior. These results indicate that bPAC-Syn1a can manipulate presynaptic cAMP signaling in vitro and in vivo. The all-optical manipulation technique developed in this study can help further elucidate the dynamic regulation of various cellular functions.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Experience-dependent changes in affective valence of taste in male mice.

Taste plays an essential role in the evaluation of food quality by detecting potential harm and benefit in what animals are about to eat and drink. While the affective valence of taste signals is supposed to be innately determined, taste preference can also be drastically modified by previous taste experiences of the animals. However, how the experience-dependent taste preference is developed and the neuronal mechanisms involved in this process are poorly understood. Here, we investigate the effects of prolonged exposure to umami and bitter tastants on taste preference using two-bottle tests in male mice. Prolonged umami exposure significantly enhanced umami preference with no changes in bitter preference, while prolonged bitter exposure significantly decreased bitter avoidance with no changes in umami preference. Because the central amygdala (CeA) is postulated as a critical node for the valence processing of sensory information including taste, we examined the responses of cells in the CeA to sweet, umami, and bitter tastants using in vivo calcium imaging. Interestingly, both protein kinase C delta (Prkcd)-positive and Somatostatin (Sst)-positive neurons in the CeA showed an umami response comparable to the bitter response, and no difference in cell type-specific activity patterns to different tastants was observed. Meanwhile, fluorescence in situ hybridization with c-Fos antisense probe revealed that a single umami experience significantly activates the CeA and several other gustatory-related nuclei, and especially CeA Sst-positive neurons were strongly activated. Intriguingly, after prolonged umami experience, umami tastant also significantly activates the CeA neurons, but the Prkcd-positive neurons instead of Sst-positive neurons were highly activated. These results suggest a relationship between amygdala activity and experience-dependent plasticity developed in taste preference and the involvement of the genetically defined neural populations in this process.

A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes.

CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.

The presynaptic active zone protein Bassoon as a marker for synapses between Type III cells and afferent nerve fibers in taste buds.

Taste buds are receptor organs for gustation. Two types of taste receptor cells have been identified in taste buds: Type II and Type III cells. Type III cells connect with afferent fibers through conventional chemical synapses. In the present study, we used immunocytochemistry to examine the distribution pattern of Bassoon, a scaffolding protein of the cytomatrix at the active zones of conventional synapses in mouse taste buds. Bassoon was predominantly detected as small puncta in Type III cells. Bassoon-immunoreactive puncta were observed in proximity to or partially overlapping with intragemmal nerve fibers. The distribution pattern of Bassoon in taste buds was similar among circumvallate, fungiform, and foliate taste buds. Immunoelectron microscopy showed Bassoon at the active zones of the conventional synapses of Type III cells in circumvallate taste buds. The present results demonstrate that Bassoon is a marker for synapses between Type III cells and afferent fibers in mouse taste buds.

ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation.

Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell-cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. In this study, we found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. We knocked-down Zswim8 in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, we detected neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP.

An engineered channelrhodopsin optimized for axon terminal activation and circuit mapping.

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. However, ChR2 variants selectively transported down a neuron's long-range axonal projections for precise presynaptic activation remain lacking. As a result, ChR2 activation is often contaminated by the spurious activation of en passant fibers that compromise the accurate interpretation of functional effects. Here, we explored the engineering of a ChR2 variant specifically localized to presynaptic axon terminals. The metabotropic glutamate receptor 2 (mGluR2) C-terminal domain fused with a proteolytic motif and axon-targeting signal (mGluR2-PA tag) localized ChR2-YFP at axon terminals without disturbing normal transmission. mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas enabling lower levels of photostimulation. Circuit connectivity mapping in vivo with the Spike Collision Test revealed that mGluR2-PA-tagged ChR2 is useful for identifying axonal projection with significant reduction in the polysynaptic excess noise. These results suggest that the mGluR2-PA tag helps actuate trafficking to the axon terminal, thereby providing abundant possibilities for optogenetic experiments.

Development of an L-type Ca2+ channel-dependent Ca2+ transient during the radial migration of cortical excitatory neurons.

Increasing evidence has shown that voltage-gated L-type Ca2+ channels (LTCCs) are crucial for neurodevelopmental events, including neuronal differentiation/migration and neurite morphogenesis/extension. However, the time course of their functional maturation during the development of excitatory neurons remains unknown. Using a combination of fluorescence in situ hybridization and in utero electroporation-based labeling, we found that the transcripts of Cacna1c and Cacna1d, which encode the LTCC pore-forming subunits, were upregulated in the intermediate zone (IZ) during radial migration. Ca2+ imaging using GCaMP6s in acute brain slices showed spontaneous Ca2+ transients in migrating neurons throughout the IZ. Neurons in the IZ upper layer, especially in the multipolar-to-bipolar transition layer (TL), exhibited more frequent Ca2+ transients than adjacent layers and responded to FPL64176, a potent activator of LTCC. Consistently, nimodipine, an LTCC blocker, inhibited spontaneous Ca2+ transients in neurons in the TL. Collectively, we showed a hitherto unknown increased prevalence of LTCC-dependent Ca2+ transients in the TL of the IZ upper layer during the radial migration of excitatory neurons, which could be essential for the regulation of Ca2+-dependent neurodevelopmental processes.

N-cadherin localization in taste buds of mouse circumvallate papillae.

Taste buds, the receptor organs for taste, contain 50-100 taste bud cells. Although these cells undergo continuous turnover, the structural and functional integrity of taste buds is maintained. The molecular mechanisms by which synaptic connectivity between taste buds and afferent fibers is formed and maintained remain ambiguous. In the present study, we examined the localization of N-cadherin in the taste buds of the mouse circumvallate papillae because N-cadherin, one of the classical cadherins, is important for the formation and maintenance of synapses. At the light microscopic level, N-cadherin was predominantly detected in type II cells and nerve fibers in the connective tissues in and around the vallate papillae. At the ultrastructural level, N-cadherin immunoreactivity appears along the cell membrane and in the intracellular vesicles of type II cells. N-cadherin immunoreactivity also is evident in the membranes of afferent terminals at the contact sites to N-cadherin-positive type II cells. At channel type synapses between type II cells and nerve fibers, N-cadherin is present surrounding, but not within, the presumed neurotransmitter release zone, identified by large mitochondria apposed to the taste cells. The present results suggest that N-cadherin is important for the formation or maintenance of type II cell afferent synapses in taste buds.

Double deletion of the active zone proteins CAST/ELKS in the mouse forebrain causes high mortality of newborn pups.

Presynaptic active zone cytomatrix proteins are essential elements of neurotransmitter release machinery that govern neural transmission. Among active zone proteins, cytomatrix at the active zone-associated structural protein (CAST) is known to regulate active zone size in retinal photoreceptors and neurotransmitter release by recruiting Ca2+ channels at various synapses. However, the role of ELKS-a protein from the same family as CAST-and the synergistic roles of CAST/ELKS have not been thoroughly investigated, particularly with regard to mouse behavior. Here, we generated ELKS conditional KO in mouse forebrain synapses by crossing ELKS flox mice with a CaMKII promoter-induced Cre line. Results showed that CAST is dominant at these synapses and that ELKS can support CAST function, but is less effective in the ELKS single KO. Pups of CAST/ELKS double KO in the forebrain were born in Mendelian rations but resulted in eventual death right after the birth. Anatomically, the forebrain neuronal compositions of CAST KO and CAST/ELKS double KO mice were indistinguishable, and the sensory neural network from whiskers on the face was identified as barrelette-like patches in the spinal trigeminal nucleus. Therefore, depletion of CAST and ELKS disrupts neurotransmission from sensory to motor networks, which can lead to deficits in exploration and failure to suckle.

VPS13D-related disorders presenting as a pure and complicated form of hereditary spastic paraplegia.

BACKGROUND: Alterations of vacuolar protein sorting-associated protein 13 (VPS13) family members including VPS13A, VPS13B, and VPS13C lead to chorea acanthocytosis, Cohen syndrome, and parkinsonism, respectively. Recently, VPS13D mutations were identified as a cause of VPS13D-related movement disorders, which show several phenotypes including chorea, dystonia, spastic ataxia, and spastic paraplegia. METHODS: We applied whole-exome analysis for a patient with a complicated form of hereditary spastic paraplegia (HSP) and her unaffected parents. Then, we screened the candidate genes in 664 Japanese families with HSP in Japan. RESULTS: We first found a compound heterozygote VPS13D mutation and a heterozygote ABHD4 variation in a sporadic patient with spastic paraplegia. Then, we found three patients with VPS13D mutations in two Japanese HSP families. The three patients with homozygous mutations (p.Thr1118Met/p.Thr1118Met and p.Thr2945Ala/p.Thr2945Ala) in the VPS13D showed an adult onset pure form of HSP. Meanwhile, the patient with a compound heterozygous mutation (p.Ser405Arg/p.Arg3141Ter) in the VPS13D showed a childhood onset complicated form of HSP associated with cerebellar ataxia, cervical dystonia, cataracts, and chorioretinal dystrophy. CONCLUSION: In the present study, we found four patients in three Japanese families with novel VPS13D mutations, which may broaden the clinical and genetic findings for VPS13D-related disorders.

UBAP1 mutations cause juvenile-onset hereditary spastic paraplegias (SPG80) and impair UBAP1 targeting to endosomes.

We aimed to find a new causative gene and elucidate the molecular mechanisms underlying a new type of hereditary spastic paraplegia (HSP). Patients with HSP were recruited from the Japan Spastic Paraplegia Research Consortium (JASPAC). Exome sequencing of genomic DNA from patients in four families was carried out, followed by Sanger sequencing of the UBAP1 gene. A mouse homolog of one UBAP1 frameshift mutation carried by one of the patients was created as a disease model. Functional properties of the UBAP1 wild type and UBAP1-mutant in mouse hippocampus neurons were examined. We identified three novel heterozygous loss of function mutations (c.425_426delAG, c.312delC, and c.535G>T) in the UBAP1 gene as the genetic cause of a new type of HSP (SPG80). All the patients presented identical clinical features of a pure type of juvenile-onset HSP. Functional studies on mouse hippocampal neurons revealed that the C-terminal deletion UBAP1-mutant of our disease model had lost its ability to bind ubiquitin in vitro. Overexpression of the UBAP1 wild type interacts directly with ubiquitin on enlarged endosomes, while the UBAP1-mutant cannot be recruited to endosome membranes. Our study demonstrated that mutations in the UBAP1 gene cause a new type of HSP and elucidated its pathogenesis. The full-length UBAP1 protein is involved in endosomal dynamics in neurons, while loss of UBAP1 function may perturb endosomal fusion and sorting of ubiquitinated cargos. These effects could be more prominent in neurons, thereby giving rise to the phenotype of a neurodegenerative disease such as HSP.

ß-N-methylamino-L-alanine (BMAA) suppresses cell cycle progression of non-neuronal cells.

ß-N-methylamino-L-alanine (BMAA), a natural non-proteinaceous amino acid, is a neurotoxin produced by a wide range of cyanobacteria living in various environments. BMAA is a candidate environmental risk factor for neurodegenerative diseases such as amyotrophic lateral sclerosis and Parkinson-dementia complex. Although BMAA is known to exhibit weak neuronal excitotoxicity via glutamate receptors, the underlying mechanism of toxicity has yet to be fully elucidated. To examine the glutamate receptor-independent toxicity of BMAA, we investigated the effects of BMAA in non-neuronal cell lines. BMAA potently suppressed the cell cycle progression of NIH3T3 cells at the G1/S checkpoint without inducing plasma membrane damage, apoptosis, or overproduction of reactive oxygen species, which were previously reported for neurons and neuroblastoma cells treated with BMAA. We found no evidence that activation of glutamate receptors was involved in the suppression of the G1/S transition by BMAA. Our results indicate that BMAA affects cellular functions, such as the division of non-neuronal cells, through glutamate receptor-independent mechanisms.

CAST: Its molecular structure and phosphorylation-dependent regulation of presynaptic plasticity.

Our brain functions rely on sophisticated communication between synapses in the nervous system. Most synapses utilize a specialized submembranous structure, the so-called 'active zone', for the efficient transmission of chemical signals. The presynaptic active zone plays pivotal roles in the precise regulation of neurotransmitter release from the nerve terminals in a temporally and spatially coordinated manner. During the last two decades, several active zone-specific proteins have been isolated and characterized, including Bassoon, Piccolo/Aczonin, RIM, Munc13-1, ELKS, and CAST. The CAST/ELKS family is capable of potent direct interactions with other active zone proteins, forming a large protein complex that seems to be a molecular basis for electron density in the presynaptic active zone. The molecular details of the integrity of the active zone been well studied, however, we are just beginning to understanding its physiological significance in higher brain functions such as learning and memory, emotion, and consciousness. Focusing on the CAST/ELKS protein family, this review describes their biochemical properties, physiological functions in brain areas such as the hippocampus, and the significance of CAST phosphorylation in presynaptic short-term plasticity.

Expression and light-dependent translocation of ß-arrestin in the visual system of the terrestrial slug Limax valentianus.

Vertebrates, cephalopods and arthropods are equipped with eyes that have the highest spatiotemporal resolution among the animal phyla. In parallel, only animals in these three phyla have visual arrestin specialized for the termination of visual signaling triggered by opsin, in addition to ubiquitously expressed ß-arrestin that serves in terminating general G protein-coupled receptor signaling. Indeed, visual arrestin in Drosophila and rodents translocates to the opsin-rich subcellular region in response to light to reduce the overall sensitivity of photoreceptors in an illuminated environment (i.e. light adaptation). We thus hypothesized that, during evolution, visual arrestin has taken over the role of ß-arrestin in those animals with eyes of high spatiotemporal resolution. If this is true, it is expected that ß-arrestin plays a role similar to visual arrestin in those animals with low-resolution eyes. In the present study, we focused on the terrestrial mollusk Limax valentianus, a species related to cephalopods but that has only ß-arrestin, and generated antibodies against ß-arrestin. We found that ß-arrestin is highly expressed in photosensory neurons, and translocates into the microvilli of the rhabdomere within 30 min in response to short wavelength light (400 nm), to which the Limax eye exhibits a robust response. These observations suggest that ß-arrestin functions in the visual system of those animals that do not have visual arrestin. We also exploited anti-ß-arrestin antibody to visualize the optic nerve projecting to the brain, and demonstrated its usefulness for tracing a visual ascending pathway.

SAD-B Phosphorylation of CAST Controls Active Zone Vesicle Recycling for Synaptic Depression.

Short-term synaptic depression (STD) is a common form of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation.

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra.

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.

Transgenic rat model of childhood-onset dermatitis by overexpressing telomerase reverse transcriptase (TERT).

Childhood-onset dermatitis is one of the most common skin disorders in children. Although various mouse models that mirror aspects of dermatitis have become available, there is still a need for an animal model that develops dermatitis in childhood and is more suitable for performing tissue transplantation experiments. There is emerging evidence that peripheral blood T lymphocytes from patients with dermatitis have significantly increased telomerase activity. Here, we developed telomerase reverse transcriptase (TERT)-expressing transgenic (Tg) rats that spontaneously developed eczematous skin inflammation in childhood. Newborn TERT-Tg rats developed visible dermatitis in 56 % of cases, and the skin lesions microscopically showed spongiosis and acanthosis with infiltration of lymphocytes, eosinophils and mast cells. TERT-Tg rats with dermatitis exhibited increased CD4 (2.5-fold) and CD8 (fivefold) T cell numbers compared with dermatitis-free TERT-Tg rats. Stronger TERT activity was observed in the peripheral lymphocytes of dermatitis-positive TERT-Tg rats than those of dermatitis-free TERT-Tg rats. RT-PCR analysis revealed that IL-4 was markedly elevated in the spleen of dermatitis-positive TERT-Tg rats, and that interferon-gamma was increased in the dermatitis lesions. Moreover, skin grafting of TERT-Tg rats with dermatitis onto T cell-deficient nude rats demonstrated that the inflamed skin lesions could not be maintained. Taken together, the results suggest that TERT activation in T lymphocytes is one of the potential predisposing factors for dermatitis. Moreover, our results demonstrated that the TERT-Tg rats mirror aspects of human childhood-onset dermatitis and that these animals represent a potential animal model system for studying childhood-onset dermatitis.