dCubilin- or dAMN-mediated protein reabsorption in Drosophila nephrocytes modulates longevity

Aging is a multifaceted process regulated by multiple cellular pathways, including the proteostasis network. Pharmacological or genetic enhancement of the intracellular proteostasis network extends lifespan and prevents age-related diseases. However, how proteostasis is regulated in different tissues throughout the aging process remains unclear. Here, we show that Drosophila homologs of Cubilinand Amnionless (dCubilin and dAMN, respectively)mediated protein reabsorption (CAMPR) from hemolymph insect blood by nephrocytes modulate longevity through regulating proteostasis in muscle and brain tissues. We find that overexpression of dAMN receptor in nephrocytes extends lifespan, whereas nephrocyte-specific dCubilin or dAMN RNAi knockdown shortens lifespan. We also show that CAMPR in nephrocytes regulates proteostasis in hemolymph and improves healthspan. In addition, we show that enhanced CAMPR in nephrocytes slows down the aging process in muscle and brain by maintaining the proteostasis network in these tissues. Altogether, our work has revealed an inter-organ communication network across nephrocytes and muscle/neuronal tissue that is essential for maintaining proteostasis, and to delay senescence in these organs. These findings provide insight into the role of renal protein reabsorption in the aging process via this tele-proteostasis network.


Introduction
Aging is an immensely complex process regulated by multiple interacting cellular pathways. Not surprisingly, numerous lines of evidence support a tight relationship between proteostasis and healthy aging (Kaushik and Cuervo, 2015;Klaips et al., 2018;Korovila et al., 2017;Labbadia and Morimoto, 2015;Santra et al., 2019;Taylor and Dillin, 2011). The proteostasis network consists of molecular chaperones, stressresponse transcription factors and protein degradation machines that sense and respond to proteotoxic stress as well as protein misfolding to ensure cell viability (Kaushik and Cuervo, 2015). Chaperones and two proteolytic systems, the ubiquitin proteasome system (UPS) and the lysosome-autophagy system, take charge of the maintenance of intracellular proteostasis. It has been shown that the activity of these systems dramatically decrease with aging (Cuervo and Wong, 2014;Revuelta and Matheu, 2017;Shirakabe et al., 2016;Wong et al., 2020). Enhancing proteasome or Disease Models & Mechanisms • DMM • Accepted manuscript autophagy activity by over-expressing proteasome subunits or essential autophagy genes has resulted in extended lifespan in model organisms, such as Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster (Cheon et al., 2019;Chondrogianni et al., 2015;Madeo et al., 2015;Pyo et al., 2013). To date, most studies on age-related changes in proteostasis consider it to be a cell-autonomous process.
However, the existence of intercellular or interorgan proteostasis networks (teleproteostasis) that help coordinate the response of tissues and organs to proteotoxic insults has been proposed (Kaushik and Cuervo, 2015). The observation of teleproteostasis, such as integrating distant networks, and its potential implications for identifying novel regulatory mechanisms and functional attributes for proteostasis are compelling. However, additional evidence of such tele-proteostasis is lacking.
Interestingly, results from a series of studies support the notion that the plasma proteome harbors key regulators of aging. Using heterochronic parabiosis method that connects the circulatory systems of young and old mice, these studies showed that multiple tissues, including heart, kidney, muscle, brain, liver, bone and pancreas, can be rejuvenated in old mice (Conboy et al., 2013;Conese et al., 2017;Eggel and Wyss-Coray, 2014;Smith et al., 2015). Plasma from old mice is sufficient to accelerate brain aging after infusion into young mice, while young plasma is able to reverse these aspects of brain aging (Smith et al., 2015). Since these initial findings, plasma proteomic changes with aging have been thoroughly exploited and changes in protein expression across the lifespan have been linked to biological pathways and diseases (Lehallier et al., 2019). However, how these plasma proteomic changes during the aging process are regulated remains unclear.

Disease Models & Mechanisms • DMM • Accepted manuscript
Drosophila has been widely used to study the aging process because of its short lifespan and easy genetic manipulation. The Drosophila excretory system is composed of nephrocytes (which regulate hemolymph composition by filtration followed by filtrate endocytosis) and Malpighian tubules (which modify and secrete urine). It has been shown that the Drosophila nephrocyte shares remarkable similarity with the glomerular podocyte for protein ultrafiltration, and the renal proximal tubule for protein reabsorption (Na and Cagan, 2013;Weavers et al., 2009;Zhang et al., 2013a). Drosophila nephrocytes can be divided into two distinct groups: the garland cells, which appear as a necklace-like structure surrounding the esophagus, and the Drosophila pericardial cells that form two rows of cells flanking the heart (Cagan, 2011;Narita et al., 1989). In the adult stage, pericardial nephrocytes serve as the primary filtration units. In our previous studies, we showed that Drosophila homologues of mammalian Cubilin and Amnionless (AMN), two major receptors for protein reabsorption in renal proximal tubules, are required for nephrocyte protein reabsorption from the hemolymph in vivo.
We also showed that dCubilin/dAMN-mediated protein reabsorption is essential for toxin removal (Zhang et al., 2013a). It has been shown that Drosophila nephrocytes remove microbiota-derived peptidoglycan from the hemolymph to maintain immune homeostasis (Guillou et al., 2016). These findings indicate nephrocytes are important for proteostasis, however gaps exist in our knowledge of nephrocytes' exact contribution and a potential role in tele-proteostasis remain unknown.

Disease Models & Mechanisms • DMM • Accepted manuscript
Drosophila pericardial nephrocyte ultrastructure changes during ageing (Psathaki et al., 2018). However, it is still not clear whether pericardial nephrocytes could also regulate lifespan. In this study, we showed that dCubilin/dAMN-mediated protein reabsorption from hemolymph by Drosophila nephrocytes modulates longevity by regulating proteostasis in muscle and brain tissues, providing evidence of a teleproteostasis network. To evaluate the role of protein reabsorption in Drosophila nephrocytes in the aging process, we manipulated the expression of AMN receptor protein specifically in nephrocytes. Our results showed that enhanced protein reabsorption in nephrocytesextended Drosophila lifespan, whereas decreased protein reabsorption resulted in shorter lifespans. Further, we showed that dCubilin/dAMNmediated protein reabsorption in nephrocytes regulates proteostasis in hemolymph and improves Drosophila healthspan. To explore the molecular mechanism through which dCubilin/dAMN-mediated protein reabsorption in nephrocytes regulates lifespan, we examined its effect on long-distance organs, such as brain and muscle. We found that enhanced dCubilin/dAMN-mediated protein reabsorption in nephrocytes slows down the aging process in muscle and brain by maintaining the proteostasis network. Therefore, our study provides evidence for the existence of a tele-proteostasis network that coordinates proteostasis across multiple organs in Drosophila melanogaster. Together, these findings suggest that renal protein reabsorption may play a major role in the regulation of the plasma proteomic changes during aging.

Increased protein accumulation in Drosophila hemolymph with aging
To test whether protein content in the plasma increases with aging, we examined protein accumulation in hemolymph extracts from 2-, 10-, 20-, 30-and 40-day old flies (20 males and 20 females). Since the hemolymph clots very quickly upon injury, 2x SDS loading buffer was added in the collecting tubule which will intervene with the Bradford protein analysis. Thus, SDS-PAGE analysis was used to assess total protein content in hemolymph. The total volume of the hemolymph extracted from 40 flies is very small (less than 5ul) and there are no standard internal markers for hemolymph, all the extracts were loaded onto SDS-PAGE to eliminate the technical artifact in our experiments. As shown in Fig.1A, the amount of total proteins in hemolymph significantly increased with aging. The 40-day old flies showed a more than two-fold increase in amount of total proteins in hemolymph compared to 2-day old flies (Fig.1B).
Our results suggested that protein accumulation in hemolymph could be used as an aging marker in Drosophila.  Fig

Drosophila hemolymph
Next, we asked whether enhancing protein reabsorption in nephrocytes ameliorates protein accumulation in Drosophila hemolymph. We overexpressed dAMN specifically in nephrocytes and examined its effect on protein reabsorption and hemolymph protein accumulation. Hemolymph proteins were extracted from controls or Disease Models & Mechanisms • DMM • Accepted manuscript flies overexpressing dAMN protein at different age groups, then subjected to SDS-PAGE analysis. As shown in Fig.2G-H, over-expression of dAMN in nephrocytes using the Dot-Gal4 driver led to increased RFP reabsorption in 3 rd instar nephrocytes.
Compared to the control group, over-expression of dAMN in nephrocytes also resulted in decreased hemolymph protein accumulation in 20-day old flies and 30-day old flies ( Fig.2I-J).

Decreased protein reabsorption in nephrocytes shortens Drosophila lifespan
To investigate whether protein reabsorption in nephrocytes affects Drosophila lifespan, we knocked down dCubilin or dAMN gene specifically in nephrocytes using Dot-Gal4/UAS-RNAi system and examined its effect on Drosophila lifespan. As shown in Fig

Increased protein reabsorption in nephrocytes extends Drosophila lifespan
To test whether enhancing protein reabsorption in nephrocytes extends lifespan, we overexpressed Drosophila and human AMN proteins (dAMN and hAMN respectively) specifically in nephrocytes and examined their effect on lifespan. As shown in Fig.3E

Defect in protein reabsorption in nephrocytes impairs Drosophila healthspan
The negative geotaxis assay, the natural tendency of flies to move against gravity when agitated, has been widely used to study genes or conditions that may hinder locomotor capacity. To test whether protein reabsorption in nephrocytes affects Drosophila healthspan, we knocked down dCubilin or dAMN proteins specifically in nephrocytes using Dot-Gal4/UAS-RNAi system and examined its effect on locomotor ability. As shown in Fig.4, dAMN or dCubilin knockdown in nephrocytes resulted in decreased climbing ability in both male and female flies ( Fig.4A (Fig.4D). These findings revealed that defect in protein reabsorption in nephrocytes impairs Drosophila healthspan.

Enhancing protein reabsorption in nephrocytes improves Drosophila healthspan
In order to examine whether protein reabsorption in nephrocytes affects Drosophila locomotor capacity, we overexpressed dAMN specifically in nephrocytes and examined its effect on healthspan as measured by geotaxis. As shown in Fig.4E

dCubilin/dAMN-mediated protein reabsorption in nephrocytes affects Drosophila brain proteostasis and aging
The formation of vacuoles in Drosophila brain was previously linked to oxidative damage and accelerated aging (Cabirol-Pol et al., 2018;Sunderhaus and Kretzschmar, 2016;Wittmann et al., 2001). To investigate how protein reabsorption in nephrocytes affects Drosophila locomotor capacities and aging, we knocked down dCubilin or dAMN gene specifically in nephrocytes and examined its effect on brain aging. As shown in Accumulation of intracellular damaged proteins is a hallmark of aging. Ubiquitinated proteins are accumulated in aging tissues because of decreased proteasome and/or autophagy activity. To investigate how protein reabsorption in nephrocytes affects Drosophila brain aging, we examined the ubiquitinated protein level in the brain using both immunofluorescence staining and western blot analysis. As shown in Fig.6A

Disease Models & Mechanisms • DMM • Accepted manuscript
We also examined the ubiquitinated protein level in the dissected muscle lysates using western blot analysis with anti-poly-ubiquitin antibody. As shown in Fig.7C-F, compared to the control group, ubiquitinated proteins significantly increased in the dissected muscle lysates of 10-or 20-day old flies with nephrocyte-specific RNAi knockdown of dAMN or dCubilin. As shown in Fig.7C and 7E, total ubiquitinated proteins significantly increased in the dissected muscle lysates of 10-day old flies with nephrocyte-specific overexpression of dAMN compared to the control flies with the same age.
Taken together, these results strongly suggested that enhancing protein reabsorption in nephrocytes improves proteostasis in muscle tissues and delays Drosophila muscle aging.

proteasome activity
dCubilin/dAMN-mediated protein reabsorption in nephrocytes affects proteostasis in Drosophila hemolymph, muscles and brain. To investigate the molecular mechanism through which dCubilin/dAMN-mediated protein reabsorption in nephrocytes affects proteostasis in muscles and brain, we measured the proteasome activity in nephrocytespecific dAMN or dCubilin knockdown and overexpressing flies. As shown in total fly extract of 2-day old flies, but not in 30-day old flies (Fig.8A). To test whether proteostasis in hemolymph affects proteasome activity in muscle tissue, we also measured the changes of proteasome activity in muscle tissues. Compared to the control group, proteasome activity in muscle tissue was significantly decreased in 2-day and 30-day old flies with nephrocyte-specific RNAi knockdown of dAMN or dCubilin.
Contrary, proteasome activity was found to be increased in muscle tissues of 30-day old flies with dAMN overexpression specifically in nephrocytes (Fig.8B). We further measured the changes of proteasome activity in brain tissues. The proteasome activity in the head was significantly decreased in 2-day and 30-day old flies with nephrocytespecific RNAi knockdown of dAMN or dCubilin compared to the control group and it was found to be increased in the head of 30-day old flies with dAMN overexpression specifically in nephrocytes (Fig.8C).

Discussion
The proteostasis network coordinates protein homeostasis intracellularly, between cells and across organs, and might lead to common age-associated diseases when it is Amnionless (dCubilin and dAMN respectively) have been shown to be required for nephrocyte protein reabsorption in vivo (Zhang et al., 2013a). In this study, using Drosophila as a model system, we uncovered that dCubilin/dAMN-mediated protein reabsorption in Drosophila nephrocytes modulates longevity by regulating proteostasis in muscle and brain tissues via tele-proteostasis mechanism.

Drosophila lifespan and regulates hemolymph proteostasis
Our previous study showed that dCubilin/dAMN-mediated protein reabsorption in In aged wild type flies, proteins accumulated in hemolymph under normal conditions. Nephrocyte-specific dCubilin or dAMN RNAi knockdown led to increased hemolymph protein accumulation, whereas overexpression of dAMN specifically in nephrocytes resulted in decreased hemolymph protein accumulation. In the future, it will be interesting to determine the composition of the hemolymph using mass spectrometry to identify the major components that show drastic changes in nephrocyte-specific dAMN knockdown or overexpressing flies across the lifespan, which can then be compared to the human findings to confirm the foundation and boundaries of Drosophila as an in vivo model system to study tele-proteostasis in general and in the context of aging.

Reduced insulin/insulin-like growth factor (IGF) signaling extends lifespan in
Drosophila (Augustin et al., 2018;Bai et al., 2012;Kannan and Fridell, 2013;Partridge, 2001). Drosophila insulin-like peptide-6 (dilp6) expression from fat body represses the secretion of Drosophila insulin-like peptide-2 (dilp2) from the brain into hemolymph and extends lifespan, suggesting that hemolymph composition is critical for lifespan regulation (Bai et al., 2012). Previous studies suggested that inter-organ proteostasis proteostasis in brain and muscle tissue, and delays Drosophila muscle aging. Defect of protein reabsorption in nephrocytes leads to ubiquitinated protein accumulation in brain and muscle tissue, and accelerates Drosophila brain and muscle aging. These results suggest that dCubilin/dAMN-mediated protein reabsorption in nephrocytes regulates proteostasis in brain and muscle tissues via the tele-proteostasis mechanism.

proteasome activity in the brain and muscle tissues
The ubiquitin-proteasome system is responsible for the removal of both normal and damaged proteins in different cell types. It has been shown that the proteasome activity  (Cagan, 2011). Our result showed that dCubilin/dAMN-mediated protein reabsorption in nephrocytes modulates Drosophila lifespan by impacting the proteostasis in hemolymph, brain and muscle tissues.
However, we cannot exclude the possibility that dCubilin/dAMN-mediated protein reabsorption in nephrocytes could indirectly impact water homeostasis because of water retention caused by protein accumulation, which further affect the aging process.
There are some limitation in our current study. As we all know, subtle differences in genetics can cause lifespan differences. In our current study, we used the UAS/Gal4 system to knockdown dAMN/ dCubilin or overexpress dAMN in nephrocytes and examined the effect of protein reabsorption on lifespan. There could be some potential genetic background differences between different genotypes, which could affect Drosophila lifespan. The GeneSwitch system is a modified Gal4/UAS system, whereby transgene expression is induced in Drosophila by adding RU486 to food. The

Disease Models & Mechanisms • DMM • Accepted manuscript
GeneSwitch system is widely used in Drosophila aging and behavioral studies to avoid confounding effects related to genetic background mutations. In this study, we did not use GeneSwitch system because currently no nephrocyte-specific GS-Gal4 is available.
In the future, it is better for us to generate a new nephrocyte-specific GS-Gal4 line and compare the phenotype difference between dAMN knockdown flies using Dot-Gal4 and GS-Gal4.
In summary, in this study, we showed that dCubilin/dAMN-mediated protein reabsorption from hemolymph by Drosophila nephrocytes modulates longevity by regulating proteostasis in muscle and brain tissues via a tele-proteostasis network. Our study provides solid evidence for the existence of the tele-proteostasis network that coordinates proteostasis across different organs in Drosophila melanogaster.

Fly Strains.
Flies were reared on diet containing 1% agar, 6.25% cornmeal, 6.25% molasses, and 3.5% Red Star yeast at 25°C. Flies were entrained to 12-hour light:dark (LD,12:12) cycles (with an average light intensity of ∼1500 lx). All UAS-Gal4 crosses were Olympus BX61 microscope with all parameters held constant throughout. Ubiquitinated proteins level was evaluated by counting the fluorescence spots in certain sized area.

Hemolymph protein extraction and SDS-PAGE analysis
20 males and 20 females of certain aged adult flies were anesthetized with carbon dioxide and a hole was gently punched using forceps. The flies were placed into a 100µm strainer which was put on top of 1.5ml tubule containing 10ul 2xSDS loading buffer. Then centrifuged at 10,000 rpm for 5min, the supernatant was collected. Total protein extracts were subjected to SDS-PAGE analysis and quantified using imageJ software.

Fruit fly Longevity Assay
Virgins round bottom polypropylene vials containing 1ml of diet. Flies were tapped to the bottom of the vials without anesthesia for diet exchange and mortality was recorded every 2-3 days. Lifespan graphs were plotted using survival curves. Some flies that escaped when we transferred flies from one vial to another vial were censored in our analyses.

Negative Geotaxis Assay
Negative geotaxis assay was used to measure the locomotor activity of flies following an established protocol (Cao et al., 2017;Madabattula et al., 2015). For each genotype tested, 10 groups of 10 flies were transferred without anesthesia into empty vials with an 8cm marker from the bottom and rested on the table for 1min. Each vial was tapped three times in rapid succession to initiate a negative geotaxis response. Climbing activity of the flies in the vials was recorded and the climbing index (CI) was calculated by dividing the number of flies passing the 8cm line mark by total fly numbers. CI 50 represents the age of flies with 50 percent of flies passing the 8cm line mark.

Hematoxylin & Eosin (H&E) staining and Brain Vacuoles Analysis
Flies of each genotype were aged to a specified age and heads were processed as previously described (Reenan and Rogina, 2008). Briefly, the fly heads were dissected at indicated time points, immediately fixed in 4% neutral buffered formalin at 4°C for 12 Disease Models & Mechanisms • DMM • Accepted manuscript hours and dehydrated using graded ethanol and then paraffin-embedded and sectioned (3µm). After slides deparaffinized and rehydrated, sections were stained with H&E staining kit (Solarbio) according to the manufacturer's instructions. Microscopic images were taken at the same level of the brain with an Olympus BX61 upright microscope.
The size of vacuoles was measured by Image J, and divided into four different sizes according to the area of vacuoles (5-20μm 2 , 20-50μm 2 , 50-150μm 2 and more than 150μm 2 ). The percentage of different size vacuoles in each fly was calculated.

Western Blot Analysis
The ubiquitinated proteins expression was analyzed by Western blot analysis. Briefly, dissected muscle or head tissue was washed twice with PBS and was then lysed and homogenized in lysis buffer and their protein concentrations were measured using BCA assay. The homogenates were then subjected to SDS-PAGE electrophoresis. After electrophoresis, the proteins were transferred to a NC (nitrocellulose filter) membrane (Merck Millipore), blocked in blocking buffer (5% skim milk) for 1 hr at room temperature, and then incubated with primary antibodies overnight at 4°C and a secondary antibody for 1 hour at room temperature. The antigen-antibody complexes were visualized automatically using Odyssey CLx Near-infrared imaging system (Li-COR, Inc., USA).

Proteasome Activity Assay
20S proteasome activity was measured using AMC-Suc-LLVY substrate (LifeSensors, PS500 For statistical analysis, a log-rank test (Mantel Cox) was applied to determine significant differences between survival curves. Some flies that escaped when we transferred flies from one vial to another vial were censored in our analyses. p< 0.05 was considered significant. Statistical significance in H was based on one-way ANOVA analysis. Data in E-F and J

Figures
were representative of at least three independent experiments. Statistical significance was accessed with a Mann-Whitney U non-parametric test. Data were shown as the mean±SD. **p<0.01, ***p<0.001, ****p < 0.0001 and ns denotes no significance.   was calculated by dividing the number of flies passing the 8cm line mark by total fly numbers. CI 50 represents the age of flies with 50 percent of flies passing the 8cm line mark. Statistical significance was based on one-way ANOVA.