Biomedical subjects

P M Salvaterra

Publications and source records attributed to P M Salvaterra.

At least 19 recordsLinked to original sources

PubMed2001

Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.

The microtubule-binding protein tau has been implicated in the pathogenesis of Alzheimer's disease and related disorders. However, the mechanisms underlying tau-mediated neurotoxicity remain unclear. We created a genetic model of tau-related neurodegenerative disease by expressing wild-type and mutant forms of human tau in the fruit fly Drosophila melanogaster. Transgenic flies showed key features of the human disorders: adult onset, progressive neurodegeneration, early death, enhanced toxicity of mutant tau, accumulation of abnormal tau, and relative anatomic selectivity. However, neurodegeneration occurred without the neurofibrillary tangle formation that is seen in human disease and some rodent tauopathy models. This fly model may allow a genetic analysis of the cellular mechanisms underlying tau neurotoxicity.

Acetylcholine↗

PubMed2001

In vivo modification of Na(+),K(+)-ATPase activity in Drosophila.

We have constructed and characterized transgenic Drosophila lines with modified Na(+),K(+)-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type alpha subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na(+),K(+)-ATPase alpha subunit hypomorphic mutant, 2206. In contrast, a mutant alpha subunit (alpha(D369N)) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled alpha transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of alpha subunit transgene. Using the Gal4-UAS system we have limited expression of alpha transgenes to cell types that normally express a particular Drosophila Na(+),K(+)-ATPase beta (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.

Animals↗

PubMed2001

Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP.

Using 7.4 kb of 5' flanking DNA from the Drosophila cholinergic gene locus to drive Gal4 expression we can visualize essentially all cholinergic neurons and neuropiles after genetic recombination with a UAS-GFP (S65T) reporter gene. In contrast to previous methods somata and neuropiles can be observed in the same samples. Fluorescence intensity is strong enough to allow observations in live animals at all developmental stages. Three-dimensional reconstructions made from confocal sections of whole-mount preparations reveal the extensive cholinergic connections among various regions of the nervous system.

Journal Article↗

PubMed2001

Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes.

The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.

3' Untranslated Regions↗

PubMed2000

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster.

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.

Alleles↗

PubMed1999

Dynamic visualization of nervous system in live Drosophila.

We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5' flanking DNA of the Drosophila Na(+), K(+)-ATPase beta-subunit gene Nervana2 (Nrv2). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS-GFP (S65T) transgenes (Nrv2-GAL4+UAS-GFP) inserted on the same chromosomes. We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults. The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila. These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.

Animals↗

PubMed1999

Organization and transcriptional regulation of Drosophila Na(+), K(+)-ATPase beta subunit genes: Nrv1 and Nrv2.

Drosophila melanogaster has two Na(+),K(+)-ATPase beta subunit genes (Nervana 1 and 2; Nrv), with tissue-specific expression patterns. Nrv1 produces a single beta subunit isoform expressed primarily in muscle tissue, whereas Nrv2 codes for two different isoforms (2.1 and 2.2) expressed in the nervous system. We have determined the complete molecular genomic organization for both Nrv genes. Only 3kb of DNA separate the 3' end of Nrv2 from Nrv1. The cDNAs from all three forms of Nrv have been mapped onto the genomic structure and all intron-exon junctions have been confirmed by direct sequencing. The genomic DNA positioned in the 5' flanking region of each Nrv gene has also been tested for tissue-specific transcriptional regulatory activity. P-element transformation vectors were constructed, which contained either 7.7kb of Nrv2 or 3.5kb Nrv1 5' flanking DNA driving expression of a lacZ reporter gene. Multiple transgenic Drosophila lines were established for each construct and analyzed for their beta-galactosidase expression pattern. The tissue-specific expression of each Nrv gene is independently regulated by the cis-element(s) present in the 5' flanking region. The Nrv2 5' flanking DNA directs expression exclusively to the nervous system, whereas Nrv1 5' flanking DNA directs expression primarily in muscle tissue.

Animals↗

PubMed1999

Localization of choline acetyltransferase-expressing neurons in Drosophila nervous system.

A variety of approaches have been developed to localize neurons and neural elements in nervous system tissues that make and use acetylcholine (ACh) as a neurotransmitter. Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of ACh and is considered to be an excellent phenotypic marker for cholinergic neurons. We have surveyed the distribution of choline acetyltransferase (ChAT)-expressing neurons in the Drosophila nervous system detected by three different but complementary techniques. Immunocytochemistry, using anti-ChAT monoclonal antibodies results in identification of neuronal processes and a few types of cell somata that contain ChAT protein. In situ hybridization using cRNA probes to ChAT messenger RNA results in identification of cell bodies transcribing the ChAT gene. X-gal staining and/or beta-galactosidase immunocytochemistry of transformed animals carrying a fusion gene composed of the regulatory DNA from the ChAT gene controlling expression of a lacZ reporter has also been useful in identifying cholinergic neurons and neural elements. The combination of these three techniques has revealed that cholinergic neurons are widespread in both the peripheral and central nervous system of this model genetic organism at all but the earliest developmental stages. Expression of ChAT is detected in a variety of peripheral sensory neurons, and in the brain neurons associated with the visual and olfactory system, as well as in neurons with unknown functions in the cortices of brain and ganglia.

Animals↗

PubMed1999

Drosophila choline acetyltransferase temperature-sensitive mutants.

We used the reverse transcription-polymerase chain reaction (RT-PCR) to amplify choline acetyltransferase (ChAT) mRNA fragments from two temperature-sensitive alleles of Drosophila melanogaster, Cha(ts1) and Cha(ts2). Single base substitutions in the mutants (T1614A in Cha(ts1) and G1596A in Cha(ts2)) would result in amino acid changes for ChAT protein (Met403Lys in Ch(ts1) and Arg397His in Cha(ts2)). These base substitutions were confirmed in mRNA extracted from homozygous mutants using a Single Nucleotide Primer Extension assay (SNuPE) and are sufficient to produce thermolabile enzyme. Our results indicate that these temperature-sensitive mutants are point mutations in the structural gene for ChAT. Using a quantitative SNuPE assay we also show that similar levels of Cha(ts) and wild type transcripts are present in heterozygous flies (Cha(ts1)/+ and Cha(Ts2)/+) at both restrictive and permissive temperatures. This contrasts with RNase protection assays of ChAT mRNA in homozygous mutant animals where the levels of mutant mRNA decrease at restrictive temperature.

Alleles↗

PubMed1998

Structure and organization of the Drosophila cholinergic locus.

The Drosophila cholinergic locus is composed of two distinct genetic functions: choline acetyltransferase (ChAT; EC 2.3.1.6), the enzyme catalyzing biosynthesis of neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter (VAChT), the synaptic vesicle membrane protein which pumps transmitter into vesicles. Both genes share a common first exon and the remainder of the VAChT gene contains a single coding exon residing entirely within the first intron of ChAT. RNase protection analysis indicates that all Drosophila VAChT specific transcripts contain the shared first exon and suggests common transcriptional control for ChAT and VAChT. Similar types of genomic organization have been evolutionarily conserved for cholinergic loci in nematodes and vertebrates, and may operate to ensure coordinate expression of these functionally related genes in the same cells. The relative levels of Drosophila ChAT and VAChT mRNA differ, however, in different tissues or in Cha mutants, indicating that independent regulation of ChAT and VAChT transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5'- and 3'-untranslated regions (UTR). The 5'-UTR contains a GTG ChAT translational initiation codon along with three other potential ATG initiation codons. These features of the VAChT 5'-UTR region suggest that a ribosome scanning model may not be used for VAChT translation initiation.

Amino Acid Sequence↗

PubMed1998

Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits.

We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.

Age Factors↗

PubMed1997

Brain-derived neurotrophic factor spares choline acetyltransferase mRNA following axotomy of motor neurons in vivo.

Choline acetyltransferase (ChAT) is a functional and specific marker gene for neurons such as primary motor neurons that synthesize and release acetylcholine as a neurotransmitter. In adult mammals, transection of the peripheral nerve results in a loss of immunoreactivity for ChAT in the injured motor neurons without affecting their cell number. Using a quantitative RNase protection assay, we have investigated dynamic changes in ChAT mRNA levels following axotomy of motor neurons in the brainstem of adult rats. One week after transection of the left hypoglossal nerve, levels of ChAT mRNA in the ipsilateral side of the hypoglossal motor nucleus decreased dramatically to around 10% when compared to the uninjured contralateral side. When cut axons were chronically exposed to brain-derived neurotrophic factor (BDNF) for 1 week, ChAT mRNA levels were maintained at 63% of control levels. Thus, BDNF can abrogate the injury-induced loss of ChAT mRNA in mature motor neurons in vivo. In contrast, neither neurotrophin 4/5 nor nerve growth factor could prevent the decrease in message. This effect of BDNF on ChAT mRNA levels following peripheral injury to motor neurons demonstrates the existence of regulatory pathways responsive to neurotrophic factors that can "rescue" or "protect" cholinergic gene expression.

Animals↗

PubMed1996

Differential regulation of choline acetyltransferase expression in adult Drosophila melanogaster brain.

Choline acetyltransferase (ChAT,E.C.2.3.1.6) catalyzes the synthesis of acetylcholine, and is considered to be a phenotypic marker specific for cholinergic neurons. In situ hybridization using a nonradioactive cRNA probe identified a large number of cell bodies expressing ChAT mRNA in the cortices of wild-type Drosophila melanogaster brain. Strong labeling is remarkable in the cortical regions associated with the lamina and antennal lobe, and also in the median neurosecretory (MNS) cells within pars intercerebralis, suggesting that some of the lamina monopolar neurons, antennal interneurons, and MNS cells are cholinergic. In two temperature-sensitive mutant alleles, Chats1 and Chats2, most hybridization signal disappears after exposure to a restrictive temperature (30 degrees C). Loss of signal is especially evident in the optic lobes. Some centrally located neurons, however, continue to express ChAT mRNA and are thus likely to have expression controlled in a different way than the majority of cholinergic neurons. Immunocytochemistry, using a ChAT specific monoclonal antibody, identified two sets of paired neurons located in the posterior cortex of the brain. These neurons persist in ChAT immunoreactivity even in the Chats mutants exposed to restrictive temperature. ChAT mRNA is also detectable in the corresponding cell bodies when Chats mutants are held at restrictive temperature. Our findings demonstrate some specific cholinergic neurons in Drosophila brain, and indicate that ChAT expression is differentially regulated in particular sets of cholinergic neurons.

Animals↗

PubMed1995

Immunocytochemical study of choline acetyltransferase in Drosophila melanogaster: an analysis of cis-regulatory regions controlling expression in the brain of cDNA-transformed flies.

We have analyzed the cis-regulatory regions in the 5' flanking DNA of the Drosophila melanogaster choline acetyltransferase (ChAT; E.C. 2.3.1.6) gene by using germline transformants. These transformants are carrying wild-type ChAT cDNA fused to different lengths of 5' flanking sequence of the ChAT gene. Appropriate genetic crosses were used to introduce the transgene into animals with a presumptive null genetic background for endogenous ChAT. Expression of ChAT protein could thus be attributed exclusively to the transgene. Using a monoclonal antibody against Drosophila ChAT, we have investigated the spatial distribution of transgenic ChAT and compared it to the normal distribution of ChAT protein in wild-type animals. The brains of 7.4 kb cDNA transformants showed a ChAT expression pattern similar to that of wild-type animals in the first- and second-order sensory neuropil but reduced expression in other highly ordered neuropil. Several lines that were transformed with 1.2 kb or 0.8 kb of 5' flanking DNA demonstrated relatively normal expression in sensory neuropil. In addition, these lines also showed ectopic expression in higher order neuropil. In the optic lobe, the expression pattern directed by 7.4 kb of 5' flanking DNA was very similar to that of wild-type ChAT expression. In contrast, 1.2 kb or 0.8 kb transformants showed reduced levels of expression and a more limited pattern of distribution in the optic lobe. Our results suggest that the 5' flanking DNA of the ChAT gene can be divided into several separable positive and negative regulatory regions, which define various subsets of cholinergic neurons in the nervous system.

Animals↗

PubMed1995

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Amino Acid Sequence↗

PubMed1995

Regulation of choline acetyltransferase/lacZ fusion gene expression in putative cholinergic neurons of Drosophila melanogaster.

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5' flanking DNA from the choline acetyltransferase (ChAT) gene and lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5' flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues.

Acetylcholine↗

PubMed1995

Localization of choline acetyltransferase-expressing neurons in the larval visual system of Drosophila melanogaster.

Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5' flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.

Animals↗

PubMed1995

Characterization of nervana, a Drosophila melanogaster neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase antibodies.

Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP-reactive Mr 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila.

Aging↗