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Loss of Sting in Parkin Mutant Flies Suppresses Muscle Defects and Mitochondria Damage

Andrew T. Moehlman,Gil Kanfer,Richard J. Youle

Abstract

The early pathogenesis and underlying molecular causes of motor neuron degeneration in Parkinson’s Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to suppress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, we investigated a potential role for STING in the onset of parkin-null phenotypes.

Introduction

Mutations in PINK1 and Parkin lead to early onset Parkinson’s disease (PD). PINK1 is a kinase imported to mitochondria and degraded, unless shunted to the outer mitochondrial membrane when mitochondrial membrane potential is impaired [1–3]. Once stabilized on the outer mitochondrial membrane (OMM) PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like domain to recruit the E3 ligase Parkin to the mitochondria, which amplifies OMM protein ubiquitination [4–7]. This ubiquitination promotes recruitment of autophagy receptors and autophagy of damaged mitochondria [8–10]. Although the molecular mechanisms of PINK1 and Parkin are well-studied, how their absence leads to Parkinsonian phenotypes is less clear [11]. Mutations in PINK1 and Parkin do not lead to substantial or PD-related phenotypes in otherwise healthy mice [12,13]. However, Drosophila melanogaster mutants lacking either pink1 or parkin (park) have severe phenotypes [14–18]. Mutants in either park or pink1 lose flight muscle, undergo degeneration of certain dopaminergic neurons, and display locomotion and flight impairment. Notably, mitochondria in the indirect flight muscles are swollen and the elongated morphology is disrupted [14,18]. Depletion of mitochondria fusion genes or expression of genes regulating fission can rescue park-/- phenotypes, supporting a role for mitochondrial dynamics in the pathophysiology of these mutant phenotypes [19–22].

Materials and method

Publicly available fly stocks (details in Table 1) were acquired from Bloomington Drosophila Stock Center (BDSC, Bloomington, IN). Experimental genotypes (see S1 Table for all genotypes) were made using classical genetics, utilizing the balancer chromosomes from w1118; wgSp-1/CyO; MKRS/TM6b, hu (BDSC stock #76357) when necessary. The null stingΔRG5 allele was gifted from Dr. Alan Goodman, Washington State University and was previously described [31]. The park25 allele was acquired from Dr. Alicia Pickrell, Virginia Tech University, and originally generated by Dr. Leo Pallanck, University of Washington [14]. All park25 mutant animals were maintained as heterozygous over the TM6b, Hu balancer and routinely checked with PCR for presence of the deletion. A second stock of stingΔRG5; park25/TM6b, hu flies were gifted to us from Dr. Alexander Whitworth. Male flies from these stocks were crossed to a w1118 background, then outcrossed for 6 further generations. After each other generation, single male flies used in crosses were checked for PCR after the cross was seeded, and only the ones carrying the park25 allele were selected. After 7 generations, single males were crossed to the double balanced stock for maintaining the outcrossed alleles, and again, PCR was used to confirm the presence of the park25 allele.

Results

Thorax indention and bent wing phenotypes in parkin mutant flies are indicative of underlying indirect flight muscle (IFM) defects and attributed to mitochondria dysfunction inducing muscle apoptosis [14,19,30]. We generated flies harboring null alleles for parkin (park25) [14] and sting (stingΔRG5) [31]. Analysis of these double knockout (DKO) flies demonstrated that loss of sting rescued both the thorax and wing phenotypes of the parkin mutant flies (Fig 1A–1C). We obtained the independently derived park1 mutant and backcrossed this allele into the stingΔRG5 mutant background [15]. These flies also demonstrated reduced penetrance of the parkin phenotypes (Fig 1A–1C). For both backgrounds, the status of the sting and park-null alleles were scored based on the presence or absence of the balancer chromosomes and fly genotypes were routinely confirmed using PCR (S1 Fig). Both park25 and park1 homozygous flies demonstrate climbing defects, due to muscle degeneration and, later, age-dependent loss of dopaminergic neurons [14,15,32]. Using the negative geotaxis assay (Fig 1D), flies homozygous for stingΔRG5 were assayed for climbing ability in parkin wild-type, park25, and park1 backgrounds. Loss of sting alone had no effect on climbing ability in young (5–7 days-old) flies. For both parkin alleles, loss of sting suppressed the climbing defects of young parkin null adults (Fig 1D and S1 Video).

Discussion

Together, our findings support a non-canonical role for Drosophila STING in the pathogenesis of mitochondria dysfunction in parkin-/- flies. Based on rescued mitochondria health and suppression of apoptosis, we propose that Drosophila STING is not responding solely to the presence of mitochondria-derived damage signals in the parkin mutants. These findings suggests that instead in flies there is an indirect role for STING or additional STING-induced genes in propagating upstream mitochondria-damage-induced signaling or indirectly promoting apoptosis. Additionally, there may also be a general dysregulation of autophagy or intraorganellar signaling in the STING-null mutants, as recent studies show that STING modulates autophagy [45,52,53] and lipid dependent starvation responses [54].

Acknowledgments

The authors thank Hong Xu (NHLBI, NIH), Rachel Cox (Uniformed Services University of the Health Sciences), and members of the Youle lab for feedback on the project and manuscript. Fly stocks were graciously provided by Alexander Whitworth (University of Cambridge), Leo Pallanck (University of Washington), Alicia Pickrell (Virginia Tech), Ed Giniger (NINDS, NIH), Jean-Luc Imler (Université de Strasbourg) and Alan Goodman (Washington State University). Dr. Helmut Krämer (UT Southwestern Medical Center) provided the polyclonal p62 antibody and valuable technical advice. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. STING plasmids were obtained from the Drosophila Genomics Resource Center (NIH 2P40OD010949). The microscopy experiments were supported by the NINDS Intramural core Light Imaging Facility (LIF).

Citation: Moehlman AT, Kanfer G, Youle RJ (2023) Loss of STING in parkin mutant flies suppresses muscle defects and mitochondria damage. PLoS Genet 19(7): e1010828. https://doi.org/10.1371/journal.pgen.1010828

Editor: Bingwei Lu, Stanford University School of Medicine, UNITED STATES

Received: January 30, 2023; Accepted: June 13, 2023; Published: July 13, 2023

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the manuscript and its Supporting information files. For RNA-seq experiments, a complete processed dataset, including normalized counts, DESeq2 results and accompanying statistics can be found in S2 Data File. The original RNA-seq read files (in FASTQ format) are available at NCBI Gene Expression Omnibus (GEO, GSE232950). Additional raw data files and original R code used for data analysis are available on Figshare (https://figshare.com/projects/Drosophila_STING_and_Parkin_Manuscript/157578).

Funding: This work was supported by the Intramural Research Program of the National Institute of Neurological Disorders and Stroke, 1ZIANS003123-11 to R.J.Y. This work was supported in part by a NIGMS Postdoctoral Research Associate Training (PRAT) fellowship, FI2GM138078-01, to A.T.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

 

Source: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010828#abstract0

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