Morphological and functional abnormalities in neuromuscular junctions of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> induced by human <Emphasis Type="Italic">APP</Emphasis> gene expression

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in increased production of amyloid-β-protein (Aβ). To gain insight into the synaptic effects of APP protein in AD patients, wild-type APP, its mutant form APP-Swedish responsible for familial AD, and human beta-secretase gene were expressed in motor neurons of Drosophila melanogaster larvae. It was found that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons caused significant morphological and functional changes in neuromuscular junctions (NMJs)-a dramatic increase in the number of synaptic boutons and altered exocytosis revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed with APP (APP-Swedish) expression, as well as for double transgenes bearing APP (APP-Swedish) and human beta-secretase (BACE), which caused secretion of amyloid beta protein (Aβ). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from Aβ neurotoxic effects.     

Reversible alteration in the neuromuscular junctions of Drosophila melanogaster bearing a temperature-sensitive mutation, shibire

In this study we report a relationship between the ultrastruct of the neuromuscular junctions of tibial muscles and the temperature-induced paralysis in shibire flies. There is a decrease in the number of synaptic vesicles of neuromuscular junctions in flies which are held at or above 29 degrees. Shortly after return to 22 degrees C, the synaptic vesicles are again present in large numbers. Prior treatment with tetrodotoxin or barbiturate protects the junctions from the temperature change in morphology.     

Translational inhibition of heat-shock induced gene expression in picornavirus-infected Drosophila melanogaster cells

Picornavirus infection of Drosophila melanogaster cells inhibited the appearance of heat-shock induced proteins. Examination of intracellular mRNAs revealed that those coding for heat shock proteins were present in a translationally competent form in infected cells. Inhibition of induced gene expression in infected Drosophila cells therefore involves, but is not necessarily solely mediated by, effects at the level of translation.     

Inbreeding by environmental interactions affect gene expression in Drosophila melanogaster

Genomewide gene expression patterns were investigated in inbred and noninbred Drosophila melanogaster lines under benign and stressful (high temperature) environmental conditions in a highly replicated experiment using Affymetrix gene chips. We found that both heat-shock protein and metabolism genes are strongly affected by temperature stress and that genes involved in metabolism are differentially expressed in inbred compared with noninbred lines, and that this effect is accentuated after heat stress exposure. Furthermore we show that inbreeding and temperature stress cause increased between-line variance in gene expression patterns. We conclude that inbreeding and environmental stress both independently and synergistically affect gene expression patterns. Interactions between inbreeding and the environment are often observed at the phenotypic level and our results reveal some of the genes that are involved at the individual gene level. Our observation of several metabolism genes being differentially expressed in inbred lines and more so after exposure to temperature stress, together with lower fitness in the investigated inbred lines, supports the hypothesis that superiority of heterozygous individuals partly derives from increased metabolic efficiency.     

Fusion between human and Drosophila melanogaster cells induced by polyethylene glycol

Polyethylene glycol was used to induce interspecific somatic cell fusion between human fibroblasts (stock F6) and Drosophila melanogaster cells from established cell lines (C1 82 and 11 P102), characterized by different ploidy levels. The present investigation defines some parameters for Drosophila cell fusion and interspecific fusion between Drosophila and human cells. The cytological analysis provided evidence of spontaneous as well as induced human-Drosophila heterokaryon formation. The presence in the same cell of two types of nuclei, distinguishable because of their different size and morphology, was confirmed autoradiographically by ³H-thymidine pre-labelling of Drosophila cells. Furthermore, the retained DNA synthetic activity and some examples of mitotic figures of both types of nuclei in the heterokaryons indicate the viability of the fused cells.     

Morphological and functional changes in the tight junctions of the bile canaliculi induced by bile duct ligation

Summary Thin sections after bile duct ligation showed that the depth of tight junctions appeared to increase and that the distance between individual punctate contacts appeared to become irregular and wider than in controls. The freeze fracture replicas clearly demonstrated these changes in the tight junction morphology. Changes were noted most conspicuously in the tight junction three weeks after ligation. Measurements of the junctional morphology in control and ligated specimens showed that the junctional depth had increased two fold in the latter, whereas the number of strands had scarcely changed. Lanthanum tracer experiments showed that the tight junctions did not permit the passage of the tracer in normal nor ligated rats. It was concluded that the mechanism of obstructive jaundice could not be related to changes in junctional morphology causing increased junctional permeability.Tight junction depth in this paper is synonymously used with Tight junction width or Tight junction thickness     

InR gene expression and octopamine metabolism in Drosophila melanogaster females

The influence of suppression of the expression of the Drosophila insulin-like receptor gene (InR) in corpus allatum (the gland-synthesizing juvenile hormone) on octopamine (OA) and juvenile hormone metabolism and on the development of the stress-reaction in Drosophila melanogaster females was studied. It was demonstrated that the suppression of InR gene expression in corpus allatum induces in D. melanogaster females an increase in the activity of the enzyme that limits the rate of octopamine synthesis (tyrosine decarboxylase), as well as in the level of juvenile hormone degradation and the intensity of the response of octopamine and juvenile hormone metabolism systems to heat stress. It was mentioned that a decrease in InR gene expression in corpus allatum does not influence the activity of OA-dependent N-acetyltransferase (the enzyme that degrades octopamine). It was established that the influence of suppression of the InR gene expression in corpus allatum on octopamine metabolism is mediated by juvenile hormone, since the treatment of flies by exogenous juvenile hormone restores the activity of tyrosine decarboxylase in flies with decreased InR expression in corpus allatum up to the normal level.     

Recombinant gene expression in cultured Drosophila melanogaster cells.

Cultured Drosophila Schneider line 2 cells provide a versatile and efficient system for the expression of recombinant gene products that retain authentic properties. An efficient method now exists for the expression of large amounts of recombinant protein from continuous cell lines. In addition, Schneider line 2 cells have proven reliable as a background for in vivo studies of gene regulation and protein function.     

Wolbachia‐induced expression of kenny gene in testes affects male fertility in Drosophila melanogaster

Wolbachia are Gram‐negative endosymbionts that are known to cause embryonic lethality when infected male insects mate with uninfected females or with females carrying a different strain of Wolbachia, a situation characterized as cytoplasmic incompatibility (CI). However, the mechanism of CI is not yet fully understood, although recent studies on Drosophila melanogaster have achieved great progress. Here, we found that Wolbachia infection caused changes in the expressions of several immunity‐related genes, including significant upregulation of kenny (key), in the testes of D. melanogaster. Overexpression of key in fly testes led to a significant decrease in egg hatch rates when these flies mate with wild‐type females. Wolbachia‐infected females could rescue this embryonic lethality. Furthermore, in key overexpressing testes terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling signal was significantly stronger than in the control testes, and the level of reactive oxygen species was significantly increased. Overexpression of key also resulted in alterations of some other immunity‐related gene expressions, including the downregulation of Zn72D. Knockdown of Zn72D in fly testes also led to a significant decrease in egg hatch rates. These results suggest that Wolbachia might induce the defect in male host fertility by immunity‐related pathways and thus cause an oxidative damage and cell death in male testes.