Polyglutamine disease in peripheral tissues.

This year is a milestone anniversary of the discovery that Huntington's disease is caused by the presence of expanded polyglutamine repeats in the huntingtin gene leading to the formation of huntingtin aggregates. 30 years have elapsed and there is still no cure and the only FDA-approved treatment to alleviate the debilitating locomotor impairments presents several adverse effects. It has long been neglected that the huntingtin gene is almost ubiquitously expressed in many tissues outside of the nervous system. Growing evidence indicates that these peripheral tissues can contribute to the symptoms of the disease. New findings in Drosophila have shown that the selective expression of mutant huntingtin in muscle or fat is sufficient to cause detrimental effects in the absence of any neurodegeneration. In addition, it was discovered that a completely different tissue distribution of Htt aggregates in Drosophila muscles is responsible for a drastic aggravation of the detrimental effects. This review examines the peripheral tissues that express huntingtin with an added focus on the nature and distribution of the aggregates, if any.

Juvenile and adult expression of polyglutamine expanded huntingtin produce distinct aggregate distributions in Drosophila muscle.

While Huntington's disease (HD) is widely recognized as a disease affecting the nervous system, much evidence has accumulated to suggest peripheral or non-neuronal tissues are affected as well. Here, we utilize the UAS/GAL4 system to express a pathogenic HD construct in the muscle of the fly and characterize the effects. We observe detrimental phenotypes such as a reduced lifespan, decreased locomotion and accumulation of protein aggregates. Strikingly, depending on the GAL4 driver used to express the construct, we saw different aggregate distributions and severity of phenotypes. These different aggregate distributions were found to be dependent on the expression level and the timing of expression. Hsp70, a well-documented suppressor of polyglutamine aggregates, was found to strongly reduce the accumulation of aggregates in the eye, but in the muscle, it did not prevent the reduction of the lifespan. Therefore, the molecular mechanisms underlying the detrimental effects of aggregates in the muscle are distinct from the nervous system.

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.

Comparison of GAL80ts and Tet-off GAL80 transgenes.

The manipulation of gene expression requires complete control of space, time and level of expression. In Drosophila , the UAS/GAL4 system has been instrumental in achieving spatial control, but the pursuit for the ideal system for temporal control is ongoing. One strategy used by many groups is to use GAL80. Two different kinds of GAL80 transgenic constructs have been reported but have never been compared directly. Here, we characterize and compare the repression ability of the GAL80ts and Tet-off GAL80 transgenes by two different assays. We found that GAL80ts was generally more efficient than Tet-off GAL80 and that the level of repression was dependent on the GAL4 driver, and did not necessarily correlate with expression level. We also investigated the level of expression upon inactivation of GAL80ts. The level and kinetics of inactivation were found to differ depending on the GAL4 driver and the stage of the life cycle, and in many cases the inactivation was incomplete. These results emphasize that it is essential to measure experimentally the level of expression under repressed and unrepressed states when multiple GAL4 drivers are used with GAL80 transgenes.

Protocol to measure ß-galactosidase in Drosophila extracts using a CPRG assay.

The quantification of ß-galactosidase activity is routinely required by laboratories worldwide. We present a cost-effective, highly replicable, simple technique for quantifying ß-galactosidase-specific activity from crude extracts made from whole organisms or dissected tissues or cells. Extracts are prepared and measured without the need for any specialized equipment, and tissue is ground manually by pestle and measured by colorimetric CPRG and Bradford assays. This protocol describes the assay using Drosophila extracts but could be applied to any biological system of interest. For complete details on the use and execution of this protocol, please refer to Seroude et al. (2002),1 Poirier et al. (2008),2 and Barwell et al. (2017).3.

Protocol for recording and analyzing spontaneous locomotion in Drosophila.

The quantitative analysis of locomotion is used to study many biological processes. Here, we describe how to record the locomotion of up to 50 Drosophila individuals and process the resulting video files using FlyTracker. We detail the use of modifiable MatLab scripts to process structure array files generated by FlyTracker. We have applied this to study Drosophila movement during aging, but it could be used to address a variety of research questions. For complete details on the use and execution of this protocol, please refer to Barwell et al. (2021).1.

Capture and light-induced release of antibiotics by an azo dye polymer.

The isomerisation of azo dyes can induce conformational changes which have potential applications in medicine and environmental protection. We developed an agar diffusion assay to test the capture and release of biologically active molecules from an azo electro-optic polymer, Poly (Disperse Red 1 methacrylate) (DR1/PMMA). The assay monitors the growth of bacteria placed in soft agar under a glass coverslip. Antibiotics can then be applied on the coverslip resulting in the clearance of the area under the coverslip due to growth inhibition. This assay demonstrates that DR1/PMMA is able to capture either tetracycline or ampicillin and the relative amount of DR1/PMMA required for capture was determined. Finally, the active antibiotics can be released from DR1/PMMA by exposure to green laser light. Exposure to white light from a torch or to heat does not release the antibiotic.

Expression of Heat Shock Protein 70 Is Insufficient To Extend Drosophila melanogaster Longevity.

It has been known for over 20 years that Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kD heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. We observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. We also observed that the down-regulation of hsp70 using RNAi did not affect longevity.

Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah.

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.

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.

Immunity and aging: the enemy within?

Functional analyses of changes in the immune response indicate that aging is associated with a decline of adaptive immunity whereas innate immunity is ramped up. Gene expression studies also support age-dependent changes in immunity. Studies using a large panel of methodologies and multiple species show that some of the most dramatic transcriptional changes that occur during aging are associated with immunity. This observation leads to two fundamental questions: (1) Why is the immune response altered with age? (2) Is this a consequence of aging or does it contribute to it? The origin of these changes and the mechanistic relationship among them as well as with aging must be identified. In mammals, this task is complicated by the interdependence of the innate and adaptive immune systems. The value of invertebrates as model organisms to help answer these questions is presented. This includes a description of the immune response in invertebrate models and how it compares with vertebrates, focusing on conserved pathways. Finally, these questions are explored in light of recent reports and data from our laboratory. Experimental alterations of longevity indicate that the differential expression of immunity-related genes during aging is linked to the rate of aging. Long-lived nematodes are more resistant to pathogens and blocking the expression of immune-related genes can prevent lifespan extension. These observations suggest that the immune response has a positive effect on longevity, possibly by increasing fitness. By contrast, it has been reported that activation of the immune system can reduce longevity upon starvation. We also observed that deregulation of the immune response has drastic effects on viability and longevity in Drosophila. These data suggest that the immune response results in a trade-off between beneficial and detrimental effects that might profoundly affect the aging process. Given this, immunity may be an ally early in life, but turns out to be an enemy as we age.

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.

Genetic approaches to study aging in Drosophila melanogaster.

The process of aging can be described as a progressive decline in an organism's function that invariably results in death. This decline results from the activities of intrinsic genetic factors within an organism. The relative contributions of the biological and environmental components to senescence are hard to measure, however different strategies have been devised in Drosophila melanogaster to isolate and identify genetic influences on aging. These strategies include selective breeding, quantitative trait loci (QTL) mapping and single gene mutant analysis. Selective breeding effectively demonstrated a genetic, heritable component to aging while QTL mapping located regions within the Drosophila genome carrying loci that influence the aging process. Within the past decade, single gene mutant analysis has facilitated the identification of specific genes whose activities play a determinative role in Drosophila aging. This review will focus on the application of selective breeding, QTL mapping and single gene mutant analysis used in Drosophila to study aging as well as the results obtained through these strategies to date.

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.

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.

Sod2 knockdown in the musculature has whole-organism consequences in Drosophila.

Oxidative damage to cell macromolecules by reactive oxygen species is associated with numerous diseases and aging. In Drosophila, RNAi-mediated silencing of the mitochondrial antioxidant manganese superoxide dismutase (SOD2) throughout the body dramatically reduces life span, accelerates senescence of locomotor function, and enhances sensitivity to applied oxidative stress. Here, we show that Sod2 knockdown in the musculature alone is sufficient to cause the shortened life span and accelerated locomotor declines observed with knockdown of Sod2 throughout the body, indicating that Sod2 deficiency in muscle is central to these phenotypes. Knockdown of Sod2 in the muscle also increased caspase activity (a marker for apoptosis) and caused a mitochondrial pathology characterized by swollen mitochondria, decreased mitochondrial content, and reduced ATP levels. These findings indicate that Sod2 plays a crucial role in the musculature in Drosophila and that the consequences of SOD2 loss in this tissue extend to the viability of the organism as a whole.

Functional analysis of the Drosophila immune response during aging.

One of the most dramatic changes associated with aging involves immunity. In aging mammals, immune function declines and chronic inflammation develops. The biological significance of this phenomenon and its relationship with aging is a priority for aging research. Drosophila is an invaluable tool in understanding the effects of aging on the immune response. Similar to the state of chronic inflammation in mammals, Drosophila exhibits a drastic up-regulation of immunity-related genes with age. However, it remains unclear whether immune function declines with age as seen in mammals. We evaluated the impact of aging on Drosophila immune function by examining across age the ability to eliminate and survive different doses of bacterial invaders. Our findings show that aging reduces the capacity to survive a bacterial infection. In contrast, we found no evidence that aging affects the ability to eliminate bacteria indicating that the mechanisms underlying immune senescence are not involved in eliminating bacteria or preventing their proliferation.

Characterization of the Drosophila gene-switch system in aging studies: a cautionary tale.

Genetic studies have shown that in many model organisms, single gene mutations can dramatically influence aging. Systems that allow researchers to control a gene's temporal and spatial expression pattern, known as inducible gene-expression systems, are a valuable asset for the study of the influence of single genes on aging. One inducible gene-expression system reported to allow temporal and tissue-specific control of gene expression in Drosophila is the Gene-Switch system. However, this system has not been extensively characterized in the context of aging research. This report uses six Gene-Switch strains to examine the tissue localization and amount of expression achievable in the major tissue types of the fly. The quantitative analysis of adult flies fed with inducer through life reveals that the levels of expression are influenced by both the inducer concentration and the age of the animal in a strain-specific manner. Furthermore, the relationship between inducer concentration and expression level is unique to each strain and, in some cases, to each gender. The analysis of the spatial expression patterns in several strains revealed expression in more tissue types than previously assumed. Finally, most Gene-Switch strains display expression in the absence of inducer during development and/or during adulthood. These findings have important implications that may reconcile contradictions reported in studies investigating the effects of dFOXO on longevity. This study is an important guide to the design and interpretation of aging studies based on the Gene-Switch system.

Tissue-specific targeting of Hsp26 has no effect on heat resistance of neural function in larval Drosophila.

Hsp26 belongs to the small heat-shock protein family and is normally expressed in all cells during heat stress. We aimed to determine if overexpression of this protein protects behavior and neural function in Drosophila melanogaster during heat stress, as has previously been shown for Hsp70. We used the UAS-GAL4 expression system to drive expression of Hsp26 in the whole animal (ubiquitously), in the motoneurons, and in the muscles of wandering third-instar larvae. There were slight increases in time to crawling failure and normalized excitatory junction potential (EJP) area for some of the transgenic lines, but these were not consistent. In addition, Hsp26 had no effect on the temperature at failure of EJPs, normalized EJP peak amplitude, and normalized EJP half-width. Overexpression larvae had a similar number of motoneuronal boutons and length of nerve terminals as controls, indicating that the occasional protective effects on locomotion were not due to changes at the synapse. We conclude that overexpression had a small thermoprotective effect on locomotion and no effect on neural function. As it has been shown that Hsp26 requires action of other Hsps to reactivate the denatured proteins to which it binds, we propose that at least in larvae, the function of Hsp26 was masked in the relative absence of other Hsps.

Targeting HSP70 to motoneurons protects locomotor activity from hyperthermia in Drosophila.

Heat shock preconditioning can enhance locomotor and synaptic performance during subsequent hyperthermia. The molecular basis underlying this neural phenotypic modification is largely unknown. Here we report that directing the expression of the 70 kDa heat shock protein (HSP70) to motoneurons protected larval locomotor activity of Drosophila. Tissue-specific expression showed that motoneurons were critical for developing HSP70-mediated thermoprotection of locomotor activity, whereas peripheral sensory neurons, dopaminergic neurons, serotonergic neurons, and muscle cells alone were insufficient. Targeting HSP70 to motoneurons caused structural plasticity of axonal terminals associated with increased transmitter release at neuromuscular junctions at high temperature. The thermoprotection induced by motoneuronal expression of HSP70 mimicked the protective effect of a prior heat shock (36 degrees C, 1 h; 25 degrees C, 1 h) but the effects of heat shock and motoneuronal expression of HSP70 were not additive. In the absence of heat shock pretreatment, ubiquitously expressed transgenic HSP70 activated the transcription of endogenous hsp70 genes. These results demonstrate that motoneurons were critical for HSP70-mediated thermoprotection, and that transgenic HSP70 activated the transcription of endogenous hsp70 in motoneurons with the result that a mix of transgenic and endogenous HSP70 conferred thermoprotection in Drosophila larva.