The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions... more The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis of windbeutel. We have found that windbeutel encodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.
Accumulation of amyloid-beta (Abeta) peptides in the brain has been suggested to be the primary e... more Accumulation of amyloid-beta (Abeta) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human Abeta40 or Abeta42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of Abeta42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of Abeta40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of Abeta42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying Abeta toxicity and the discovery of novel therapeutic targets for AD.
We report the isolation, full sequence characterization, amplification and expression properties ... more We report the isolation, full sequence characterization, amplification and expression properties of medfly chorion genes corresponding to the autosomal chorion locus of Drosophila. These genes are found adjacent to the paramyosin gene and are organized in the same order and tandem orientation as their Drosophila homologues, although they are spaced further apart. They show substantial sequence divergence from their Drosophila homologues, including novel peptide repeats and a new spacing of the tyrosines, which are known to be cross-linked in Dipteran chorion. The genes are amplified and expressed during oogenesis, as in Drosophila. Three of them are expressed in the same relative temporal order as in Drosophila but the fourth gene, the homologue of s15, shows a clear shift to an earlier expression period. This is the first known instance of changed temporal regulation in dipteran chorion genes.
Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restr... more Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade which defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. Here, we show that this ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the Epidermal Growth Factor Receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered ...
An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is suf... more An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is sufficient for directing proper temporal and spatial expression of a reporter gene in three domains of the follicle: anterior, posterior, and main body. Here we show that the fidelity of PRR-directed s36 expression is dependent on the proper dorsal-ventral differentiation of the follicular epithelium, which requires the Drosophila epidermal growth factor receptor homolog. Transgenic analysis of site-directed mutants of the PRR suggests that s36 expression is regulated by the concerted action of multiple positive activators. Several cis-acting transcriptional elements have been identified: some appear to function in a quantitative manner, while others either are essential or appear to regulate expression in particular spatial domains. The approximate locations of these regulatory elements have been defined; some map within sequences that are strongly conserved in widely divergent dipteran sp...
We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected int... more We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT. Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration. The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo. This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable. Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos.
AD phases. Between 6 and 30 months all phases were found in APP23, while APP24 (lower expression)... more AD phases. Between 6 and 30 months all phases were found in APP23, while APP24 (lower expression) and APP51 only reach phase 4 and 3, respectively. Crossbreeding with PS mice accelerated deposition to phase 5. Diffuse, compact and vascular amyloid was found in all lines with age and region dependent differences in the proportion of the various forms. Compact plaques (bigger and more dense than in AD) and vascular amyloid was most pronounced in APP23, whereas APP24 and APP51 showed primarily diffuse and less compact or vascular A[3 deposits. Cored plaques were relatively more abundant in APP51 than in both other lines. These differences remained largely unchanged after crossbreeding with PS mice. Conclusions: A[3 deposition in all APP transgenic mice analyzed follows a similar regional hierarchy as seen in AD. Although the different forms of amyloid can be found in all lines there is a considerable variation in abundance. This seems to dependent on the APP expression level and APP mutation used. Different APP transgenic mouse lines are required to study the various aspects of amyloidosis in AD.
Proceedings of The National Academy of Sciences, 2000
Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event i... more Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human A40 or A42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of A42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of A40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of A42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying A toxicity and the discovery of novel therapeutic targets for AD.
Proceedings of the National Academy of Sciences, 2004
Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event i... more Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human A40 or A42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of A42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of A40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of A42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying A toxicity and the discovery of novel therapeutic targets for AD.
FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that p... more FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Ab toxicity. Towards this goal, we generated Ab transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Ab and increased lifespan in Ab flies, whereas loss of function of FKBP52 exacerbated these Ab phenotypes. Interestingly, the Ab pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (2/2) cells have increased intracellular copper and higher levels of Ab. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Ab peptides. Citation: Sanokawa-Akakura R, Cao W, Allan K, Patel K, Ganesh A, et al. (2010) Control of Alzheimer's Amyloid Beta Toxicity by the High Molecular Weight Immunophilin FKBP52 and Copper Homeostasis in Drosophila. PLoS ONE 5(1): e8626.
Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction ... more Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction of older individuals. Although a proteolytic product of the Amyloid precursor protein, the Ab42 polypeptide, has been directly implicated in the disease, the genes and biological pathways that are deployed during the process of Ab42 induced neurodegeneration are not well understood and remain controversial. To identify genes and pathways that mediated Ab42 induced neurodegeneration we took advantage of a Drosophila model for AD disease in which ectopically expressed human Ab42 polypeptide induces cell death and tissue degeneration in the compound eye. One of the genes identified in our genetic screen is Toll (Tl). It encodes the receptor for the highly conserved TlRNFkB innate immunity/inflammatory pathway and is a fly homolog of the mammalian Interleukin-1 (Ilk-1) receptor. We found that Tl loss-of-function mutations dominantly suppress the neuropathological effects of the Ab42 polypeptide while gain-of-function mutations that increase receptor activity dominantly enhance them. Furthermore, we present evidence demonstrating that Tl and key downstream components of the innate immunity/inflammatory pathway play a central role in mediating the neuropathological activities of Ab42. We show that the deleterious effects of Ab42 can be suppressed by genetic manipulations of the TlRNFkB pathway that downregulate signal transduction. Conversely, manipulations that upregulate signal transduction exacerbate the deleterious effects of Ab42. Since postmortem studies have shown that the Ilk-1RNFkB innate immunity pathway is substantially upregulated in the brains of AD patients, the demonstration that the TlRNFkB signaling actively promotes the process of Ab42 induced cell death and tissue degeneration in flies points to possible therapeutic targets and strategies.
We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recove... more We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recover its homologue from genomic and cDNA libraries of the medfly, Ceratitis capitata. The same gene has also been recovered from a genomic library of D. virilis. The medfly s36 gene shows similar developmental specificity as in Drosophila (early choriogenesis). It is also specifically amplified in ovarian follicles; this is the first report of chorion gene amplification outside the genus Drosophila. Alignments of s36 sequences from three species show that, in addition to its regulatory conservation, the s36 gene is extensively conserved in sequence, in a region corresponding to a central protein domain, and in short regions of 5' flanking DNA that might correspond to cis-regulatory elements.
suppressor loci that modify the tan induced phenotype have been isolated. The molecular identity ... more suppressor loci that modify the tan induced phenotype have been isolated. The molecular identity of the modifier genes should give insights into the mechanisms of tan-induced neurodegeneration. 1. Kamath et al. Nature 421(6920), 231-7. 2003.
Alzheimer's disease is a neurological disorder re... more Alzheimer's disease is a neurological disorder resulting in the degeneration and death of brain neurons controlling memory, cognition and behavior. Although overproduction of Abeta peptides is widely considered a causative event in the disease, the mechanisms by which Abeta peptides cause neurodegeneration and the processes of Abeta clearance and degradation remain unclear. To address these issues, we have expressed the Abeta peptides in Drosophila melanogaster. We show that overexpression of Abeta42 peptides in the nervous system results in phenotypes associated with neuronal degeneration in a dose- and age-dependent manner. We further show that a mutation in a Drosophila neprilysin gene suppresses the Abeta42 phenotypes by lowering the levels of the Abeta42 peptide, supporting the role of neprilysin in the catabolism of Abeta peptides in vivo. We propose that our Drosophila model is suitable for the study and elucidation of Abeta metabolism and toxicity at the genetic level.
ABSTRACT The purpose of this research was to investigate post-harvest heat treatment of Valenci... more ABSTRACT The purpose of this research was to investigate post-harvest heat treatment of Valencia oranges as an effective disinfestation protocol (fast, no fruit damage) for the Mediterranean fruit fly Ceratitis capitata (Wiedemann). Forced high-temperature air was applied under the following conditions: (a) exposure to air temperature of 56°C, for fast increase of temperature in the interior of the fruit, (b) reduction in air temperature to 47°C when the fruit centre reached 47°C and (c) maintenance of fruits in the chamber for another 30 min. Relative humidity in the treatment chamber was kept between 50% and 65% during treatment. Forced air at 47°C applied for 30 min on eggs before hatch or late third instar larvae (the most heat-tolerant stages) resulted in complete kill. Egg and larval sensitivity to high temperature differed between a wild strain and a laboratory genetic-sexing strain based on white pupa mutation. In this strain males emerge from brown pupae and females from white pupae. In particular, mature eggs from the wild strain were significantly more temperature resistant than eggs from the laboratory strain. Exposure of Valencia oranges of a diameter of 7–7.5 cm to 56°C forced air for about 86 and 99 min was required to increase temperature to 47°C at 1.5 cm depth and the fruit centre respectively. Treated oranges showed no substantial peel or interior deterioration, or change in colour and taste when kept at 25°C and 50–60% RH for a period of up to 1 month following treatment. Treatment in 1% O2 atmosphere, produced by flushing of CO2 into the treatment chamber, resulted in about 1°C reduction in killing temperature and faster increase in temperature inside the fruit to a lethal level.
International Journal of Biological Macromolecules, 1991
Evidence from amino acid composition, Fourier transform analysis of primary structure and seconda... more Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking 'arms'. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide -X-X-X-Z-Z-Z-Z-Z-(where X = large hydrophobic residue and Z = t-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel t-pleated sheet structure of t-sheet strands regularly alternating with t-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different, The 'arms' of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal 'arm' is formed by, almost precise, tandem repeats of an octapeptide -. The latter are predicted as t-sheet strands alternating with t-turns, whereas, for the former the evidence is conflicting. The presence of these motifs suggests periodical patterns of dityrosine crosslinks, which harden the eggshell rendering it insoluble. Fourier transform infrared spectroscopy data from intact Ceratitis capitata eggshells support the validity of prediction.
Sustained increases in life expectancy have underscored the importance of managing diseases with ... more Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of b-amyloid (Ab) peptides, the complete network of interactions regulating Ab metabolism and toxicity still eludes us. To address this, we have conducted genetic interaction screens using transgenic Drosophila expressing Ab and we have identified mutations that affect Ab metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Ab effects. Several of the mutations that we identified have not been linked to Ab toxicity before and thus constitute novel potential targets for AD intervention. We additionally tested these mutations for interactions with tau and expanded-polyglutamine overexpression and found a few candidate mutations that may mediate common mechanisms of neurodegeneration. Our data offer insight into the toxicity of Ab and open new areas for further study into AD pathogenesis
The gamma-secretase complex is involved in cleaving transmembrane proteins such as Notch and one ... more The gamma-secretase complex is involved in cleaving transmembrane proteins such as Notch and one of the genes targeted in Alzheimer's disease known as amyloid precursor protein (APP). Presenilins function within the catalytic core of gamma-secretase, and mutated forms of presenilins were identified as causative factors in familial Alzheimer's disease. Recent studies show that in addition to Notch and APP, numerous signal transduction pathways are modulated by presenilins, including intracellular calcium signaling. Thus, presenilins appear to have diverse roles. To further understand presenilin function, we searched for Presenilin-interacting genes in Drosophila by performing a genetic modifier screen for enhancers and suppressors of Presenilin-dependent Notch-related phenotypes. We identified 177 modifiers, including known members of the Notch pathway and genes involved in intracellular calcium homeostasis. We further demonstrate that 53 of these modifiers genetically interacted with APP. Characterization of these genes may provide valuable insights into Presenilin function in development and disease.
ABSTRACT We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library... more ABSTRACT We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.
We hybridized cloned DNA segments to salivary gland polytene chromosomes of the medfly, Ceratitis... more We hybridized cloned DNA segments to salivary gland polytene chromosomes of the medfly, Ceratitis capitata, and thus established molecular markers for 24 sites on 6 out of 10 autosomal arms. An additional marker identified a medfly repetitive element that hybridizes to approximately 100 autosomal sites as well as a granular network that is thought to represent the X chromosome. Some of the markers correspond to 9 characterized transcription units, while 17 remain anonymous; at least 3 of the latter are restriction fragment length polymorphism (RFLP) markers. The characterized transcription units document that chromosomal arm 5L of C. capitata is homologous to the Drosophila melanogaster X chromosome, in agreement with previous inferences based on the extensive conservation of linkage groups in Diptera.
The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions... more The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis of windbeutel. We have found that windbeutel encodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.
Accumulation of amyloid-beta (Abeta) peptides in the brain has been suggested to be the primary e... more Accumulation of amyloid-beta (Abeta) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human Abeta40 or Abeta42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of Abeta42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of Abeta40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of Abeta42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying Abeta toxicity and the discovery of novel therapeutic targets for AD.
We report the isolation, full sequence characterization, amplification and expression properties ... more We report the isolation, full sequence characterization, amplification and expression properties of medfly chorion genes corresponding to the autosomal chorion locus of Drosophila. These genes are found adjacent to the paramyosin gene and are organized in the same order and tandem orientation as their Drosophila homologues, although they are spaced further apart. They show substantial sequence divergence from their Drosophila homologues, including novel peptide repeats and a new spacing of the tyrosines, which are known to be cross-linked in Dipteran chorion. The genes are amplified and expressed during oogenesis, as in Drosophila. Three of them are expressed in the same relative temporal order as in Drosophila but the fourth gene, the homologue of s15, shows a clear shift to an earlier expression period. This is the first known instance of changed temporal regulation in dipteran chorion genes.
Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restr... more Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade which defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. Here, we show that this ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the Epidermal Growth Factor Receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered ...
An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is suf... more An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is sufficient for directing proper temporal and spatial expression of a reporter gene in three domains of the follicle: anterior, posterior, and main body. Here we show that the fidelity of PRR-directed s36 expression is dependent on the proper dorsal-ventral differentiation of the follicular epithelium, which requires the Drosophila epidermal growth factor receptor homolog. Transgenic analysis of site-directed mutants of the PRR suggests that s36 expression is regulated by the concerted action of multiple positive activators. Several cis-acting transcriptional elements have been identified: some appear to function in a quantitative manner, while others either are essential or appear to regulate expression in particular spatial domains. The approximate locations of these regulatory elements have been defined; some map within sequences that are strongly conserved in widely divergent dipteran sp...
We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected int... more We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT. Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration. The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo. This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable. Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos.
AD phases. Between 6 and 30 months all phases were found in APP23, while APP24 (lower expression)... more AD phases. Between 6 and 30 months all phases were found in APP23, while APP24 (lower expression) and APP51 only reach phase 4 and 3, respectively. Crossbreeding with PS mice accelerated deposition to phase 5. Diffuse, compact and vascular amyloid was found in all lines with age and region dependent differences in the proportion of the various forms. Compact plaques (bigger and more dense than in AD) and vascular amyloid was most pronounced in APP23, whereas APP24 and APP51 showed primarily diffuse and less compact or vascular A[3 deposits. Cored plaques were relatively more abundant in APP51 than in both other lines. These differences remained largely unchanged after crossbreeding with PS mice. Conclusions: A[3 deposition in all APP transgenic mice analyzed follows a similar regional hierarchy as seen in AD. Although the different forms of amyloid can be found in all lines there is a considerable variation in abundance. This seems to dependent on the APP expression level and APP mutation used. Different APP transgenic mouse lines are required to study the various aspects of amyloidosis in AD.
Proceedings of The National Academy of Sciences, 2000
Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event i... more Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human A40 or A42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of A42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of A40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of A42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying A toxicity and the discovery of novel therapeutic targets for AD.
Proceedings of the National Academy of Sciences, 2004
Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event i... more Accumulation of amyloid- (A) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human A40 or A42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of A42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of A40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of A42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying A toxicity and the discovery of novel therapeutic targets for AD.
FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that p... more FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Ab toxicity. Towards this goal, we generated Ab transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Ab and increased lifespan in Ab flies, whereas loss of function of FKBP52 exacerbated these Ab phenotypes. Interestingly, the Ab pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (2/2) cells have increased intracellular copper and higher levels of Ab. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Ab peptides. Citation: Sanokawa-Akakura R, Cao W, Allan K, Patel K, Ganesh A, et al. (2010) Control of Alzheimer's Amyloid Beta Toxicity by the High Molecular Weight Immunophilin FKBP52 and Copper Homeostasis in Drosophila. PLoS ONE 5(1): e8626.
Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction ... more Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction of older individuals. Although a proteolytic product of the Amyloid precursor protein, the Ab42 polypeptide, has been directly implicated in the disease, the genes and biological pathways that are deployed during the process of Ab42 induced neurodegeneration are not well understood and remain controversial. To identify genes and pathways that mediated Ab42 induced neurodegeneration we took advantage of a Drosophila model for AD disease in which ectopically expressed human Ab42 polypeptide induces cell death and tissue degeneration in the compound eye. One of the genes identified in our genetic screen is Toll (Tl). It encodes the receptor for the highly conserved TlRNFkB innate immunity/inflammatory pathway and is a fly homolog of the mammalian Interleukin-1 (Ilk-1) receptor. We found that Tl loss-of-function mutations dominantly suppress the neuropathological effects of the Ab42 polypeptide while gain-of-function mutations that increase receptor activity dominantly enhance them. Furthermore, we present evidence demonstrating that Tl and key downstream components of the innate immunity/inflammatory pathway play a central role in mediating the neuropathological activities of Ab42. We show that the deleterious effects of Ab42 can be suppressed by genetic manipulations of the TlRNFkB pathway that downregulate signal transduction. Conversely, manipulations that upregulate signal transduction exacerbate the deleterious effects of Ab42. Since postmortem studies have shown that the Ilk-1RNFkB innate immunity pathway is substantially upregulated in the brains of AD patients, the demonstration that the TlRNFkB signaling actively promotes the process of Ab42 induced cell death and tissue degeneration in flies points to possible therapeutic targets and strategies.
We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recove... more We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recover its homologue from genomic and cDNA libraries of the medfly, Ceratitis capitata. The same gene has also been recovered from a genomic library of D. virilis. The medfly s36 gene shows similar developmental specificity as in Drosophila (early choriogenesis). It is also specifically amplified in ovarian follicles; this is the first report of chorion gene amplification outside the genus Drosophila. Alignments of s36 sequences from three species show that, in addition to its regulatory conservation, the s36 gene is extensively conserved in sequence, in a region corresponding to a central protein domain, and in short regions of 5' flanking DNA that might correspond to cis-regulatory elements.
suppressor loci that modify the tan induced phenotype have been isolated. The molecular identity ... more suppressor loci that modify the tan induced phenotype have been isolated. The molecular identity of the modifier genes should give insights into the mechanisms of tan-induced neurodegeneration. 1. Kamath et al. Nature 421(6920), 231-7. 2003.
Alzheimer's disease is a neurological disorder re... more Alzheimer's disease is a neurological disorder resulting in the degeneration and death of brain neurons controlling memory, cognition and behavior. Although overproduction of Abeta peptides is widely considered a causative event in the disease, the mechanisms by which Abeta peptides cause neurodegeneration and the processes of Abeta clearance and degradation remain unclear. To address these issues, we have expressed the Abeta peptides in Drosophila melanogaster. We show that overexpression of Abeta42 peptides in the nervous system results in phenotypes associated with neuronal degeneration in a dose- and age-dependent manner. We further show that a mutation in a Drosophila neprilysin gene suppresses the Abeta42 phenotypes by lowering the levels of the Abeta42 peptide, supporting the role of neprilysin in the catabolism of Abeta peptides in vivo. We propose that our Drosophila model is suitable for the study and elucidation of Abeta metabolism and toxicity at the genetic level.
ABSTRACT The purpose of this research was to investigate post-harvest heat treatment of Valenci... more ABSTRACT The purpose of this research was to investigate post-harvest heat treatment of Valencia oranges as an effective disinfestation protocol (fast, no fruit damage) for the Mediterranean fruit fly Ceratitis capitata (Wiedemann). Forced high-temperature air was applied under the following conditions: (a) exposure to air temperature of 56°C, for fast increase of temperature in the interior of the fruit, (b) reduction in air temperature to 47°C when the fruit centre reached 47°C and (c) maintenance of fruits in the chamber for another 30 min. Relative humidity in the treatment chamber was kept between 50% and 65% during treatment. Forced air at 47°C applied for 30 min on eggs before hatch or late third instar larvae (the most heat-tolerant stages) resulted in complete kill. Egg and larval sensitivity to high temperature differed between a wild strain and a laboratory genetic-sexing strain based on white pupa mutation. In this strain males emerge from brown pupae and females from white pupae. In particular, mature eggs from the wild strain were significantly more temperature resistant than eggs from the laboratory strain. Exposure of Valencia oranges of a diameter of 7–7.5 cm to 56°C forced air for about 86 and 99 min was required to increase temperature to 47°C at 1.5 cm depth and the fruit centre respectively. Treated oranges showed no substantial peel or interior deterioration, or change in colour and taste when kept at 25°C and 50–60% RH for a period of up to 1 month following treatment. Treatment in 1% O2 atmosphere, produced by flushing of CO2 into the treatment chamber, resulted in about 1°C reduction in killing temperature and faster increase in temperature inside the fruit to a lethal level.
International Journal of Biological Macromolecules, 1991
Evidence from amino acid composition, Fourier transform analysis of primary structure and seconda... more Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking 'arms'. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide -X-X-X-Z-Z-Z-Z-Z-(where X = large hydrophobic residue and Z = t-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel t-pleated sheet structure of t-sheet strands regularly alternating with t-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different, The 'arms' of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal 'arm' is formed by, almost precise, tandem repeats of an octapeptide -. The latter are predicted as t-sheet strands alternating with t-turns, whereas, for the former the evidence is conflicting. The presence of these motifs suggests periodical patterns of dityrosine crosslinks, which harden the eggshell rendering it insoluble. Fourier transform infrared spectroscopy data from intact Ceratitis capitata eggshells support the validity of prediction.
Sustained increases in life expectancy have underscored the importance of managing diseases with ... more Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of b-amyloid (Ab) peptides, the complete network of interactions regulating Ab metabolism and toxicity still eludes us. To address this, we have conducted genetic interaction screens using transgenic Drosophila expressing Ab and we have identified mutations that affect Ab metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Ab effects. Several of the mutations that we identified have not been linked to Ab toxicity before and thus constitute novel potential targets for AD intervention. We additionally tested these mutations for interactions with tau and expanded-polyglutamine overexpression and found a few candidate mutations that may mediate common mechanisms of neurodegeneration. Our data offer insight into the toxicity of Ab and open new areas for further study into AD pathogenesis
The gamma-secretase complex is involved in cleaving transmembrane proteins such as Notch and one ... more The gamma-secretase complex is involved in cleaving transmembrane proteins such as Notch and one of the genes targeted in Alzheimer's disease known as amyloid precursor protein (APP). Presenilins function within the catalytic core of gamma-secretase, and mutated forms of presenilins were identified as causative factors in familial Alzheimer's disease. Recent studies show that in addition to Notch and APP, numerous signal transduction pathways are modulated by presenilins, including intracellular calcium signaling. Thus, presenilins appear to have diverse roles. To further understand presenilin function, we searched for Presenilin-interacting genes in Drosophila by performing a genetic modifier screen for enhancers and suppressors of Presenilin-dependent Notch-related phenotypes. We identified 177 modifiers, including known members of the Notch pathway and genes involved in intracellular calcium homeostasis. We further demonstrate that 53 of these modifiers genetically interacted with APP. Characterization of these genes may provide valuable insights into Presenilin function in development and disease.
ABSTRACT We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library... more ABSTRACT We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.
We hybridized cloned DNA segments to salivary gland polytene chromosomes of the medfly, Ceratitis... more We hybridized cloned DNA segments to salivary gland polytene chromosomes of the medfly, Ceratitis capitata, and thus established molecular markers for 24 sites on 6 out of 10 autosomal arms. An additional marker identified a medfly repetitive element that hybridizes to approximately 100 autosomal sites as well as a granular network that is thought to represent the X chromosome. Some of the markers correspond to 9 characterized transcription units, while 17 remain anonymous; at least 3 of the latter are restriction fragment length polymorphism (RFLP) markers. The characterized transcription units document that chromosomal arm 5L of C. capitata is homologous to the Drosophila melanogaster X chromosome, in agreement with previous inferences based on the extensive conservation of linkage groups in Diptera.
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Papers by Mary Konsolaki