A genetic basis for the variable effect of smoking/nicotine on Parkinson's disease.

Prior studies have established an inverse association between cigarette smoking and the risk of developing Parkinson's disease (PD), and currently, the disease-modifying potential of the nicotine patch is being tested in clinical trials. To identify genes that interact with the effect of smoking/nicotine, we conducted genome-wide interaction studies in humans and in Drosophila. We identified SV2C, which encodes a synaptic-vesicle protein in PD-vulnerable substantia nigra (P=1 × 10(-7) for gene-smoking interaction on PD risk), and CG14691, which is predicted to encode a synaptic-vesicle protein in Drosophila (P=2 × 10(-11) for nicotine-paraquat interaction on gene expression). SV2C is biologically plausible because nicotine enhances the release of dopamine through synaptic vesicles, and PD is caused by the depletion of dopamine. Effect of smoking on PD varied by SV2C genotype from protective to neutral to harmful (P=5 × 10(-10)). Taken together, cross-validating evidence from humans and Drosophila suggests SV2C is involved in PD pathogenesis and it might be a useful marker for pharmacogenomics studies involving nicotine.

Cystamine and intrabody co-treatment confers additional benefits in a fly model of Huntington's disease.

Huntington's disease (HD) is a lethal, neurodegenerative disorder caused by expansion of the polyglutamine repeat in the Huntingtin gene (HTT), leading to mutant protein misfolding, aggregation, and neuronal death. Feeding a Drosophila HD model cystamine, or expressing a transgene encoding the anti-htt intracellular antibody (intrabody) C4-scFv in the nervous system, demonstrated therapeutic potential, but suppression of pathology was incomplete. We hypothesized that a combinatorial approach entailing drug and intrabody administration could enhance rescue of HD pathology in flies and that timing of treatment would affect outcomes. Feeding cystamine to adult HD flies expressing the intrabody resulted in a significant, additional rescue of photoreceptor neurodegeneration, but no additional benefit in longevity. Feeding cystamine during both larval and adult stages produced the converse result: longevity was significantly improved, but increased photoreceptor survival was not. We conclude that cystamine-intrabody combination therapies can be effective, reducing neurodegeneration and prolonging survival, depending on administration protocols.

Combinational approach of intrabody with enhanced Hsp70 expression addresses multiple pathologies in a fly model of Huntington's disease.

Intracellular antibodies (intrabodies) and the chaperone, heat shock protein 70 (Hsp70), have each shown potential as therapeutics for neurodegenerative diseases in vitro and in vivo. Investigating combinational therapy in an established Drosophila model of Huntington's disease (HD), we show that Hsp70 and intrabody actually affect different aspects of the disease. Overexpression of human Hsp70 resulted in improved survival of HD flies to eclosion and prolonged adult life compared with intrabody treatment alone. An additive effect on adult survival was observed when the two therapies were combined. Intrabody was more successful at suppressing neurodegeneration in photoreceptors than was Hsp70. Furthermore, Hsp70 treatment alone did not block aggregation of mutant huntingtin, a process slowed by intrabody. Expression of each is restricted to the nervous system, which implies different neuronal populations respond distinctly to these treatments. Importantly, a role for endogenous Hsp70 in suppression of mutant huntingtin pathology was confirmed by a separate set of genetic studies in which HD flies deficient for Hsp70 showed significantly increased pathology. We conclude that a combinational approach of intrabody with enhanced Hsp70 expression is beneficial in addressing multiple pathologies associated with HD and has potential application for other neurodegenerative disorders.

The pupal cuticle of Drosophila: differential ultrastructural immunolocalization of cuticle proteins.

Precise ultrastructural localization of Drosophila melanogaster pupal cuticle proteins (PCPs) was achieved by the immunogold labeling of frozen thin sections. PCPs were found in lamellate cuticle and intracellular vesicles but, curiously, were absent from the assembly zone of the cuticle. Antibodies that distinguish between the two classes of PCPs--low molecular weight (L-PCPs) and high molecular weight (H-PCPs)--revealed that the morphologically distinct outer lamellae contained L-PCPs and the inner lamellae contained H-PCPs. The sharp boundary between these two antigenic domains coincides with the transition from the outer to the inner lamellae, which in turn is correlated with the cessation of L-PCP synthesis and the initiation of H-PCP synthesis in response to 20-hydroxyecdysone (Doctor, J., D. Fristrom, and J.W. Fristrom, 1985, J. Cell Biol. 101:189-200). Hence, differences in protein composition are associated with differences in lamellar morphology.

Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.

Modulation of the light response by cAMP in Drosophila photoreceptors.

Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C transduction cascade in which each absorbed photon generates a discrete electrical event, the quantum bump. In whole-cell voltage-clamp recordings, cAMP, as well as its nonhydrolyzable and membrane-permeant analogs 8-bromo-cAMP (8-Br-cAMP) and dibutyryl-cAMP, slowed down the macroscopic light response by increasing quantum bump latency, without changes in bump amplitude or duration. In contrast, cGMP or 8-Br-cGMP had no effect on light response amplitude or kinetics. None of the cyclic nucleotides activated any channels in the plasma membrane. The effects of cAMP were mimicked by application of the non-specific phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin; zaprinast, a specific cGMP-phosphodiesterase inhibitor, was ineffective. Bump latency was also increased by targeted expression of either an activated G(s) alpha subunit, which increased endogenous adenylyl cyclase activity, or an activated catalytic protein kinase A (PKA) subunit. The action of IBMX was blocked by pretreatment with the PKA inhibitor H-89. The effects of cAMP were abolished in mutants of the ninaC gene, suggesting this nonconventional myosin as a possible target for PKA-mediated phosphorylation. Dopamine (10 microM) and octopamine (100 microM) mimicked the effects of cAMP. These results indicate the existence of a G-protein-coupled adenylyl cyclase pathway in Drosophila photoreceptors, which modulates the phospholipase C-based phototransduction cascade.

Genetic analysis of the Drosophila Gs(alpha) gene.

One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells.

Posterior localization of the Drosophila Gi alpha protein during early embryogenesis requires a subset of the posterior group genes.

Shortly after fertilization in Drosophila embryos, the G-protein alpha subunit, Gi alpha, undergoes a dramatic redistribution. Initially granules containing Gi alpha are present throughout the embryonic cortex but during nuclear cleavage they become concentrated at the posterior pole and are lost by the blastoderm stage. Mutations that eliminate anterior structures bicoid, swallow, and exuperantia did not prevent the posterior accumulation of Gi alpha. Likewise, embryos from mothers with dominant gain of function mutations in the Bicaudal D gene show normal polarization of Gi alpha granules. By contrast, a subset of mutations which eliminate posterior structures, cappuccino, spire, staufen, mago nashi, valois, and oskar, prevented the posterior accumulation of Gi alpha. It is important to note that mutations in posterior genes lower in the putative hierarchy vasa, tudor nanos, and pumilio did not affect Gi alpha redistribution. From these results we conclude that Gi alpha redistribution to the posterior pole depends on maternal factors involved in the localization of the posterior morphogen nanos.

Cuticular morphogenesis during continuous growth of the final instar larva of a moth.

The larvae of the tobacco hornworm, Manduca sexta, grow continuously. During the feeding period of the fifth larval instar their weight increases ten-fold (ca. 1.2-12 g) accompanied by a four-fold expansion of the surface area of the abdominal cuticle. We have found that this cuticle contains structures which facilitate its expansion. Folds in the epicuticle (papillae) flatten as the cuticle expands. The endocuticle, in contrast, does not unfold but rather is plastically deformed. This plastic deformation is assisted by vertical structures in the cuticle (cuticular columns) which are more easily deformed than the surrounding lamellate cuticle. The head capsule cuticle, which does not expand as the larva grows, lacks papillae and cuticular columns. Thus, these are specialized structures that are reserved for cuticle that must expand as the larva grows.

An assembly zone antigen of the insect cuticle.

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.

Cuticular mechanics during larval development of the tobacco hornworm, Manduca sexta.

Tensile properties of the larval cuticle of Manduca sexta were measured during the fifth instar. It was found that as the larvae grew and the cuticle thickened, the tangent modulus (intrinsic stiffness) for the cuticle declined rapidly. The extensibility of the cuticle during the growth period remained relatively high and fairly constant, while the flexural stiffness remained low. Subsequently, during the wandering and burrowing stage the extensibility decreased dramatically. Finally, in the prepupal stage extensibility remained low while flexural stiffness was highest. Using the cuticle deposition inhibitor diflubenzuron we demonstrated that the increase in larval cuticular flexural stiffness was required for normal pupation to proceed. Thus, during larval growth the cuticle remains flexible and extensible. Once growth is completed, the cuticle becomes much less extensible and more rigid, converting the previously hydrostatic skeleton into a self-supporting skeleton. This conversion was associated with changes in cuticular structure, hydration and protein composition.

Larval cuticular morphogenesis in the tobacco hornworm, Manduca sexta, and its hormonal regulation.

The sequential synthesis and deposition of larval cuticular proteins was followed during the final larval molt and the final larval instar of the tobacco hornworm Manduca sexta and correlated with changes in cuticular structure. On the final day of feeding (Day 3) before the onset of metamorphosis many endocuticular proteins were no longer synthesized and new isoelectric variants of 27,000-Da polypeptides were deposited into the cuticle coincident with the formation of lamellae 5- to 10-fold thinner than those previously deposited. Application of a juvenile hormone analog methoprene on Day 1 prevented this change in protein synthesis and in lamellar structure by preventing the observed rise in the intermolt ecdysteroid titer on Day 2. These changes could be induced in vitro by 25-100 ng/ml 20-hydroxyecdysone in the absence of juvenile hormone. Thus, the intermolt change in the lamellar assembly process appears to result from hormone-induced changes in cuticular protein synthesis.

Expression of acetylcholinesterase during visual system development in Drosophila.

As in other insects acetylcholine (ACh) and acetylcholinesterase (AChE) function in synaptic transmission in the central nervous system of Drosophila. Studies on flies mutant for AChE indicate that in addition to its synaptic function of inactivating acetylcholine, this neural enzyme is required for normal development of the nervous system (J.C. Hall, S.N. Alahiotis, D.A. Strumpf, and K. White, 1980, Genetics 96, 939-965; R.J. Greenspan, J.A. Finn, and J.C. Hall, 1980, J. Comp. Neurol. 189, 741-774). In order to understand what role AChE may play in neural development, it is necessary to know, in detail, where and when the enzyme appears. The use of monoclonal antibodies to localize AChE in the developing visual system of wild type Drosophila has yielded the novel observation that AChE appears in photoreceptor (retinula) cells 4-6 hr after they differentiate and 3 to 4 days before they are functional. Three days later the staining in the cell body of these cells is reduced. Because retinula cells have no functional connections at the time when AChE is first detected, AChE can not be performing its standard synaptic function. Subsequent to the reduction of AChE in the retinula cells, midway through the pupal stage, the enzyme accumulates rapidly in the neuropils of the optic lobes of the brain. Thus, there is a biphasic accumulation of AChE in the developing visual system with the enzyme initially being expressed in the retinula cells and accumulating later in the optic lobes.

Restricted spatial and temporal expression of G-protein alpha subunits during Drosophila embryogenesis.

Of the known signal transduction mechanisms, the most evolutionarily ancient is mediated by a family of heterotrimeric guanine nucleotide binding proteins or G proteins. In simple organisms, this form of sensory transduction is used exclusively to convey signals of developmental consequence. In metazoan organisms, however, the developmental role of G-protein-coupled sensory transduction has been more difficult to elucidate because of the wide variety of signals (peptides, small molecules, odorants, hormones, etc.) that use this form of sensory transduction. We have begun to examine the role of G-protein-coupled signaling during development by investigating the expression during Drosophila embryogenesis of a limited set of G proteins. Since these proteins are a common component of all G-protein-coupled signaling systems, their developmental pattern of expression should indicate when and where programmed changes in gene activity are initiated by, or involve the participation of, G-protein-coupled signaling events. We have focused on the spatial and temporal expression pattern of three different Drosophila G-protein alpha subunits by northern blot analysis, in situ hybridization and immunocytochemistry using antibodies directed to peptides specifically found in each alpha subunit. From the spatial and temporal restriction of the expression of each protein, our results suggest that different forms of G-protein-coupled sensory transduction may mediate developmental interactions during both early and late stages of embryogenesis and may participate in a variety of specific developmental processes such as the establishment of embryonic position, the ontogeny of the nervous system and organogenesis.

The Drosophila gene coding for the alpha subunit of a stimulatory G protein is preferentially expressed in the nervous system.

In mammals, the alpha subunit of the stimulatory guanine nucleotide-binding protein (Gs alpha) functions to couple a variety of extracellular membrane receptors to adenylate cyclase. Activation of Gs alpha results in the stimulation of adenylate cyclase and an increase in the second messenger cAMP. A 1.7-kilobase cDNA has been identified and characterized from Drosophila that codes for a protein 71% identical to bovine Gs alpha. The similarity is most striking in the regions thought to be responsible for the interactions with receptors and effectors, suggesting that the basic components of this signal-transduction pathway have been conserved through evolution. RNA blot hybridization and DNA sequence analysis suggest that a single transcript, expressed predominantly in the head, is present in Drosophila. In situ hybridization studies indicate that the Drosophila Gs alpha transcript is localized primarily in the cells of the central nervous system and in the eyes.

A Drosophila G-protein alpha subunit, Gf alpha, expressed in a spatially and temporally restricted pattern during Drosophila development.

Heterotrimeric guanine nucleotide binding proteins (G proteins) couple receptors for extracellular signals to intracellular second messenger-generating systems. Previous studies have shown that G-protein alpha subunits (G alpha) are expressed in a spatially and temporally restricted manner during Drosophila embryogenesis and, thus, may be responsible for mediating developmental interactions. We have used the polymerase chain reaction to search for specific G alpha subunits that function primarily during development and not in the adult fly. Using poly(A)+ RNA isolated from early pupae (day 1), we have isolated a cDNA coding for a fly G alpha subunit, designated Gf alpha. This subunit is 30-38% identical to previously described vertebrate and Drosophila G alpha subunits and appears to define an additional family of G alpha proteins. Gf alpha transcripts are expressed primarily during embryonic, larval, and early pupal stages and only at low levels in adult flies. In situ hybridization studies indicate that Gf alpha transcripts are expressed maternally and later during embryogenesis primarily in the developing midgut and transiently in the amnioserosa. The Gf alpha gene has been characterized and mapped to position 73B of the Drosophila genome.

Immunological and molecular characterization of Go alpha-like proteins in the Drosophila central nervous system.

The alpha subunits of heterotrimeric G proteins are responsible for the coupling of receptors for a wide variety of stimuli to a number of intracellular effector systems. In the nervous system of vertebrates, high levels of a specific class of G protein (Go alpha) are expressed. The alpha subunit of Go serves as a substrate for modification by pertussis toxin (PTX). In this report, we demonstrate that the Drosophila heads contain high levels of a 40-kDa PTX substrate. Modification of this protein by PTX is modulated in a manner similar to that observed for vertebrate G proteins. The PTX substrate in Drosophila is also recognized specifically by antibodies raised against peptide sequences found specifically in vertebrate Go alpha. Vertebrate Go alpha probes were used to identify a Drosophila cDNA coding for a potential PTX substrate with high sequence identity (82%) to vertebrate Go alpha. An additional cDNA coding for a related Go alpha has also been isolated. The two cDNAs differ only in the 5'-untranslated and amino-terminal regions of the protein. This observation, in addition to Northern analysis, suggests that alternate splicing may generate a variety of Go alpha-like proteins in Drosophila. In situ hybridization of specific probes to tissue sections indicates that the mRNAs coding for Go alpha-like proteins in Drosophila are expressed primarily in neuronal cell bodies and, at lower levels, in the eyes.

A unique Kex2-like endoprotease from Drosophila melanogaster is expressed in the central nervous system during early embryogenesis.

Complementary DNA sequences were cloned from a Drosophila library encoding a 1,101 amino acid polypeptide that we have named dKLIP-1. The deduced protein is structurally similar to the yeast KEX2 prohormone endoprotease including the conserved Asp, His, and Ser catalytic triad residues characteristic of the subtilisin family. When coexpressed in vivo with pro-beta-NGF, dKLIP-1 greatly enhanced the endoproteolytic conversion of the precursor to mature beta-NGF by cleavage at a -Lys-Arg- doublet. In adults, dKLIP-1 transcripts were detected in cortical regions of the CNS and fat body. Most striking, however, was the high level of maternal transcripts deposited into developing oocytes. The temporal and spatial expression of dKLIP-1 mRNAs during embryonic development indicates a potential role for this novel Kex2p-like endoprotease in early embryogenesis and neurogenesis.

Activation of protein kinase A-independent pathways by Gs alpha in Drosophila.

One of the best-described transmembrane signal transduction mechanisms is based on receptor activation of the alpha subunit of the heterotrimeric G protein Gs, leading to stimulation of adenylyl cyclase and the production of cAMP. Intracellular cAMP is then thought to mediate its effects largely, if not entirely, by activation of protein kinase A and the subsequent phosphorylation of substrates which in turn control diverse cellular phenomena. In this report we demonstrate, by two different methods, that reduction or elimination of protein kinase A activity had no effect on phenotypes generated by activation of Gs alpha pathways in Drosophila wing epithelial cells. These genetic studies show that the Gs alpha pathway mediates its primary effects by a novel pathway in differentiating, wing epithelial cells. This novel pathway may in part be responsible for some of the complex, cell-specific responses observed following activation of this pathway in different cell types.

Immunolocalization of G protein alpha-subunits in the Drosophila CNS.

In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, we have begun a multidisciplinary study of the G proteins present in the fruit fly, Drosophila melanogaster. In this report, we describe the distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit. Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning (Quan et al., 1989; Thambi et al., 1989). The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA (Provost et al., 1988). However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E. coli. Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina contained low levels of these alpha-subunits in the CNS. Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina. Little or no Gi alpha was detected in other brain regions or in the thoracic ganglion. Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila.