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.

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.

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.

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.

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.