Systemic innate immune response induces death of olfactory receptor neurons in Drosophila.

Neural functions are known to decline during normal aging and neurodegenerative diseases. However, the mechanisms of functional impairment owing to the normal aging of the brain are poorly understood. Previously, we reported that caspase-3-like protease, the protease responsible for inducing apoptosis, is activated in a subset of olfactory receptor neurons (ORNs), especially in Drosophila Or42b neurons, during normal aging. Herein, we investigated the molecular mechanism underlying age-related caspase-3-like protease activation and cell death in Or42b neurons. Gene expression profiling of young and aged fly antenna showed that the expression of antimicrobial peptides was significantly upregulated, suggesting an activated innate immune response. Consistent with this observation, inhibition or activation of the innate immune pathway caused delayed or precocious cell death, respectively, in Or42b neurons. Accordingly, autonomous cell activation of the innate immune pathway in Or42b neurons is not likely required for their age-related death, whereas the systemic innate immune response induces caspase-3-like protease activation in Or42b neurons; this indicated that the death of these neurons is regulated non-cell autonomously. We propose a possible link between the innate immune response and the death of olfactory neurons during normal aging.

Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila.

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

Suppression of a phosphatidylinositol 3-kinase signal by a specific spliced variant of Drosophila PTEN.

Drosophila PTEN (dPTEN) plays indispensable roles in the development of Drosophila melanogaster by controlling cell size and number. Although three potential spliced forms of dPTEN have been isolated, functional distinction among these forms remains elusive. In this study, we demonstrate that all spliced forms of dPTEN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)); however, PI(3,4,5)P(3)-dependent activation of Drosophila Akt is suppressed specifically by one of three spliced forms, dPTEN3. Further, dPTEN3 dramatically changes its expression during the Drosophila development, while the other forms are expressed throughout the development. Our results suggest that dPTEN3 is the predominant spliced form that participates in PI(3,4,5)P(3)-mediated signaling pathways.

Identification and functional characterization of nadrin variants, a novel family of GTPase activating protein for rho GTPases.

Nadrin is a GTPase-activating protein (GAP) for the rho family of GTPases that controls Ca2+-dependent exocytosis in nerve endings. In this study, three novel splice variants of nadrin were identified and the variants were designated as nadrin-102, -104, -116 and -126 according to their relative molecular masses. All nadrin variants share the GAP domain, coiled-coil domain, serine/threonine/proline-rich domain, SH3-binding motif, and a successive repeat of 29 glutamines. Tissue distribution analyses using polyclonal antibodies that can discriminate each variant showed that the expression of nadrin-102, -104 and -116 was dominant in neuronal tissues and correlates well with the differentiation of neurons while nadrin-126 was strongly expressed in embryonic brain. Expression of nadrin-116 in PC12 cells strongly inhibited NGF-dependent neurite outgrowth and this effect was dependent on its GAP activity. In contrast, no significant effect on either cell morphology or neurite outgrowth was observed with other variants. All variants showed punctate appearance throughout the cytoplasm, while the 66-kDa carboxyl-terminal fragment of nadrin-102 and/or nadrin-116 was localized to the nucleus and its nuclear translocation was accelerated by NGF-induced differentiation of the cells. These results suggested that nadrin variants are different in their ability to regulate rho-mediated signaling and that, in addition to being a GTPase-activating protein, nadrin-102 and -116 have other distinct functions in the nucleus of the cell, implying a possible role in the cross-talk between the cytoskeleton and the nucleus.

Characterization of the promoter for the mouse alpha 3 integrin gene.

The alpha 3 beta 1 integrin is an adhesion receptor for extracellular matrix proteins including isoforms of laminin, and the changes of its expression level in various cancer cells are thought to cause their malignant phenotypes. We have cloned an approximately 4 kb DNA fragment of the 5'-flanking region of the murine alpha 3 integrin gene and analyzed its promoter activity. Transfection of MKN1 gastric carcinoma cells with serially truncated segments of the 5'-flanking region linked to a luciferase gene indicated that a 537-bp SalI/SacI fragment upstream of exon 1 was sufficient to promote high level gene expression. By 5'-rapid amplification of cDNA ends (5'-RACE) using a cap site-labeled cDNA library, we determined one major and one minor transcription start sites in this region. The murine alpha 3 integrin gene was found to contain a CCAAT box, but to lack a TATA box. Luciferase assay following transfection with a series of deletion constructs of the SalI/SacI fragment revealed that the sequence between positions -260 and -119 bp (relative to the major transcription start site) is required for efficient transcription in gastric carcinoma cells. The sequence analysis of this segment showed the presence of several consensus sequences for transcription factors including Ets, GATA and MyoD/E-box binding factors. The introduction of mutation in one of the Ets-binding sequences greatly decreased its promoter activity, suggesting that the transcription of the alpha 3 integrin gene in these cells is regulated by the Ets-family of transcription factors.

trans-2,2'-Bi(1-phenyladamantylidene): the most twisted biadamantylidene.

The Grignard coupling of 2,2-dibromo-1-phenyladamantane gave trans-2,2'-bi(1-phenyladamantylidene) (1-Ph). Single-crystal X-ray analysis indicated that 1-Ph has a 23.2 degrees twisted double bond, which is much more distorted than that of parent 2,2'-biadamantylidene (1-H) and that of the ethyl-substituted derivative (1-Et). A cyclic voltammogram showed a reversible electron oxidation wave at 0.87 V vs Fc/Fc(+), which is 0.19 V lower than 1-H, indicating a significant increase in the HOMO energy level due to the distortion. The reaction of 1-Ph with 0.9 equiv of bromine gave an intramolecular Friedel-Crafts alkylation product, while bromination of 1-H and 1-Me has been reported to give a bridged bromonium ion and a rearranged product, 2-(1-methyl-2-adamantylidene)-4-bromotricyclo[5,3,1,0(3.9)]undec-4-ene, respectively.

Regulation of melanoma cell migration and invasion by laminin-5 and alpha3beta1 integrin (VLA-3).

We evaluated the role of soluble factors produced from epidermal cells in melanoma cell motility by using the Boyden chamber chemoinvasion system. The migration of two melanoma cell lines, A375 and Mewo, was potentiated by conditioned media of A431 epidermoid cells in a concentration-dependent manner. The enhancement of A375 melanoma cell motility induced by the conditioned medium was blocked by antibodies against either alpha3 or beta1 integrin subunit. The motility-stimulating activity was recovered in the same fraction as the alpha3 integrin-dependent adhesion-promoting activity in a high-molecular-weight (>200 kDa) fraction on Superose 12 gel chromatography, and adsorbed with an anti-laminin-5 antibody. Purified laminin-5 was capable of potentiating melanoma cell migration as measured in either the chemotaxis assay with a soluble form of laminin-5 or the haptotaxis assay with membranes coated with a mixture of laminin-5 and Matrigel. Furthermore, immobilized laminin-5 induced A375 melanoma cells to secrete matrix metalloproteinase-9 (type IV collagenase) into the culture medium. These results strongly suggest that the interaction of laminin-5 produced in the epidermis with alpha3beta1 integrin on melanoma cells is involved in cell migration, invasion, and degradation of extracellular matrix proteins.

Nucleophilic substitution of alkylchlorodihydro[60]fullerenes: thermodynamic stabilities of alkylated C60 cation intermediates.

Thermodynamic stabilities of alkylated C60 cations (RC60+) were determined based on the activation free energies of SN1 solvolysis of the title compounds, indicating that these cations have stabilities comparable to that of the tert-butyl cation.

Orbital Interactions and Solvent Effects Determining the Stability of Condensed Cyclopentadienides in Solution.

The structure and the stability of various cyclopentadienides, which involve 6pi, 10pi, 14pi, and 22pi electrons, are investigated from computations at various levels of theory as well as from orbital interaction analyses. The reason that some of the cyclopentadienides are stabilized and others are destabilized by the introduction of aromatic rings is discussed in terms of absolute hardness and orbital interaction. Cyclopentadienide, a special 6pi-electron system, has the largest value of absolute hardness among the condensed cyclopentadienides investigated; thus this carbanion resists both oxidation and reduction most strongly. The absolute hardness decreases when aromatic rings are introduced to cyclopentadienide to form condensed cyclopentadienides, depending on the way they are connected. Computed values of the ionization potential and oxidation potentials measured in solution have a linear correlation within isomers of the same size, but are not in agreement for different sets of isomers. Solvent effects on the ionization potential are assessed by performing self-consistent reaction field calculations, the results being in excellent agreement with experiments. It is demonstrated that the solvent effects are significant in small cyclopentadienides of 6pi- and 10pi-electron systems, compared to larger ones and that addition of condensed aromatic rings intrinsically stabilizes the formed condensed cyclopentadienides with respect to ionization potential.