Molecular Neurobiology (2019) 56:8306–8322 https://doi.org/10.1007/s12035-019-01667-w Dysregulation in the Brain Protein Profile of Zebrafish Lacking the Parkinson’s Disease-Related Protein DJ-1 Amanda J. Edson1 & Helena A. Hushagen1 & Ann Kristin Frøyset1 & Inga Elda1 & Essa A. Khan1 & Antonio Di Stefano2 & Kari E. Fladmark1 Received: 4 February 2019 /Accepted: 31 May 2019 /Published online: 19 June 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract DJ-1 is a protein with a wide range of functions importantly related to redox regulation in the cell. In humans, dysfunction of the PARK7 gene is associated with neurodegeneration and Parkinson’s disease. Our objective was to establish a novel DJ-1 knockout zebrafish line and to identify early brain proteome changes, which could be linked to later pathology. The CRISPR-Cas9 method was used to target exon 1 of the park7-/- gene to produce a transgenic DJ-1-deficient zebrafish model of Parkinson’sdisease. Label-free mass spectrometry was employed to identify altered protein expression in the DJ-1 null brain of early adult animals. The park7−/− line appears to develop normally at young adult and larval stages. With aging however, DJ-1 null fish exhibit lower tyrosine hydroxylase levels, respiratory failure in skeletal muscle, and lower body mass which is especially prevalent among male fish. By proteomic analysis of early adult brains, we determined that less than 5% of the 4091 identified proteins were influenced by the lack of DJ-1. The dysregulated proteins were mainly proteins known to be involved in mitochondrial metabolism, mitophagy, stress response, redox regulation, and inflammation. This dysregulation in protein networks of our novel DJ-1- deficient zebrafish model occurs in the early adult stage preceding a Parkinson’s disease-related phenotype and the reduction of tyrosine hydroxylase level. The identified protein changes provide new mechanistic background for DJ-1 function. The experimental power of zebrafish makes this model a highly valuable tool to understand and modulate cellular signaling leading to neurodegeneration. Keywords DJ-1 . park7 . Parkinson’s disease . Zebrafish . Proteomics . CRISPR Background PARK7 gene, and loss-of-function mutations are linked to a rare familial type of early onset parkinsonism [2]. However, The Parkinson’s disease (PD)-associated protein DJ-1 is a also in idiopathic cases of PD, post-mortem analysis shows multifunctional oxidative stress response protein ubiquitously accumulation of oxidatively modified DJ-1 [3]. Multiple roles expressed in all human cell types [1]. DJ-1 is encoded by the and functions have been assigned to DJ-1, including Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12035-019-01667-w) contains supplementary material, which is available to authorized users. * Kari E. Fladmark Essa A. Khan [email protected] [email protected] Amanda J. Edson [email protected] Antonio Di Stefano [email protected] Helena A. Hushagen [email protected] 1 Department of Biological Sciences, University of Bergen, Ann Kristin Frøyset 5020 Bergen, Norway [email protected] Inga Elda 2 Department of Pharmacy, University of BG. D’Annunzio^, [email protected] 66100 Chieti, Italy Mol Neurobiol (2019) 56:8306–8322 8307 transcriptional and post-transcriptional regulation [4], chaper- deletion into exon 1 of the zebrafish park7 gene. Here, we one activity [5], maintenance of mitochondrial function and have identified the early protein dysregulation pattern of the motility [6], proteasome activity [7], participation in synaptic DJ-1 null brain occurring prior to tyrosine hydroxylase loss or vesicle recycling [8], stress response regulation, and anti- phenotypic change. Our line should therefore be a highly valu- oxidative defense [9]. Whether this multifacial function is able model for pinpointing possible links, sequence, and im- controlled by a single overarching function is not yet known. portance in the complexity of cellular events leading to PD Upon oxidative stress, a highly conserved cysteine (C106) and other neurological pathologies and to identify future drug becomes reversibly oxidized converting DJ-1 to an oxidative targets. sensor that translocates a subset of DJ-1 to mitochondria [10]. Oxidative stress may also lead to irreversible oxidative mod- ification of DJ-1, which has been linked to idiopathic PD and Alzheimer’sdisease[3]. DJ-1 also seems to be able to elicit its Materials and Methods antioxidant function through a C106 oxidation independent pathway involving nuclear factor (erythroid-derived 2)-like 2 Animal Maintenance (Nrf2)-dependent signaling [7]. DJ-1-deficient mice have been shown to be hypersensitive All the animals used in experimentation were housed at the to oxidative stress-inducing neurotoxicants [11]. Also, alter- Zebrafish Facility located in the Department of Biological nations in both motor and dopaminergic functions have been Sciences at the University of Bergen. The facility is run ac- observed in DJ-1-deficient mouse and rat models, although cording to the European Convention for the Protection of loss of nigral neurons and reduced tyrosine hydroxylase Vertebrate Animals used for Experimental and Other levels, which are hallmarks of PD, have not always been de- Scientific Purposes. Adult zebrafish were maintained at 26– tected [12, 13]. The lack of consensus regarding nigrostriatal 28 °C with a 14/10 light cycle and were fed twice daily. changes might be related to the variation in experimental an- Embryos were maintained at 28 °C and raised in E3 buffer imal age. (5 mM NaCl, 0.17 mM KCl, and 0.33 mM MgSO4) until Deciphering the mechanisms underlying DJ-1 function is 14 days post-fertilization (dpf). Establishment of the park7−/ particularly appealing since its broad functional regulatory − (KO) line and euthanization of adult fish were approved by action overlaps with a number of aspects associated to PD the Norwegian National Animal Research Authority at pathogenesis, i.e., mitochondrial dysfunction, neuroinflam- Mattilsynet (FOTS ID8039 and ID14039). mation, and oxidative/nitrosative stress [14–16]. Though tra- ditionally linked with PD, DJ-1 also has implications in sev- eral other neurological diseases and disorders which have pa- Guide RNA and Cas9 Preparation thologies underpinned by dysregulation of oxidative stress levels, e.g., multiple sclerosis inflammatory lesions [17], Target selection for the guide RNA (gRNA) was performed Alzheimer’s disease [3], and amyotrophic lateral sclerosis using CHOPCHOPv1 [22]. The 20-base pair (bp) region 5′- [18]. Thus, understanding how DJ-1 controls multiple path- GGTGGATGTGATGCGCAGAG-3′ with the PAM site CGG ways important for neuronal protection may offer new oppor- was chosen, no off-targets were identified in CHOPCHOPv1 tunities for disease therapy. Mice treated with the DJ-1-based with an allowance for up to 2 bp mismatches. The gRNA was peptide, ND-13, showed protection from oxidative stress- synthesized with a non-cloning PCR based method as previ- induced dopaminergic loss in both DJ-1 knockout and control ously described [23]. The resulting product was analyzed on mice, possibly via an Nrf2 activation pathway independent of 2% agarose gel and cleaned with the QIAquick PCR purifica- the full-length DJ-1 protein [19]. More recently, ND-13 was tion kit (Qiagen P/N 28106). The PCR product was extracted shown to be neuroprotective in a mouse model of focal ische- once with phenol:chloroform:isoamyl alcohol (25:24:1) and mic injury and restore the absence of response in DJ-1 knock- ethanol precipitation of the DNA followed by in vitro tran- out mice [20]. In contrast with rodent models, zebrafish are scription with MEGAshortscript T7 kit (Ambion particularly suited for drug screening and cell signaling stud- P/NAM1354). The RNA product was purified with mirVana ies due to optical transparency for live imaging, ease of uptake (Ambion P/N 1560) diluted and stored at − 80 °C. of drug dissolved in water, and efficiency of establishing The nCas9 mRNA was prepared from the pCS2-nCas9n transgenes [21]. Additionally, well-characterized neuronal cir- (generously received from Max Suster Addgene No. cuitries make zebrafish excellent vertebrate models for move- 47929) by first linearizing with NotIdigestion(New ment disorders. England Biolabs) and purifying with phenol/chloroform We have generated a DJ-1-deficient zebrafish line using DNA extraction. Ambion’s mMESSAGE mMACHINE clustered regularly spaced palindromic repeat-associated pro- SP6 kit (P/NAM1340) was used for in vitro transcription tein 9 (CRISPR-Cas9) technology to introduce a 5-base pair of the mRNA. 8308 Mol Neurobiol (2019) 56:8306–8322 CRISPR and Line Generation In order to validate the knockout at the level of transcrip- tion, the gene was amplified from cDNA using Expand High Tübingen AB wild-type (WT) embryos were collected from Fidelity PCR System and the primers Fwd 5′-GCAT natural matings and cell injected at the single cell embryo TGCAGACACGCACAGG-3′ and Rev 5′-TCAG stage with 1.76 nl of the CRISPR injection mix containing: ACTGACAGAGCGGTGC-3′ with an annealing temperature 100 ng/μl, park7 gRNA, 150 ng/μl nCas9n mRNA, 50 ng/μl of 60 °C for 30 cycles. Gel bands were purified with the eGFP mRNA, and 0.05% phenol red. At 1 dpf, unsuccessful UltraClean 15 DNA purification kit (Mo-Bio 12100-300) injections determined by lack of eGFP expression were re- and sequenced at the Sequencing Facility