Posterior structural brain volumes differ in maltreated youth with and without chronic posttraumatic stress disorder.

Magnetic resonance imaging studies of maltreated children with posttraumatic stress disorder (PTSD) suggest that maltreatment-related PTSD is associated with adverse brain development. Maltreated youth resilient to chronic PTSD were not previously investigated and may elucidate neuromechanisms of the stress diathesis that leads to resilience to chronic PTSD. In this cross-sectional study, anatomical volumetric and corpus callosum diffusion tensor imaging measures were examined using magnetic resonance imaging in maltreated youth with chronic PTSD (N = 38), without PTSD (N = 35), and nonmaltreated participants (n = 59). Groups were sociodemographically similar. Participants underwent assessments for strict inclusion/exclusion criteria and psychopathology. Maltreated youth with PTSD were psychobiologically different from maltreated youth without PTSD and nonmaltreated controls. Maltreated youth with PTSD had smaller posterior cerebral and cerebellar gray matter volumes than did maltreated youth without PTSD and nonmaltreated participants. Cerebral and cerebellar gray matter volumes inversely correlated with PTSD symptoms. Posterior corpus callosum microstructure in pediatric maltreatment-related PTSD differed compared to maltreated youth without PTSD and controls. The group differences remained significant when controlling for psychopathology, numbers of Axis I disorders, and trauma load. Alterations of these posterior brain structures may result from a shared trauma-related mechanism or an inherent vulnerability that mediates the pathway from chronic PTSD to comorbidity.

A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis.

In insects, the neuropeptide prothoracicotropic hormone (PTTH) stimulates production of ecdysone (E) in the prothoracic glands (PGs). E is the precursor of the principal steroid hormone, 20-hydroxyecdysone (20E), that is responsible for eliciting molting and metamorphosis. In this study, we used quantitative phosphoproteomics to investigate signal transduction events initiated by PTTH. We identified Spook (CYP307A1), a suspected rate-limiting enzyme for E biosynthesis, and components of the mitogen-activated protein kinase (MAPK) pathway, as major phosphorylation targets of PTTH signaling. Further, proteins not previously linked to PTTH and ecdysone biosynthesis were identified as targets of PTTH signaling. These include proteins involved in signal transduction, endosomal trafficking, constituents of the cytoskeleton and regulators of transcription and translation. Our screen shows that PTTH likely stimulates E production by activation of Spook, an integral enzyme in the E biosynthetic pathway. This directly connects PTTH signaling to the pathway that produces E. A new mechanism for regulation of E biosynthesis in insects is proposed.

Manduca sexta prothoracicotropic hormone: evidence for a role beyond steroidogenesis.

Prothoracicotropic hormone (PTTH) is a homodimeric brain peptide hormone that positively regulates the production of ecdysteroids by the prothoracic gland of Lepidoptera and probably other insects. PTTH was first purified from heads of adult domestic silkworms, Bombyx mori. Prothoracic glands of Bombyx and Manduca sexta undergo apoptosis well before the adult stage is reached, raising the recurring question of PTTH function at these later stages. Because Bombyx has been domesticated for thousands of years, the possibility exists that the presence of PTTH in adult animals is an accidental result of domestication for silk production. In contrast, Manduca has been raised in the laboratory for only five or six decades. The present study found that Manduca brains contain PTTH at all stages examined post-prothoracic gland apoptosis, i.e., pharate adult and adult life, and that PTTH-dependent changes in protein phosphorylation and protein synthesis were observed in several reproductive and reproduction-associated organs. The data indicate that PTTH indeed plays a role in non-steroidogenic tissues and suggest possible future avenues for determining which cellular processes are being so regulated.

Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera.

Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each is expressed at all stages of development and shows no closely related homolog in their respective genomes. In contrast, we show here that several dipteran genomes encode two novel, highly related, microsomal P450 enzymes, Cyp307A1 and Cyp307A2, that likely participate as stage-specific components of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development.

Protein kinase C modulates ecdysteroidogenesis in the prothoracic gland of the tobacco hornworm, Manduca sexta.

The prothoracic gland is the primary source of ecdysteroid hormones in the immature insect. Ecdysteroids coordinate gene expression necessary for growth, molting and metamorphosis. Prothoracicotropic hormone (PTTH), a brain neuropeptide, regulates ecdysteroid synthesis in the prothoracic gland. PTTH stimulates ecdysteroid synthesis through a signal transduction cascade that involves at least four protein kinases: protein kinase A (PKA), p70 S6 kinase, an unidentified tyrosine kinase, and the extracellular signal-regulated kinase (ERK). In this report, the participation of protein kinase C (PKC) in PTTH signalling is demonstrated and characterized. PTTH stimulates PKC activity through a PLC and Ca(2+)-dependent pathway that is not cAMP regulated. Inhibition of PKC inhibits PTTH-stimulated ecdysteroidogenesis as well as PTTH-stimulated phosphorylation of ERK and its upstream regulator, MAP/ERK kinase (MEK). These observations reveal that the acute regulation of prothoracic gland steroidogenesis is dependent on a web of interacting kinase pathways, which probably converge on factors that regulate translation.

Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta.

The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.

Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis.

The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active insect molting hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.

Insulin signaling is necessary for vitellogenesis in Drosophila melanogaster independent of the roles of juvenile hormone and ecdysteroids: female sterility of the chico1 insulin signaling mutation is autonomous to the ovary.

It has been suggested that insulin signaling mutations of Drosophila melanogaster are sterile and long-lived because of juvenile hormone (JH) and ecdysteroid deficiency. However, female sterility of an insulin/IGF-like signaling mutant (chico(1)) of D. melanogaster is not mediated by downstream systemic signaling in terms of major alterations in JH or ecdysteroid levels. chico(1) is a null mutation in the insulin substrate protein (CHICO) gene of D. melanogaster. Homozygous chico(1) females are sterile and their oocytes do not mature beyond the last previtellogenic stage. Homozygous chico(1) females exhibit approximately wild-type rates of JH biosynthesis, ovarian release of ecdysteroids and haemolymph ecdysteroid levels, suggesting that these two major hormone systems play no role in producing the sterility. Previtellogenic wild-type ovaries transplanted into homozygous chico(1) females underwent vitellogenesis, showing that systemic factors present in mutant females are sufficient to support normal vitellogenesis. chico(1) ovaries transplanted into wild-type females did not undergo vitellogenesis indicating that CHICO is necessary in the ovary for vitellogenic maturation. The ovary transplant experiments corroborate the endocrine results and demonstrate that insulin/insulin-like signaling (IIS) is necessary for vitellogenesis even when sufficient levels of JH, ecdysteroids or other factors are present.

Ca2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: evidence for mobilization and entry mechanisms.

Prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.

Control and biochemical nature of the ecdysteroidogenic pathway.

Molting is elicited by a critical titer of ecdysteroids that includes the principal molting hormone, 20-hydroxyecdysone (20E), and ecdysone (E), which is the precursor of 20E but also has morphogenetic roles of its own. The prothoracic glands are the predominate source of ecdysteroids, and the rate of synthesis of these polyhydroxylated sterols is critical for molting and metamorphosis. This review concerns three aspects of ecdysteroidogenesis: (a) how the brain neuropeptide prothoracicotropic hormone (PTTH) initiates a transductory cascade in cells of the prothoracic gland, which results in an increased rate of ecdysteroid biosynthesis (upregulation); (b) how the concentrations of 20E in the hemolymph feed back on the prothoracic gland to decrease rates of ecdysteroidogenesis (downregulation); and (c) how the prothoracic gland cells convert cholesterol to the precursor of E and then 20E, a series of reactions only now being understood because of the use of a combination of classical biochemistry and molecular genetics.