Table of contents

The multiple adenylate cyclase isoforms of Drosophila

A novel isoform of adenylyl cyclase, DAC78C, has been identified in Drosophila that encodes two structurally distinct proteins in a developmentally restricted manner. The protein corresponding to one transcript is potently activated by G protein and protein kinase C and is expressed ubiquitously. The protein corresponding to the second transcript is expressed in a dynamic pattern in gastrulation stage embryos; it is restricted to the cephalic furrow and dorsal transverse folds, active regions of cell movement of unknown function in the Drosophila embryo. It is proposed that DAC78C and the cAMP pathway play an important role in directing these morphogenetic movements: this gene may provide clues to the functional significance of these structures in gastrulation (Cann, 2000a).

The hydrophobicity profile of the ORF has revealed a proposed structure resembling the known transmembrane adenylyl cyclase (tmAC) isoforms, consisting of 12 putative transmembrane domains; homology searches have identified two presumptive catalytic domains. This novel Drosophila tmAC displays additional structural characteristics distinct from other known tmACs. The ORF has a large, approximately 100 amino acid extracellular loop between transmembrane domains 9 and 10 containing four potential N-linked glycosylation sites. The ORF also has a 300 amino acid long intracellular N terminal domain of unknown function. Both regions are considerably longer than the analogous domains in previously identified mammalian and Drosophila tmACs (Cann, 2000a).

To determine whether DAC78C is the ortholog of a previously identified mammalian AC gene extensive phylogenetic analysis was performed. CAC79C is not a clear ortholog of any previously identified mammalian AC. DAC78C is the sole member of a distinct evolutionary clade. Other Drosophila tmAC isoforms have clear mammalian orthologs based on similar analysis; Rutabaga is orthologous to bovine AC1 (38.3% similar), DAC39E is orthologous to rat AC3 (33.4% similar), and DAC9 is orthologous to mouse AC9 (28.7% similar). In each case the mammalian tmAC is more similar to its Drosophila ortholog than it is to the other mammalian isoforms. Among the known mammalian tmACs, DAC78C is most similar to rat AC8 (30.4% similar) and rat AC5 (29.1% similar); however, rat AC8 and rat AC5 are more similar to other mammalian tmACs than to DAC78C, making relationships based purely on primary sequence data problematic. Another Drosophila tmAC has been identified, DAC76E, corresponding to mammalian tmAC types 2, 4, and 7. Drosophila therefore possesses a single tmAC isoform corresponding to each clade of related mammalian tmACs: Rutabaga with AC1, AC5, and AC6; DAC39E with AC3; DAC9 with AC9, and DAC76E with AC2, AC4, and AC7. In contrast DAC78C is the sole member of a distinct clade among the higher eukaryotic tmACs (Cann, 2000a).

The larger, approximately 6.5 kb transcript is ubiquitously expressed in embryos, larvae, and adults, and the smaller, approximately 4.5 kb transcript is present, seemingly, only in embryos. Using developmentally staged embryonic total RNA, the 6.5 kb transcript has been shown to be ubiquitous from hour 8 of embryogenesis while the 4.5 kb transcript is expressed specifically in 2-4 h embryos. To characterize the molecular difference between the 6.5 and 4.5 kb transcripts, the genomic structure of the entire DAC78C locus was solved. The 4.5 kb transcript encodes an N terminal truncation of the full-length DAC78C protein, missing transmembrane domains 1-6. The 6.5 kb transcript was thereafter denoted DAC78C L, and the 4.5 kb transcript was denoted DAC78C S. Transmembrane domains 1-6 are intact in the DAC78C L transcript. Despite possessing two catalytic domains, which have been shown to be both necessary and sufficient for catalytic activity, AC activity could not be demonstrated for DAC78C S using a variety of heterologous expression systems (Cann, 2000a).

The DAC78C L transcript is expressed ubiquitously in late embryos. DAC78C S, which is temporally restricted to the earliest (2-4) hours of embryogenesis, is first detected at stage 4 just prior to cellularization, throughout the embryo. Stage 4 corresponds approximately to a 2-h-old embryo, and its initial appearance at this stage implies that DAC78C S is zygotically transcribed rather then maternally deposited in the embryo. As the embryo cellularizes, DAC78C S mRNA levels rise and become excluded from the anterior and posterior poles. As germ band extension commences, expression decreases along the entire ventral surface of the embryo and is restricted to the CF and DTFs, active regions of cell involution of unknown function. By late stage 7, strong expression of DAC78C S is present only in the CF and DTFs on the dorsal and lateral surfaces of the embryo. A view of the dorsal surface of a late stage 7 embryo demonstrates the restriction of mRNA expression to the cephalic furrow (CF) and dorsal transverse folds (DTFs). Expression in the CF persists longer dorsally than ventrally. This may reflect distinct mechanisms that control normal CF formation and DAC78C S expression dorsally and laterally. At stage 8, corresponding to an approximately 4-h-old embryo, DAC78C S mRNA is no longer detectable. Thus DAC78C S represents the first example of an AC specifically expressed during development. The function of these gastrulating regions and the mechanisms that direct these morphogenetic movements are unknown (Cann, 2000a).

The cloning and characterization of a new gene family of adenylyl cyclase related genes in Drosophila is described. The five adenylyl cyclase-like genes that define this family are clearly distinct from previously known adenylyl cyclases. One member forms a unique locus on chromosome 3 whereas the other four members form a tightly clustered, tandemly repeated array on chromosome 2. The genes on chromosome 2 are transcribed in the male germline in a doublesex dependent manner and are expressed in postmitotic, meiotic, and early differentiating sperm. These genes therefore provide the first evidence for a role for the cAMP signaling pathway in Drosophila spermatogenesis. Expression from this locus is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required for the G2/M transition of meiosis I during spermatogenesis, implying a mechanism for the coordination of differentiation and proliferation. Evidence is also provided for positive selection at the locus on chromosome 2, which suggests this gene family is actively evolving and may play a novel role in spermatogenesis (Cann, 2000b).

cDNAs have been isolated that correspond to two previously identified transmembrane adenylyl cyclase (tmAC) related genomic DNA fragments, AC34A and AC62D. Their predicted protein products are highly related to each other, and closely related to known mammalian and Drosophila tmACs. PCR with degenerate primers designed to selectively amplify similar genes has identified two additional highly related sequences that were subsequently cloned. The four genes have been renamed Drosophila AC 'X' (DACX)A (formerly called AC62D); DACXB (formerly called AC34A); DACXC, and DACXD, to reflect the fact that they are a related family of genes distinct from the known tmACs. The reconstructed full-length cDNA clones each possess a single open reading frame (ORF) homologous to previously characterized mammalian and Drosophila tmACs. The hydrophobicity profiles of each ORF reveal a proposed structure resembling the known tmAC isoforms consisting of 12 putative transmembrane domains; homology searches have identified two presumptive catalytic domains. To determine whether DACXA-D are orthologs of any previously identified mammalian AC gene phylogenetic analysis was performed using the complete amino acid sequences. The phylogenetic tree reveals that the DACX ORFs represent a novel gene family distinct from any previously identified tmACs (70% similar to one another compared to 25%-35% similarity to all other tmACs in the catalytic regions). The proteins are only distantly related to Drosophila Rutabaga (E-value = e-42). Examination of the Caenorhabditis elegans genome sequence and the GenBank Expressed Sequence Tag database does not reveal any likely orthologous sequences. Comparison of the amino acid sequences of DACXA-D with the known tmACs reveals that all residues shown by the tmAC crystal structure to be essential for catalytic activity are intact; however, contact residues for Gsalpha.GTPgammaS and forskolin are not conserved (Cann, 2000b).

Since DACXA and DACXB had been previously cytologically localized to polytene bands 62D and 34A, respectively, P1 clones were obtained that encompassed these loci: 62D (DS00542) and 34A (DS03829). Surprisingly, DACXC also resides on P1 DS03829 (34A) and DACXD on P1 DS00542 (62D). Further analysis reveals that DACXA is also found on P1 DS03829 (34A) and not at 62D, as previously described. This false cytology is thought to reflect the extensive cross-hybridization observed between the DACX family members. The intron/exon structures of the loci at 34A and 62D were mapped by a combination of subcloning, PCR, and DNA sequence analysis. These analyses have revealed a fifth member of the gene family, DACXE, which resides on P1 DS03829 (34A). A putative ORF was reconstructed for DACXE that demonstrates that it is highly related to the other DACX family members. This gene was not analyzed further. DACXD is found as a single gene at 62D whereas DACXA, DACXB, DACXC, and DACXE form a tandem array of directly repeated genes at 34A with 39 bp (DACXC to DACXB), 84 bp (DA-CXB to DACXA), and 166 bp (DACXA to DACXE) between transcription units. All 11 intron/exon boundaries are conserved between the DACX family members at 34A although there is some variation in intron size. Five of these intron/exon boundaries are also conserved with DACXD (Cann, 2000b).

The expression profiles of DACXA-D were examined by Northern blotting of developmentally staged RNA. DACXD is expressed predominantly during late embryogenesis, in late larvae, and in male and female adults. In contrast, DACXA, DACXB, and DACXC, which reside in the tandem array at 34A, are expressed at low levels in late larvae, increase during the pupal stage, and exclusively in adult males. Presumably the expression in late larvae and pupae is restricted to males of these mixed populations of males and females. Spatial expression of DACXA, DACXB, and DACXC mRNA was examined by in situ hybridization to whole Drosophila testes with antisense riboprobes. The expression profile is similar for all three genes. Expression is absent in the stem cells at the distal tip of the testis and in the mitotic cells that undergo four rounds of division within somatically derived cyst cells. Expression is first evident in prophase pre-meiotic spermatocytes that undergo considerable growth before meiosis. mRNA is detectable throughout meiosis and in early haploid postmeiotic spermatids (Cann, 2000b).

Although cAMP has been known to play an essential role in fruit fly oogenesis, these AC-like genes provide the first evidence for a potential role of cAMP signaling in Drosophila spermatogenesis. Developing sperm undergo a number of predefined developmental processes that are tightly controlled. Perhaps the best described of these processes is the checkpoint that controls the transition through the G2/M phase of meiosis I. aly is the master gene essential for the normal function of can, sa, mia, and the transcriptional activation of the cdc25 phosphatase ortholog, twine. The expression of DACXA, DACXB, and DACXC is downregulated in adult males mutant for aly, can, mia, or sa. The control of the DACX genes by the G2/M checkpoint is likely to provide a powerful mechanism for coordinating the precise requirements for cell cycle progression with postmeiotic differentiation. Mammals also possess a distinct AC-related gene, sAC, which is specifically expressed in the male germline. Previous studies have demonstrated that dsx in Drosophila and mab-3, a dsx homolog in C. elegans, direct the transcription of yolk proteins that are entirely distinct between species. The DACX family and sAC may also represent convergent evolution for a specific and novel role for cAMP in Drosophila and mammalian spermatogenesis (Cann, 2000b).

Adenylate cyclase structure and kinetics

The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2'-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2'-deoxyadenosine per C1/C2 heterodimer. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PPi). A substrate analog, Ap(CH2)pp (alpha,beta-methylene adenosine 5'-triphosphate), and cyclic AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate binding (in the absence of PPi). Kinetic analysis indicates that the release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which kinetic support is provided. It is proposed that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPi is slow and partially rate-limiting (Dessauer, 1997).

Mammalian adenylyl cyclases contain two conserved regions, C1 and C2, which are responsible for forskolin- and G-protein-stimulated catalysis. The structure of the C2 catalytic region of type II rat adenylyl cyclase has an alpha/beta class fold in a wreath-like dimer, which has a central cleft. Two forskolin molecules bind in hydrophobic pockets at the ends of cleft. The central part of the cleft is lined by charged residues implicated in ATP binding. Forskolin appears to activate adenylyl cyclase by promoting the assembly of the active dimer and by direct interaction within the catalytic cleft. Other adenylyl cyclase regulators act at the dimer interface or on a flexible C-terminal region (Zhang, 1997).

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K+ channels. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. Nothing is known of the function or organization of the 12 transmembrane segments. In the present study a range of strategies is adopted to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains -- largely independent of any cytosolic region -- formed a tight complex that is delivered to the plasma membrane. This cooperation between the two intact transmembrane domains is essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, is also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell (Gu, 2001).

Adenylate cyclases in different species: Identification and tissue distribution

Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis, a mutant has been isolated that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus reveals a new gene, DdMYB2, whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues the differentiation and morphogenesis of Ddmyb2-null mutants. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP (Otsuka, 1998).

The role of the adenylyl cyclase ACA in Dictyostelium discoideum chemotaxis and streaming was studied. In this process, cells orient themselves in a head to tail fashion as they are migrating to form aggregates. Cells lacking ACA are capable of moving up a chemoattractant gradient, but are unable to stream. Imaging of ACA-YFP reveals plasma membrane labeling highly enriched at the uropod (a slender posterior appendage) of polarized cells. This localization requires the actin cytoskeleton but is independent of the regulator CRAC and the effector PKA. A constitutively active mutant of ACA shows dramatically reduced uropod enrichment and has severe streaming defects. It is proposed that the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant, thereby providing a unique mechanism to amplify chemical gradients. This could represent a general mechanism that cells use to amplify chemotactic responses (Kriebel, 2003).

A variety of extracellular signals leads to the accumulation of cAMP, which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA- mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI), a novel adenylyl cyclase gene, acrA, was discovered. acrA is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA- mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation; plateaus during the slug stage, and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA- cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk (Söderbom, 1999).

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA is highly active during aggregation, with negligible activity in the slug stage. ACG activity is not present at significant levels until mature spores have formed. ACB activity increases strongly after slugs have formed with optimal activity at early fruiting body formation. The same high activity is observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it is not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation (Meima, 1999).

Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. This prediction is tested directly in this study. During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. Twenty-six genes required for Drosophila melanogaster sleep were identified. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, a conserved genetic pathway was delineated by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. The comprehensive analysis presented in this study suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry (Singh, 2014).

There are at least six mammalian adenylcyclases. Type I, to which rutabaga is homologous, is a Calmodulin-sensitive form found in the brain and other tissues. Calmodulin is a ubiquitious receptor of Ca++ that senses the intracellular Ca++ concentration: based on these levels it will bind to and activate other proteins. Types II, III and IV are found in brain and lung, olfactory tissue and testis respectively. All these are Calmodulin insensitive. Each of these proteins share a similar predicted structure. Each has two clusters of six transmembrane segments separated by two homologous cytoplasmic domains. Sequences within each cytoplasmic domain display significant homology with the catalytic portion of guanylyl cyclases, suggesting their involvement in cyclase activity. Two large cyclase domains have been found in Rutabaga with homology to bovine type I cyclase (Levin, 1992).

The distribution of type I calmodulin-sensitive adenylyl cyclase in bovine and rat tissues was examined by northern blot analysis and in situ hybridization. Messenger RNA for type I adenylyl cyclase is found only in brain, retina, and adrenal medulla, suggesting that this enzyme is neural specific. In situ hybridization studies using bovine, rabbit, and rat retina indicate that mRNA for type I adenylyl cyclase is found in all three nuclear layers of the neural retina and is particularly abundant in the inner segment of the photoreceptor cells. The neural-specific distribution of type I adenylyl cyclase mRNA and its restricted expression in areas of brain implicated in neuroplasticity are consistent with the proposal that this enzyme plays an important role in various neuronal functions, including learning and memory (Xia, 1993).

The distribution of mRNA encoding the Calmodulin-sensitive Adenylate cyclase in rat brain have been examined by in situ hybridization. mRNA for this enzyme is expressed in specific areas of brain that have been implicated in learning and memory, including the neocortex, the hippocampus, and the olfactory system. The presence of mRNA for this enzyme in the pyramidal and granule cells of the hippocampal formation provides evidence that it is found in neurons. These data are consistent with the proposal that the Calmodulin-sensitive Adenylate cyclase plays an important role in learning and memory (Xia, 1991).

Anchoring of adenylate cyclase

Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Evidence is provided for interactions between AKAP79/150 and AC isoenzymes ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production (Bauman, 2006).

Mutation of adenylate cyclase

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. Expression of a modified acaA gene rescues acrA- mutant cells, indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive, acrA was disrupted in a acaA- PKA-Cover strain. When developed at high cell densities, acrA- acaA- PKA-Cover cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high (Anjard, 2001).

Mutant mice in which type I Ca(2+)-sensitive adenylyl cyclase has been inactivated by targeted mutagenesis show deficient spatial memory and altered long term potentiation. Long term potentiation in the CA1 region of the rat hippocampus develops during the first 2 weeks after birth and reaches maximal expression at postnatal day 15 with a gradual decline at later stages of development. Ca(2+)-stimulated adenylyl cyclase activity in rat hippocampus, cerebellum, and cortex increases significantly between postnatal days 1-16. This increase appears to be due to enhanced expression of type I adenylyl cyclase rather than type VIII adenylyl cyclase, the other adenylyl cyclase that is directly stimulated by Ca2+ and calmodulin. Type I adenylyl cyclase mRNA in the hippocampus increases 7-fold during this developmental period. The developmental expression of Ca(2+)-stimulated adenylyl cyclase activity in mouse brain is attenuated in mutant mice lacking type I adenylyl cyclase. Changes in expression of the type I adenylyl cyclase during the period of long term potentiation development are consistent with the hypothesis that this enzyme is important for neuroplasticity and spatial memory in vertebrates (Villacres, 1995).

The somatosensory (SI) cortex of mice displays a patterned, nonuniform distribution of neurons in layer IV termed the 'barrelfield' because of the barrel shapes formed by cylindrical arrays of neurons within the region. Thalamocortical afferents (TCAs) that terminate in layer IV are segregated such that each 'barrel' (the readily visible cylindrical array of neurons surrounding a cell-sparse center) represents a distinct receptive field. TCA arbors are confined to the hollow part of the so-called barrel; the arbors synapse on barrel-wall neurons whose dendrites are oriented toward the center of the barrel. Mice homozygous for the barrelless (brl) mutation, which occurred spontaneously in ICR stock at Universite de Lausanne (Switzerland), fail to develop this patterned distribution of neurons, but still display normal topological organization of the SI cortex. Despite the absence of barrels in these mutants, and the overlapping zones of TCA arborization, the size of individual whisker representations, as judged by 2-deoxyglucose uptake, is similar to that of wild-type mice. Adenylyl cyclase type I (Adcy1) has been identified as the gene disrupted in brl mutant mice, by using fine mapping of proximal chromosome 11, enzyme assay, mutation analysis and by examining mice homozygous for a targeted disruption of Adcy1. These results provide the first evidence for involvement of cAMP signaling pathways in brain pattern formation, and they suggest that an activity dependent process is involved in the wiring of the barrel cortex (Abdel-Majid, 1998).

Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice are indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, are reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Allodynia is the inflammation-related behavioral sensitization to a non-noxious stimulus. Intra-ACC administration of forskolin rescues behavioral allodynia defective in the AC1&8 DKO mice. These studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia (Wei, 2002).

The ACC plays important roles in the cognitive, motor, and emotional functions of the brain. It has been suggested to contribute to the perception of pain, to the learning processes associated with the prediction and avoidance of noxious sensory stimuli, as well as to pathological phantom pain. Recent studies from animals and humans demonstrate that ACC neurons play key roles in behavioral nociceptive responses to injury in animals and pain perception or unpleasantness in humans. In humans, results from electrophysiological recordings from the ACC and functional imaging studies show that the ACC neurons respond to noxious stimuli. In animals, ACC neurons respond to peripheral noxious stimuli or electrical shocks at high intensities. It is proposed that activity in the ACC may underlie the unpleasantness or discomfort associated with some somatosensory stimuli. Consistently, lesions in the ACC can reduce chronic pain in patients. In animal models of acute pain and persistent pain, lesions of the ACC produce antinociceptive effects. In freely moving animals, local administration of various opioid receptor agonists in the ACC produces powerful antinociceptive effects. In the present studies, it was found that AC1 and AC8 both are highly expressed in the ACC neurons and that genetic deletion of AC1 and AC8 leads to a complete abolishment of the behavioral allodynia caused by tissue injury and inflammation. Consistent with these findings, behavioral nociceptive responses in the formalin test were also significantly reduced. These results support a role for the ACC in the processing of pain-related information. Behavioral responses to acute noxious stimuli are normal in these mutant mice. This strongly suggests that AC1 and AC8 are selectively involved in mediating the behavioral responses to injury. These results are consistent with findings from in vitro brain slices that the activity of adenylyl cyclases is primarily required for plastic changes, while basal synaptic transmission is unaffected by deletion of AC1 and AC8. The pharmacological rescue of behavioral allodynia by local forskolin microinjection into the ACC provides further evidence for an important role of the ACC in persistent pain (Wei, 2002).

What is the possible synaptic mechanism for the action of AC1 and AC8 within the ACC? It is thought that activation of adenylyl cyclases in the ACC may lead to long-lasting changes in synaptic transmission. Glutamate is a major fast excitatory transmitter within the ACC. Theta burst stimulation causes long-term potentiation of synaptic responses in ACC slices from adult mice. The potentiation is completely absent in mice lacking both AC1 and AC8, suggesting that Ca2+-CaM sensitive adenylyl cyclases are important for synaptic potentiation. cAMP clearly contributes to the synaptic potentiation observed 5-40 min after the induction. These results suggest that the enhancement of synaptic responses within the ACC may serve as a synaptic mechanism contributing to injury-related behavioral sensitization. The possible presynaptic effects of forskolin within the ACC cannot be ruled out. It is quite possible that both pre- and postsynaptic changes occur within the ACC during forskolin treatment. Future studies are clearly needed to dissect the detailed synaptic mechanisms within the ACC (Wei, 2002).

Evidence is presented that AC1 and AC8 are important for CREB activation following tissue injury and inflammation in the ACC and insular cortex. Deletion of AC1 or AC8 causes significant reduction of CREB activated by inflammation. Interestingly, no further reduction was found in the AC1&8 DKO mice. Furthermore, injury-triggered CREB activation is not completely blocked in any mice, suggesting that other signaling pathways also contribute to CREB activation in the forebrain. These findings are slightly different from those in the spinal cord. In spinal cord dorsal horn, injury-induced activation of CREB is completely blocked in AC1&8 DKO mice. Future studies are needed to identify other signaling molecules for injury-related CREB activation in the ACC and insular cortex. In both the spinal cord and ACC, signaling molecules downstream of activated CREB remain to be identified (Wei, 2002).

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they 'capacitate,' i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, since the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but these 'rescued' null sperm are not hyperactive, display flagellar angulation, and remain unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, a specific sAC inhibitor was identified that allows temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC (Hess, 2005).

The vertebrate olfactory bulb is a remarkably organized neuronal structure, in which hundreds of functionally different sensory inputs are organized into a highly stereotyped topographical map. How this wiring is achieved is not yet understood. This study shows that the olfactory bulb topographical map is modified in adenylyl cyclase 3 (adenylate cyclase 3)-deficient mice. In these mutants, axonal projection targets corresponding to specific odorant receptors are disorganized, are no longer exclusively innervated by functionally identical axonal projections and shift dramatically along the anteroposterior axis of the olfactory bulb. Moreover, the cyclase depletion leads to the prevention of neuropilin 1 (Nrp1) expression in olfactory sensory neuron axonal projections. Taken together, these data point to a major role played by a crucial element of the odorant-induced transduction cascade, adenylyl cyclase 3, in the targeting of olfactory sensory neuron axons towards the brain. This mechanism probably involves the regulation of receptor genes known to be crucial in axonal guidance processes (Col, 2007).

Table of contents

rutabaga: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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