A hyperpolarizing neuron recruits undocked innexin hemichannels to transmit neural information in Caenorhabditis elegans.

While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

AWC thermosensory neuron interferes with information processing in a compact circuit regulating temperature-evoked posture dynamics in the nematode Caenorhabditis elegans.

Elucidating how individual neurons encode and integrate sensory information to generate a behavior is crucial for understanding neural logic underlying sensory-dependent behavior. In the nematode Caenorhabditis elegans, information flow from sensory input to behavioral output is traceable at single-cell level due to its entirely solved neural connectivity. C. elegans processes the temperature information for regulating behavior consisting of undulatory posture dynamics in a circuit including two thermosensory neurons AFD and AWC, and their postsynaptic interneuron AIY. However, how the information processing in AFD-AWC-AIY circuit generates the posture dynamics remains elusive. To quantitatively evaluate the posture dynamics, we introduce locomotion entropy, which measures bandwidth of the frequency spectrum of the undulatory posture dynamics, and assess how the motor pattern fluctuates. We here found that AWC disorders the information processing in AFD-AWC-AIY circuit for regulating temperature-evoked posture dynamics. Under slow temperature ramp-up, AWC adjusts AFD response, whereby broadening the temperature range in which animals exhibit fluctuating posture undulation. Under rapid temperature ramp-up, AWC increases inter-individual variability in AIY activity and the fluctuating posture undulation. We propose that a compact nervous system recruits a sensory neuron as a fluctuation inducer for regulating sensory-dependent behavior.

OLA-1, an Obg-like ATPase, integrates hunger with temperature information in sensory neurons in C. elegans.

Animals detect changes in both their environment and their internal state and modify their behavior accordingly. Yet, it remains largely to be clarified how information of environment and internal state is integrated and how such integrated information modifies behavior. Well-fed C. elegans migrates to past cultivation temperature on a thermal gradient, which is disrupted when animals are starved. We recently reported that the neuronal activities synchronize between a thermosensory neuron AFD and an interneuron AIY, which is directly downstream of AFD, in well-fed animals, while this synchrony is disrupted in starved animals. However, it remained to be determined whether the disruption of the synchrony is derived from modulation of the transmitter release from AFD or from the modification of reception or signal transduction in AIY. By performing forward genetics on a transition of thermotaxis behavior along starvation, we revealed that OLA-1, an Obg-like ATPase, functions in AFD to promote disruption of AFD-AIY synchrony and behavioral transition. Our results suggest that the information of hunger is delivered to the AFD thermosensory neuron and gates transmitter release from AFD to disrupt thermotaxis, thereby shedding light onto a mechanism for the integration of environmental and internal state to modulate behavior.

Neural Coding of Thermal Preferences in the Nematode Caenorhabditis elegans.

Animals are capable to modify sensory preferences according to past experiences. Surrounded by ever-changing environments, they continue assigning a hedonic value to a sensory stimulus. It remains to be elucidated however how such alteration of sensory preference is encoded in the nervous system. Here we show that past experiences alter temporal interaction between the calcium responses of sensory neurons and their postsynaptic interneurons in the nematode Caenorhabditis elegansC. elegans exhibits thermotaxis, in which its temperature preference is modified by the past feeding experience: well-fed animals are attracted toward their past cultivation temperature on a thermal gradient, whereas starved animals lose that attraction. By monitoring calcium responses simultaneously from both AFD thermosensory neurons and their postsynaptic AIY interneurons in well-fed and starved animals under time-varying thermal stimuli, we found that past feeding experiences alter phase shift between AFD and AIY calcium responses. Furthermore, the difference in neuronal activities between well-fed and starved animals observed here are able to explain the difference in the behavioral output on a thermal gradient between well-fed and starved animals. Although previous studies have shown that C. elegans executes thermotaxis by regulating amplitude or frequency of the AIY response, our results proposed a new mechanism by which thermal preference is encoded by phase shift between AFD and AIY activities. Given these observations, thermal preference is likely to be computed on synapses between AFD and AIY neurons. Such a neural strategy may enable animals to enrich information processing within defined connectivity via dynamic alterations of synaptic communication.

Age-dependent changes in response property and morphology of a thermosensory neuron and thermotaxis behavior in Caenorhabditis elegans.

Age-dependent cognitive and behavioral deterioration may arise from defects in different components of the nervous system, including those of neurons, synapses, glial cells, or a combination of them. We find that AFD, the primary thermosensory neuron of Caenorhabditis elegans, in aged animals is characterized by loss of sensory ending integrity, including reduced actin-based microvilli abundance and aggregation of thermosensory guanylyl cyclases. At the functional level, AFD neurons in aged animals are hypersensitive to high temperatures and show sustained sensory-evoked calcium dynamics, resulting in a prolonged operating range. At the behavioral level, senescent animals display cryophilic behaviors that remain plastic to acute temperature changes. Excessive cyclase activity of the AFD-specific guanylyl cyclase, GCY-8, is associated with developmental defects in AFD sensory ending and cryophilic behavior. Surprisingly, loss of the GCY-8 cyclase domain reduces these age-dependent morphological and behavioral changes, while a prolonged AFD operating range still exists in gcy-8 animals. The lack of apparent correlation between age-dependent changes in the morphology or stimuli-evoked response properties of primary sensory neurons and those in related behaviors highlights the importance of quantitative analyses of aging features when interpreting age-related changes at structural and functional levels. Our work identifies aging hallmarks in AFD receptive ending, temperature-evoked AFD responses, and experience-based thermotaxis behavior, which serve as a foundation to further elucidate the neural basis of cognitive aging.

Systems and Synthetic microRNA Biology: From Biogenesis to Disease Pathogenesis.

MicroRNAs (miRNAs) are approximately 22-nucleotide-long, small non-coding RNAs that post-transcriptionally regulate gene expression. The biogenesis of miRNAs involves multiple steps, including the transcription of primary miRNAs (pri-miRNAs), nuclear Drosha-mediated processing, cytoplasmic Dicer-mediated processing, and loading onto Argonaute (Ago) proteins. Further, miRNAs control diverse biological and pathological processes via the silencing of target mRNAs. This review summarizes recent findings regarding the quantitative aspects of miRNA homeostasis, including Drosha-mediated pri-miRNA processing, Ago-mediated asymmetric miRNA strand selection, and modifications of miRNA pathway components, as well as the roles of RNA modifications (epitranscriptomics), epigenetics, transcription factor circuits, and super-enhancers in miRNA regulation. These recent advances have facilitated a system-level understanding of miRNA networks, as well as the improvement of RNAi performance for both gene-specific targeting and genome-wide screening. The comprehensive understanding and modeling of miRNA biogenesis and function have been applied to the design of synthetic gene circuits. In addition, the relationships between miRNA genes and super-enhancers provide the molecular basis for the highly biased cell type-specific expression patterns of miRNAs and the evolution of miRNA-target connections, while highlighting the importance of alterations of super-enhancer-associated miRNAs in a variety of human diseases.

Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast.

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

Multistage slow relaxation in a Hamiltonian system: The Fermi-Pasta-Ulam model.

The relaxation process toward equipartition of energy among normal modes in a Hamiltonian system with many degrees of freedom, the Fermi-Pasta-Ulam (FPU) model is investigated numerically. We introduce a general indicator of relaxation σ which denotes the distance from equipartition state. In the time evolution of σ, some long-time interferences with relaxation, named "plateaus," are observed. In order to examine the details of the plateaus, relaxation time of σ and excitation time for each normal mode are measured as a function of the energy density ε0=E0/N. As a result, multistage relaxation is detected in the finite-size system. Moreover, by an analysis of the Lyapunov spectrum, the spectrum of mode energy occupancy, and the power spectrum of mode energy, we characterize the multistage slow relaxation, and some dynamical phases are extracted: quasiperiodic motion, stagnant motion (escaping from quasiperiodic motion), local chaos, and stronger chaos with nonthermal noise. We emphasize that the plateaus are robust against the arranging microscopic state. In other words, we can often observe plateaus and multistage slow relaxation in the FPU phase space. Slow relaxation is expected to remain or vanish in the thermodynamic limit depending on indicators.

MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation.

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.

miR-135b mediates NPM-ALK-driven oncogenicity and renders IL-17-producing immunophenotype to anaplastic large cell lymphoma.

Many transformed lymphoma cells show immune-phenotypes resembling the corresponding normal lymphocytes; thus, they provide a guide for proper diagnosis and present promising routes to improve their pathophysiologic understanding and to identify novel therapeutic targets. However, the underlying molecular mechanism(s) of these aberrant immune-phenotypes is largely unknown. Here, we report that microRNA-135b (miR-135b) mediates nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-driven oncogenicity and empowers IL-17-producing immunophenotype in anaplastic large cell lymphoma (ALCL). NPM-ALK oncogene strongly promoted the expression of miR-135b and its host gene LEMD1 through activation of signal transducer and activator of transcription (STAT) 3. In turn, elevated miR-135b targeted FOXO1 in ALCL cells. miR-135b introduction also decreased chemosensitivity in Jurkat cells, suggesting its contribution to oncogenic activities of NPM-ALK. Interestingly, miR-135b suppressed T-helper (Th) 2 master regulators STAT6 and GATA3, and miR-135b blockade attenuated IL-17 production and paracrine inflammatory response by ALCL cells, indicating that miR-135b-mediated Th2 suppression may lead to the skewing to ALCL immunophenotype overlapping with Th17 cells. Furthermore, antisense-based miR-135b inhibition reduced tumor angiogenesis and growth in vivo, demonstrating significance of this "Th17 mimic" pathway as a therapeutic target. These results collectively illuminated unique contribution of oncogenic kinase-linked microRNA to tumorigenesis through modulation of tumor immune-phenotype and microenvironment.

Autophagy is activated by TGF-beta and potentiates TGF-beta-mediated growth inhibition in human hepatocellular carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell growth, differentiation, and apoptosis of various types of cells. Autophagy is emerging as a critical response of normal and cancer cells to environmental changes, but the relationship between TGF-beta signaling and autophagy has been poorly understood. Here, we showed that TGF-beta activates autophagy in human hepatocellular carcinoma cell lines. TGF-beta induced accumulation of autophagosomes and conversion of microtubule-associated protein 1 light chain 3 and enhanced the degradation rate of long-lived proteins. TGF-beta increased the mRNA expression levels of BECLIN1, ATG5, ATG7, and death-associated protein kinase (DAPK). Knockdown of Smad2/3, Smad4, or DAPK, or inhibition of c-Jun NH(2)-terminal kinase, attenuated TGF-beta-induced autophagy, indicating the involvement of both Smad and non-Smad pathway(s). TGF-beta activated autophagy earlier than execution of apoptosis (6-12 versus 48 h), and reduction of autophagy genes by small interfering RNA attenuated TGF-beta-mediated growth inhibition and induction of proapoptotic genes Bim and Bmf, suggesting the contribution of autophagy pathway to the growth-inhibitory effect of TGF-beta. Additionally, TGF-beta also induced autophagy in some mammary carcinoma cells, including MDA-MB-231 cells. These findings show that TGF-beta signaling pathway activates autophagy in certain human cancer cells and that induction of autophagy is a novel aspect of biological functions of TGF-beta.

Modulation of regulatory factors involved in cholesterol metabolism in response to feeding of pravastatin- or cholesterol-supplemented diet in chickens.

mRNA transcripts encoding multiple proteins from the cholesterol biosynthetic and uptake pathways in livers are controlled by sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound mammalian transcription factor. The aims of the present study were to investigate whether SREBP-2 responds to plasma cholesterol levels and modulates expression of factors involved in the cholesterol metabolism of chickens. Supplementing the diets of chickens with 0.1% pravastatin, a drug used to control hypercholesterolemia, decreased plasma LDL-cholesterol concentrations. It also increased levels of the nuclear forms of SREBP-2 and increased gene expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and low-density lipoprotein receptor (LDLr) in livers, relative to a control group. In contrast, feeding of 3% cholesterol-supplemented diet increased plasma total- and LDL-cholesterol concentrations but decreased levels of nuclear forms of SREBP-2 and reduced gene expression of HMGR and LDLr. However, the LDL-binding activities of chicken liver membranes were not affected by plasma cholesterol concentrations or by hepatic levels of the nuclear form of SREBP-2. LDL-binding proteins were detected as bands of 90 and 515 kDa on a ligand blot and the intensity of these bands was unaffected by pravastatin and cholesterol supplementation. These findings suggest that the cholesterol biosynthetic pathway is regulated by the nuclear form of SREBP-2 in chickens as well as in mammals, but that there may be species-specific differences in the regulatory mechanisms of hepatic cholesterol uptake.