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

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 signicantly 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 signicant, 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 signicant 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 ubiquitinproteasome 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-offunction 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) RNAi 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.

Longevity Assays
The survival of Drosophila was analyzed to examine the lifespan of experimental ies in comparison to control ies. 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 25º C for the duration of the experiment. The ies were scored every two days to examine if any death had occurred. A y was considered dead when no movement was observed. Males were transferred onto fresh media every four 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 signi cant.

Locomotor Assays
Approximately seventy critical class male progeny to be collected from crosses between female D42-Gal4, TH-Gal4, and ddc-Gal4 HL4.3D ies and male UAS-Ref(2)P-RNAi HMS00551 , UAS-Ref(2)P-RNAi HMS00938 and UAS-lacZ ies. This assay was used to measure the ability of ies to climb up a narrow glass tube over the course of their lifespan, with fty male ies from each genotype being evaluated once every seven days, beginning at the seventh day post-eclosion. Critical class males were maintained in vials with ten ies per vial, stored at 25º 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 tted using nonlinear regression. 95% con dence intervals were used to test for signi cance with the curves considered to be signi cantly different if P < 0.05.

Inhibition of Ref(2)P in the motor neurons increases longevity and reduces locomotor ability
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 ies 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) (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 ies lost their ability to climb at week 4 (Fig. 2b).
Similarly, the inhibition of Ref (2) (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 ies 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 ies 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 ies 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)