Glial Hsp70 Protects K+ Homeostasis in the Drosophila Brain during Repetitive Anoxic Depolarization

Neural tissue is particularly vulnerable to metabolic stress and loss of ion homeostasis. Repetitive stress generally leads to more permanent dysfunction but the mechanisms underlying this progression are poorly understood. We investigated the effects of energetic compromise in Drosophila by targeting the Na+/K+-ATPase. Acute ouabain treatment of intact flies resulted in subsequent repetitive comas that led to death and were associated with transient loss of K+ homeostasis in the brain. Heat shock pre-conditioned flies were resistant to ouabain treatment. To control the timing of repeated loss of ion homeostasis we subjected flies to repetitive anoxia while recording extracellular [K+] in the brain. We show that targeted expression of the chaperone protein Hsp70 in glial cells delays a permanent loss of ion homeostasis associated with repetitive anoxic stress and suggest that this is a useful model for investigating molecular mechanisms of neuroprotection

Differential Gene Expression and Aging

It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging

Dataset: Comparison of GAL80ts and Tet-off GAL80 transgenes

Grim workflow and datase

Comparison of tetracycline and temperature sensitive GAL80 transgenes with UAS-grim

In order to assess the ability of GAL80 to repress the expression of a UAS transgene, lethality tests were performed in presence of a UAS-grim reporter (Genesis 34:34, 2002, PeerJ 5:e4167, 2017). The Grim reporter gene encodes a strong pro-apoptotic factor, thus the lethality across development (embryonic, larval, pupal) can be scored to assess the repression ability of GAL80. The UAS-grim lethality test facilitates the determination of the developmental period during which GAL4 activity is not inhibited. Two different muscle-specific GAL4 drivers, Mef2-Gal4 (BDSC:27390) and DJ694 (BDSC: 8176), were each crossed with a second chromosome insertion of UAS-grim (Cell Death Differ 5:930,1998), UAS-grim recombined with third chromosome insertions of GAL80TET (3.3+1077, PeerJ 5:e4167), and UAS-grim recombined with third chromosome insertions of GAL80ts (BDSC:7017). Negative control (no lethality) was generated by crossing the UAS-grim strain with a strain without any GAL4 transgene (w1118,Cell 36:469, 1984 ). Crosses were maintained, and eggs were collected at 20˚C (Experimental workflow). Parents were obtained from multiple independent cultures to set-up independent crosses (biological replicates, Set indicates parents from the same culture). Crosses were kept 24 to 48h in culture tubes to allow mating before transferring them to egg collectors. Multiple egg collections were done for each parental set (technical replicates, Date indicates dates of collection). Parents were allowed to lay eggs for 12h to 16h. Up to 25 eggs were aligned on a slice of food and up to 4 slices were used for a given collection from a set. Slices of food are then transferred to culture vials and incubated at the indicated temperature. The scoring of the number of first-instar larvae (L1) was done by scoring the number of empty eggs 25-30h (29˚C) or 43-48h (20˚C) after egg alignment. The number of pupae and adults was scored 5-6 days (29˚C) or 8-10 days (20˚C) after the scoring of the previous stage

Versatile method to measure locomotion in adult Drosophila

Many studies require the ability to quantify locomotor behavior over time. The list of tracking softwares and their capabilities are constantly growing. At the 2019 CanFly Conference, we presented preliminary results from an investigation of the effects of expressing polyglutamine repeats in fly muscles on longevity, locomotion, and protein aggregation. Numerous requests have been received regarding our protocol to measure locomotion and how to use the FlyTracker MatLab software. This report describes a versatile locomotion measuring device and custom MatLab scripts for the extraction, analysis, and compilation of FlyTracker data in a format compatible with spreadsheet softwares. The measurement and analysis of multiple genotypes of both sexes across age demonstrates that this method yields reproducible results that confirm that normal aging is associated with a progressive decline in locomotion as indicated by increased immobility and reduced velocity.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Regulating the UAS/GAL4 system in adult Drosophila with Tet-off GAL80 transgenes

The UAS/GAL4 system is the most used method in Drosophila melanogaster for directing the expression of a gene of interest to a specific tissue. However, the ability to control the temporal activity of GAL4 with this system is very limited. This study constructed and characterized Tet-off GAL80 transgenes designed to allow temporal control of GAL4 activity in aging adult muscles. By placing GAL80 under the control of a Tet-off promoter, GAL4 activity is regulated by the presence or absence of tetracycline in the diet. Almost complete inhibition of the expression of UAS transgenes during the pre-adult stages of the life cycle is obtained by using four copies and two types of Tet-off GAL80 transgenes. Upon treatment of newly emerged adults with tetracycline, induction of GAL4 activity is observed but the level of induction is influenced by the concentration of the inducer, the age, the sex and the anatomical location of the expression. The inhibition of GAL4 activity and the maintenance of induced expression are altered in old animals. This study reveals that the repressive ability of GAL80 is affected by the age and sex of the animal which is a major limitation to regulate gene expression with GAL80 in aged Drosophila

Extended Life-Span and Stress Resistance in the Drosophila Mutant methuselah

Toward a genetic dissection of the processes involved in aging, a screen for gene mutations that extend life-span in Drosophila melanogaster was performed. The mutant line methuselah(mth) displayed approximately 35 percent increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat, a free-radical generator. The mth gene predicted a protein with homology to several guanosine triphosphate–binding protein–coupled seven–transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span

Spatio-temporal analysis of gene expression during aging in Drosophila melanogaster

The relationship between gene expression and the regulation of longevity is poorly understood. Previous studies focusing on microarray or tissue‐specific changes in gene expression as a function of age have provided evidence that gene expression is a dynamic process which is regulated, even late in an organism's lifespan. Using the enhancer‐trap technique, a systematic analysis of the spatio‐temporal regulation of gene expression in tissues of adult Drosophila is presented. As many as 80% of enhancer traps analysed displayed (some form of) transcriptional change with age. In some cases the rate of change in expression was found to correlate with changes in longevity under various conditions, suggesting that they may be indicators of ‘physiological age’ and therefore valuable markers for dissecting the aging process. Molecular analysis of enhancer traps that showed increased activity with age was performed to identify candidate genes that may be important in the regulation of longevity; we identified changes in reporters associated with immunity, microtubule organization and muscle function