Inhibition of Ref(2)P, the Drosophila homologue of the p62/SQSTM1 gene, increases lifespan and leads to a decline in motor function

Objective Sequestosome 1 (p62/SQSTM1) is a multifunctional scaffold/adaptor protein encoded by the p62/SQSTM1 gene with function in cellular homeostasis. Mutations in the p62/SQSTM1 gene have been known to be associated with patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson disease (PD). The aim of the present study was to create a novel model of human neurogenerative disease in Drosophila melanogaster by altering the expression of Ref(2)P, the Drosophila orthologue of the human p62/SQSTM1 gene. Ref(2)P expression was altered in all neurons, the dopaminergic neurons and in the motor neurons, with longevity and locomotor function assessed over time. Results Inhibition of Ref(2)P resulted in a significantly increased median lifespan in the motor neurons, followed by a severe decline in motor skills. Inhibition of Ref(2)P in the dopaminergic neurons resulted in a significant, but minimal increase in median lifespan, accompanied by a drastic decline in locomotor function. Inhibition of Ref(2)P in the ddc-Gal4-expressing neurons resulted in a significant increase in median lifespan, while dramatically reducing motor function.


Introduction
The elucidation of the cellular mechanisms that are altered during the progression of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD), is an ongoing subject of current research. The protein, sequestosome1, which is also known as p62 (p62/SQSTM1), has been suggested to be a potential contributor to in the pathogenesis of a number of neurodegenerative diseases [1]. The p62/SQSTM1 protein is a multifunctional scaffold/adaptor protein encoded by the p62/SQSTM1 gene [2]. Alternatively designated as Refractory to Sigma P" (Ref(2)P) in Drosophila, p62/SQSTM1 is involved in various aspects of selective autophagy-such as mitophagy, the ubiquitin-proteasome system (UPS) and in some signal transduction pathways [3]. The p62/SQSTM1 protein is localized throughout the cell in the cytoplasm, in the cytosol, and the endoplasmic reticulum, among other places such as autophagosomes, aggresomes, and autolysosomes, as this protein functions during the process of autophagy [4,5]. The role which p62/SQSTM1 has in autophagy is critical as it seems to regulates the removal of protein aggregates and damaged organelles through the activities of several of its many functional domains [3]. Structurally the p62/ SQSTM1 protein has many functional domains, several of which are essential to autophagic activities. These domains include the Phox and Bem1 (PB1) domain, the LC3 interacting region (LIR) domain, and the UBA domain at the C-terminus [6]. During autophagy, the LIR domain and the UBA domains of p62/SQSTM1 have vital roles in the pathway [1,7,8], as p62/SQSTM1 promotes autophagic degradation by binding to LC3 via its LIR region [9]. The PB1 and the UBA domains function together to form protein aggregates [10], and are known to be critical for mitochondrial clustering [11]. Not only does p62/SQSTM1 function in quality control through roles in autophagy, mitophagy and UPS, but it is known to have roles in age-related diseases. Mutations in p62/SQSTM1 are known to be associated with ALS and frontotemporal dementia (FTD) [2], as well as with some forms of Parkinson disease through a role in mitophagy [12]. Where during PINK1-parkin-mediated mitophagy, p62/SQSTM1 among other adaptor proteins are recruited to damaged mitochondria [13]. Studies in Drosophila have demonstrated that loss-of-function of Ref (2)P results in poor locomotor function related to mitochondrial dysfunction and accumulation of mitochondrial DNA [11,14]. Through the investigation of p62/SQSTM1 as a candidate gene may further insight into our understanding of p62/SQSTM1 function in both ageing and neurodegenerative disease. It was predicted that inhibiting the Ref(2)P gene in Drosophila would impair median organism lifespan and locomotor ability, as the loss-of-function mutations in the orthologous p62/SQSTM1 gene have been observed in ALS, FTD and PD patients. The aim of the present study was to create a p62/SQSTM1-based model of human neurodegenerative disease in D. melanogaster by altering the expression of the orthologous Ref(2)P gene.

Drosophila stocks and culture
The UAS-LacZ has been used as a benign control in a number of experiments produced in our lab as other controls investigated to date do not have a benign response and may reduce median lifespan. Drosophila melanogaster was maintained on a standard media comprised of 65 g/L cornmeal, 50 ml/L fancy grade molasses, 10 g/L yeast and 5.5 g/L agar which was then treated with 2.5 ml propionic acid and 5 ml of 0.1 g/ml methylparaben. This mixture was then allowed to solidify at the bottom of vials and stored in 4 to 6 °C until use. Stocks were stored at room temperature (~ 21 °C), while crosses and experiments were performed at 25 °C. Quantification of inhibition efficiency was not conducted in this study, however, the UAS-Ref(2)P-RNAi HMS00551 has been quantified by Liu et al. (2018).

Drosophila crosses
Virgin female flies from the D42-Gal4, TH-Gal4, ddc-Gal4 HL4.3D lines were bred with male flies which expressed Ref(2)P inhibition. The consistent material and paternal sources allow for minimal genetic differences. HMS00938 and the control line UAS-lacZ were used. Critical class male progeny from these crosses were assessed for longevity and locomotor ability through ageing and climbing assays. While UAS-LacZ does not produce a non-silencing RNAi control it is still a valid control for the GAL4 and responding transgenes. Future experiments may include the addition of a RNAisilencing control if shown to be reasonable.

Longevity assays
The survival of Drosophila was analyzed to examine the lifespan of experimental flies in comparison to control flies. Critical class male progeny were collected daily and placed in vials with fresh medium. A sample size of approximately three hundred males was collected in total and stored at 25 °C for the duration of the experiment. The flies were scored every two days to examine if any death had occurred. A fly was considered dead when no movement was observed. Males were transferred onto fresh media every 4 days to obtain a healthy environment. Graphpad Prism 8 (Graphpad Software Inc.) was used to analyze longevity data, and survival curves were analyzed and compared using the Log-rank (Mantel-Cox) test, with a P-value less than or equal to 0.05 with Bonferroni correction being considered statistically significant. Longevity data is a representation of the summation of independent replicates.

Locomotor assays
Approximately 70 critical class male progeny to be collected from crosses between female D42-Gal4, TH-Gal4, and ddc-Gal4 HL4.3D flies and male UAS- HMS00938 and UAS-lacZ flies. This assay was used to measure the ability of flies to climb up a narrow glass tube over the course of their lifespan, with 50 male flies from each genotype being evaluated once every 7 days, beginning at the 7th day post-eclosion. Climbing analysis was conducted until flies had a minimum climbing score for 2 consecutive weeks, or less than 10 flies remained alive for that genotype. Class males were maintained in vials with ten flies per vial, stored at 25 °C, and placed on new medium once per week throughout the experiment. Climbing analysis followed the standard protocol outlined by our laboratory. Graphpad Prism 8 (Graphpad Software Inc.) was used to analyze the data, and to generate climbing curves fitted using non-linear regression. 95% confidence intervals were used to test for significance with the curves considered to be significantly different if P < 0.05. Locomotive data is a representation of the summation of independent replicates.

Inhibition of Ref(2)P in the motor neurons increases longevity and reduces locomotor ability
The inhibition of Ref (2) (2)P-RNAi HMS00551 lead to a median lifespan of 86 days (Fig. 1a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the Ref (2)P-RNAi HMS00551 flies lost their ability to climb at week 4 (Fig. 1b). Similarly, the inhibition of Ref (2) (2)P-RNAi HMS00938 lead to a median lifespan of 82 days (Fig. 1a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the Ref (2)P-RNAi HMS00938 flies lost their ability to climb at week 3 (Fig. 1b).

Inhibition of Ref(2)P in the dopaminergic neurons increases longevity and reduces locomotor ability
The inhibition of Ref (2) (2)P-RNAi HMS00551 lead to a median lifespan of 88 days (Fig. 2a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the Ref(2)P-RNAi HMS00551 flies lost their ability to climb at week 4 (Fig. 2b). Similarly, the inhibition of Ref(2)P via   (2)P-RNAi HMS00938 lead to a median lifespan of 86 days (Fig. 2a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the Ref(2)P-RNAi HMS00938 flies lost their ability to climb at week 4 (Fig. 2b).

Inhibition of Ref(2)P in the ddc-Gal4 HL4.3D -expressing neurons increases longevity and reduces locomotor ability
The inhibition of Ref (2) (2)P-RNAi HMS00551 lead to a median lifespan of 74 days (Fig. 3a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the Ref (2)P-RNAi HMS00551 flies lost their ability to climb at week 4 (Fig. 3b) (2)P-RNAi HMS00938 lead to a median lifespan of 94 days (Fig. 3a). Locomotor ability was reduced over time compared to the control UAS-lacZ which maintained strong climbing ability well into the 8 weeks the Ref(2)P-RNAi HMS00938 flies lost their ability to climb at week 4 (Fig. 3b).

Discussion
The p62/SQSM1 gene is known to be associated with the human neurodegenerative disease ALS, FTD, and more recently in Parkinson Disease. We found that the inhibition of the Ref (2) HMS00938 in the ddc-Gal4 HL4.3D -expressing neurons gave a significant increase in median lifespan by approximately 34%, accompanied by a severe reduction locomotor ability over time. This significant reduction in motor ability and increase in lifespan seen when Ref (2) P expression is altered may also be interpreted as a type of compensation, and seen as an imperfect model of neurodegenerative disease.
Investigating the inhibition of Ref(2)P expression in a tissue-specific manner demonstrates the phenotypic consequences to the organism in a more direct manner. By inhibiting expression in the motor neuron, dopaminergic neuron and ddc-GAL4-expressing neurons, rather than the generating mutant Ref(2)P critical class which express Ref(2)P inhibition throughout the whole body, less restrictive results are produced that show the consequences of tissue-specific loss-of-function. While the loss-of-function of Ref(2)P has shown a reduction in climbing ability, which can be a promising model of neurodegenerative disease, but yet increase in median lifespan, this cannot be explained through this study, and instead can be seen as a compensational trade-off.

Limitations
To significantly add to our experimental design, the directed overexpression of Ref(2)P would be highly desirable and essential for a "gene replacement" study of Ref (2) P. Although several Gal-4 transgenes were employed in this set of experiments, a number of others could prove to informative. Our future plans include combining the inhibition of Ref (2)P with other gene manipulations to identify interactions including synergies that further increase lifespan and improve locomotor activity over time. Although UAS-LacZ is not the best control it has been used as benign control in our lab, we aim in the future to explore other controls that may be better. Finally, as critical class males were analysed, broadening the study to critical females might be very provide useful data.