MTOR is a nutrient sensor and hence is central to cells growth. MTOR also is a regulator of the autophagy pathway (Noda and Ohsumi, 1998; Loewith and Hall, 2011). Rapamycin, an inhibitor of MTOR, activates autophagy pathway (Abraham and Wiederrecht, 1996). This understanding has been widely utilized

aggregate proteins or rapamycin treatment. This cytostatic effect exhibited by yeast can be used as a platform to screen compounds that rescue the growth lag through autophagy induction. After compound treatments, analysis of yeast growth curves identifies the compounds that rescued the growth lag. (B) Fluorescence/Luminescence based screening: fluorescent or luminescent reporters are tagged to autophagy proteins for transfection in yeast or mammalian model systems. Modulators of autophagy from chemical libraries are obtained by analyzing the fluorescent/luminescent signal intensities or by visualizing the autophagic vesicle formation by microscopy. (C) In silico screening: structures of autophagy proteins/motifs of interest can be obtained from data sources like Protein Data Bank and can be used as a model system to identify chemical molecules that bind using in silico modeling softwares. The selected lead molecules are then verified in biological system to validate its ability to modulate the process.

#### TABLE 2 | Summary of HTS assays for compound libraries.

fcell-06-00160 November 24, 2018 Time: 16:29 # 5

to develop assays to monitor autophagy via MTOR activity. Butcher et al., developed an assay that monitored the growth of yeast cells each harboring a different plasmid from a pool of 3900 overexpression plasmids in the presence of rapamycin, which is an inhibitor of MTOR (Butcher et al., 2006). Yeast cells when cultured in the presence of rapamycin, undergo growth inhibition, because of block in TOR pathway. From the pool of overexpression plasmids, candidate gene products were identified that suppressed the cytostatic effect of rapamycin and were involved in the TOR pathway. They also characterized the mechanism of LY-83583. LY-83583 is a novel molecule that suppressed the rapamycin-induced growth inhibition and its several candidate targets were also implicated. Sarkar et al. (2007), used yeast to identify small molecule enhancers (SMERs) and inhibitors (SMIRs) of rapamycin using the same strategy. From the screening, 21 SMIRs and 12 SMERs were listed that were structurally non-redundant. They identified SMERs that could enhance autophagy independently of MTOR, and these SMERs (SMER 10, 18, and 28) when tested in mouse and Drosophila models decreased the toxicity associated with mutant Huntington protein, also reflecting on the therapeutic potential of these compounds (Sarkar et al., 2007). The HTS utilized a chemical genetic suppressor platform to rescue or elevate the growth inhibitory properties of rapamycin on wild type yeast cells (Huang et al., 2004). Therefore, because of the involvement of MTOR pathway in regulating autophagy, a simple screen based on the growth of yeast was able to give therapeutically potent small molecule hits.

A growth-based neurotoxicity assay in yeast was also utilized by Suresh et al., to identify novel autophagy enhancer 6-Bio that ameliorates α-synuclein toxicity. The compound 6-Bio effectively cleared toxic aggregates in an autophagy dependent manner in both yeast as well as mammalian cells. More importantly, the action of the compound was conserved and showed neuroprotection in a pre-clinical mouse model of Parkinson's disease (Suresh et al., 2017).

### Fluorescence Based High Throughput Assays

Fluorescence based microscopy assays are the most commonly used techniques to monitor autophagic flux. Autophagy, being a multistep process involving several molecular players, presents with a number of markers that can be tagged with a fluorescent probe and the autophagy rates can be monitored. Interestingly, this has also been exploited to design several high content-based

imaging strategies to screen for novel autophagy modulators (**Figure 1**).

Clearance of toxic poly glutamine aggregates in cell culture was also demonstrated by using autophagy enhancers obtained from an image based HTS of GFP tagged LC3 puncta representing autophagosomes (Zhang et al., 2007). The number, size and intensity of the autophagosomes were analyzed and quantified using high throughput fluorescence microscopy. GFP-LC3 was used as a probe in an automated microscopy cellbased assay to identify chemical enhancers that rapidly led to an increase in autophagosome content (Balgi et al., 2009). The same reporter was also used by Jo et al. (2011), to identify ARP101, that inhibits matrix metalloproteinase-2 (MMP-2) selectively; as an inducer of autophagy- associated cell death in cancer cells. A high content, flow cytometry based screening approach was used to screen Prestwick Chemical Library containing FDA approved drugs by looking at autolysosome formation and degradation and also endolysosomal activities under basal and stimulated autophagy conditions (Hundeshagen et al., 2011). This study used three different probes to investigate different stages of autophagic flux (GFP-LC3 for autophagosome, mCherry-GFP-LC3 for autophagosome and autolysosome) and endocytic activity (GFP-Rab7). From the screening, cardiac glycosides were validated as potent enhancers of autophagic flux. The same GFP-LC3 probe has also been used by Kuo et al. (2015), to screen 59,541 small molecules prepared by stereoselective diversityoriented chemical synthesis and identification of enhancers of autophagy.

Larsen et al. (2010), followed the degradation of three fluorescent tagged autophagy markers: GFP-p62, GFP-NBR1, or GFP-LC3 by flow cytometry of live cells after their promoter has been turned off. Relative degradation rates of these three promoters was analyzed under basal autophagy conditions. Through single cell analysis, GFP-LC3B was found to be the most stable protein whereas GFP-NBR1 was the reporter that was most effectively degraded. The degradation of GFP-p62 was observed to show the strongest response to nutrient limitation condition and was reported to be the best reporter out of the three. Chemical screening strategies have also been used to identify novel target processes that activate autophagy (Chauhan et al., 2015). In this study, LC3B puncta in HeLa cells stably expressing mRFP-GFP-LC3B were analyzed using high-content (HC) image analysis of and revealed a novel role of microtubules, which when altered resulted in autophagy induction.

Autophagy dependent degradation of lipid droplets (LDs) was also used for the development of a high content screening platform to discover novel autophagy modulators (Lee et al., 2013). In this study, an indolizine-based fluorescent skeleton called Seoul-Fluor (SF) (Kim et al., 2011) that stains the hydrophobic LDs was used and its subsequent degradation via autophagy was followed.

Two anticonvulsants were discovered as mTOR independent autophagy enhancers, from a functional cell-based screening of FDA-approved drugs that were further shown to clear intracellular population of Mycobacterium tuberculosis (Schiebler et al., 2015). In this screen, a library of 214 compounds was screened for its ability to kill intracellular luminescent strain of M. bovis BCG (bacille-Calmette-Guerin, live attenuated strain of M. bovis) within macrophages. These hits were further validated both in primary macrophages and autophagy null cells and also for their effect on autophagy in an mTOR independent manner. The probe GFP-LC3-RFP-LC31G developed by Kaizuka et al. (2016), serves as a cumulative index for autophagy activity. The probe utilizes the protease activity of the ATG4 family of proteins. Upon cleavage of the fusion protein by ATG4, GFP-LC3 gets associated with the autophagosomes and then degraded upon subsequent fusion to lysosomes. RFP-LC31G on the other hand is cytosolic due to the deletion of glycine at the C-terminal of LC3. This probe can be utilized in different settings like high throughput microscopy, flow cytometry and microplate readers and is also amenable to screening small molecule modulators of autophagy by comparing the ratio of GFP/RFP.

A high content screening in HeLa cells using EGFP-LC3 reporter identified several autophagy inhibitors. These compounds were then further analyzed using an array of phenotypic cell-based assays. The screening strategy identified several hitherto unknown target proteins amongst the well defined targets like Vps34 and ULK1 (Peppard et al., 2014). In a first of a kind, a high content screening using the fluorescent LC3 reporter, a library of 1539 chemical compounds was aimed to identify modulators that affected the nuclear localization of LC3. Potent modulators were identified that may help in the understanding of LC3 nuclear-cytoplasmic localization (Kolla et al., 2018).

Parkinson's disease associated protein A30P α-synuclein is a substrate for autophagy and has been used to study aggregate clearance by autophagy in the past. One such study used A30P α-synuclein clearance by autophagy as a primary screen to identify novel autophagy enhancers. Using this screen L-type Ca2+ channel antagonists, the K+ ATP channel opener minoxidil, and the Gi signaling activator clonidine were identified as autophagy inducers that work independent of MTOR. This important discovery revealed that MTOR is dispensable for autophagy induction. The authors showed that cAMP can modulate autophagy by controlling IP3 activity (Williams et al., 2008). As MTOR is central to several other pathways as well, identification of an alternative pathway opened the scope of controlling autophagy independent of MTOR.

### Luminescence Based High Throughput Assays

Luciferase being a sensitive reporter protein comes in handy when an assay has to be scaled to a high throughput format (**Figure 1**). Availability of different luciferase variants further helps in the design of an assay according to the needs. These luciferase variants have different degrees of sensitivity (Nanoluc is more sensitive to Firefly luciferase), different sizes and spectra (Renilla luciferase is smaller in size to Firefly luciferase) or different properties (Gaussian luciferase is secretory in nature while Renilla luciferase is cytosolic and Firefly luciferase naturally has a peroxisomal targeting signal). Depending on the need of the assay and the process to be studied, an appropriate luciferase

variant may be used in the study. A Gaussian luciferase reporter based assay that quantitatively measures the autophagy rate by monitoring proteolytic activity of ATG4B, can be done at a large scale and is quantifiable (Ketteler and Seed, 2008). This luciferase release assay is well suited for upstream signaling events that either increase or decrease the rates of autophagy. A luciferase-based assay that exploited the property of long lived proteins to be solely degraded via autophagy pathway provided a direct relevance of the autophagy modulation in aggregate prone cells. This assay demonstrated autophagic clearance of an expanded polyglutamine in vitro and in vivo conditions (Ju et al., 2009). This assay takes into account the selective degradation of autophagy cargo using a sensitive luciferase-based reporter. Dynamic and sensitive assay could be achieved by following the cargo that is selective for degradation through autophagy. Peroxisomes provide highly inducible cargo with high turnover rates which are specifically degraded through autophagy machinery under starvation conditions (Sakai et al., 2006). This high turnover of peroxisomes when combined to the sensitivity of luciferase reporter, provides a very sensitive assay to monitor autophagic flux which is also amenable to high throughput setting. Based on this principle, Mishra et al., designed a screening strategy that allows measurement of autophagic cargo (facultative organelle, peroxisomes) clearance rather than ATG8 based changes in autophagosome number. The principle of the assay is based on detection of the levels of firefly and Renilla luciferase activities to monitor the flux of selective and general autophagy, respectively, in S. cerevisiae (Mishra et al., 2017b). Reporter strains were constructed that expressed Renilla luciferase and firefly luciferase with a peroxisome targeting signal (PTS1) under a fatty acid responsive promoter. These cells when grown in the presence of fatty acid or glycerol containing media, leads to the expression of peroxisomal firefly luciferase and Renilla luciferase which is cytosolic. These cells are then subjected to starvation to induce autophagy. Induction of autophagy leads to selective autophagic degradation of peroxisomes (pexophagy) and also non-selective bulk degradation of cytoplasm. The rate of decay in firefly luciferase activity depicts pexophagy whereas Renilla levels depict general autophagy. The dual luciferase assay provides the added advantage of monitoring autophagy in real time, is more sensitive and gives kinetic assessment of two different types of autophagy processes simultaneously. Interestingly, the action of the autophagy modulators identified from the screen was conserved across higher eukaryotes (Mishra et al., 2017a). The autophagy inhibitor Bay11 identified from the screen acted at the autophagosome biogenesis step and ZPCK inhibited the degradation of cargo inside the vacuole/lysosome. These inhibitors had a conserved mode of action across yeast, animals and plants.

Luciferase based HTS autophagy assay has been reported for mammalian cells as well. In a study by Min et al. (2018), a luciferase variant Luc2p was fused with the wild type p62/SQSTM1 or a deletion version of p62 (p62 lacking the ubiquitin binding domain) and transfected into glioma cells. The lysates from the two populations (wild type and mutant p62) were compared to monitor the autophagic flux. The performance of this probe was reported to be comparable to GFP-LC3-RFP-LC31G probe described earlier in the review (Min et al., 2018).

### In vitro and in silico Assays

In recent years, many groups have also carried out a target driven autophagy screen using purified proteins and substrates. To identify substrates for ULK1 that might be involved in the process of autophagy, Egan et al. (2015), screened degenerate peptide libraries to identify a consensus motif for ULK1 mediated phosphorylation. After identifying novel phosphorylation sites, multiple targets for ULK1 were discovered. These substrates were then used to screen for potent inhibitors of ULK1 phosphorylation.

Renilla luciferase based turnover of LC3 was used to screen two kinase inhibitor libraries for identifying inhibitors of autophagic flux (Farkas et al., 2011). This study identified specific and more potent inhibitors of the upstream signaling component; class III phosphatidylinositol 3-kinase. Inhibitors specific to Ulk1 kinase activity, an upstream protein involved in autophagy initiation were obtained from a screen that utilized purified stress-activated Ulk1 and then looked at the phosphorylation of its substrate, Atg13 at Serine 318 position (Rosenberg et al., 2015).

Iorio et al. (2010) used the large dataset of drug expression pattern integrated into "drug network" and identified the previously hitherto unknown functions of several well characterized drugs. This is a dataset of expression profiles constructed while comparing the transcriptional responses induced by different small molecules in human cell lines. Through data mining, they identified fasudil as a novel autophagy enhancer taking the help of the same drug network (Iorio et al., 2010).

### DISCUSSION

Although the core autophagy machinery and the proteins involved in disease conditions might be known, but the exact mechanism of action and how the autophagic flux is regulated is not completely understood which leads to many unanswered questions. Understanding and controlling the autophagic flux either through a genetic or pharmacological approach is a highly promising approach and of great scientific interest. Studies with genetic modulations of autophagic flux have been carried out in the past with immense success. Yoshinori Ohsumi, a pioneer in autophagy field was awarded the Nobel Prize in 2016 for his contribution to the study of autophagic flux. However, chemical modulation has an advantage over genetic manipulations that the phenotype could be observed just on the addition of the compound and the action could be reversed on its withdrawal. The method is less laborious, and the putative modulators could be used as leads for pharmacological purposes in certain disease conditions. However, there are limitations associated with the chemical approach because of the bioavailability issues, toxicity and the secondary or off-target effects associated with the chemical compound. Also, tissue specific effects are difficult to monitor.

To identify novel small molecule modulators of autophagy having a robust and sensitive screening system is the primary step. Therefore, HTS assays for autophagy are of utmost importance as they enable us to screen several small molecules in a small space of time with the inclusion of all possible biological and technical replicates. The data obtained from these assays should be amenable for direct comparison between the control and test groups and statistical analysis. Several high throughput assays have been developed in the recent past to identify small molecule modulators of autophagy. But some limitations associated with these assays must be overcome for a highly potent and effective HTS assay system. Many of these assays have issues with sensitivity and range. They do not directly look at the cargo or possess a higher physiological working range to detect smaller changes in

### REFERENCES

autophagic flux. Although these assays are quantitative but may lack in one of the many parameters required to attain an ideal autophagy assay. An ideal assay would incorporate all these properties such as cargo build up, high sensitivity, ease of experimentation, broader physiological range, and live cell readout in a single high throughput format. Dynamic, sensitive and highly effective assay could be achieved by following the cargo that is inducible and selective for degradation through autophagy.

### AUTHOR CONTRIBUTIONS

PM and RM conceived the idea and wrote the manuscript. VA conceptualized and contributed to the figure.

through chemical genetics and proteome chips. Proc. Natl. Acad. Sci. U.S.A. 101, 16594–16599. doi: 10.1073/pnas.0407117101

for monitoring autophagy (3rd edition). Autophagy 12, 1–222. doi: 10.1080/ 15548627.2015.1100356

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Mishra, Ammanathan and Manjithaya. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Chemical Screening Approaches Enabling Drug Discovery of Autophagy Modulators for Biomedical Applications in Human Diseases

#### Edited by:

Brian Storrie, University of Arkansas for Medical Sciences, United States

#### Reviewed by:

Vytaute Starkuviene, Vilnius University, Lithuania Satoshi Kametaka, Nagoya University, Japan

#### \*Correspondence:

Sovan Sarkar<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 12 December 2018 Accepted: 01 March 2019 Published: 19 March 2019

#### Citation:

Panda PK, Fahrner A, Vats S, Seranova E, Sharma V, Chipara M, Desai P, Torresi J, Rosenstock T, Kumar D and Sarkar S (2019) Chemical Screening Approaches Enabling Drug Discovery of Autophagy Modulators for Biomedical Applications in Human Diseases. Front. Cell Dev. Biol. 7:38. doi: 10.3389/fcell.2019.00038 Prashanta Kumar Panda1† , Alexandra Fahrner1† , Somya Vats1,2, Elena Seranova1 , Vartika Sharma3 , Miruna Chipara1 , Priyal Desai1 , Jorge Torresi1,4, Tatiana Rosenstock1,4 , Dhiraj Kumar3 and Sovan Sarkar1 \*

1 Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom, 2 Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India, 3 Cellular Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India, 4 Department of Physiological Science, Santa Casa de São Paulo School of Medical Sciences, São Paulo, Brazil

Autophagy is an intracellular degradation pathway for malfunctioning aggregation-prone proteins, damaged organelles, unwanted macromolecules and invading pathogens. This process is essential for maintaining cellular and tissue homeostasis that contribute to organismal survival. Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases, and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance. A number of chemical screening approaches have been established for the drug discovery of autophagy modulators based on the perturbations of autophagy reporters or the clearance of autophagy substrates. These readouts can be detected by fluorescence and high-content microscopy, flow cytometry, microplate reader and immunoblotting, and the assays have evolved to enable high-throughput screening and measurement of autophagic flux. Several pharmacological modulators of autophagy have been identified that act either via the classical mechanistic target of rapamycin (mTOR) pathway or independently of mTOR. Many of these autophagy modulators have been demonstrated to exert beneficial effects in transgenic models of neurodegenerative disorders, cancer, infectious diseases, liver diseases, myopathies as well as in lifespan extension. This review describes the commonly used chemical screening approaches in mammalian cells and the key autophagy modulators identified through these methods, and highlights the therapeutic benefits of these compounds in specific disease contexts.

Keywords: autophagy, autophagy reporter, autophagy substrate, autophagy modulator, screening method, neurodegenerative diseases, cancer, lifespan extension

## INTRODUCTION

fcell-07-00038 March 16, 2019 Time: 17:5 # 2

Macroautophagy, herein referred to as autophagy, is an intracellular degradation process essential for ensuring cellular homeostasis. This well-conserved catabolic process mediates the targeted degradation of unwanted or excess cytoplasmic materials, such as aggregation-prone proteins, pathogens and damaged organelles like mitochondria, amongst others (Ravikumar et al., 2010). This process is also involved in the bulk degradation of cytoplasmic macromolecules and recycling of the breakdown products especially during nutrient deprivation to provide energy homeostasis, thereby forming a crucial connection between anabolism and catabolism (Boya et al., 2013; Kaur and Debnath, 2015). Due to its vital function as a homeostatic regulator, impairment of the autophagy is implicated in several human pathologies including certain cancer, metabolic syndromes, infectious diseases, liver diseases, myopathies, aging and neurodegenerative disorders (Mizushima et al., 2008). Therefore, therapeutic modulation of autophagy holds great potential in the development of treatment strategies for these diseases (Rubinsztein et al., 2012).

Autophagy is evolutionarily-conserved from yeast to humans. The de novo formation of phagophores, the double-membrane structures that expand to form double-membrane vesicles called autophagosomes, require multiple autophagy-related (Atg) genes in the autophagic machinery, such as the Atg5-Atg12- Atg16 complex and the phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3 (LC3-II) (Kabeya et al., 2000; Mizushima et al., 2011; Ktistakis and Tooze, 2016). Maturation of autophagosomes into the degradative autolysosomes occurs either via the multi-step route involving the fusion of autophagosomes with late endosomes to form amphisomes which subsequently fuse with the lysosomes, or via the direct route involving the fusion between autophagosomes and the lysosomes (Nakamura and Yoshimori, 2017). The autophagic cargo engulfed by the autophagosomes are ultimately degraded in the acidic autolysosomes by the lysosomal hydrolases, which are only active at the low pH maintained by the vacuolar-type H+-ATPase (V-ATPase) on the lysosomal membrane (Saftig and Klumperman, 2009). Finally, the breakdown products are recycled and utilized as inputs to cellular metabolism for energy generation (Rabinowitz and White, 2010). The rate at which this dynamic turnover of cellular contents occurs through the process of autophagy is referred to as autophagic flux. Autophagic flux encompasses all stages of autophagy which includes autophagosome formation, fusion with the lysosomes and cargo degradation in the autolysosomes (**Figure 1**).

Key upstream modulators of autophagy include the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which promotes cellular biosynthesis and inhibits autophagy (Saxton and Sabatini, 2017). Regulation of autophagosome formation by mTORC1 is mediated via the ULK1–Atg13–FIP200 complex; mTORC1 suppresses autophagy under nutrient-rich conditions by phosphorylation-dependent inactivation of ULK1 and Atg13 (Mizushima, 2010; Zachari and Ganley, 2017). Various signals such as growth factors and nutrients impinge on mTORC1 to negatively influence autophagy (Kim and Guan, 2015). Conversely, during nutrient starvation, autophagy is promoted by inhibition of the mTORC1 activity (Carroll et al., 2014; Russell et al., 2014). Furthermore, ULK1 can be directly phosphorylated and activated by the energy sensor AMPK to stimulate autophagy (Egan et al., 2011; Kim et al., 2011). In addition, several mTORC1-independent pathways have been described where autophagy is negatively regulated by the elevation in intracellular inositol, Ca2+ and nitric oxide levels, amongst others (Sarkar, 2013b). Molecular mediators of the late stage of autophagy involving autophagosome maturation include Rab7, SNAREs (N-ethylmaleimide-sensitive factor-attachment protein receptors), GABARAPs, BRUCE and Beclin1-interacting partners such as Atg14L, UVRAG and Ambra1 (He and Levine, 2010; Nguyen et al., 2016; Wang et al., 2016; Reggiori and Ungermann, 2017; Ebner et al., 2018). At a transcriptional level, autophagy is governed by the transcription factor EB (TFEB) (Settembre et al., 2011), which in itself is activated by lysosomal Ca2+ (Medina et al., 2015).

Chemical modulation of autophagy by targeting the mTORdependent and mTOR-independent pathways has proven to be of potential biomedical relevance due to therapeutic advantages, especially in neurodegenerative disorders as well as in diverse human pathological conditions such as in certain liver diseases, myopathies, infectious diseases, metabolic diseases, cancer and aging (Rubinsztein et al., 2012; Sarkar, 2013b; Levine et al., 2015). Hence, the discovery of potent small molecules regulating autophagy is of great interest. Here we review the chemical screening strategies for autophagy drug discovery, and highlight the potential benefits of autophagy modulators in human diseases.

### CHEMICAL SCREENING STRATEGIES FOR IDENTIFYING AUTOPHAGY MODULATORS

A number of in vitro screening methods have been designed for identifying compounds (Sarkar, 2013a; Joachim et al., 2015; Seranova et al., 2019). The assays are primarily based on the perturbations of autophagy reporters or autophagy cargoes as readouts (**Figure 1**), which can be measured via fluorescence or high-content imaging, immunoblotting, flow cytometry and microplate reader (Mizushima et al., 2010; Klionsky et al., 2016; **Figure 2** and **Table 1**). Some of these screening methods can be subjected to high-throughput applications. Below are descriptions of the common screening approaches in mammalian cells, and the identification and therapeutic benefits of key autophagy modulators.

### CHEMICAL SCREENING METHODS BASED ON AUTOPHAGY REPORTERS

Screening methods based on autophagy reporters are the most commonly used approaches to detect changes in the numbers of autophagosomes and autolysosomes (**Table 1**). The protein

FIGURE 1 | Autophagy reporter and substrate based screening strategies and the impact of autophagy modulators at different stages of the autophagy process. Autophagy is regulated by the mechanistic target of rapamycin complex 1 (mTORC1) or mTORC1-independent pathways. This process initiates by the formation of phagophores that expand and engulf autophagy substrates to form autophagosomes, which then fuse with the lysosomes to form autolysosomes where the autophagic cargo is degraded. Autophagy inducers and inhibitors increase or decrease autophagosome formation, respectively, at the early stages of autophagy, whereas autophagy blockers prevent lysosomal degradation and/or autophagosome maturation at late stages of autophagy. Autophagic flux is thus enhanced by autophagy inducers but is retarded by autophagy inhibitors and blockers. Chemical screening methods for identifying autophagy modulators are commonly based on the readouts of perturbations in autophagy reporters such as LC3-II, or autophagy substrate clearance such as aggregation-prone proteins or p62/SQSTM1.

TABLE 1 | Chemical screening methods for identifying autophagy modulators in mammalian cells.

The detection methods, strengths and limitations of the autophagy reporter and substrate based screening assays are highlighted.

reporter that is widely used to study autophagy is microtubuleassociated protein 1 (MAP1) light chain 3 (LC3). The nascent LC3 is cleaved at its C-terminal arginine residue by Atg4 to form the cytoplasmic LC3-I, which is then post-translationally conjugated with phosphatidylethanolamine at its C-terminal glycine residue by Atg7 to form the autophagosome-associated LC3-II (Kabeya et al., 2000). The lipidated LC3-II remains associated to the autophagosomes throughout their lifespan, and is present on

both the outer and inner membranes. Following the maturation of autophagosomes with lysosomes to form autolysosomes, the LC3-II on the inner surface is degraded whereas the LC3-II on the outer surface is delipidated and removed by Atg4B for recycling (Tanida et al., 2004). A number of fluorescent-tagged reporters of LC3, such as GFP-LC3 (Kabeya et al., 2000), mRFP-GFP-LC3 (Kimura et al., 2007) and GFP-LC3-RFP-LC31G (Kaizuka et al., 2016), have been used to study autophagy and undertake chemical screening.

### Identification of Autophagy Modulators by GFP-LC3 Screening Method

The most common LC3-based reporter that has been used in several studies is GFP-LC3, which labels autophagosomes, autolysosomes as well as phagophores (Kabeya et al., 2000). For the GFP-LC3 screening method, image-based analysis is done by quantifying the GFP+ puncta per cell to measure perturbations in autophagosome number. In general, an autophagy inducer as well as an autophagy blocker will increase GFP-LC3 puncta whereas an autophagy inhibitor will decrease GFP-LC3 puncta (**Figure 2**). A number of high-throughput and small-scale screens have been undertaken with this strategy that has been also utilized to assess the key hits arising from other screening methods; and some of the primary chemical screens utilizing GFP-LC3 readout are highlighted below.

Using GFP-LC3 as the primary screening method in a stable human glioblastoma H4 cell line, an image-based chemical screen with 480 bioactive compounds was performed wherein the number, size and intensity of GFP-LC3 spots were taken into consideration while selecting potent autophagy modulators (Zhang et al., 2007). Compounds were treated at 3–12 µM concentrations for 24 h. This screen identified 8 autophagy inducers, which included a number of FDAapproved drugs such as fluspirilene, trifluoperazine, pimozide (antipsychotic drugs), niguldipine, nicardipine, amiodarone (drugs used for cardiovascular conditions) and loperamide (used in diarrhea). While fluspirilene, trifluoperazine are dopamine antagonists, the other drugs are Ca2+ channel antagonists that lower intracellular Ca2+; all of which induced

FIGURE 2 | Autophagy chemical screening strategies in mammalian cells. Chemical screening methods that are commonly used for identifying autophagy modulators are based on autophagy reporters (LC3) or autophagy substrates (p62 or aggregation-prone proteins). The detection methods for the respective assays and the expected readouts for autophagy inducers, blockers or inhibitors are indicated as a general guidance.

autophagy independently of mTOR (Zhang et al., 2007). Another image-based chemical screen was performed with a library of 3584 pharmacologically active compounds in human breast cancer MCF-7 cells stably expressing GFP-LC3 (Balgi et al., 2009). Treatment of compounds was done at ∼15 µM concentration for 4 h. This screen identified 3 FDA-approved drugs such as perhexilene, niclosamide and amiodarone, as well as rottlerin, as autophagy inducers; all of which were shown to inhibit mTORC1 (Balgi et al., 2009). However, other screens have reported amiodarone (Ca2+ channel antagonist) to act independently of mTORC1 for inducing autophagy at a much lower dose than what is required to inhibit mTORC1 (Williams et al., 2008); and likewise, perhexilene is a Ca2+ channel blocker that could be also mTORindependent. Furthermore, one of the largest chemical screens for identifying autophagy modulators was undertaken in HeLa cells stably expressing GFP-LC3 with 59541 stereochemically and skeletally diverse compounds derived from diversityoriented synthesis (Kuo et al., 2015). Compounds were treated for 4 h in 8-point dose with a maximal concentration of 10 µM. Several hits were subjected to a secondary screen at 10 µM concentration from which BRD5631 was identified as the potent autophagy inducer along with other hits like BRD2716 and BRD34009; all of which did not affect mTOR activity. Interestingly, the hit rate in the primary screen for compounds having an alkyl amine was higher than that for all of the compounds. This effect was augmented by the additional presence of a single lipophilic group, such as diphenyl alkyne, biphenyl, cyclohexane or naphthalene (Kuo et al., 2015). While the above screens were undertaken in immortalized human cell lines, another chemical screen was done with 1280 pharmacologically active compounds in mouse embryonic fibroblasts (MEFs) stably expressing GFP-LC3 (Li et al., 2016). Compounds were treated at 0.02–46 µM concentrations for 16 h in the presence or absence of chloroquine (autophagy blocker) to determine their effects on autophagic flux. Out of the 27 autophagy inducers identified, few were characterized further. These include anti-psychotic drugs such as indatraline hydrochloride (dopamine inhibitor), chlorpromazine hydrochloride and fluphenazine dihydrochloride (dopamine receptor antagonists). Fluphenazine was found to inhibit mTORC1 whereas indatraline and chlorpromazine were mTORindependent (Li et al., 2016).

Although GFP-LC3 is a straightforward, widely-used screening assay, its inability to distinguish between autophagosomes and autolysosomes is a major inadequacy of this reporter. Accumulation of autophagosomes can occur either due to induction of autophagosome formation (by autophagy inducers) or due to block in autophagosome maturation (by autophagy blockers) in the early and late stages of autophagy, respectively (Rubinsztein et al., 2009). Since autophagy is a dynamic, multi-step process, it is imperative to measure autophagosome flux in order to assess the status of autophagy. Therefore, the hits from the primary GFP-LC3 screen are subjected to rigorous secondary assays (such as autophagosome formation and maturation, and autophagic substrate clearance, amongst others) (Mizushima et al., 2010; Klionsky et al., 2012) for characterizing autophagy modulators.

### Identification of Autophagy Modulators by mRFP-GFP-LC3 Screening Method

In order to overcome the problem of the GFP-LC3 reporter, a tandem fluorescent-tagged mRFP-GFP-LC3 reporter can be employed to determine autophagosome maturation for distinguishing between the autophagosomes and the autolysosomes. This mRFP-GFP-LC3 reporter is pH-sensitive. When overexpressed in cells, the autophagosomes exhibit both mRFP and GFP signals, whereas the autolysosomes emit only mRFP signal because the acid-labile GFP signal is quenched in the acidic environment (Kimura et al., 2007). For the mRFP-GFP-LC3 screening method, image-based analysis is done by quantifying the mRFP+ and GFP+ puncta per cell to measure perturbations in the number of autophagosomes (mRFP+/GFP+) and autolysosomes (mRFP+/GFP−). In general, an autophagy inducer (acting at early stage) will increase autophagosomes and autolysosomes, an autophagy inhibitor (acting at early stage) will decrease both these compartments, whereas an autophagy blocker (acting at late stage) will increase autophagosomes and decrease autolysosomes (**Figure 2**). Alternative versions of the mRFP-GFP-LC3 reporter have been described that may provide better readouts. These include replacing mRFP with mCherry that has superior photostability over mRFP (Pankiv et al., 2007), and substituting GFP with mWasabi that is more acid-sensitive than GFP (Zhou et al., 2012).

This pH-sensitive reporter has been primarily utilized as a secondary screening strategy following primary screens utilizing the more simpler GFP-LC3 method. In a high-throughput screen with 59541 compounds in GFP-LC3 platform, 400 screen hits were subjected to additional screening in stable HeLa cells expressing mCherry-GFP-LC3 (Kuo et al., 2015). These compounds were treated at 10 µM concentration for 24 h, after which 250 compounds increased (putative inducers) and 80 compounds decreased (putative inhibitors/blockers) the number of mCherry+/GFP− autolysosomes. Following further characterization, potent mTOR-independent autophagy inducers identified were BRD5631, BRD2716, and BRD34009 (Kuo et al., 2015). In another study, HeLa cells stably expressing mRFP-GFP-LC3 was subjected to three drug libraries such as the Prestwick Chemical Library, Microsource Spectrum 2000 library and Johns Hopkins Library that encompass 3791 compounds including FDA-approved drugs and bioactive molecules (Chauhan et al., 2015). Compounds were treated at 10 µM concentration for 4 h. However, high-content image analysis was done based only on GFP-LC3 puncta and total integrated area per cell, but not together with mRFP-LC3 that was utilized later during secondary characterization. 80 compounds were identified, out of which 55 were novel and 25 were previously reported as autophagy modulators. Further characterization of the hits including the mRFP-GFP-LC3 analysis identified flubendazole as a novel autophagy inducer that is also an antihelminthic drug. Flubendazole was shown to impact on dynamic and

acetylated microtubules to inhibit mTOR and disrupt Bcl2- Beclin 1 complex for inducing autophagy (Chauhan et al., 2015). More recently, a primary screen with mRFP-GFP-LC3 has been performed in U343 glioma cell spheroids (3D tumor spheroids) by dynamic live-cell imaging (Pampaloni et al., 2017). A subset of the Enzo Life Sciences Screen-Well Natural Compounds library comprising of 94 compounds were used at 1, 12.5, and 50 µM concentrations, followed by long-term time-lapse fluorescence imaging over 24 h at an interval of 1 h. Instead of measuring puncta formation, this study quantified the readout based on the ratio of mRFP and GFP emission intensities over time. Apart from validating this approach with the Enzo Life Sciences Screen-Well Autophagy library consisting of known autophagy modulators, the screen with selected natural compounds identified six potent autophagy inducers and four inhibitors. The autophagy-inducing natural compounds include PI-103, nonactin, valinomycin, quercetin, ivermectin, and harmine (Pampaloni et al., 2017).

The mRFP-GFP-LC3 reporter or its alternative versions can be subjected to high-throughput image-based screens to analyse autophagosome flux. This assay requires proper acidification of the lysosomes that could be affected by lysosomotrophic agents. However, autophagic substrate clearance along with other secondary assays should be assessed following the primary screen in order to assess the overall autophagic flux.

### Identification of Autophagy Modulators by GFP-LC3-RFP-LC31G Screening Method

A novel autophagy probe, GFP-LC3-RFP-LC31G, has been recently developed for evaluating autophagic flux that can be used for high-throughput screening approaches (Kaizuka et al., 2016). When overexpressed in cells, the Atg4 family proteases can cleave this reporter into equimolar amounts of GFP-LC3 and RFP-LC31G. While GFP-LC3 on the autophagosomes is degraded or recycled after fusion with the lysosomes, RFP-LC31G cannot be lipidated due to a deletion in its C-terminal glycine and thus remains in the cytosol serving as an internal control. This GFP-LC3-RFP-LC31G reporter can be subjected to both qualitative (by ratiometric imaging via fluorescence microscopy) and quantitative (via microplate reader or flow cytometry) analyses by measuring the fluorescence of GFP-LC3 and RFP-LC31G, and then calculating the GFP/RFP ratio (Kaizuka et al., 2016). Autophagy inducers are expected to decrease GFP/RFP ratio by enhancing autophagic flux, whereas autophagy inhibitors or blockers will increase GFP/RFP ratio by reducing autophagic flux (**Figure 2**).

Two chemical screens employing the GFP-LC3-RFP-LC31G screening method have been undertaken using a selected library of 34 known autophagy-regulating compounds and 1054 approved drugs under basal or starvation conditions in HeLa cells stably expressing this reporter (Kaizuka et al., 2016). The GFP/RFP ratio was calculated from fluorescence measurement via a microplate reader. For the first screen with known autophagy-regulating compounds, cells were treated for 6, 12 or 24 h with concentrations previously shown to modulate autophagy. A number of known autophagy modulators, but not all, acted as expected primarily after 12 or 24 h treatment. Specifically, autophagy inducers such as rapamycin (Blommaart et al., 1995) and Torin 1 (Thoreen et al., 2009) decreased GFP/RFP ratio whereas autophagy blockers like bafilomycin A1 (Yamamoto et al., 1998) and chloroquine (Seglen et al., 1979) increased GFP/RFP ratio (Kaizuka et al., 2016). For the second screen with approved drug library, cells were treated for 24 h at 10 µM concentration with few exceptions at 5 µM. The screen hits included 47 autophagy-inducing drugs (comprising of certain anti-cancer drugs, antibiotics and cardiotonic drugs) and 43 autophagy inhibitory drugs. Although many of these hits were previously reported, 13 inducers and 18 inhibitors/blockers were identified as novel autophagy modulators, of which some of the novel autophagy inducers were adefovir pivoxil, methyltestosterone, norethisterone, oxaprozin, and zidovudine (Kaizuka et al., 2016). This GFP-LC3-RFP-LC31G probe has been demonstrated to be capable of measuring basal and induced autophagic flux in Zebrafish and in tissues of transgenic mice (Kaizuka et al., 2016), and is thus valuable for monitoring autophagic flux in vivo.

Although this reporter can be used for high-throughput applications and in vivo studies to measure the overall autophagic flux, it is not ideal for investigating the distinct stages of autophagy such as autophagosome formation and maturation. Importantly, the two LC3 sequences of GFP-LC3-RFP-LC31G in retrovirally transfected cells can undergo homologous recombination, which will generate GFP-LC31G that is incapable of being degraded by autophagy. In addition, the expression levels of this reporter define the accuracy of the readout, and hence analysis in different cell lines or tissues will require comparable expression (Kaizuka et al., 2016; Geng and Klionsky, 2017).

### CHEMICAL SCREENING METHODS BASED ON AUTOPHAGY SUBSTRATES

In addition to the screening approaches based on LC3 reporters, autophagy substrate clearance has also been utilized as a primary screening assay for identifying autophagy modulators (**Table 1**). This method measures the autophagic cargo flux, which together with LC3-based secondary assays for autophagosome flux can indicate the overall autophagic flux.

### Identification of Autophagy Modulators by Clearance of Aggregation-Prone Proteins

A number of neurodegeneration-associated aggregationprone proteins are predominantly degraded by autophagy (Menzies et al., 2017), and hence screening methods can be based on their clearance as readouts (Sarkar, 2013a). The well-established substrates undergoing autophagic degradation include mutant huntingtin (with expanded polyglutamine repeats) and mutant α-synuclein (A53T or A30P mutants) associated with Huntington's and Parkinson's disease,

respectively (Webb et al., 2003; Ravikumar et al., 2004). Since the steady-state level of proteins is not ideal for accurately reflecting any impact on their degradation, stable inducible cell lines are required for analyzing autophagic substrate clearance where the transgene product is temporally synthesized by doxycycline followed by treatment with compounds after the expression is turned off (Wyttenbach et al., 2001; Webb et al., 2003; Sarkar et al., 2009). In general, autophagy inducers will enhance the clearance of aggregation-prone proteins, whereas autophagy inhibitors or blockers will retard their clearance (**Figure 2**).

Independent studies using a stable inducible PC12 cell line expressing EGFP-tagged mutant huntingtin (EGFP-HDQ74) identified mTOR-independent autophagy inducers such as trehalose (Sarkar et al., 2007a) as well as inositol-lowering agents (lithium, carbamazepine, valproic acid, L-690330) (Sarkar et al., 2005) and nitric oxide synthase inhibitors (L-NAME) (Sarkar et al., 2011). These studies also identified autophagy inhibitory compounds such as agents increasing inositol or inositol 1,4,5-trisphosphate (IP3) levels (myo-inositol, prolyl endopeptidase inhibitor 2) (Sarkar et al., 2005) and nitric oxide donors (DEA NONOate, DETA NONOate) (Sarkar et al., 2011). Utilizing stable inducible PC12 cell line expressing hemagglutinin (HA)-tagged A53T α-synuclein (HA-α-syn(A53T) ) as the primary screening method, a chemical screen was undertaken with 72 hits arising from an yeast screen involving 50729 compounds (Sarkar et al., 2007b). Cells were treated with compounds at 2 mg mL−1 concentration for 24 h after the initial doxycycline-induced synthesis of the transgene product (A53T α-synuclein), followed by immunoblotting analysis to measure its clearance. A number of novel autophagy modulators were identified which enhanced the autophagy substrate clearance. These include 4 small molecule enhancers of rapamycin (SMERs) and 13 small molecule inhibitors of rapamycin (SMIRs), of which SMER10, SMER18, and SMER28 were characterized to be autophagy inducers acting independently of mTOR. Further screening of the chemical analogs of these SMERs identified 18 additional autophagy inducers, such as 1 SMER10, 7 SMER18 and 10 SMER28 analogs that are capable of enhancing substrate clearance; although not substantially better than the respective parent compounds (Sarkar et al., 2007b). Another screen also utilizing a stable inducible PC12 cell line expressing HA-tagged A30P α-synuclein (HA-α-syn(A30P) ) was undertaken with a library of 253 compounds including FDAapproved drugs and pharmacological probes (Williams et al., 2008). Drug treatment was done at 1 µM for 24 h after the synthesis of the transgene product, followed by immunoblotting analysis. This study elucidated a cyclic mTOR-independent autophagy pathway with multiple drug targets, in which cAMP regulates IP3 levels that impact on calpain activity, which in turn activates Gsα that regulates cAMP levels. Some of the autophagy-inducing compounds identified include L-type Ca2+ channel blockers (verapamil, loperamide, amiodarone), calpain inhibitors (calpastatin), ATP-sensitive K+ channel agonist (minoxidil), cAMP reducing agents (rilmenidine, clonidine) and inositol lowering agents (valproic acid), whereas Ca2+ channel openers [(±)-Bay K8644] and agents elevating cAMP (dibutyryl cAMP, forskolin) and cytosolic Ca2+ (thapsigargin) levels were autophagy inhibitory (Williams et al., 2008). In addition to these immunoblotting based methods, the effects of autophagy modulators on autophagy-dependent clearance of EGFP-tagged mutant huntingtin aggregates can be validated by fluorescence microscopy in wild-type (Atg5+/+) and autophagy-deficient (Atg5−/−) mouse embryonic fibroblasts (MEFs) (Kuma et al., 2004; Sarkar et al., 2009).

Although autophagic clearance of aggregation-prone proteins is informative for autophagic flux, only low-throughput approaches are possible that creates a major hurdle for highthroughput applications. Nonetheless, this method could be used as a secondary assay for characterization of selected hits arising from screens with LC3-based reporters.

### Identification of Autophagy Modulators by p62/SQSTM1 Clearance

An alternative approach to the clearance of aggregation-prone proteins is to monitor the autophagic degradation of a known autophagy substrate, p62/SQSTM1, which also functions as an adaptor protein during selective autophagy for recruiting specific autophagic cargo to the autophagosomes (Bjorkoy et al., 2005; Pankiv et al., 2007). Similarly, to the method involving aggregation-prone proteins, screening approaches based on p62 clearance would ideally require a stable inducible cell line where the transgene product is temporally expressed before the treatment with compounds. The p62 reporters, such as GFPp62 (Larsen et al., 2010) or luciferase-tagged p62 (Brown et al., 2016; Min et al., 2018), could be utilized for medium- to highthroughput screens by flow cytometry or microplate reader (for analyzing p62 levels) or by fluorescence imaging (for analyzing p62 aggregates). Genetic screens have been undertaken with p62-based reporters (Pietrocola et al., 2015; Strohecker et al., 2015; DeJesus et al., 2016; Hale et al., 2016), and therefore, similar chemical screening approaches are also possible. In addition, analyzing the steady-state levels of endogenous p62 by immunoblotting is often used as a secondary assay for characterization of autophagy modulators (Klionsky et al., 2012). It is expected that an autophagy inducer will decrease p62 levels or aggregates, whereas an autophagy inhibitor or blocker will cause its accumulation (**Figure 2**). Recently, an assay based on LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) has been described to monitor autophagy independent of any exogenous labels. This method is based on the proximity of the donor and the acceptor antibodies of LC3- II and p62, in which autophagy inducers increase LC3-II signal and decrease p62 signal, autophagy inhibitors do not display any turnover of either signals, whereas autophagy blockers will increase LC3-II signal without any turnover of p62 signal (Bresciani et al., 2018).

Although p62 is a specific autophagy substrate in most mammalian cell lines (Klionsky et al., 2012), its autophagic degradation should be confirmed in the cell-type and the timepoints to be used in the screens. Moreover, transcriptional upregulation of p62 has been reported during some instances of autophagy activation, such as under prolonged starvation or with certain pharmacological inducers (Klionsky et al., 2012;

Sahani et al., 2014; Kuo et al., 2015), and therefore, any perturbation in p62 protein levels needs to be accompanied by qPCR assessment of its mRNA levels.

malfunctioning autophagy in these contexts, are highlighted.

### BIOMEDICAL APPLICATIONS OF AUTOPHAGY MODULATORS IN HUMAN DISEASES

Autophagy plays an essential role for tissue homeostasis and cellular survival by removing unwanted materials like malfunctioning aggregated proteins and damaged organelles from the cells; however, deregulation of this process could contribute to cytotoxicity (Mizushima et al., 2008). Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases (Levine and Kroemer, 2008; Jiang and Mizushima, 2014), and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance (**Figure 3**). A number of independent studies and chemical screens have identified several autophagy modulators, which have been shown to impart beneficial effects in various transgenic disease models (**Table 2**; Rubinsztein et al., 2012; Sarkar, 2013b; Levine et al., 2015). Some of the key studies in specific disease contexts are highlighted below.

## AUTOPHAGY MODULATORS IN NEURODEGENERATIVE DISEASES

Basal autophagy in the brain is critical for maintaining cellular homeostasis in post-mitotic cells like neurons, which is evident from the genetic studies in mice where brain-specific deletion of essential autophagy genes resulted in neurodegenerative phenotypes (Hara et al., 2006; Komatsu et al., 2006). Particularly, autophagy is the primary degradation pathway for several aggregation-prone proteins associated with neurodegeneration (Rubinsztein, 2006; Nixon, 2013). However, defective autophagy has been reported in several neurodegenerative diseases, including neurodegenerative lysosomal storage disorders, and is considered a major causative factor for neurodegeneration (Nixon, 2013; Sarkar, 2013b; Menzies et al., 2017; Seranova et al., 2017). Therefore, induction of autophagy for enhancing the clearance of mutant aggregation-prone proteins is considered a potential treatment strategy. The therapeutic benefits of autophagy inducers have been robustly demonstrated in the context of neurodegeneration where upregulation of autophagy was protective in several in vitro and in vivo transgenic models of neurodegenerative diseases (Rubinsztein et al., 2012; Sarkar, 2013b; Levine et al., 2015; Seranova et al., 2017).   The most widely used mTOR-independent autophagy inducer in vivo is trehalose (Sarkar et al., 2007a), a disaccharide that stimulates autophagy by inhibiting SLC2A family of glucose transporters and activating AMPK (DeBosch et al., 2016), which in turn can directly influence the phosphorylation of the autophagyinitiating kinase ULK1 (Egan et al., 2011; Kim et al., 2011).   A combinatorial approach in enhancing autophagy has been shown with rapamycin and mTOR-independent autophagy inducers such as lithium, trehalose or SMERs. Higher efficacy was achieved via the additive effects of dual treatment on autophagy induction and cytoprotection in cell and fly models of HD than the effects of single compounds (Sarkar et al., 2007a,b, 2008).

### AUTOPHAGY MODULATORS IN CANCER

The ability of autophagy in the maintenance of metabolic homeostasis has drawn considerable attention as a potential target for cancer therapy via its pro-survival and pro-death mechanisms (Rabinowitz and White, 2010; Levy et al., 2017). Autophagy plays tumor suppressive role by mitigating oxidative stress, removing superfluous mitochondria and preventing DNA damage and genome instability; and on the other hand, shows pro-tumor activity by preventing the induction of tumor suppressors, increasing resistance to apoptosis and maintaining tumor metabolism through recycling of nutrients (Mathew et al., 2007; Galluzzi et al., 2015; Kimmelman and White, 2017). Depending on the cancer context and the opposing effects of autophagy, either inhibitors or inducers of autophagy could be exploited for cancer therapy (Galluzzi et al., 2017; Levy et al., 2017). Since autophagy promotes tumorigenesis in most contexts, inhibition of autophagy has gathered considerable interest for cancer therapy. Accumulating evidence demonstrate that autophagy inhibitors/blockers exerted therapeutic benefits in cancer models. The clinically- approved autophagy inhibitors chloroquine or hydroxychloroquine (HCQ), which impair lysosomal acidification and block autophagic flux (Murakami et al., 1998; Boya et al., 2005), caused tumor shrinkage in preclinical studies; and thus HCQ being more potent with lesser side-effects is used in ongoing clinical trials either alone or in combination with other treatments (Briceno et al., 2003; Amaravadi et al., 2007; Cook et al., 2014; Chude and Amaravadi, 2017; Levy et al., 2017; Onorati et al., 2018). Autophagy inhibitory compounds, such as Lys05 and ROC-325, which exhibited anti-tumor activity in mice have been suggested to be more potent than HCQ (McAfee et al., 2012; Carew et al., 2017). In addition, autophagy inhibitors preventing autophagosome formation such as ATG4B antagonists (compounds NSC185058 and UAMC-2526), Vps34 (vacuolar protein sorting protein 34) inhibitor (compound SAR405), ULK1 (Unc-51-like kinase 1) inhibitor (compound SBI-0206965), USP10/USP13 (ubiquitin-specific peptidases) inhibitor (Spautin-1) and agents causing transcriptional inhibition of autophagy genes (pyrvinium pamoate), also exerted anti-proliferative and anti-tumor effects in cellular and in vivo models of cancer (Liu et al., 2011; Deng et al., 2013; Akin et al., 2014; Ronan et al., 2014; Shao et al., 2014; Egan et al., 2015; Kurdi et al., 2017). On the contrary, various chemical agents or natural products exerting antiproliferative or anti-tumor activity either alone or in combination with chemotherapeutic agents could induce autophagy or autophagic cell death, which include Torin 1, AC-73, MC-4, metformin, silibinin, Abrus agglutinin, curcumin, liensinine, spermidine, vitamin D3, and imatinib (Buzzai et al., 2007; Ertmer et al., 2007; Wang et al., 2008; Thoreen et al., 2009; Qian et al., 2011; Francipane and Lagasse, 2013; Law et al., 2014; Jiang et al., 2016; Pietrocola et al., 2016; Panda et al., 2017; Son et al., 2018; Spinello et al., 2018).

### AUTOPHAGY MODULATORS IN INFECTIOUS DISEASES

Autophagy plays an important role in innate defense mechanism by removing intracellular pathogens; a process termed xenophagy (Levine et al., 2011; Deretic et al., 2013). The role of autophagy in regulating intracellular infections initially emerged through studies on Mycobacterium tuberculosis (Mtb) (Gutierrez et al., 2004; Singh et al., 2006). Subsequently, several other bacterial pathogens like Salmonella and Listeria, and viral pathogens like HIV and Dengue were shown to utilize host autophagy pathways for their own advantage (Jia et al., 2009; Kyei et al., 2009; Yoshikawa et al., 2009; Heaton and Randall, 2010). A genome-wide siRNA screen to identify host factors required for intracellular Mtb survival within macrophages revealed that a large number of host factors acted via regulation of autophagy to help the bacteria (Kumar et al., 2010). Induction of autophagy with rapamycin, carbamazepine, SMER28, and vitamin D3 were shown to prevent bacterial survival or HIV replication in macrophages (Gutierrez et al., 2004; Floto et al., 2007; Yuk et al., 2009; Kumar et al., 2010; Campbell and Spector, 2011, 2012; Schiebler et al., 2015). Notably, carbamazepine reduced bacterial burden, improved lung pathology and stimulated adaptive immunity in mice infected with multidrug-resistant Mtb (Schiebler et al., 2015). Rapamycin also controlled viral and

TABLE 2 | Therapeutic benefits of autophagy modulators in diverse human diseases.

(Continued)

#### TABLE 2 | Continued

fcell-07-00038 March 16, 2019 Time: 17:5 # 11

Autophagy modulators have shown beneficial effects in a number of transgenic disease models, such as but not limited to, neurodegenerative disorders, cancer, infectious diseases, liver diseases and myopathies as well as in lifespan extension. Selected examples of autophagy modulators are highlighted in specific pathological contexts. AATD, α1 antitrypsin deficiency; AD, Alzheimer's disease; AFLD, Alcoholic fatty liver disease; ALS, Amyotrophic lateral sclerosis; AMPK, 50 adenosine monophosphateactivated protein kinase; Atg, Autophagy-related genes; cAMP, 30 ,50 -cyclic adenosine monophosphate; DMD, Duchenne muscular dystrophy; FSD, Fibrinogen storage disease, FTD, Frontotemporal dementia; GAPR-1, Golgi-associated plant pathogenesis-related protein 1; HD, Huntington's disease; HIV, Human immunodeficiency virus; IP3, Inositol 1,4,5-trisphosphate; iPSC, Induced pluripotent stem cells; LD, Lafora disease; LMNA, Lamin A/C gene; MTMR14, Myotubularin related protein 14; mTORC1, Mechanistic target of rapamycin complex 1; NAFLD, Non-alcoholic fatty liver disease; NPC1, Niemann-Pick type C1 disease; PBMC, Peripheral blood mononuclear cells; PD, Parkinson's disease; PI(3,5)P2, Phosphatidylinositol 3,5-bisphosphate; RCC, Renal cell carcinoma; SCA, Spinocerebellar ataxia; SIRT1 Sirtuin 1; SLC2A, Solute carrier 2A; TRPML1, Transient receptor potential cation channel mucolipin subfamily member 1.

bacterial pathogens both in vitro and in vivo (Donia et al., 2010). In an integrated chemical and RNAi screening for modulators of intracellular mycobacteria, one of the top three compounds was nortriptyline which significantly suppressed Mtb survival within macrophages and induced autophagy (Sundaramurthy et al., 2013). Other compounds limiting bacterial or HIV infections

through activation of autophagic flux were nitazoxanide (antiprotozoan drug) and flubendazole (antihelminthic drug) (Lam et al., 2012; Chauhan et al., 2015). Similarly, the naturally occurring disaccharide trehalose, a potent mTOR-independent enhancer of autophagy in diverse cell-types (Sarkar et al., 2007a), can also induce autophagy and xenophagy in Mtb-infected

Panda et al. Drug Discovery of Autophagy Modulators

macrophages that resulted in the killing of bacteria (Sharma et al., 2017). In this study, trehalose was found to act as a PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) agonist for activating the lysosomal Ca2+ channel TRPML1 (Sharma et al., 2017), which in turn released lysosomal Ca2+ that caused nuclear translocation of TFEB to induce autophagy (Medina et al., 2015). Excitingly, trehalose also seemed to be effective during HIV-Mtb co-infection and limits Mtb survival by reversing the HIVmediated block in autophagy flux (Sharma et al., 2017). Similarly, vitamin D3 could also kill Mtb during HIV co-infection by inducing autophagy (Campbell and Spector, 2012). Several host factors currently being tested for anti-Mtb therapeutics function by regulating host autophagy and xenophagy. For example, inhibition of host Src kinase by the compound AZD0530 induced autophagy and lysosomal maturation to clear Mtb (Chandra et al., 2016). A pioneering anti-infective, autophagy-inducing agent is Tat-Beclin 1, which is a peptide representing a region of the autophagy regulator Beclin 1 that interacts with the HIV-1 accessory protein NEF, and this domain is linked with the HIV-1 Tat transduction domain to make it cell permeable (Shoji-Kawata et al., 2013). Tat-Beclin 1 prevented the replication of a number of viral and bacterial pathogens in vitro in autophagydependent manner, as well as induced autophagy and anti-viral activity in mice infected with chikungunya or West Nile virus (Shoji-Kawata et al., 2013). Thus, it is evident that regulators of autophagy and xenophagy have tremendous potential for novel therapeutics against various infectious diseases. It is now clear that within an infected host cell, there is a possibility of uncoupling between homeostatic autophagy and anti-bacterial xenophagy (Chandra et al., 2015; Sharma et al., 2018). Therefore, it is desirable to perform chemical screening pertaining to infection-specific xenophagy flux for identifying novel regulators of bacterial/viral survival within the host cells through the autophagy pathway.

### AUTOPHAGY MODULATORS IN LIVER DISEASES

Liver autophagy is essential for various hepatic functions and is implicated in various liver conditions including α1-antitrypsin (AAT) deficiency, non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma and viral hepatitis (Rautou et al., 2010; Ueno and Komatsu, 2017). Chemical modulation of autophagy has been shown to have beneficial effects in some of these diseases. Carbamazepine, an mTOR independent autophagy inducer acting by reducing inositol levels (Sarkar et al., 2005), reduced hepatic load of mutant α1-antitrypsin Z and hepatic fibrosis in a mouse model of AAT deficiency (Hidvegi et al., 2010), as well as decreased hepatocellular aggregate-related toxicity in patients suffering from fibrinogen storage disease (Puls et al., 2013). A high-throughput drug screen in hepatocyte-like cells derived from iPSC lines of patients with AAT deficiency also revealed inositol-lowering autophagy-inducing agents, such as carbamazepine, lithium, and valproic acid, in facilitating the clearance mutant AAT (Choi et al., 2013). Carbamazepine as well as the mTOR inhibitor rapamycin also rescued dysfunctional autophagic flux and improved cell viability in hepatic-like cells differentiated from patient-derived iPSC lines of Niemann-Pick type C1 (NPC1) disease (Maetzel et al., 2014). In addition, autophagy induction with trehalose, carbamazepine, rapamycin or hydrogen sulfide reduced steatosis, lipid accumulation and liver injury in high-fat diet-induced NAFLD in mice (Lin et al., 2013; Sun et al., 2015; DeBosch et al., 2016). Furthermore, the anti-diabetic drug metformin, which indirectly inhibits mTOR, induced SIRT1-mediated autophagy in primary hepatocytes and ameliorated hepatic steatosis in vivo (Song et al., 2015). Overall, these studies indicate that activation of autophagy via inhibition of mTOR, lowering inositol levels or with trehalose are effective modes of inducing autophagy in the liver.

### AUTOPHAGY MODULATORS IN MYOPATHIES

Basal autophagy is required for maintaining muscle mass and myofiber integrity (Masiero et al., 2009), and thus deregulation of muscle autophagy is implicated in myopathies and muscular dystrophies (Sandri et al., 2013). Sustained activation of mTORC1 in skeletal muscle of TSC1-deficient mice could cause late-onset myopathy related to suppression of autophagy (Castets et al., 2013). Upregulation of autophagy, primarily by inhibiting the mTORC1 pathway, has been reported to have beneficial effects in certain transgenic disease models. Autophagy induction by rapamycin or low-protein diet increased myofiber survival and attenuated dystrophic phenotype in a mouse model of collagen type VI muscular dystrophy (Grumati et al., 2010). Likewise, activation of autophagy by dietary changes or with the AMP-activated protein kinase (AMPK) agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside),

improved dystrophic phenotypes in mouse models of Duchenne muscular dystrophy (DMD) (De Palma et al., 2012; Pauly et al., 2012). A potential role of simvastatin, which has been reported to induce autophagy by inhibiting the Rac1-mTOR pathway (Wei et al., 2013), has been suggested in improving the physiological function of skeletal muscle in DMD transgenic mice (Whitehead et al., 2015). In addition, rapamycin or its analog, temsirolimus, ameliorated cardiomyopathy and improved skeletal and cardiac muscle function in mouse models of LMNA (lamin A/C gene) cardiomyopathy that recapitulate Emery-Dreifuss muscular dystrophy (EDMD) (Choi et al., 2012; Ramos et al., 2012).

## AUTOPHAGY MODULATORS IN LIFESPAN EXTENSION

The functionality of autophagy declines with aging (Rubinsztein et al., 2011), and thus restoring adequate autophagy is considered a possible anti-aging strategy for lifespan extension. There are a number of lifespan expanding strategies, and in many of such approaches, autophagy acts as a common denominator

for promoting longevity (Madeo et al., 2010; Hansen et al., 2018). Pharmacological treatment with autophagy inducers has been linked to increasing longevity in transgenic in vivo models (Madeo et al., 2015). Lifespan extension via induction of autophagy with naturally- occurring polyamines such as spermidine, which is an acetyltransferase inhibitor, was shown in yeast, flies, worms and mice (Eisenberg et al., 2009, 2016); and likewise also reported with the natural phenol resveratrol, which is a deacetylase activator, in yeast, flies, worms as well as in mice on high-fat diet (Howitz et al., 2003; Wood et al., 2004; Baur et al., 2006; Morselli et al., 2010). Although both spermidine and resveratrol impacts on the acetylproteome, stimulation of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) whereas the effect of spermidine was SIRT1 independent (Morselli et al., 2010, 2011). Inhibition of mTOR by rapamycin also extended lifespan in yeast, flies and mice (Alvers et al., 2009; Harrison et al., 2009; Bjedov et al., 2010; Lamming et al., 2013). In addition, lifespan extension in multiple organisms including mice and apes could be achieved by caloric restriction, which is a physiological inducer of autophagy via AMPK activation, mTORC1 inhibition and SIRT1 activation (Mair and Dillin, 2008; Colman et al., 2009; Mercken et al., 2014; Mattison et al., 2017). In some of these studies reporting lifespan extension by autophagy activation, the role of autophagy has been specifically determined by abolishing the anti-aging effects via knockdown of essential autophagy genes (Madeo et al., 2015; Nakamura and Yoshimori, 2018).

### CONCLUSION

The methodologies for measuring autophagy have evolved over the past decade and it is now feasible to undertake highthroughput chemical screens for identifying modulators of autophagic flux. A number of pharmacological modulators of autophagy have been identified via screening approaches or individual studies; some of which have been demonstrated to exert therapeutic benefits in diverse human diseases. Most of the key autophagy modulators have been identified either by the GFP-LC3 screening method in HeLa cells or via assessing the clearance of aggregation-prone proteins in inducible PC12 cell lines. While analysis of changes in autophagosome number with GFP-LC3 reporter requires shorter treatment period (such as 8–24 h), analysis of clearance of aggregation-prone proteins requires longer treatment duration (such as 24–72 h) depending on the nature of the transgene product. Following the primary screen, it is pertinent to characterize the highconfidence screen hits with secondary autophagy assays because there are no single assays to determine autophagic flux. These normally include analysis of autophagosome formation with bafilomycin A1 via immunoblotting with anti-LC3 antibody, analysis of autophagosome maturation with mRFP-GFP-LC3 reporter, and analysis of autophagy substrate (p62) clearance via immunoblotting with anti-p62 antibody (Mizushima et al., 2010; Klionsky et al., 2016).

Although the methods described in this review are those that have been generally used in the field, alternative autophagy assays could also be employed for chemical screening. One potential approach is the use of Keima, a fluorescent acid-stable protein that exhibits bimodal excitation spectra in neutral and acidic pH, such as in autophagosomes and autolysosomes, respectively (Katayama et al., 2011). The cumulative fluorescence readout can be used to measure bulk autophagic flux. This protein can also be utilized for selective autophagic flux, such as with mitochondriatargeted Keima to measure mitophagy (Katayama et al., 2011; Sun et al., 2017). However, Keima-based assays solely depend upon the lysosomal acidity and thus cannot be performed in fixed cells where the pH gradient across lysosomal membranes is lost. In addition, other screening approaches could be based on fluorescent-tagged early markers of autophagy initiation, such as with WIPI-1 (Proikas-Cezanne and Pfisterer, 2009) and DFCP1 (Axe et al., 2008); however, these methods will not capture the late events of autophagy pathway involving autophagosome maturation and cargo degradation.

For the therapeutic exploitation of autophagy modulators, mTOR-independent autophagy inducers are generally favorable and considered to have lesser side-effects than the mTOR inhibitors like rapamycin. This is because mTOR controls vital cellular functions like cell growth and translation and thus its inhibition can lead to undesirable side-effects unrelated to autophagy induction. For clinical translation to patients, it is important to determine the efficacy and penetrance of the autophagy modulators in the target organs. Future directions could include identifying specific inducers of autophagy acting at the level of autophagic machinery rather than the upstream signaling pathways.

### AUTHOR CONTRIBUTIONS

PP, AF, SV, DK, and SS wrote the manuscript. ES and SS made the figures. PP and SS made the tables. PP, AF, SV, ES, VS, MC, PD, JT, TR, DK, and SS reviewed the manuscript.

## FUNDING

SS is funded by Wellcome Trust Seed Award (109626/Z/15/Z), UKIERI (UK-India Education and Research Initiative) DST Thematic Partnership Award (2016-17-0087) with DK, FAPESP-Birmingham-Nottingham Strategic Collaboration Fund with TR, and Birmingham Fellowship from the University of Birmingham (UoB). SV is also a Newton Bhabha Ph.D. Placement Fellow (funded by British Council) and TR is also a Brazil Visiting Fellow (funded by UoB) and Rutherford Fellow in SS lab at UoB. SS is also a Former Fellow for life at Hughes Hall, University of Cambridge, United Kingdom.

### ACKNOWLEDGMENTS

We thank the funding agencies for supporting our research.

### REFERENCES

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Panda, Fahrner, Vats, Seranova, Sharma, Chipara, Desai, Torresi, Rosenstock, Kumar and Sarkar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Generation and Characterization of Germline-Specific Autophagy and Mitochondrial Reactive Oxygen Species Reporters in Drosophila

Kiran Nilangekar1,2, Nidhi Murmu1,2, Govind Sahu1 and Bhupendra V. Shravage1,2 \*

1 Developmental Biology Group, Agharkar Research Institute, Pune, India, 2 Department of Biotechnology, Savitribai Phule Pune University (SPPU), Pune, India

#### Edited by:

Ioannis Nezis, University of Warwick, United Kingdom

#### Reviewed by:

Peter Nagy, Cornell University, United States Sharon Gorski, British Columbia Cancer Agency, Canada Kimberly McCall, Boston University, United States

> \*Correspondence: Bhupendra V. Shravage<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology Received: 15 July 2018 Accepted: 15 March 2019 Published: 03 April 2019

#### Citation:

Nilangekar K, Murmu N, Sahu G and Shravage BV (2019) Generation and Characterization of Germline-Specific Autophagy and Mitochondrial Reactive Oxygen Species Reporters in Drosophila. Front. Cell Dev. Biol. 7:47. doi: 10.3389/fcell.2019.00047 Oogenesis is a fundamental process that forms the egg and, is crucial for the transmission of genetic information to the next generation. Drosophila oogenesis has been used extensively as a genetically tractable model to study organogenesis, nichegermline stem cell communication, and more recently reproductive aging including germline stem cell (GSC) aging. Autophagy, a lysosome-mediated degradation process, is implicated in gametogenesis and aging. However, there is a lack of genetic tools to study autophagy in the context of gametogenesis and GSC aging. Here we describe the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2- Orp1 that are specifically expressed in the germline compartment including GSCs during Drosophila oogenesis. These transgenes are expressed from the nanos promoter and present a better alternative to UASp mediated overexpression of transgenes. These fluorescent reporters can be used to monitor and quantify autophagy, and the production of reactive oxygen species during oogenesis. These reporters provide a valuable tool that can be utilized in designing genetic screens to identify novel regulators of autophagy and redox homeostasis during oogenesis.

Keywords: GFP-Ref(2)P, mCherry-Atg8a, mito-roGFP2-Orp1, autophagic flux, redox, germline stem cell, autophagy, Atg8a

### INTRODUCTION

In multicellular organisms, the production of functional gametes depends on the activity of specialized stem cells called "germline stem cells" (GSCs) located in the gonads (reviewed in Fuller and Spradling, 2007; Dansereau and Lasko, 2008). Like most other types of stem cells, GSCs are also subject to cellular damage that causes premature aging and render them inactive leading to their depletion within the gonads (López-Otín et al., 2013; Signer and Morrison, 2013; Oh et al., 2014). Cellular damage caused by genotoxic agents and reactive oxygen species can damage organelles, proteins, and DNA within the stem cells. Thus, it is crucial to maintaining the integrity and quality of GSCs, as cellular and genetic defects may be passed onto the next

generation which can be detrimental to the survival of the species. The prolonged sustenance of GSCs, therefore, require efficient homeostasis mechanisms to be operational constitutively within these cells. Such homeostasis mechanism(s) must function to significantly reduce cellular damage. Macroautophagy (autophagy) is the cellular mechanism that is involved in removing toxic protein aggregates and damaged organelles such as mitochondria from within the cytoplasm (Takeshige et al., 1992; Mizushima, 2007; Yu et al., 2018). Autophagy is necessary during animal development and impaired autophagy has been implicated in several diseases including cancer, neurodegenerative diseases, infectious diseases, cardiopathy and autoimmunity (Jiang and Mizushima, 2014; Schneider and Cuervo, 2014). However, the involvement of autophagy during gametogenesis, reproduction and germline aging is not extensively studied.

Autophagy proceeds with the sequestration of cytoplasmic contents within a double-membraned vesicle termed as autophagosome which fuses with the lysosome to form an autolysosome. The cargo within the autolysosome is degraded by the lysosomal enzymes. The degradation products are exported from the autolysosome to the cytoplasm where they can be reused in several metabolic processes (He and Klionsky, 2009; Boya et al., 2013; Bento et al., 2016). The complex process of autophagosome formation and its subsequent fusion to the lysosome is highly regulated by Autophagy-related (Atg) proteins (Mizushima et al., 1998, 2011; Klionsky, 2012; Mulakkal et al., 2014).

In metazoans, 16 Atg proteins have been shown to be essential for the autophagy. The process is initiated at the preautophagosome structure/phagophore assembly site (PAS) where Atg1 kinase complex consisting of Atg1, FIP200, Atg13, and Atg101 assembles (reviewed in Scott et al., 2004; Nagy et al., 2014b; Hurley and Young, 2017). PAS formation can occur at the ER and is termed as omegasome in mammals (Axe et al., 2008). The activity of Atg1 kinase is required for recruitment of Vps34 complex comprising of Beclin-1 (Atg6 in Drosophila), Vps34, Vps15 and Atg14 to the phagophore where it catalyzes the formation of PI3P which is essential for vesicle nucleation (Kihara et al., 2001; Itakura et al., 2008; Juhász et al., 2008). Several membrane sources can contribute to the formation of the phagophore/isolation membrane (Chan and Tang, 2013). The formation of Phosphatidylinositol 3-phosphate (PI3P) at the phagophore site is crucial for the recruitment of WIPI family proteins (Burman and Ktistakis, 2010). WIPI2 (Atg18 in Drosophila) family proteins interact with Atg16L1 (Atg16 in Drosophila) that subsequently allow for recruitment of Atg12- Atg5-Atg6L1 complex at the PAS (Romanov et al., 2012; Walczak and Martens, 2013). The formation of the Atg12-Atg5-Atg16 complex is catalyzed by several enzymes. Atg12 is a ubiquitinlike protein that is activated by Atg7 E1-like enzyme and subsequently transferred to E2 like enzyme Atg10 allowing for the formation of Atg12-Atg5 complex. The Atg12-Atg5 complex can form a ternary complex with Atg16L1 (Atg16 in Drosophila), finally catalyzing lipidation of Atg8 on the autophagosome membrane (Mizushima et al., 1998; Kim et al., 2016; Nagy et al., 2017). Atg8/MAP1LC3, a ubiquitin-like molecule (microtubule associated protein 1 light-chain 3, LC3, in mammals) is a component of conjugation system that is also essential for elongation and completion of the autophagosome (Ichimura et al., 2000; Kabeya et al., 2000). C-terminal amino acid residues of Atg8 are removed by ATG4 protease to expose a C-terminal glycine that is covalently bound to phosphatidylethanolamine (PE) by series of enzymes Atg7 (E1-like enzyme), Atg3 (E2-like enzyme), and the Atg12 complex (consisting of Atg12-Atg5- Atg16). The Atg8-PE (Atg8-II) is localized to the autophagosome membrane and this lipidation event has been exploited to study autophagy. Typically, Atg8 is fused with fluorescent proteins such as GFP or RFP/mCherry at its amino terminus and are driven by tissue-specific promoters, endogenous Atg8a promoters, by UAS and UASp sequences (Scott et al., 2004; Denton et al., 2009, 2012; Nelson et al., 2014; Hegedus et al., 2016; Jacomin and Nezis, 2016; Bali and Shravage, 2017). However, a few of these transgenes do not express in the germline or are expressed at very high levels in the germline complicating autophagy assays. An alternative to monitoring autophagy is to assay for lipid conjugation of Atg8 proteins by immunoblotting (Barth et al., 2011; Mauvezin et al., 2014; Nagy et al., 2015; Lörincz et al., 2017). The lipidated protein Atg8-II migrates at a faster rate on polyacrylamide gels than its unprocessed form, Atg8-I. However, immunoblotting techniques typically use protein extracts prepared from the entire tissue or group of cells which eliminate individual differences in autophagy status in individual cell types within the tissue (Klionsky et al., 2016).

p62/SQSTM1 is an autophagy adaptor first described in mammals and subsequently shown to function during autophagy in Drosophila (Ref(2)P in Drosophila) (Bartlett et al., 2011). The ability of p62/Ref(2)P to interact with ubiquitin or polyubiquitin chains in different proteins via the UBA domain enables it to deliver cargo to autophagosome via the LIR domain (which is also known as the Atg8 interacting domain in Drosophila). Interestingly, p62 is capable of both homo-oligomerization and hetero-oligomerization via PB1 domains thus allowing the formation of complex protein aggregates that can be recognized by the autophagy machinery and targeted for degradation (Mulakkal et al., 2014; Nagy et al., 2015; Lörincz et al., 2017). Ref(2)P has been used to measure autophagic activity in Drosophila fat body cells where an increase in the size of Ref(2)P punctae under stress conditions was observed followed by a reduction in number due to the clearance of protein aggregates by autophagy (Pircs et al., 2012). The increased levels of Ref(2)P which marks the protein aggregates during the inhibition of autophagy serves as an in vivo measure of long term autophagic activity. Thus, it can be used as a tool to detect the protein inclusion formation and changes in autophagic activity under various physiological conditions (Bartlett et al., 2011; Pircs et al., 2012; Devorkin and Gorski, 2014; Mauvezin et al., 2014). In Drosophila, there are no transgenic lines that can detect Ref(2)P in GSCs and any assays performed in the germline are based on the use of anti-Ref(2)P antibody which becomes a limiting factor for designing forward genetic screens.

The elongation of phagophore is catalyzed by Atg9/Atg9L1 complex in addition to the Atg1 and Vps34 complexes, and Atg12 and Atg8 conjugation systems. The Atg9 complex consists of Atg9, a transmembrane protein, Atg18, and Atg2 (Nagy

et al., 2014a). Atg9 containing vesicles shuttle between the PAS/phagophore and membrane source (Golgi/endosomes). Membrane scission occurs at the end of the elongation process resulting in the generation of a closed autophagosome laden with cargo. Following this event, most Atg proteins are removed or disassembled from the autophagosome membrane catalyzed by PI3-P phosphatases which aid in the elimination of PI3-P from the membrane. In most metazoans, autophagosomes fuse with endosomes to form amphisomes before being fused to lysosomes by kiss-and-run like process. Several proteins which are involved in vesicle fusion including SNAP29, Syntaxin17, Rab7, Atg14, HOPS complex and PLEKHM1 are shown to be required for this process (Takáts et al., 2013; Guo et al., 2014; Takats et al., 2014; Liu et al., 2015; McEwan et al., 2015; Hegedus et al., 2016). The outer membrane of the autophagosome fuses to the lysosome to form the autolysosome. The cargo along with the inner membrane of the autophagosome is degraded in the autolysosome by the lysosomal hydrolases. The degradation products are exported from the autolysosome back into the cytoplasm where they are used for anabolic processes (Mizushima, 2007; Bento et al., 2016; Nakamura and Yoshimori, 2017; Yu et al., 2018).

Autophagy is also triggered in response to reactive oxygen species (Scherz-Shouval and Elazar, 2011; Filomeni et al., 2015). Hydrogen peroxide (H2O2), one of the metabolites of the intracellular redox reaction, is an important signaling molecule, is required for peroxisomal catabolism and used by cells in a controlled manner to oxidize substrates. However, it is also the main source of peroxide ions which oxidize and damage proteins, lipids, and DNA (Sies, 2017). One of the main sources of H2O2 is the mitochondrial electron transport chain (Dröge, 2002; Sena and Chandel, 2012; Shadel and Horvath, 2015). Superoxide ions O2 − are produced by electron transport chain complex I and III present in different compartments of the mitochondria. O2 − ions are reduced by superoxide dismutase (SOD) enzymes. Mndependent SOD2 in the mitochondrial matrix is responsible for the conversion of O2 − to H2O2. H2O2 is detoxified to water by catalase and glutathione peroxidase (Dröge, 2002). However, H2O2 can be damaging when it reacts with the thiol group (−SH) within the proteins to form sulphenic acid (−SOH) altering their activity or rendering them inactive (Finkel, 2012). Such cellular damage within mitochondria leads to depolarization of mitochondria which results in a decrease of ATP synthesis and metabolic activities. Multiple proteins within the cells have been demonstrated to sense production of H2O2 and react with it (Ma et al., 2007; Gutscher et al., 2008).

Oxidant receptor peroxidase 1 (Orp1), a component of redox relay in yeast Saccharomyces cerevisiae, is homologous to glutathione peroxidase and senses H2O2 (Ma et al., 2007). Orp1 is shown to be able to successfully oxidize roGFP2 based on H2O2 sensing in vitro. This was first demonstrated by Gutscher et al. (2008), by making an Orp1 roGFP2 fusion protein. This construct was then modified by adding a mitochondrial localization signal (mito-roGFP2-Orp1) by Albrecht and colleagues enabling measurement of H2O2 production within the mitochondria (Albrecht et al., 2011, 2014). The probe senses the redox state of Orp1 through roGFP2. Thiol groups in Orp1 are oxidized by H2O2. This alters the redox state of Orp1 that causes reduction or oxidation of the roGFP2 which results in the switch in excitation of fluorescence from 488 to 405 nm. The shift in excitation of roGFP2 is a reliable measure of the H2O2 production.

Here we describe generation and characterization of transgenic lines that express mCherry-Atg8a, GFP-Ref(2)P and mito-ro-GFP2-Orp1 under the nanos gene promoter. Our data show that these transgenes could be used reliably to monitor autophagy and H2O2 production within the germ cells in Drosophila during gametogenesis. These transgenes lack the problems associated with UASp driven transgenes which can overexpress the Atg proteins complicating and affecting the interpretation of autophagy. We believe that these transgenes will aid in conducting screens designed to identify genes affecting autophagy and redox status within the germ cells in Drosophila.

### MATERIALS AND METHODS

### Fly Maintenance

All the transgenic fly stocks were maintained at 25◦C on standard cornmeal sucrose malt agar. The stocks were maintained homozygous for all transgenes. All insertions were tested for expression of the transgene using confocal microscopy. Insertion with the highest expression (strong fluorescence) was chosen for characterization. mCherry-Atg8a and GFP-Ref(2)P were mapped on to the chromosome, balanced and crossed to get the following combination; ywhsFLP1; mCherry-Atg8a/CyO; GFP-Ref(2)P/TM6b. The following flies were used; yw; UASp.mCherry.Atg8a; Dr/ TM3, Ser (BL37750, RRID:BDSC\_37750), w; +; nosGal4VP16 (BL4937, RRID:BDSC\_4937), y sc v; +; Atg8a-RNAi (BL34340, RRID:BDSC\_34340), y sc v; +; Atg5-RNAi (BL 34899, RRID:BDSC\_34899), y Atg8aKG07569/FM7c; +; + (BL14639, RRID:BDSC\_14639).

### Feeding, Starvation and Pharmacological Treatments

For each treatment, 24–96 h old flies in a group of 15 females and 10 males were housed in a vial.

### Feeding

Flies were transferred every day into a fresh vial containing cornmeal sucrose malt agar supplemented with yeast paste/pellets.

### Starvation

Flies were starved on vials containing 20% sucrose in 2.5% agar.

#### Chloroquine Treatment

Flies were transferred every day into a vial containing sucrose agar with chloroquine to a final concentration of 3 mg/ml.

#### Rapamycin Treatment

Rapamycin was added into food just before pouring food (60◦C) to a final concentration of 200 µM.

To determine the optimal time of starvation where most number of mCherry-Atg8a punctae would occur

(**Supplementary Figure S3D**), nosP-mCherry-Atg8a line was subjected to starvation for 1, 2, 3, and 4 days by transferring them daily into a vials containing 20% sucrose in 2.5% agar. nosP-GFP-Ref(2)P flies after being exposed to pharmacological treatments were dissected in Grace's medium and imaged immediately on Nikon SMZ 1270 microscope fitted with Nikon DS-Fi2 camera. All images were acquired at the same magnification (**Supplementary Figure S1C**).

### Cloning of pC4 nosP-nos 30UTR

nanos30UTR was PCR amplified from CantonS genome using primers nos30UTRF250 -tctagaagagggcgaatccagctctggagcaga and nos30UTRR 50 -tctagaccattttgggagacgccttgaacctaagtg and digested with XbaI and PstI. The resulting fragment of 1236 bp was cloned in XbaI, PstI digested pCasper4 to obtain pC4 nos30UTR. Nanos promoter was amplified using primers nosPF 50 -aagcttcgaccgttttaacctcgaaatatg and newnosPR 50 tggcgaaaatccgggtcgaaagttacg to obtained 935 bp fragment. This fragment was cloned in pGEMt-Easy to obtain pGEMt-nosP. pGEMt-nosP was digested using EcoRI and the resulting 963bp fragment was cloned in EcoRI digested pC4-nos30UTR to obtain pC4-nosP-nos30UTR.

### nosP-GFP-Ref(2)P-nos30UTR

2537 bp NotI-XbaI fragment from pUASt-GFP-Ref(2)P (Chang and Neufeld, 2009) was cloned in NotI-XbaI digested pC4-nosPnos3'UTR to generate pC4-nosP-GFP-Ref(2)P-nos3'UTR.

Chang and Neufeld, 2009 have referred pUASt-GFP-Ref(2)P as GFP-Ref(2)P in their published article. However, GFP-Ref(2)P is generated using EGFP sequence from pEGFP (Clonetech, United States; Scott et al., 2004).

### nosP-mCherry-Atg8a-nos30UTR

1162 bp XbaI fragment from pmCherry-Atga (Bali and Shravage, 2017) was cloned in XbaI digested pC4-nosP-nos30UTR to generate pC4-nosP-mCherry-Atg8a-nos30UTR.

### nosP-mito-roGFP2-Orp1-nos30UTR

1241 bp NotI-XbaI fragment from pUASt-mito-roGFP2-Orp1 (Albrecht et al., 2011, 2014) was cloned in NotI-XbaI digested pC4-nosP-nos30UTR to generate pC4-nosP-mito-roGFP2-Orp1 nos30UTR.

All constructs were sequenced (1st Base, Malaysia), the sequences analyzed for accuracy and were verified to match the published sequence. Transgenic lines were generated at C-CAMP, Bangalore, India, using standard microinjection techniques.

### Fat Body Dissection

Third instar larvae were floated using 20% sucrose and transferred to a Petri dish. The larvae were dissected in 1xPBS, the cuticle was torn along the length of the larvae using forceps to expose all the internal organs including the fat body.

### Immunostaining

Third instar larvae were dissected in 1xPBS and fixed with 4% paraformaldehyde (PFA) overnight at 4◦C. Fixed larval carcasses were washed with 0.1% PBTx (1xPBS + 0.1% Triton X-100) four times for 5 min each and blocked in PBTxGS (0.1% PBTx + 5% Normal Goat Serum) for 2–4 h at RT. After blocking, carcasses were incubated in primary antibody solution overnight at 4◦C. The next day samples were washed with 0.1% PBTx four times for 20 min each and blocked in PBTxGS for 1–2 h at RT. Carcasses were incubated in secondary antibody solution for 2–3 h at RT (protected from light) followed by four washes with 0.1% PBTx for 20 min each. One µg/ml DAPI solution prepared in 0.1% PBTx was added to the sample and incubated for 10 min followed by washing with 0.1% PBTx three times for 10 min each. In the final step, the fat body along with the larval gonads was separated from the carcasses and mounted in Prolong Gold anti-fade reagent (Invitrogen Inc, United States).

Dissected adult ovaries were fixed in 4% PFA for 15 min, washed three times with 0.1% PBTx for 5 min each and blocked with 1% PBTx containing 0.5% BSA for 1 h at room temperature. The ovaries were incubated with primary antibody in 0.3% PBTx containing 0.5% BSA overnight at 4◦C. The next day primary antibody was removed and the sample was washed with 0.1% PBTx for 15 min then blocked with 10% NGS in 0.1% PBTx for 2 h at room temperature. Ovaries were then stained with secondary antibody in 0.1% PBTx containing 10% NGS for 2 h at room temperature protected from light. Excess antibody was removed by three washes with 0.1%PBTX for 15 min each. One µg/ml DAPI solution in 0.1% PBTx was added to the samples and incubated at room temperature for 10 min followed by washing with 0.1% PBTx solution 3 times for 5 min each. Ovarioles were mounted in Prolong Gold.

The following antibodies and dilutions were used; anti-Cathepsin L (Abcam Cat# ab58991, RRID:AB\_940826) 1:400 for larval tissues and 1:300 for adult ovaries; anti-ATP5α 1:100 (Thermo Fisher Scientific Cat# 43-9800, RRID:AB\_2533548), anti-GABARAP 1:100 (Cell Signaling Technology Cat# 13733), anti-Ref(2)p 1:1000 (Abcam Cat# ab178440), anti-GFP 1:10 (DSHB Cat# DSHB-GFP-12A6, RRID:AB\_2617417) anti-α spectrin 1:20 (DSHB Cat# 3A9 (323 or M10-2), RRID:AB\_528473). Secondary antibodies used were Alexa fluor 555 goat anti-rabbit 1:250 (Molecular Probes Cat# A-21429, RRID:AB\_141761) and Alexa fluor 488 goat anti-rabbit 1:250 (Molecular Probes Cat# A-11070, RRID:AB\_142134).

### Redox Chemical Treatment

Ovaries of mito-roGFP2-Orp1 flies were dissected in Grace's medium and washed in 1xPBS for 2 min. The ovaries were incubated in 4 mM diamide (Sigma Cat # D3648) in 1xPBS in order to oxidize or 40 mM DTT (SRL Cat# 17315) in 1xPBS in order to reduce for 10 min at room temperature with gentle shaking. They were washed once in 1xPBS for 2 min before being treated with the redox conservative reagent; 20 mM N-ethyl maleimide (Sigma Cat# E3876) in 1xPBS for 10 min at room temperature. The untreated set and pharmacologically treated samples were proceeded directly for redox conservation. They were washed once in 1xPBS before being fixed in 4% PFA for 15 min at room temperature. Following fixation, two washes of 1xPBS for 5 min each were given. The ovaries were mounted in 80% glycerol and imaged the same day. The redox treatment procedure is modified from Albrecht et al., 2011; (Nilangekar and Shravage, 2018).

### Imaging and Analysis

fcell-07-00047 April 1, 2019 Time: 18:5 # 5

All imaging was performed on Leica SP8 Confocal microscope using 63x oil objective. Images acquired were 8 bit, 1024 × 1024 pixel resolution at 100 Hz scanning. Frame accumulation was performed with 6 frames for mCherry-Atg8a and GFP-Ref(2)P. Images were analyzed using ImageJ. For mCherry-Atg8a mean intensity measurements, an ROI was drawn around the GSCs, identified by the location and size of their nuclei. mCherry-Atg8a and GFP-Ref(2)P punctae were counted manually and the area of germarium were measured using ImageJ. For colocalization analysis, JACoP plugin was used (Bolte and Cordelières, 2006). Both the channels were thresholded in the plugin and Pearson's coefficient were recorded. Microsoft Excel was used for statistical analysis. Student's T-Test of two samples assuming unequal variance was performed for all comparisons. Graphs were plotted in GraphPad Prism 7.

### Imaging and Image Analysis of Mito-roGFP2-Orp1

roGFP2 was excited at 405 and 488 nm "line by line" and its emission from both of these excitations was collected between 500 to 530 nm. Frame accumulation was performed with 6 frames for both the channels.

The ratiometric analysis was performed using ImageJ by the following steps; the background was subtracted (roll ball = 50 pixels), images were converted to 32 bit, the 488 channel was thresholded and the background pixels were set as "NaN," the intensity in the GSCs from the same region in both channels was measured. To generate the ratio image, "Ratio Plus" plugin was used and the resultant image was displayed in the "Fire" lookup table.

### Western Blotting

For lysate preparation, 25–30 flies were dissected in a buffer containing 1 M NaCl, 50 mM Tris and protease inhibitors (Merck Cat #<PHONE_NUMBER>1). The ovaries were transferred to 1.5 ml tube and the buffer was replaced with RIPA buffer (1 M NaCl, 50 mM Tris, 1% Nonidet-P 40, 5% Sodium deoxycholate, 1% SDS) containing protease inhibitors. The tissue was homogenized on ice using plastic pestles. The homogenate was centrifuged at 20,000 rcf for 3 min at 4◦C, the supernatant was transferred to a fresh tube and this step was repeated two more times. This lysate was quantified using BCA (Thermo Fisher Cat # 23227) and final concentration with addition to 2x Laemmli buffer (4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, 125 mM Tris) was adjusted so that equal quantities of protein could be loaded for the sets.

### SDS-PAGE and Western Blotting

Total of 66, 50, and 55 µg/well protein sample was loaded for 12, 24, and 48 h treated samples respectively. Protein samples were run in 4–20% gradient SDS polyacrylamide gel (Bio-Rad Cat # 4561096) and transferred onto a PVDF membrane (Bio-Rad Cat # 1620177) by the wet transfer method. The membranes were blocked in 5% non-fat milk in TBST (50 mMTris, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, washed thrice with TBST for 5 min each. The membranes were then incubated with primary antibody solution at 4◦C with gentle shaking overnight. The membranes were washed thrice with TBST for 5 min each. HRP linked secondary antibody binding was performed at room temperature for 1 h or 4◦C overnight. The membranes were then washed thrice with TBST for 5 min each. Detection was done using ECL kit (Bio-Rad Cat # 1705062) and chemiluminescence was detected on the Bio-Rad ChemiDoc XRS+ system.

### RESULTS

### Expression Analysis of GFP-Ref(2)P

Ref(2)P (SQSTM1/p62 in mammals) is one of the cargo receptors that binds polyubiquitylated substrates and aids in their recruitment into autophagosomes that are marked for degradation. In Drosophila, Ref(2)P was first characterized for its role in sigma rhabdovirus multiplication (Dezelee et al., 1989). Recently, it was shown that Ref(2)P is the Drosophila ortholog of mammalian p62, and the conserved PB1 and UBA domains are necessary for protein aggregate formation (Nezis et al., 2008). Genetic and pharmacological experiments in Drosophila demonstrate that reduction of autophagy activity leads to an accumulation of Ref(2)P-positive protein aggregates, suggesting that it can be used as a marker of autophagic activity (Nezis et al., 2008; Bartlett et al., 2011; Devorkin and Gorski, 2014).

We created a pCasper4 based plasmid that possesses 935 bp nanos promoter and a 1236 bp 30 UTR of nanos that stabilizes transcripts in the germline to generate GFP-Ref(2)P (Doren et al., 1998; Rørth, 1998; Nilangekar and Shravage, 2018; **Figure 1**). Nanos is a maternally expressed and is required for the process of oogenesis and egg production. Nanos expression is detected very early in primordial germ cells of the embryo which later become the germline cells of larval gonads and of the adult ovaries (Wang et al., 1994; Rørth, 1998; Dansereau and Lasko, 2008). We tested larval and adult ovaries for GFP-Ref(2)P expression and its subcellular localization. GFP-Ref(2)P puncta could be readily detected in the GSCs of both larval ovaries and adult ovaries (**Figure 2** and **Supplementary Figures S1A,B**). Larval ovaries from these transgenic lines showed high levels of GFP-Ref(2)P in the developing GSCs. GFP-Ref(2)P expression was weaker and diffused in the support cells and in the region where

niche cells would develop (**Supplementary Figure S1A**). Several of the germaria and late egg chambers possessed rod-shaped distribution of GFP-Ref(2)P which was described previously (Nezis, 2012; **Supplementary Figure S1D**). The distribution of GFP-Ref(2)P puncta within the germarium exhibited differences. Interestingly, majority of GFP-Ref(2)P puncta were found to be localized in the region 2 (2a and 2b) of germarium while region 1 appeared to have very few or no punctate GFP structures. In our analyses upto 30% germaria did not possess GFP-Ref(2)P punctae (**Supplementary Figure S2D**). Antibodies against GFP and p62 were used to validate the expression of GFP-Ref(2)P during oogenesis. Significant overlap between anti-GFP and GFP-Ref(2)P punctate structures was observed and further supported by high Pearson's coefficient. Approximately 60% of the GFP-Ref(2)P puncta were positive for anti-p62 as indicated by Pearson's correlation coefficient (**Supplementary Figures S2A–C**).

The transgenic line was subjected to nutrient stress and pharmacological treatments to validate their utility in various autophagy assays. Females expressing GFP-Ref(2)P were grown on sucrose only diet (nitrogen starvation), rapamycin (autophagy inducer) and 4-hydroxy-chloroquine (CQ) (autophagosomelysosome fusion inhibitor; for details please refer to methods sections) for varying periods of time (Klionsky et al., 2016). The treatments were carried out for 6, 12, 24, 48, and 96 h. The ovary morphology changes in response to these stress stimuli are shown for each treatment in **Supplementary Figure S1C**. The number of GFP-Ref(2)P puncta decreased significantly in germaria obtained from starved females as compared to females reared on nutrientrich food indicating induction of autophagy. CQ neutralizes lysosomal pH which subsequently prevents autophagosomelysosome fusion (Ahlberg et al., 1985; Yoon et al., 2010). Upon CQ treatment, the number of GFP-Ref(2)P puncta significantly increased, as compared to germaria obtained from starved females indicating a disruption autophagic degradation of Ref2P (**Figure 2A**). Rapamycin induces autophagy by inhibiting mTOR kinase (Noda and Ohsumi, 1998; Klionsky et al., 2016). Females grown on food containing rapamycin exhibited a significant reduction of GFP-Ref(2)P puncta suggesting a robust induction of autophagy in germaria (**Figures 2A,B**).

Autophagy dependent cleavage of GFP-Ref(2)P is one of the ways to monitor its degradation. GFP is comparatively resistant to lysosomal hydrolases due to its compact globular structure. The liberation of free GFP from GFP-Ref(2)P following its delivery to the lysosome and can be reliably assayed using an immunoblot assay (Pircs et al., 2012; Devorkin and Gorski, 2014; Klionsky et al., 2016). We subjected GFP-Ref(2)P transgenic females to starvation induced autophagy and CQ treatment, extracted protein from the ovaries and tested if free GFP is liberated (**Figure 2C** and **Supplementary Figure S8**). As seen from **Figure 2C**, the anti-GFP antibody could detect changes in the levels of GFP-Ref(2)P fusion protein (∼130 kDa) in fed, starved and CQ treated conditions. For instance, high molecular weight bands > 130 kDa [GFP-Ref(2)P aggregates] were detected in starved and CQ treated conditions. Interestingly, a number of bands ranging from ∼ 60kDa–25kDa, intermediate degradation products of GFP-Ref(2)P, could be detected with the anti-GFP antibody. In particular, the predominant GFP-Ref(2)P intermediate degradation product was detected at ∼60 kDa. This GFP-Ref(2)P intermediate degradation product was seen to be enriched in starved condition and its abundance was found to be decreased upon CQ treatment. Free GFP (∼27 kDa) was not detected in any of the conditions tested and possible reasons are discussed in later section (Pircs et al., 2012; **Figure 2C** and **Supplementary Figures S8A,B**). Taken together, our data suggest that the GFP-Ref(2)P transgenic lines could be used as a reporter of autophagy in the female germline of Drosophila.

### Expression Analysis of mCherry-Atg8a

Due to its ubiquitous expression in most tissues, Atg8a has been routinely used to monitor autophagy using various techniques including western blotting and immunofluorescence microscopy. Atg8a has been shown to be induced in germline cells as well as follicle cells in response to starvation during Drosophila oogenesis (Nezis et al., 2010a; Barth et al., 2011; Hegedus et al., 2016; Bali and Shravage, 2017).

Transgenic lines expressing mCherry-Atg8a under the nanos promoter were generated as described in materials and methods. mCherry-Atga8a expression was monitored in larval ovaries. mCherry-Atg8a was found to be diffused in larval GSCs but

∗∗∗∗p < 0.0001. (C) Western blot analysis using anti-GFP antibodies of ovarian extracts expressing GFP-Ref(2)P. 130 kDa GFP-Ref(2)P band could be detected in fed, starved and starved + CQ treated ovaries for 48 h. Loading control actin is shown below for the same samples.

was punctate in the differentiated cells of larval ovaries. Unlike in GFP-Ref(2)P transgenic lines, no signal was detected in the fat body cells surrounding the larval ovaries (**Supplementary Figure S3A**). mCherry-Atg8a puncta could be detected in germaria and late egg chambers in adult ovaries (**Figure 3A** and **Supplementary Figures S3B**, **4A**).

The most dramatic change in localization of Atg8a appears when autophagy is induced, where cytoplasmic Atg8a in fed conditions localizes to autophagosomes and autolysosomes and is observed as punctate structures in fluorescence microscopy. Ovaries from adult females reared on 20% sucrose (nitrogen deprivation for 1–4 days) and fed flies (yeast) were dissected and assayed for mCherry-Atg8a expression (**Supplementary Figure S3C**). Our analyses suggested upregulation of autophagy by 24 h of starvation and a significant upregulation of autophagy by 48 h of starvation (**Supplementary**

fed with rapamycin treatment. Dotted ovals mark the GSCs and the asterisks mark the cap cells. Scale bar 10 µm. (B) Interleaved scatter graph showing distribution of mCherry-Atg8a punctae in germarium of flies subjected to fed (for 6 days), fed + starved (2 days fed + 4 days), starved + Chloroquine (2 days fed + 4 days), fed + rapamycin (2 days fed + 4 days). Error bars represent SD in red and the mean is blue. n = 25, 26, 22 and 22 for fed, starved, starved and chloroquine and fed with rapamycin respectively. ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Western blot analysis using anti-mCherry antibodies of ovarian extracts expressing mCherry-Atg8a. 44, 41, and 29 kDa bands corresponding to mCherry-Atg8a-I, mCherry-Atg8a-II, and free mCherry respectively could be detected in fed, starved and starved + CQ treated ovaries for 48 h. Loading control actin is shown below for the same samples.

**Figure S3D**). However, we could detect an increase in total mCherry-(Atg8a) intensity in the GSCs in starved vs. fed GSCs (**Supplementary Figure S4E**). A significant increase in the number of mCherry-Atg8a punctae was detected in the entire germaria of ovaries dissected from fed vs. starved females (**Figures 3A,B**). The increase in mCherry-Atg8a punctae was concentrated in region 2 of germarium (**Figure 3A**). We next tested if disrupting autophagosome-lysosome fusion led to the accumulation of mCherry-Atg8a puncta. As expected, mCherry-Atg8a positive punctate structures accumulated within the germaria isolated from females grown on food containing CQ. In addition, germaria from rapamycin-treated transgenic females exhibited significantly higher number of mCherry-Atg8a puncta (autophagosomes and autolysosomes) as compared to transgenic females reared on nutrient rich food (**Figures 3A,B**). These data suggest that nosP-mCherry-Atg8a transgenic line could be used to monitor autophagy in the germarium.

Immunoblot analyses of mCherry-Atg8a (Atg8a) is recommended as it provides independent confirmation of autophagy induction (Mauvezin et al., 2014; Mulakkal et al., 2014; Nagy et al., 2015; Klionsky et al., 2016; Lörincz et al., 2017).

The degradation of mCherry-Atg8a II fusion protein within the lysosome is differential. The Atg8a-II part of the fusion protein is degraded rapidly while the mCherry part is relatively resistant to destruction by the lysosomal enzymes. The liberation of free mCherry can be reliably assayed using immunoblot analysis to infer autophagic flux (Mauvezin et al., 2014; Nagy et al., 2015; Klionsky et al., 2016). Transgenic females expressing mCherry-Atg8a were subjected to nutrient limitation and CQ treatment and, the protein from the ovaries was assayed for mCherry liberation using an immunoblot assay. In fed, starved and CQ treatment, both mCherry-Atg8a-I (∼44 kDa) and mCherry-Atg8a-II (∼41 kDa) forms were detected. As expected there was an increase in the formation of mCherry-Atg8a-II and corresponding increase in liberation of free mCherry (∼29 kDa) in starved conditions. As compared to fed and starved conditions, in CQ treated animals, the mCherry-Atg8a-II form was detected at significantly higher levels, however, the liberation of free mCherry was inhibited indicating impaired lysosomal destruction of mCherry-Atg8a (**Figure 3C** and **Supplementary Figures S8C,D**). These data suggest that the mCherry-Atg8a transgenic lines could be used to monitor autophagy during oogenesis.

We tested if mCherry positive punctate structures expressed during oogenesis from the transgene are positive for Atg8a. To test this, anti-GABARAP antibodies which also detect Drosophila Atg8a were utilized (Lörincz et al., 2017; Tusco et al., 2017). mCherry positive puncta colocalized with anti-GABARAP positive puncta both in the germarium and late stage egg chamber confirming expression of mCherry-Atg8a fusion protein (**Supplementary Figure S4A**).

UASp-mCherry-Atg8a autophagy reporter has been previously described and has been demonstrated to monitor autophagy in the nurse cells during oogenesis (Jacomin and Nezis, 2016). In this study, we compared UASp-mCherry-Atg8a (nanos-Gal4VP16 X UASp-mCherry-Atg8a) and nosP-mCherry-Atg8a simultaneously in starvation-induced autophagy assay in the ovaries. mCherry-Atg8a punctae are not significantly different between germaria dissected from nanos-Gal4 driven UASp-mCherry-Atg8a females and nosP-mCherry-Atg8a females (**Supplementary Figures S4D,E**). nanos-Gal4VP16 X UASp-mCherry-Atg8a germaria possess higher levels of cytoplasmic mCherry-Atg8a in region 2 (4, 8 and 16 cell cysts) of germaria. Upon nutrient limitation, the increase in the number and fluorescence intensity of mCherry-Atg8a punctae in both nanos-Gal4 X UASp-mCherry-Atg8a expressing germaria and nanosP-mCherry-Atg8a germaria is comparable (**Supplementary Figures S4D,E**).

We checked if nosP-mCherry-Atg8a transgene can rescue lethality associated with Atg8aKG07569 transposon insertion. Homozygous Atg8aKG07569 insertion mutants lack detectable levels of Atg8a protein as tested by western blotting technique (Chang et al., 2013). The rescue experiment is designed to test if the expression level of mCherry-Atg8a from nanos promoter is adequate enough for the complementing the deficiency of Atg8a in homozygous Atg8aKG07569 mutant. Two separate nosP-mCherry-Atg8a insertions were tested for rescue of lethality of Atg8aKG07569 mutant. Our data indicate that both transgenes were capable of complementing the deficiency of Atg8a in homozygous Atg8aKG07569 mutant. Mendelian ratios of inheritance were observed (**Supplementary Figure S5B**). Taken together, these data suggest that nosP-mCherry-Atg8a transgenic line could be used to monitor autophagy in GSCs, germaria and nurse cells during oogenesis.

### Measurement of Autophagy Flux

Autophagic flux is a measure of degradation of autophagic cargo within the lysosomes. There are several methods to measure autophagic flux. Atg8a based assays measure autophagic carrier flux and not autophagic cargo/substrate flux per se (Tanida et al., 2005; Klionsky et al., 2016). While Ref(2)P based assays are considered to be a better measure of autophagic flux. This is due to the fact that Ref(2)P has ability to bind to polyubiquitinated proteins/substrates which allows for their delivery to the autophagosome. In addition, upon starvation and rapamycin treatment, Ref(2)P displays the largest degree of change which can be quantified reliably (Klionsky et al., 2016). To test if transgenic lines expressing GFP-Ref(2)P and mCherry-Atg8a individually could be used for assaying flux we stained them for CathepsinL. CathepsinL is cysteine protease that is a component of the lysosomal acid proteases and used as a lysosomal marker (Klionsky et al., 2016). GFP-Ref(2)P (green) puncta localized close to CathepsinL (red) dots in the germarium and nurse cell cytoplasm in late egg chambers (**Figure 4A** and **Supplementary Figure S6A**). As expected three different types of puncta were visible viz. green puncta (GFP-Ref(2)P aggregates alone or vesicle-bound), and red puncta (lysosomes) could be detected along with yellow puncta (autolysosomes). Pearson's coefficient of GFP-Ref(2)P and CathepsinL colocalization was found to be significantly lower in starved conditions as compared to fed conditions (**Figures 4A,C**). In contrast, mCherry-Atg8a (red) puncta colocalized with CathepsinL (green dots) stained lysosomes. In this experiment, three different puncta were visible: red puncta which represent autophagosomes, yellow puncta which correspond to autolysosomes, and green dots that depict lysosomes. Pearson's coefficient showed a significant increase of mCherry-Atg8a and CathepsinL colocalization in starved vs. fed conditions (**Figures 4B,D** and **Supplementary Figure S6B**).

To get a better estimate of autophagic flux transgenic lines expressing both GFP-Ref(2)P and mCherry-Ag8a were crossed together and ovaries were dissected and stained for CathepsinL. Several puncta positive for GFP-Ref(2)P, mCherry-Atg8a and CathepsinL were visible in fed germaria (**Figure 4E** and **Supplementary Figure S6C**). Taken together, our analyses suggest that these transgenic lines could be used to measure changes in autophagic flux in combination with CathepsinL.

### The Utility of Transgenes in Genetic Screens

One of the advantages of the Drosophila model system is the ability to conduct rapid forward and reverse genetic screens (St Johnston, 2002). We examined whether the GFP-Ref(2)P and mCherry-Atg8a reporters could be of utility in genetic screens. A double-stranded inverse-repeat (IR)

fed + 4 days starved). ∗∗∗p < 0.001. (D) Induction of autophagy upon starvation. Interleaved scatter graph showing Pearson's coefficient as measure of colocalization (autolysosome) of Cathepsin-L (lysosome) and mCherry-Atg8a (autophagosomes) in germarium of fed and starved nosP-mCherry-Atg8a transgenic flies immunostained with anti-Cathepsin-L antibody. Error bars represent SD in red and the mean is blue. n = 9 for both fed and starved. ∗p < 0.05. (E) Ref(2)P sequestered by autophagosomes and fused to lysosomes. Germarium of mCherry-Atg8a; GFP-Ref(2)P flies immunostained for Cathepsin-L. Inset shows enlarged region having a puncta (arrow) positive for GFP, mCherry as well as Cathepsin-L. Dotted ovals mark the GSCs and the asterisks mark the cap cells. Scale bar 10 µm.

construct designed to target and knockdown Atg5 (Atg5IR) was expressed specifically in the germline cells using nanos-Gal4VP16 (Doren et al., 1998; Ni et al., 2008). The expression and cytoplasmic localization of GFP-Ref(2)P and mCherry-Atg8a was monitored in Atg5 knockdown cells. Indeed, as compared to control, germaria expressing Atg5IR failed to degrade GFP-Ref(2)P as seen from the accumulation of GFP positive punctate structures. Moreover, these GFP-Ref(2)P puncta appeared to be larger (GFP-Ref(2)P aggregates) as compared to GFP-Ref(2)P in control germaria indicating

impaired autophagy (**Figures 5A,B**). Knockdown of Atg5 in germaria led to the disruption of punctate localization of mCherry-Atg8a indicating reduced autophagosome formation when compared to control germaria. mCherry-Atg8a was predominantly cytoplasmic in Atg5IR expressing germaria further supporting a decrease in autophagosome formation (**Figures 5A,C**). Similar results were obtained when Atg8aIR was expressed in the germ cells (**Supplementary Figure S5A**). Taken together, these data suggest that both GFP-Ref(2)P and mCherry-Atg8a could be used in conducting genetic screens

fed + rapamycin (2 days fed + 4 days) treatment. Scale bar 5 µm. (D) Interleaved scatter graph showing ratiometric shift of excitation fed (for 6 days), fed + starved (Continued)

#### FIGURE 6 | Continued

fcell-07-00047 April 1, 2019 Time: 18:5 # 13

(2 days fed + 4 days), starved + Chloroquine (2 days fed + 4 days), fed + rapamycin (2 days fed + 4 days) treatment. Error bars represent SD in red and the mean is blue. n = 20, 18, 19, and 21 for fed, fed + starved, starved + Chloroquine, fed + rapamycin respectively. ∗∗p < 0.01. (E) Germarium of mito-roGFP2-Orp1 transgenic flies immunostained with anti-ATP5α antibody that marks the mitochondria. Dotted ovals mark the GSCs and the asterisks mark the cap cells. Scale bar 10 µm. (F) mito-roGFP2-Orp1 (green) in the germarium and a stage 7 egg chamber shows co-localization with lysosomes marked by Cathepsin-L (red). Some lysosomes are GFP positive (yellow) marked by arrow heads while the arrows mark GFP negative lysosomes. Inset shows the enlarged region of the fusion of mitochondrion (presumably in the autophagosome) and lysosome. Dotted ovals mark the GSCs and the asterisk marks the cap cell. Scale bar 10 µm.

to identify modules contributing to the maintenance and induction of autophagy.

### Expression Analysis of mitoroGFP2-Orp1

Mitochondria are susceptible to oxidative damage due to the production of mitochondrial reactive oxygen species (mROS) which include OH−, O2 − and H2O2 (Dröge, 2002; Dan Dunn et al., 2015). Damaged mitochondria lose their redox potential and are subsequently cleared via mitophagy. mROS are products of normal as well as altered cell physiology and can provide valuable information of cellular health (Dröge, 2002; Shadel and Horvath, 2015; Tan et al., 2017). We generated a germline mitochondria-specific sensor for H2O2, termed here as mitoroGFP2-Orp1.

The expression of this H2O2 sensor was tested in the larval and adult ovaries. mito-roGFP2-Orp1 was detected in the entire germarium with very strong expression in the GSCs (Tan et al., 2017). mito-roGFP2-Orp1 distribution in GSCs appeared to be predominantly at the periphery of the GSC nucleus with random distribution in cysts present in region 2 of the germarium. In late stage egg chambers, mito-roGFP2-Orp1 was also detected surrounding the nurse cell nucleus. The follicle cells were devoid of any GFP expression (**Figures 6A,F**). mito-roGFP2-Orp1 can be detected in the larval ovaries with predominant expression within the developing GSCs. GFP signal was not detected in differentiating cells of the larval ovaries or in the fat tissue surrounding the larval ovaries (**Supplementary Figure S7**).

We tested if mito-roGFP2-Orp1 could be used to sense the mitochondrial redox potential within the germarium and GSCs as the mito-roGFP2-Orp1 cassette carries a mitochondrial localization signal at the N-terminal. This reporter senses H2O2 first via Orp1 which oxidizes when exposed to H2O2 and relays the oxidation state to roGFP2 which leads to shift in its excitation maxima of roGFP2 from 488 to 405 nm (Albrecht et al., 2011, 2014). The ovarian tissue was subjected to complete oxidation and reduction using chemical agents, and the excitation maxima of the reporter was tested in the GSCs. As shown in **Figures 6A,B** this redox sensor has the ability to sense both reduced and oxidation states of mitochondria within the GSCs. The dynamic range of the reporter that measure the maximum oxidation and maximum reduction and was found to be 2.9 in the GSCs (**Figure 6B**).

To determine if mito-roGFP2-Orp1 expression or its distribution changes in response to different stress stimuli, we subjected mito-roGFP2-Orp1 expressing females to nutrient limitation, CQ treatment and rapamycin exposure. As compared to nutrient-rich conditions, starvation of the mito-roGFP2-Orp1 expressing females led to reduced roGFP2 expression within the GSCs indicating turnover of mitochondria. In contrast, exposure to CQ during nutrient limitation restored the roGFP2 expression in the GSCs indicating disruption of mitophagy. Rapamycin treated germaria exhibited reduced roGFP2 expression indicating upregulation of mitophagy leading to clearance of mitochondria within the GSCs (**Figure 6C**). We further examined the shift in excitation maxima of roGFP2 from 488 to 405 nm in response to these stress stimuli. A shift in excitation maxima of roGFP2 was not detected when females expressing the transgene were grown on nutrient-rich, nutrient-deprived or in presence of CQ. However, we observed a significant shift in excitation maxima of roGFP2 from 488 to 405 nm indicating increased oxidative stress in GSCs upon rapamycin exposure (**Figure 6D**).

We confirmed that the mito-roGFP2-Orp1 expression is indeed mitochondrial by co-staining with an anti-ATP5α antibody. Our data suggest near complete overlap of the roGFP2 signal with anti-ATP5α suggesting mitochondrial-specific expression of the mito-roGFP2-Orp1 transgene (**Figure 6E**). To test if mito-roGFP2-Orp1 could be used to track mitophagy we stained ovaries dissected from flies expressing mito-roGFP2- Orp1 with CathepsinL. As seen in **Figure 6F** we could detect colocalization of GFP (green mitochondria) and CathepsinL (red lysosomes) in germarium and stage 7 egg chambers suggesting occurrence of mitophagy.

### DISCUSSION

Here we describe generation and characterization of three transgenic lines that aid in assaying for autophagy, mitochondrial H2O2 production and mitophagy specifically in the GSCs. The purpose of generating these lines was multifold. First, to avoid problems linked with Gal4/UASp mediated overexpression of GFP-Ref(2)P, mCherry-Atg8a and mito-roGFP2-Orp1. Second, to validate expression and localization of GFP-Ref(2)P, mCherry-Atg8a and assay for H2O2 production in the GSC and their progeny. Third, to document any differences in expression in different cell types within the germarium including GSCs. And finally, to test the utility of the transgenes in the measurement of autophagy flux and mitophagy.

Although Gal4/UASp is a powerful tool to express genes of interest in the germline cells, it has limitations similar to those documented in the case of GAL4/UAS. Gal4/UASp driven transgenes have mosaic/patchy expression within the tissue (Brand and Perrimon, 1993). This leads to significant differences in the expression levels of the transgenes and in addition the variance is dependent on cell type within the tissue (Duffy, 2002). Our analyses with nanosGal4/UASp-mCherry-Atg8a in