Yuko Matsuo

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray1

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display‐positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5‐trisphosphate (IP3) 5‐phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large‐type Kenyon cells, suggesting that IP3‐mediated Ca2+ signaling is enhanced in these neurons.

Gene expression of ecdysteroid-regulated gene E74 of the honeybee in ovary and brain

To facilitate studies of hormonal control in the honeybee (Apis mellifera L.), a cDNA for a honeybee homologue of the ecdysteroid‐regulated gene E74 (AmE74) was isolated and its expression was analysed. Northern blot analysis indicated strong expression in the adult queen abdomen, and no significant expression in the adult drone and worker abdomens. In situ hybridization demonstrated that this gene was expressed selectively in the ovary and gut in the queen abdomen. Furthermore, this gene was also expressed selectively in subsets of mushroom body interneurones in the brain of the adult worker bees. These findings suggest that AmE74 is involved in neural function as well as in reproduction in adult honeybees.

Prepro-tachykinin gene expression in the brain of the honeybee Apis mellifera

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization. The prepro-AmTRP gene is expressed predominantly in the MBs and in some neurons located in the optic and antennal lobes. cDNA microarray studies have revealed that AmTRP expression is enriched in the MBs compared with other brain regions. There is no difference in AmTRP-expressing cells among worker, queen, and drone brains, suggesting that the cell types that express the prepro-AmTRP gene do not change according to division of labor, sex, or caste. The unique expression pattern of the prepro-AmTRP gene suggests that AmTRPs function as neuromodulators in the MBs of the honeybee brain.

Discovery of Mitocryptide-1, a Neutrophil-activating Cryptide from Healthy Porcine Heart

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing β-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and Gi2-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce β-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.

CLAC-P/Collagen Type XXV Is Required for the Intramuscular Innervation of Motoneurons during Neuromuscular Development

Formation of proper neuromuscular connections is a process coordinated by both motoneuron-intrinsic and target-dependent programs. Under these programs, motoneurons innervate target muscles, escape programmed cell death during fetal development, and form neuromuscular junctions (NMJ). Although a number of studies have revealed molecules involved in axon guidance to target muscles and NMJ formation, little is known about the molecular mechanisms linking intramuscular innervation and target-derived trophic factor-dependent prevention of motoneuron apoptosis. Here we studied the physiological function of CLAC-P/collagen XXV, a transmembrane-type collagen originally identified as a component of senile plaque amyloid of Alzheimers disease brains, by means of generating Col25a1-deficient (KO) mice. Col25a1 KO mice died immediately after birth of respiratory failure. In Col25a1 KO mice, motor axons projected properly toward the target muscles but failed to elongate and branch within the muscle, followed by degeneration of axons. Failure of muscular innervation in Col25a1 KO mice led to excessive apoptosis during development, resulting in almost complete and exclusive loss of spinal motoneurons and immaturity in skeletal muscle development. Bax deletion in Col25a1 KO mice rescued motoneurons from apoptosis, although motor axons remained halted around the muscle entry site. Furthermore, these motoneurons were positive for phosphorylated c-Jun, an indicator of insufficient supply of target-derived survival signals. Together, these observations indicate that CLAC-P/collagen XXV is a novel essential factor that regulates the initial phase of intramuscular motor innervation, which is required for subsequent target-dependent motoneuron survival and NMJ formation during development.

Isolation and Identification of Novel Neutrophil-Activating Cryptides Hidden in Mitochondrial Cytochrome c

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on β-hexosaminidase (β-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced β-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

P1-114: Pathological effects of CLAC in the brains of APP/CLAC-P double transgenic mice

Massive deposition of senile plaques (SP) in neocortices and hippocampus is the hallmark pathological lesion in Alzheimer’s disease (AD). SP are composed of amyloid fibrils formed by amyloid beta peptide (A ). We have identified CLAC (collagenous Alzheimer amyloid plaque component) in amyloid fractions of cortices from patients with AD. CLAC co-deposits with A in SP amyloid, especially in primitive-type SP in AD brains, whereas amyloid cores, vascular amyloid deposits or diffuse type plaques are CLAC-negative. We also reported that recombinant CLAC binds to fibrillized, but not soluble form of A and that recombinant CLAC inhibits the elongation phase of -amyloid fibril formation in vitro. To elucidate the pathological effects of CLAC in vivo, we generated transgenic (TG) mice overexpressing CLAC-P (CLAC precursor protein) in neurons under the control of Thy1.2 promoter. In the brains of CLAC-P TG mice, CLAC-P was expressed in neurons and extracellular deposits comprised of CLAC (i.e., extracellular fragment of CLAC-P) were observed in the neocortical neuropil. We next crossed CLAC-P TG mice with APP TG mice (i.e., J20 and TgCRND8). In the neocortices and hippocampus of the double TG mice, -amyloid plaques were strongly positive for CLAC, whereas vascular amyloid deposits. In 12-month-old double TG mice, diffuse-type plaques and huge cored plaques were markedly decreased compared to the APP TG mice, whereas compact or multi-cored plaques of medium size were predominant. The amyloid burden (i.e., percent A -positive area) in the brains of double TG mice were reduced by 30% (TgCRND8) or 50% (J20) compared to those in the littermate APP TG mice, although the levels of SDS-insoluble A quantitated by ELISA, as well as the protein expression of APP, were similar between APP TG and double TG mice. These observations suggest that the binding of CLAC to A may modify the process of -amyloid deposition.