ORIGINAL RESEARCH published: 28 July 2020 doi: 10.3389/fcell.2020.00660 Mitochondrial Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh Syndrome Alejandro Gella 1,2 , Patricia Prada-Dacasa 1,3 , Montserrat Carrascal 4 , Andrea Urpi 1 , Melania González-Torres 1 , Joaquin Abian 4 , Elisenda Sanz 1,3* and Albert Quintana 1,3* 1 Mitochondrial Neuropathology Lab, Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain, 2 Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Bellaterra, Spain, 3 Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain, 4 Proteomics Edited by: Laboratory CSIC/UAB, Institute of Biomedical Research of Barcelona, Spanish National Research Council Pablo M. Garcia-Roves, (IIBB-CSIC/IDIBAPS), Barcelona, Spain University of Barcelona, Spain Reviewed by: Defects in mitochondrial function lead to severe neuromuscular orphan pathologies Alba Gonzalez-Franquesa, University of Copenhagen, Denmark known as mitochondrial disease. Among them, Leigh Syndrome is the most common Simon C. Johnson, pediatric presentation, characterized by symmetrical brain lesions, hypotonia, motor University of Washington, and respiratory deficits, and premature death. Mitochondrial diseases are characterized United States by a marked anatomical and cellular specificity. However, the molecular determinants *Correspondence: Elisenda Sanz for this susceptibility are currently unknown, hindering the efforts to find an effective elisenda.sanz@uab.cat treatment. Due to the complex crosstalk between mitochondria and their supporting Albert Quintana albert.quintana@uab.cat cell, strategies to assess the underlying alterations in affected cell types in the context of mitochondrial dysfunction are critical. Here, we developed a novel virus-based Specialty section: tool, the AAV-mitoTag viral vector, to isolate mitochondria from genetically defined cell This article was submitted to Molecular Medicine, types. Expression of the AAV-mitoTag in the glutamatergic vestibular neurons of a a section of the journal mouse model of Leigh Syndrome lacking the complex I subunit Ndufs4 allowed us Frontiers in Cell and Developmental to assess the proteome and acetylome of a subset of susceptible neurons in a well Biology characterized model recapitulating the human disease. Our results show a marked Received: 19 November 2019 Accepted: 01 July 2020 reduction of complex I N-module subunit abundance and an increase in the levels Published: 28 July 2020 of the assembly factor NDUFA2. Transiently associated non-mitochondrial proteins Citation: such as PKCδ, and the complement subcomponent C1Q were also increased in Gella A, Prada-Dacasa P, Carrascal M, Urpi A, Ndufs4-deficient mitochondria. Furthermore, lack of Ndufs4 induced ATP synthase González-Torres M, Abian J, Sanz E complex and pyruvate dehydrogenase (PDH) subunit hyperacetylation, leading to and Quintana A (2020) Mitochondrial decreased PDH activity. We provide novel insight on the pathways involved in Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh mitochondrial disease, which could underlie potential therapeutic approaches for Syndrome. these pathologies. Front. Cell Dev. Biol. 8:660. doi: 10.3389/fcell.2020.00660 Keywords: Leigh syndrome, animal models, neuroscience, proteomics, cell type-specific, mitochondrial isolation Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome INTRODUCTION Hence, we provide insight on the specific proteomic changes driven by NDUFS4 deficiency, which may underlie the molecular Severe alterations of the mitochondrial machinery involved in and metabolic alterations elicited by mitochondrial dysfunction, energy generation lead to a group of progressive and usually fatal with the overarching goal of identifying novel molecular targets pathologies collectively known as primary mitochondrial disease with therapeutic potential for LS and MD. (MD), affecting 1:4,000 births (Gorman et al., 2015). Currently, there is no cure and the treatments available are mostly ineffective (Schon et al., 2010). MD predominantly affects high energy- MATERIALS AND METHODS requiring organs such as the brain (Vafai and Mootha, 2012). Indeed, neurological damage plays a prominent role in MD Animal Husbandry pathology and lethality (DiMauro and Schon, 2008). However, Mice were maintained with Teklad Global rodent diet No. not all neuronal populations are equally vulnerable to MD, 2018S (HSD Teklad Inc., Madison, Wis.) and water available but rather show a striking anatomical and cellular specificity ad libitum in a vivarium with a 12-h light/dark cycle at 22◦ C. (Dubinsky, 2009). Recent reports have evidenced the remarkable All experiments were conducted following the recommendations regional heterogeneity in mitochondrial protein composition in in the Guide for the Care and Use of Laboratory Animals and the brain, which may account for the cell type specificity of MD were approved by the Animal Care and Use Committee of the (Pagliarini et al., 2008). Universitat Autònoma de Barcelona (Barcelona, Spain). Humans harboring mutations in NDUFS4, a subunit of the mitochondrial complex I involved in the assembly and stability Mouse Genetics of the complex (Scacco et al., 2003; Kruse et al., 2008; Calvaruso Slc17a6Cre (BAC-Vglut2::Cre) mice were generated by Ole et al., 2012) develop Leigh Syndrome (LS), a severe form of Kiehn (Borgius et al., 2010). Ndufs4lox/lox and Ndufs41/+ MD (DiMauro and Schon, 2008). These individuals die at an were previously generated by our group (Kruse et al., 2008; early age, commonly presenting brain lesions, hypotonia and Quintana et al., 2010). Mice with conditional deletion of respiratory deficits (Leshinsky-Silver et al., 2009; Lombardo Ndufs4 in Vglut2-expressing glutamatergic neurons (Slc17a6Cre , et al., 2014). Mice lacking this protein (NDUFS4KO) recapitulate Ndufs41/lox or Vglut2:Ndufs4cKO) were generated by crossing the human symptoms and are an excellent correlate of the mice with one Ndufs4 allele deleted and expressing a codon- human pathology (Quintana et al., 2010; Breuer et al., 2013). improved Cre recombinase (iCre) under the Slc17a6 promoter As observed in LS patients, NDUFS4 deficiency in mice leads (Slc17a6Cre , Ndufs41/+ ) to mice with two floxed Ndufs4 alleles to prominent bilateral lesions in the dorsal brainstem, more (Ndufs4lox/lox ). Littermate controls were Slc17a6Cre , Ndufs4lox/+ specifically in the vestibular nucleus (VN). Conditional NDUFS4 (Vglut2:Ndufs4cCT). ablation restricted to the VN causes a pathology similar to global Ndufs4KO mice. Conversely, viral-mediated post-weaning Cell Cultures re-expression of Ndufs4 in the VN is sufficient to ameliorate HEK293T cells were obtained from the American Type the pathology and to extend the median lifespan of Ndufs4KO Culture Collection (ATCC). Cells were grown in high glucose mice by 30% (Quintana et al., 2012a). In addition, NDUFS4 Dulbecco’s Modified Eagle Medium (Gibco) supplemented with deficiency reveals a cell type-specific susceptibility to the disease. 10% (v/v) fetal calf serum (Gibco), L-glutamine (Gibco) and NDUFS4 ablation in glutamatergic neurons (Vglut2-expressing) penicillin/streptomycin (Gibco) and maintained at 37 ◦ C with recapitulates most of the phenotype present in the global 5% CO2 . Cells were seeded at 200,000 cells/mL and transfected NDUFS4-deficient mice (Bolea et al., 2019), thus warranting with plasmids with calcium phosphate and/or transduced with further study of glutamatergic neurons in the VN. adeno-associated viral vectors (AAVs) for 4 days prior to Here, we have developed and implemented a novel viral immunostaining for hemagglutinin (HA), western blot analysis vector approach (AAV-mitoTag) to purify cell type-specific or mitochondria immunoisolation (mitoTag) assays. mitochondria in a fast and minimally disruptive manner. Other genetic tools to isolate mitochondria with cell-type specificity Immunofluorescence have recently been developed (Bayraktar et al., 2019; Fecher For immunofluorescence analysis of cultured cells, HEK293T et al., 2019). However, these rely on breeding strategies with cells were fixed in 4% paraformaldehyde (PFA) for 10 min mouse lines that have the potential disadvantage of introducing at RT, and further washed two times with PBS before experimental confounds, such as germline recombination or permeabilization with 0.25% Triton X-100 in PBS for 10 min. transient expression of Cre recombinase during development, After permeabilization, cells were washed three times for 5 min therefore compromising the cell type specificity of these with PBS, blocked with 1% BSA in PBS-Tween 20 (0.05%) approaches (Lam et al., 2011; Sanz et al., 2015). Cre-dependent for 60 min, and incubated with an anti-HA antibody (at viral vector approaches overcome these limitations since only 1:1,000 dilution, #901514 BioLegend) overnight at 4◦ C. After adult cells expressing Cre recombinase are targeted. incubation, cells were washed three times with PBS for 5 min The implementation of this approach in glutamatergic and further incubated with anti-mouse Alexa 555 antibody neurons in the VN has allowed us to gain a better understanding (1:500, Thermo Fisher Scientific). Finally, cells were washed three of both mitochondrial proteome and acetylome specifically in a times with PBS for 5 min, counterstained with Hoechst 33258 subset of cells highly susceptible to mitochondrial dysfunction. (2 µg/mL, Sigma-Aldrich) in PBS for 10 min and mounted Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome onto slides using Fluoromount G (Electron Microscopy Sciences) (ECL) detection system (Thermo Fisher Sci.) in a C-DiGit Blot before microscopic analysis. Immunofluorescence analysis was Scanner with Image Studio Ver 5.2 (Licor). Bands were quantified accomplished in mouse brains fixed overnight in 4% PFA using Image Studio (Licor). in PBS (pH 7.4). Subsequently, brains were cryoprotected in a PBS solution containing 30% (w/v) sucrose and frozen in Double-Labeled in situ Hybridization dry ice. Frozen brains were embedded in OCT, sectioned at (ISH)/ Immunofluorescence (IF) 30 µm in a cryostat and rinsed in PBS-Tx buffer (Phosphate- For double-labeling in situ hybridization buffered saline containing 0.2% (v/v) Triton X-100). Non- (ISH)/immunofluorescence (IF) assays, fresh brainstem sections specific binding was blocked with 10% (v/v) normal donkey containing the VN were embedded in OCT, frozen in dry ice and serum (NDS) in a PBS-Tx solution for 60 min at room stored at −80◦ C until sectioning. Coronal sections (16 µm) were temperature, followed by overnight incubation at 4◦ C with obtained using a cryostat and used for analysis. Double-labeled primary antibodies diluted in 1% NDS-PBS-Tx (1:1,000 for ISH/IF was accomplished by performing an RNAscope assay for mouse anti-HA, #901514 BioLegend; 1:1,000 for rabbit anti- Slc17a6 (Vglut2) transcripts followed by immunofluorescence TOM20, #sc-11415 Santa Cruz Biotech; 1:1,000 for rabbit anti- analysis for HA. RNAscope assay for Slc17a6 (#319171 Advanced VDAC, #ab154856 Abcam; 1,000 for Alexa Fluor 488 anti-HA, Cell Diagnostics) was performed according to manufacturer’s #901509 Biolegend). Sections were then washed in PBS-Tx and directions. After Slc17a6 detection, sections were incubated incubated for 1 h at room temperature with the corresponding overnight at 4◦ C with an anti-HA antibody (1:1,000; #715500 Alexa Fluor-conjugated secondary antibodies (1:500, Thermo Invitrogen) followed by a 1 h incubation at room temperature Fisher Scientific) in 1% NDS-PBS-Tx. Sections were finally with an anti-rabbit Alexa Fluor 488-conjugated secondary washed in PBS-Tx and rinsed in water before mounting onto antibody (1:500, Thermo Fisher Scientific). Sections were finally slides with Fluoromount G with DAPI (Electron Microscopy washed in PBS-Tx and rinsed in water before mounting onto Sciences) for microscopic analysis. slides with Prolong Diamond with DAPI (Thermo Fisher Scientific) for microscopic analysis. Western Blotting For western blot analysis of cultured cells, HEK293T cells were Transmission Electron Microscopy lysed with SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% Vestibular nuclei mitochondria were fixed in 2.5% (v/v) SDS, 10% glycerol, all from Sigma-Aldrich) without dithiothreitol glutaraldehyde (Merck) in 0.1 M phosphate buffer (PB), pH 7.4 at (DTT) and bromophenol blue, to avoid interference with 4◦ C overnight. Postfixed for 2 h with 1% (w/v) osmium tetroxide protein quantification. Prior to protein determination, samples (TAAB Lab) containing 0.8% (w/v) potassium hexacyanoferrate were sonicated to shear genomic DNA and reduce the (Sigma-Aldrich) in PB, dehydrated in gradual ethanol (50–100%), viscosity of the lysates. Protein concentration of the lysates embedded in Eponate 12 resin (Ted Pella Inc.), and polymerized was determined using the BCA protein assay (Thermo Fisher at 60◦ C for 48 h. Ultrathin sections of 50 nm were collected Sci.) according to the manufacturer’s instructions. After protein onto carbon-coated copper grids of 200 mesh and contrasted with quantification, 50 mM DTT and 0.1% bromophenol blue conventional uranyl acetate and lead citrate solutions. Sections (Sigma-Aldrich) were added to the samples. Lysates from were visualized in a TEM Jeol JEM-1400 (Jeol Ltd., Tokyo, Japan) mitoTag assays were obtained as described below. Western operating at 80 kV and equipped with a CCD Gatan ES1000W blot analysis of whole tissue lysates or immunoprecipitates Erlang Shen camera (Gatan Inc.). from mitoTag assays was accomplished by adding 4X Laemmli sample buffer to samples prior to heat-denaturing. Protein lysates (10 µg for HEK293T total cell lysates, 0.125% whole MitoTag Adeno-Associated Viral Vector tissue lysates and 10% immunoprecipitates for mitoTag assays (AAV) Generation in HEK293T cells, and 2 µg for whole tissue lysates and We generated an adeno-associated virus (AAV) that would immunoprecipitates for mitoTag assays in tissue were heated initiate Cre-inducible mitoTag expression using a double floxed at 99◦ C for 3 minutes and subjected to 4–20% gradient inverted open reading frame (DIO) approach. An NheI/AscI SDS-PAGE prior to transfer to nitrocellulose membranes fragment containing a construct coding for the mouse Tomm20 (Bio-Rad Laboratories, Inc.). Membranes were then blocked gene fused to three hemagglutinin (HA) sequences (Tomm20 for 1 h with 5% (w/v) dried skimmed milk in TBS-T ·3HA) was cloned into a NheI/AscI-digested pAAV-EF1a-DIO- buffer (Tris-buffered saline containing 0.1% (v/v) Tween- WPRE-hGH polyA plasmid to obtain a pAAV1-EF1a-DIO- 20) and incubated overnight at 4◦ C with primary antibodies Tomm20·3HA- WPRE-hGH polyA construct (AAV-mitoTag). against HA (1:1,000; #715500 Invitrogen), TOM20 (1:5,000; An AAV (AAV1 serotype) vector was produced and CsCl- #sc11415 Santa Cruz Biotech), NDUFS4 (1:500: #87399 Abcam), gradient purified as described previously (Quintana et al., 2012a). ATPIF1 (1:1,000; #A-21355 Invitrogen), GAPDH (1:1,000; The TOM20·HA fusion protein was generated by mutation of #GTX627408 GeneTex), Calreticulin (1:1,000; #ab2707 Abcam) Tomm20 stop codon to glutamic acid (E) and phenylalanine (F). and PDHA1 (1:1,000; ab155096 Abcam). After incubation with the corresponding HRP-conjugated secondary antibodies AAV-mitoTag Delivery (1:10,000; Jackson ImmunoResearch), membranes were washed Mice (n = 28, 37.4 ± 7 days, 22.1 ± 3.2 g) were anesthetized with in TBS-T and developed using an enhanced chemiluminescence isoflurane (5.0 % induction, 1.5–0.7 % maintenance) and placed Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome in a stereotaxic frame (Kopf Instruments). Ketoprofen analgesic Isolation of Mitochondria by Double solution (5 mg/kg s.c; Sanofi-aventis) and ocular protective gel Centrifugation (Viscotears ) were applied before stereotaxic procedure. Mice R Mitochondria were isolated by differential centrifugation using received intracranial AAV injections into the vestibular nucleus protocols previously described (Brown et al., 2004; Pagliarini (VN) at the following coordinates: AP −0.6, ML ± 0.125, DV et al., 2008). Briefly, following decapitation, vestibular nuclei −0.39 mm from the Bregma, using the correction factor (Bregma were dissected according to the Paxinos mouse brain atlas and - Lambda/4.21). A total of 1.0 µL of AAV-mitoTag vector homogenized using a glass dounce homogenizer containing (0.7 × 1012 viral genomes/mL; 0.5 µL per side) was injected into MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM each hemisphere at a rate of 0.25 µL/min using a 5 µL Hamilton KH2 PO4 , 5 mM MgCl2 , 1 mM EGTA, 2 mM HEPES- syringe with a 32G blunt needle. Following injection, the needle KOH, pH 7.2). Homogenates were centrifuged at 900 × g was kept in place for 4 min post-delivery to allow proper viral for 10 min at 4◦ C. Supernatants were further centrifuged vector diffusion and, subsequently, removed at 1 mm/min. at 9,000 × g for 10 min at 4◦ C. Resulting supernatants were used for L-lactate measurements (Biosystems S.A.), Isolation of Mitochondria by and pellets containing mitochondria were resuspended in MAS buffer. Protein concentration was determined using the Immunoaffinity Purification BCA protein assay (Thermo Fisher Sci.) according to the For HEK293T cells, 200,000 cells/mL were transduced with manufacturer’s instructions. AAV-mitoTag and a viral vector expressing Cre recombinase (AAV-Pkg-Cre; UNC Vector Core) or left untransduced for 4 days prior to lysis in mitochondria isolation kit lysis buffer Pyruvate Dehydrogenase (PDH) Activity PDH activity was measured in VN homogenates by using the (1 × 107 cells/mL; Miltenyi Biotec) with protease inhibitors Pyruvate Dehydrogenase (PDH) Enzyme Activity Microplate (Sigma-Aldrich) and homogenized by passing the lysate 25 times Assay Kit (Abcam) according to manufacturer’s instructions. through a 1 mL syringe with a 25G needle. Homogenized Briefly, mitochondria isolated by double centrifugation (50 µg) lysates were next centrifuged at 700 x g for 10 min at were loaded into the wells of a 96-well plate that was specific 4◦ C, and supernatants collected and incubated with anti- for PDH activity assay. PDH enzyme was immunocaptured by HA or anti-TOM22 microbeads (Miltenyi Biotec) according the monoclonal antibodies coated on the wall of each well. to manufacturer’s directions. Mitochondria from glutamatergic The enzymatic activity of PDH was determined based on the cells in the vestibular nucleus (VN) were isolated from freshly production of NADH coupled to a reporter dye whose formation collected mouse brain tissue. Tissue homogenates were prepared is monitored by measuring the increase in absorbance at 450 nm using the Mitochondria Extraction Kit, tissue (Miltenyi Biotec) on a Varioskan LUX plate reader (Thermo Fisher Sci.). according to manufacturer’s instructions. Briefly, following decapitation, brains were weighted, and vestibular nuclei dissected according to the Paxinos mouse brain atlas and digested Measurement of Respiration in Isolated with extraction buffer for 30 min at 4◦ C. After centrifugation Mitochondria at 300 × g for 5 min at 4◦ C, pellets were resuspended in Oxygen consumption rate (OCR) was measured in MAS buffer Solution 2 supplemented with EDTA-free protease inhibitor supplemented with 0.5 % (w/v) fatty acid–free BSA, using a cocktail (Roche Diagnostics), and, for acetylome analysis assays, Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies, deacetylase inhibitors (10 mM nicotinamide, 10 µM TSA and Inc). Freshly isolated mitochondria (5–20 µg of protein) obtained 10 mM sodium butyrate). Digested tissue was homogenized using by double centrifugation were plated in each well of an XFp a glass dounce homogenizer. Homogenates were centrifuged plate in 25 µL of MAS-BSA Buffer. To attach mitochondria, at 500 × g for 5 min at 4◦ C. Subsequently, supernatants the plate was centrifuged at 2,000 × g at 4◦ C for 20 min containing mitochondria were further purified as directed in and then 155 µL of MAS-BSA buffer containing 10 mM the Mitochondria Isolation Kit (Miltenyi Biotec). For magnetic pyruvate/5 mM malate, 100 µM palmitoyl-L-carnitine/5 mM labeling and isolation, supernatants diluted in Separation malate, or 20 mM succinate/4 µM rotenone was added to each Buffer were incubated with 50 µL of anti-HA Microbeads well. First, basal measurements of oxygen consumption rate (state on a shaker (60 rpm) for 1 h at 4 ◦ C. Subsequently, II) were obtained. Next, ADP (5 mM) was injected (state III), LS columns (Miltenyi Biotec) were placed in a magnetic followed by sequential injections of oligomycin (2 µM, state IV), QuadroMACSTM Separator (Miltenyi Biotec) and equilibrated FCCP (5 µM, state IIIu) and antimycin A/rotenone (2.5 µM with 3 mL Separation Buffer (Miltenyi Biotec). Microbead- and 2 µM, respectively) to disrupt mitochondrial respiration. coated mitochondria were applied onto the LS column, Together, this injection series allowed for the determination of followed by three 3-mL washing steps with Separation Buffer. ADP-uncoupled respiration (ADP), proton leak (oligomycin), Columns were removed from the separator and mitochondria maximal respiration (FCCP) and non-mitochondrial residual gently flushed out in 1.5 mL of Separation Buffer with a oxygen consumption (antimycin A/rotenone). plunger. Mitochondria were pelleted by centrifugation for 2 min at 12,000 × g and washed twice with Storage Buffer. Protein Extraction and Digestion Two immunoprecipitation (from 4–5 mice) were pooled for Mitochondria from glutamatergic neurons in the vestibular proteomic analysis. nucleus were immunocaptured as described above. These Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome mitochondria were then suspended in lysis buffer (4% (w/v) Separations were done at 200 nL/min using a linear ACN gradient SDS, 100 mM Tris–HCl, pH 7.6, 0.1 M DTT) and incubated from 0 to 40% B in 240 min (solvent A = 0.1% formic acid at 95◦ C for 5 min. Protein extracts were quantified using RC- in water, solvent B = 0.1% formic acid in acetonitrile). The DCTM Protein Assay (Bio-Rad). Three biological replicates from mass spectrometer was set up in the positive ion mode and Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice mitochondria the analysis was performed in an automatic data dependent were processed. Protein was digested with sequencing grade mode (a full scan followed of 10 HCD scan for the most modified trypsin (Promega) using the FASP (Filter Aided Sample abundant signals). Preparation) digestion protocol (Wisniewski et al., 2009). Briefly, 150 µg of protein from each sample were loaded onto a Database Search and Proteomic Data 10-kDa Amicon Ultra-0.5 centrifugal filter (Millipore). The Analysis protein mixture was washed three times by adding 200 µL Database search was done using Proteome Discoverer v2.1 of UA buffer (8 M urea, 0.1 M Tris–HCl pH 8.5) to the (Thermo-Instruments) with a 1% false discovery rate (FDR) filter and centrifuging at 14,000 × g for 10 min at 13◦ C. and the UniProt 2018-10 database restricted to Mus musculus Next, proteins were alkylated with 100 µL of alkylation buffer (mouse) and contaminants. Search parameters were precursor (0.05 M IAA, 8 M urea, 0.1 M Tris–HCl pH 8.5) in the and fragment tolerance, 20ppm; enzyme, trypsin; missed dark for 20 min at 25◦ C. Subsequently, the protein extract cleavages, 1; fixed modifications, TMTsixplex (N-terminal, K), was washed three times with 100 µL of UA buffer and three carbamidomethyl (C); and variable modifications, oxidation (M). times with 100 µL of 200 mM triethylammonium bicarbonate For proteomic data analysis, DanteR (Taverner et al., (TEAB). Trypsin digestion was performed at 37◦ C for 18 h 2012) was used for relative quantification. Only unique using an enzyme-to-protein ratio of 5:100. Tryptic peptides peptides were considered for the analysis. Tandem mass were eluted by the addition of 3 × 100 µL of 200 mM tag (TMT) reporter intensity data were normalized using TEAB followed by a centrifugation at 14,000 × g for 15 min the total intensity for each label. ANOVA was performed at 13◦ C. using a linear model and p-values were adjusted by using the Benjamini-Hochberg FDR correction. In the acetylome Peptide Labeling by Isobaric Tandem quantification, the ratio of the quantified peptides was Mass Tag normalized using the ratio of the corresponding protein. Each tryptic peptide mixture obtained from 150 µg of Differential proteins and peptides were selected using a p-value protein extract was isotopically labeled with the corresponding (adjusted) cutoff of 0.1 and a fold change lower than 0.8 (down) TMT6plex reagent (Thermo Fisher Sci.) based on the standard or higher than 1.2 (up). procedure. TMT-labeled peptide mixtures were combined in a The mass spectrometry proteomic data have been deposited low-bind 1.5 mL Eppendorf tube, evaporated, and desalted using to the ProteomeXchange Consortium1 via the PRIDE a C18 SPE cartridge (Agilent Technologies). The SPE eluates were (Vizcaino et al., 2016) partner repository with the dataset evaporated and resuspended in 50 mM MOPS pH 7.2, 10 mM identifier PXD016321. Na2 HPO4 , 50 mM NaCl. Bioinformatics Analysis Acetylated Peptide Enrichment Functional enrichment analysis using overrepresentation analysis The TMT-labeled peptide mixtures (900 µg) were enriched for (ORA) with the Gene Ontology (Biological process) and Pathway acetylated peptides by immunoaffinity purification using an anti- (Reactome) databases was accomplished using WebGestalt acetyl lysine antibody following the PTMScan Pilot Acetyl- R (Liao et al., 2019)2 . The STRING (Search Tool for the Lysine Motif (Ac-K) protocol (Cell Signaling Technologies). Retrieval of Interacting Genes/Proteins3 ) algorithm (version Peptides eluted from the enrichment were desalted using 11) was used to build protein–protein interaction networks. PolyLC C18 tips (PolyLC Inc., MD, United States), and Active interaction sources, including text mining, experiments, resuspended in 5% methanol (1% formic acid) for mass databases, co-expression, neighborhood, gene fusion and co- spectrometry analysis. occurrence were applied. Only high-confidence interactions (score 0.7) were chosen. LC–MSn Analysis Peptides were analyzed using an Orbitrap Fusion Lumos Statistics Tribrid mass spectrometer (Thermo Fisher Sci.) equipped with Data are shown as the mean ± SEM. GraphPad Prism v6.0 a Thermo Scientific Dionex Ultimate 3000 ultrahigh-pressure software was used for statistical analyses. Appropriate tests were chromatographic system (Thermo Fisher Sci.) and Advion selected depending on the experimental design as stated in the TriVersa NanoMate (Advion Biosciences Inc.) as the nanospray figure legends. Statistical significance, when reached, is stated in interface. Peptide mixtures were loaded to a µ-precolumn the figure legend. Acclaim C18 PepMap100 (100 µm × 2 cm, 5 µm, 100 Å, C18 Trap column; Thermo Fisher Sci.) at a flow rate of 1 http://proteomecentral.proteomexchange.org 15 µL/min and separated using a C18 Acclaim PepMap RSLC 2 http://webgestalt.org/ analytical column (75 µm × 50 cm, 2 µm, 100 Å, nanoViper). 3 https://string-db.org/ Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome RESULTS double-label in situ hybridization/immunofluorescence (ISH/IF) assays using probes against Slc17a6 mRNA and an anti-HA Generation and Validation of the antibody (Figure 2C). Immunocapture of mitochondria from Vglut2-expressing cells in Vglut2-Cre mice injected with the AAV-mitoTag Viral Vector mitoTag viral vector was accomplished from lysates of pooled To allow the isolation of mitochondria from specific neuronal vestibular nuclei using anti-HA coated magnetic microbeads cell types, a Cre-dependent mitoTag adeno-associated viral (Figure 2D). Western blot analysis of whole lysates (input of the vector (AAV-mitoTag) was generated by inserting the cDNA immunoprecipitation) and immunoprecipitates showed specific of an HA-tagged Tomm20 into a double-floxed inverse open enrichment for the fusion protein TOM20-HA (as assessed using reading frame (DIO) AAV construct (Figure 1A). Analysis anti-HA and anti-TOM20 antibodies) and the mitochondrial for Cre recombinase-dependency of the construct (Figure 1B) markers NDUFS4 and ATPIF1 in the immunoprecipitates. and mitochondrial targeting of the resulting TOM20·HA The cytosolic marker GAPDH appeared depleted in the fusion protein (Figure 1C) was first assessed in HEK293T/17 immunoprecipitates. In contrast, the endoplasmatic reticulum cells. Transfection of the viral vector construct showed that (ER) marker calreticulin did not show a significant depletion in the TOM20·HA fusion protein was only expressed in cells this fraction, which is consistent with the fact that mitochondria transduced with a viral vector expressing Cre recombinase. In can form contacts with the ER in neurons (Wu et al., these cells, fusion protein showed the expected molecular weight. 2017; Figure 2D). Electron microscopy (EM) analysis of the Immunofluorescence analysis for HA in cells transfected with the immunoprecipitates from VN lysates of Vglut2Cre mice injected mitoTag viral vector construct and transduced with viral vector with the mitoTag viral vector confirmed binding of the anti- particles expressing Cre recombinase showed a localization HA magnetic microbeads to the outer membrane of whole of the fusion protein consistent with incorporation into mitochondria (Figure 2E). mitochondria (Figure 1C). To test whether whole mitochondria isolation from Cre-expressing cells could be accomplished by an immunocapture method, lysates from HEK293T/17 Identification and Quantitation of the cells transduced with the AAV-mitoTag viral vector (AAV- Global and Acetyl-Proteome in DIO-Tomm20·HA) along with a Cre recombinase-expressing Mitochondria From Glutamatergic viral vector (AAV-Cre) were used to immunoprecipitate HA-tagged mitochondria with anti-HA coated magnetic Neurons in the VN Next, we sought to define the specific protein changes microbeads (Figure 1D). Anti-TOM22 coated magnetic induced by complex I deficiency in mitochondria from microbeads were also used as control to immunoprecipitate glutamatergic neurons in the VN. To accomplish this, mice unlabeled mitochondria from non-transduced cells. Western conditionally deficient for the complex I subunit NDUFS4 blot analysis of the immunoprecipitates showed specific in glutamatergic neurons (Vglut2:Ndufs4cKO mice) and their immunoisolation of mitochondria without contamination from controls (Vglut2:Ndufs4cKO mice) were injected with the AAV- cytosolic proteins, confirming the suitability of the mitoTag viral mitoTag viral vector to isolate mitochondria from glutamatergic vector for the specific purification of mitochondria from Cre neurons in the VN (Figure 2F). Western blot analysis recombinase-expressing cells. of immunoprecipitates revealed specific isolation of HA- tagged, NDUFS4-deficient mitochondria (Figure 2G), which Cell Type-Specific Isolation of confirms Ndufs4 deletion in Cre-expressing cells. Cell type- Mitochondria From Glutamatergic specific analysis of the global mitochondrial proteome and Neurons in the VN acetylproteome from Vglut2:Ndufs4cKO and Vglut2:Ndufs4cCT Specific deletion of Ndufs4 in Glutamatergic neurons (Vglut2- mice was accomplished by liquid chromatography-tandem mass expressing) in the VN has been shown to drive motor and spectrometry (LC–MSn). Global proteome and acetylome were breathing deficits in mice (Bolea et al., 2019). To define the simultaneously identified and quantified from pooled mitoTag underlying mitochondrial alterations induced by complex I preparations from the VN of Vglut2:Ndufs4cKO (n = 3) and deficiency in these cells, we first confirmed the feasibility of Vglut2:Ndufs4cCT mice (n = 3). Each replicate was a pool of two the AAV-mitoTag approach for the isolation of mitochondria mitoTag preparations. 95% of labeled peptides were dedicated to from this genetically defined neuronal population in vivo. analysis of the acetyl-proteome, the remaining 5% was used for To show expression of the mitoTag in mitochondria from global proteome analysis. the cell type of interest, AAV-mitoTag viral vector was For global proteome analysis, a total of 34,297 spectra injected bilaterally into the VN of Slc17a6Cre (Vglut2Cre ) corresponding to 17,636 non-redundant peptides were identified mice. Subsequently, brain sections containing the area were through database search (1% FDR). Only peptides identified analyzed by immunofluorescence using an anti-HA antibody as unique (i.e., peptide sequences belonging to one single (Figure 2A). HA staining was restricted to specific cells protein in the database) were considered. Overall, a total of in the VN, and double-labeling analysis using anti-HA and 2,998 proteins were quantified from 15,102 non-redundant anti-TOM20/VDAC antibodies confirmed the mitochondrial unique peptides. Among these, 22 proteins showed increased localization of the mitoTag (Figure 2B). Specific expression of abundance (> 1.2-fold, P adj < 0.1), while 34 proteins presented the mitoTag in Slc17a6-expressing cells was further confirmed by reduced levels (< 0.8-fold, P adj < 0.1) in mitoTag preparations Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome FIGURE 1 | Generation of the mitoTag viral vector (AAV-mitoTag). (A) A TOM20·hemagglutinin (HA) fusion protein was generated by mutation of Tomm20 stop codon (red). The resulting construct was cloned in a Cre-dependent viral vector plasmid (pAAV-DIO). (B) Western blot analysis showing the expression and Cre-dependency of the pAAV-DIO-TOM20·HA construct in HEK293T cells. Immunoblot (IB) for HA and TOM20 shows expression of the fusion protein TOM20·HA at the expected molecular weight only in cells transduced with the Cre-expressing viral vector (AAV-Cre). (C) Immunofluorescence analysis showing an HA staining consistent with mitochondrial localization of TOM20·HA in HEK293T cells. Cells were transfected with the pAAV-DIO-TOM20·HA construct and transduced with a viral vector expressing Cre recombinase (AAV-Cre) prior to fixation and immunostaining using an anti-HA antibody. (D) Western blot analysis showing specific immunoprecipitation of mitochondria using anti-HA magnetic microbeads. GAPDH was used as a cytosolic marker. TOM20 was used as a mitochondrial marker. Immunoprecipitation assays with anti-TOM22 microbeads were used as a control for mitochondria isolation in non-transduced cells. I: input of the immunoprecipitation; IP: immunoprecipitation; NT: non-transduced. from Vglut2:Ndufs4cKO mice when compared to controls (Reactome; Figure 3D) confirmed complement-mediated (Supplementary Table 1). Remarkably, most complex I signaling as the most enriched protein sets (terms) in the analysis subunits (36 out of 45 detected proteins) showed significantly of the proteins with increased abundance in mitochondria from reduced levels in NDUFS4-deficient mitochondria, except the VN of Vglut2:Ndufs4cKO mice. Analysis of downregulated for the assembly factor NDUFAF2, which showed increased proteins in NDUFS4-deficient mitochondria resulted in highly abundance in these mitochondrial preparations (Figures 3A,B). significant categories confirming a role in mitochondrial Constituents of the NADH dehydrogenase N-module (NDUFA2, function, and more specifically in respiratory electron transport, NDUFA12, NDUFV1, NDUFV2, NDUFV3, NDUFS1, NDUFS6 ATP synthesis and complex I biogenesis. and NDUFS4) were the most significantly decreased proteins in To explore the functional relevance of the proteomic these mitochondria (Figure 3B). Constituents of the Q-module, alterations observed in Vglut2:Ndufs4cKO mice, we assessed which is located adjacent to N-module, and specific subunits mitochondrial respiration in mitochondria preparations from of the P-module, were also reduced in Vglut2:Ndufs4cKO VN the VN. Mitochondrial oxygen consumption rate (OCR) mitochondria (Figure 3B). Proteins with increased abundance in was assessed by using a Seahorse XFp extracellular flux these mitochondria included the A, B and C-chain polypeptides analyzer (Figure 3E). Mitochondria were first incubated with of the complement subcomponent C1q (C1QA, C1QB, and pyruvate and malate as respiratory complex I substrates (state C1QC), which are known to bind to the mitochondrial protein II). Then, ADP was injected to induce phosphorylation gC1qR (globular head C1q receptor or C1QBP) to modulate (state III), followed by oligomycin to inhibit the ATP mitochondrial metabolism (Ling et al., 2018), and protein kinase synthase (state IV) and the chemical uncoupler FCCP to C delta (PKCδ), a member of the protein kinase C family of induce maximal respiration (state IIIu). VN mitochondria serine- and threonine-specific protein kinases that translocates from Vglut2:Ndufs4cKO mice exhibited a profound to mitochondria upon different stimuli (Majumder et al., 2001). decrease in the OCR (state II, III and IIIu) compared to Functional enrichment analysis for overrepresented gene Vglut2:Ndufs4cCT mice. A more detailed analysis of VN ontology terms (Biological Process; Figure 3C) and pathways mitochondria respiration (Figure 3F) revealed significantly Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome FIGURE 2 | In vivo validation of the mitoTag approach. (A) Diagram of the targeted injection site (VN; coronal and sagittal view) and immunofluorescence analysis using an anti-HA antibody to confirm transduced neurons in the VN of Vglut2Cre mice. Scale bar: 1 mm. (B) Double immunostaining for TOM20/HA and VDAC/HA showing mitochondrial localization of the fusion protein. Scale bar HA/TOM20: 50 µm; Scale bar HA/VDAC: 25 µm; (C) Double-label in situ hybridization/ Immunofluorescence (ISH/IF) assay showing restricted expression of the mitoTag (TOM20·HA) in glutamatergic (Slc17a6-expressing) cells. Scale bar: 25 µm; (D) Western blot analysis and quantification for the relative abundance of the fusion protein TOM20·HA and organelle-specific proteins in whole-cell lysates (W) and lysates from mitoTag immunoprecipitates (IP). (E) Electron micrographs of mitoTag preparations from the VN of Vglut2:Ndufs4cCT mice. Electron dense spots (black arrows) are anti-HA magnetic microbeads bound to the outer mitochondrial membrane. M, mitochondria. (F) Cartoon showing the experimental approach. (G) Western blot analysis for HA, TOM20, and NDUFS4 levels and quantification of NDUFS4 in mitoTag immunoprecipitates (IP) from the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice. Data are presented as the mean ± SEM. Statistical analysis was performed using an unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001). reduced spare capacity, proton leak and ATP-linked respiration β-oxidation (palmitoyl-carnitine/malate) (Figures 3G,H). in Vglut2:Ndufs4cKO compared to control mice. Finally, OCR results revealed that state III (ADP-induced) and state whether the decreased VN mitochondria respiration was IIIu (FCCP-induced) respirations were significantly different complex I mediated was investigated. To that end, we used between Vglut2:Ndufs4cKO and control mice for complex saturating concentrations of specific substrates for complex I I substrates, and similar for complex II and β-oxidation (pyruvate/malate), complex II (succinate/rotenone) and substrates. Collectively, these data show a selective complex Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome FIGURE 3 | Analysis of the mitochondrial proteome and respiration in mitochondria from the VN of Vglut2:Ndufs4cKO mice. (A) Volcano plot showing proteomic changes induced by NDUFS4-deficiency in mitoTag preparations from Vglut2:Ndufs4cKO and control mice. Blue area contains proteins (n = 34) with decreased abundance (< 0.8-fold, P adj < 0.1) and red area contains proteins (n = 22) with increased abundance (> 1.2-fold, P adj < 0.1). (B) Schematic illustration of mitochondrial complex I structure. Heat map shows the changes induced by NDUFS4-deficiency in complex I proteins from panel (A). Hexagons represent complex I subunits and ovals represent complex I assembly factors. For the sake of clarity, prefix “NDUF” has been omitted. (C) Enriched biological process GO terms for proteins with decreased (blue) or increased (red) abundance (FDR < 0.05) in mitoTag preparations from the VN of Vglut2:Ndufs4cKO mice. (D) Enriched pathways (Reactome) for proteins with decreased (blue) or increased (red) abundance (FDR < 0.05) in mitoTag preparations from the VN of Vglut2:Ndufs4cKO mice. (E) Representative pharmacological profile of oxygen consumption rate (OCR) in mitochondria from the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice. Seahorse assay was run with the assay media containing 10 mM pyruvate and 5 mM malate. OCR was normalized to the total protein amount. (F) Respiratory capacities for mitochondria from the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice (calculated from Figure 3E). Data are presented as the mean ± SEM. Statistical analysis was performed using an unpaired t-test (*P < 0.05, **P < 0.01, ****P < 0.0001). (G,H) OCR of VN mitochondria was measured in the presence of pyruvate/malate, succinate/rotenone, palmitoyl-carnitine/malate, following the addition of ADP, oligomycin, FCCP, and antimycin. Representative OCR results obtained with the addition of ADP (G) and FCCP (H) are shown. Data are presented as the mean ± SEM. Statistical analysis was performed using an unpaired t-test (*P < 0.05). Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome I-mediated dysfunction in mitochondria from the VN of Vglut2:Ndufs4cCT mice (Supplementary Table 2). A total of Vglut2:Ndufs4cKO mice. 3,445 spectra corresponding to 2,364 non-redundant peptides (928 proteins) were identified through database search (1% FDR). From these, 742 lysine acetylated peptides in 332 protein Hyperacetylation of PDH and Complex V groups were accurately quantified. A total of 28 differentially Subunits in VN Neurons of acetylated peptides corresponding to 22 proteins were identified Vglut2:Ndufs4cKO Mice in mitoTag preparations from the VN of Vglut2:Ndufs4cKO Mitochondrial complex I deficiency has been shown to lead to mice when compared to controls (Figure 4A). Among these, protein hyperacetylation in the heart (Karamanlidis et al., 2013). 7 peptides showed decreased lysine acetylation (< 0.8-fold, P Hence, we next sought to assess changes in protein acetylation adj < 0.1), and most matched to complex I subunits including due to NDUFS4 deficiency in mitochondria from glutamatergic NDUFS1 (K709), NDUFAB1 (K88), NDUFA5 (K36 and K56), neurons in the VN. Acetylated proteins and their modification NDUFA9 (K189) and NDUFB3 (K40). On the other hand, sites were identified after peptide labeling and affinity enrichment 20 peptides showed increased lysine acetylation (> 1.2-fold, of lysine-acetylated (Kac) peptides by high-resolution LC- P adj < 0.1), 4 of them corresponding to subunits of the MSn in mitoTag preparations from Vglut2:Ndufs4cKO and pyruvate dehydrogenase (PDH) complex (K321 on PDHA1, FIGURE 4 | Analysis of the acetylome in NDUFS4-deficient mitochondria from the VN of Vglut2:Ndufs4cCT mice. (A) Volcano plot showing acetyl-peptides with decreased (< 0.8-fold, P adj < 0.1; blue area; n = 7) or increased abundance (> 1.2-fold, P adj < 0.1; red area; n = 20) in mitoTag preparations from the VN of Vglut2:Ndufs4cKO mice. (B) Protein-protein interaction network of proteins with differential increased acetylation in mitoTag preparations from the VN of Vglut2:Ndufs4cKO mice using STRING. (C) Pyruvate dehydrogenase activity in mitochondria isolated from the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice. Statistical analysis was performed using an unpaired t-test (*P < 0.05). (D) Western blot analysis for PDHA1 protein in mitochondria isolated from the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice. (E) Metabolic alterations in PDH activity signaling in the VN of Vglut2:Ndufs4cKO mice. Reduced PDH activity results in elevated levels of lactate. Green arrows show up-regulated pathways and red arrows denotes down-regulated pathways. LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid cycle. (F) L-Lactate concentrations in mitochondria isolated from the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice. Statistical analysis was performed using an unpaired t-test (*P < 0.05). Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome K354 on PDHB and K444 PDHX) and 9 corresponding to limiting the application of these tools (Lam et al., 2011; Sanz subunits of the ATP synthase complex (K48 and K54 on et al., 2015). Cre-dependent viral vector-based approaches such MT-ATP8, K239 and K539 on ATP5A1, K426 on ATP5B, as our mitoTag assay combined with cell type-specific Cre K32 on ATP5E, and K162, K225, and K244 on ATP5F1). driver mouse lines allow delivery of the transgene into the Functional protein association network analysis of proteins with adult brain, therefore overcoming this important limitation. increased acetylation using STRING (Figure 4B) showed that In addition, viral vector-based approaches avoid complex and these fit into different Reactome pathway nodes, confirming time-consuming breeding strategies and enable temporal and regulation of PDH complex and respiratory electron transport regional control, therefore allowing rapid and anatomically and ATP synthesis. To correlate this increased acetylation in restricted mitoTag assays. subunits of the PDH complex with changes in the activity Our proteomics data shows that NDUFS4 deficiency leads of the enzyme, we next assessed mitochondrial preparations to mitochondria depleted of most of the subunits of the from Vglut2:Ndufs4cKO and control mice for PDH activity. respiratory chain NADH dehydrogenase (complex I; CI). Mitochondrial preparations from the VN of Vglut2:Ndufs4cKO Complex I (CI; NADH: ubiquinone oxidoreductase; EC 1.6.5.3) mice showed reduced PDH activity when compared to controls is essential for oxidative energy metabolism as it contributes (Figure 4C), which was not due to reduced PDHA1 levels, as to the generation of the proton motive force needed for assed by western blot analysis (Figure 4D). Reduced PDH activity ATP synthesis (Hirst, 2013). This complex has a boot-shaped may shunt pyruvate to lactate instead of entering mitochondria structure with a hydrophilic matrix arm and a hydrophobic (Figure 4E), therefore, we next sought to define the concentration membrane arm that is embedded in the inner mitochondrial of lactate in the supernatants of mitochondria isolated from membrane. These arms are subdivided into three structurally the VN of Vglut2:Ndufs4cCT and Vglut2:Ndufs4cKO mice and functionally defined modules: the N-module (involved (Figure 4F). Analysis showed significantly increased levels of in NADH binding and oxidation; where NDUFS4 is located), lactate in samples from Vglut2:Ndufs4cKO mice, suggesting the Q-module (for transfer of electrons along Fe-S clusters to that glutamatergic neurons in Vglut2:Ndufs4cKO are more ubiquinone), and the P-module (mediating proton pumping) glycolytic than controls. (Formosa et al., 2018). Our results evidence that components of the N-module were the most significantly downregulated in mitochondria from the VN of Vglut2:Ndufs4cKO mice, DISCUSSION indicating a critical role of this accessory subunit in the assembly and/or stability of the N-module component of CI, and The mechanisms conferring neuronal vulnerability to LS are confirming previous studies in LS patients harboring NDUFS4 currently unknown. Mitochondrial dysfunction in unique cell mutations (Scacco et al., 2003) and Ndufs4-deficient mice populations leads to distinct clinical and pathological outcomes (Calvaruso et al., 2012; Leong et al., 2012; Valsecchi et al., 2012). (Chen et al., 2017; Bolea et al., 2019). Furthermore, the Furthermore, our data also shows an increase abundance of type of dysfunction and the cellular metabolic status of a specific assembly factor, NDUFAF2 (B17.2L), a molecular the cell have been shown to initiate different stress-induced chaperone associated with a large subassembly of ∼830- pathways (Mick et al., 2020). Therefore, cell type-specific kDa form of complex I and required in the late stage of analyses are needed to define the underlying cellular pathological CI assembly that is essential for the efficient assemblage of mechanisms induced by mitochondrial dysfunction. Here, we complex I (Ogilvie et al., 2005; Lazarou et al., 2007; Sanchez- have developed a tool that allows rapid, viral vector-based Caballero et al., 2016). This increase may result from a isolation of mitochondria with cell type specificity and in compensatory attempt to keep complex I assembled upon a spatiotemporally restricted fashion, with the overarching NDUFS4 deficiency, and confirms recently published data goal of identifying the underlying cellular mechanisms driving (Adjobo-Hermans et al., 2020). neuronal susceptibility to mitochondrial dysfunction. Recently, Our analysis also revealed mitochondrial association of the urgent need to develop approaches to assess mitochondrial characterized non-mitochondrial proteins upon NDUFS4 (dys)function in susceptible cell types has led to the generation deficiency, which would remain unnoticed in a global analysis of different genetic tools to isolate mitochondria with cell- of the cellular proteome. Vglut2:Ndufs4cKO mitochondria type specificity (Bayraktar et al., 2019; Fecher et al., 2019). showed increased abundance of the three different polypeptide These are based on mouse lines that conditionally express, chains that compose the complement subcomponent C1q. C1q in a Cre recombinase-dependent manner, a tag (GFP or has been shown to be present within the cell (Ten et al., 2010; 3XHA-EGFP) that is targeted to the outer mitochondrial Ghebrehiwet et al., 2012), and one of the receptors for C1q, membrane to enable immunocapture of mitochondria from the globular head C1q receptor (gC1qR), is a mitochondrial specific cell types. However, these approaches are limited by the cell-surface protein known to modulate metabolism through possibility of unexpected transient expression of Cre recombinase association with C1q (Dedio et al., 1998; Kolev and Kemper, during development in defined cell types (Song and Palmiter, 2017). In this regard, an effect on spare respiratory capacity 2018). A significant number of Cre-driver cell lines rely on and oxygen consumption has been shown by gC1qR-bound temporally dynamic promoters that may be transiently active C1q, which is known to enhance nuclear transcription of during development, leading to off-target recombination events mitochondrial biogenesis genes (Ling et al., 2018). Association of that compromise the cell type specificity and significantly C1q with NDUFS4-deficient mitochondria may be a mechanism Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome of the cell to promote mitochondrial biogenesis to overcome Vander Heiden et al., 2009). Hence, the reduction in PDH mitochondrial dysfunction. Conversely, C1q has also been shown activity may be involved in the increased glycolysis observed to mediate mitochondrial ROS production by cortical neurons in in Ndufs4KO mice (Johnson et al., 2013) and contribute to neonatal hypoxic-ischemic brain injury and that C1q-deficient the metabolic demise of Ndufs4-deficient cells. Noteworthy, brain mitochondria present preserved activity of the respiratory mutations in the PDH gene are associated with X-linked Leigh chain (Ten et al., 2010), suggesting tissue- and likely cell type- syndrome (Ruhoy and Saneto, 2014), indicating its central specific mitochondrial roles of complement factors. Therefore, role in mitochondrial function. Several ATP synthase complex the role of C1q association to NDUFS4-deficient mitochondria subunits also showed increased acetylation in specific residues, warrants further research. including K239 and K539 on ATP5A1, and K426 on ATP5B. Furthermore, our direct proteomics approach has detected SIRT3 has also been shown to target these residues to regulate increased abundance of PKCδ in mitoTag preparations from ATP synthase activity (Vassilopoulos et al., 2014), which will Vglut2:Ndufs4cKO mice, suggesting mitochondrial translocation likely contribute to reduce ATP production in mitochondria of this kinase in NDUFS4-deficient vulnerable neuronal from glutamatergic neurons in the VN of Vglut2:Ndufs4cKO populations. In this regard, PKCδ has been shown to translocate mice (Wu et al., 2013). to mitochondria upon different stressors and in different To date, the marked heterogeneity in mitochondrial neurodegenerative pathologies, including oxidative (Majumder composition and function has limited our understanding et al., 2001) and genotoxic stress (Lasfer et al., 2006), or cerebral of the underlying molecular determinants of the neuronal ischemia (Dave et al., 2011). Targeting of PKCδ to mitochondria susceptibility to mitochondrial dysfunction. Therefore, it is alters the mitochondrial membrane potential and induces paramount to study mitochondrial function in a genetically apoptotic responses of cells to oxidative stress. Consistently, defined manner to unravel the pathogenic mechanisms treatment with pan-PKC inhibitors increases survival and of mitochondrial disease. Here, by combining mouse delays the onset of neurological symptoms in NDUFS4KO genetics and cell type-specific mitochondrial isolation, we mice (Martin-Perez et al., 2019). Administration of isoform- have assessed the mitochondrial proteome of a neuronal specific PKCδ inhibitors to NDUFS4-deficient mice will define population shown to play a key role in the pathophysiology the relevance of this protein as a target for the treatment of of a well-established mouse model of mitochondrial disease. mitochondrial disease. Our results underscore the marked consequences of CI- CI-deficiency in the heart of conditional NDUFS4KO deficiency in the proteome of affected neurons, identifying mice results in a NAD(H) redox imbalance, with a the metabolic consequences of such alterations. Furthermore, decreased NAD+ /NADH ratio, inhibiting the activity of the we have identified a potential role for PDH, complex V, the mitochondrial sirtuin deacetylase SIRT3, and leading to protein complement factor C1q, and PKCδ in the pathology, paving hyperacetylation (Karamanlidis et al., 2013). However, the the way for future studies of the therapeutic potential of effect of NDUFS4-deficiency on the mitochondrial acetylome in modulating these pathways in the quest for effective treatments neurons was unknown. Analysis of the mitochondrial acetylome of mitochondrial disease. in mitoTag preparations greatly reduces the complexity of the sample by avoiding the interference of heavily acetylated cytosolic and nuclear proteins. This results in increased identification of acetylated peptides corresponding to mitochondrial proteins. We DATA AVAILABILITY STATEMENT report increased abundance of acetylated peptides corresponding The datasets generated for this study can be found in to the pyruvate dehydrogenase (PDH) complex in NDUFS4- the ProteomeXchange Consortium (PRIDE) repository, deficient mitochondria. PDH is involved in transforming http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID pyruvate into acetyl-CoA that is subsequently used by both =PXD016321. the citric acid cycle and oxidative phosphorylation to generate ATP. Increased acetylation at different subunits of the PDH complex anticipated reduced activity of the enzyme since acetylation is typically reported to inhibit the catalytic activity of ETHICS STATEMENT mitochondrial enzymes (Ghanta et al., 2013). More specifically, it has been described that acetylation of PDHA1 at K321 The animal study was reviewed and approved by Animal Care reduce the activity of the enzyme. Noteworthy, K321 residue and Use Committee of the Universitat Autònoma de Barcelona is a SIRT3 substrate (Fan et al., 2014; Wei et al., 2019), and (Barcelona, Spain). SIRT3-mediated deacetylation of PDHA1 K321 increases PDH activity (Ozden et al., 2014), therefore evidencing that modifications in the acetylation status of this sole residue can AUTHOR CONTRIBUTIONS direct the activity of the PDH complex. As suggested by our data, reduced PDH activity may be contributing to the Warburg AG, PP-D, MC, AU, MG-T, JA, ES, and AQ conducted the effect by enhancing glycolysis over mitochondrial oxidation experiments and acquired and analyzed the data. AG, MC, JA, of pyruvate (Warburg, 1956; Kroemer and Pouyssegur, 2008; ES, and AQ designed the research studies. AG, ES, and AQ Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 July 2020 | Volume 8 | Article 660 Gella et al. Neuronal Mitochondrial Proteome in Leigh Syndrome wrote the manuscript. AQ and JA provided reagents. ES and AQ NEUROMITO, ERC-2014-StG-638106), MINECO Proyectos coordinated the work. All authors contributed to the article and I+D de Excelencia (SAF2014-57981P and SAF2017-88108-R), approved the submitted version. and AGAUR (2017SGR-323). FUNDING ACKNOWLEDGMENTS This work was supported by a Marie Skłodowska-Curie We are indebted to Dr. Alejandro Sànchez and Ms. Núria Barba COFUND action (H2020-MSCA-COFUND-2014-665919; AG), from “Servei de Microscopia” from Universitat Autònoma de a Marie Skłodowska-Curie Individual Fellowship (H2020- Barcelona (UAB) for helpful technical assistance (http://sct.uab. MSCA-IF-2014-658352; ES), pre-doctoral fellowships (BES- cat/microscopia). 2015-073041 to PP-D; 2018FI_B 00452 to AU), and a Ramón y Cajal fellowship (RyC-2012-11873; AQ). The Proteomics Laboratory CSIC/UAB is a member of Proteored, PRB3-ISCIII, SUPPLEMENTARY MATERIAL and is supported by Grant PT17/0019/0008, funded by ISCIII and FEDER. ES received funds from MICIU Proyectos I+D+i The Supplementary Material for this article can be found online “Retos Investigacion” (RTI2018-101838-J-I00). AQ received at: https://www.frontiersin.org/articles/10.3389/fcell.2020.00660/ funds from the European Research Council (Starting grant full#supplementary-material REFERENCES Fan, J., Shan, C., Kang, H.-B., Elf, S., Xie, J., Tucker, M., et al. (2014). Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to Adjobo-Hermans, M. J. W., de Haas, R., Willems, P., Wojtala, A., van Emst-de regulate the pyruvate dehydrogenase complex. Mol. Cell. 53, 534–548. doi: Vries, S. E., Wagenaars, J. 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Copyright © 2020 Gella, Prada-Dacasa, Carrascal, Urpi, González-Torres, Abian, Sanchez-Caballero, L., Guerrero-Castillo, S., and Nijtmans, L. (2016). Unraveling Sanz and Quintana. This is an open-access article distributed under the terms the complexity of mitochondrial complex I assembly: a dynamic process. of the Creative Commons Attribution License (CC BY). The use, distribution or Biochim. Biophys. Acta 1857, 980–990. doi: 10.1016/j.bbabio.2016. reproduction in other forums is permitted, provided the original author(s) and the 03.031 copyright owner(s) are credited and that the original publication in this journal Sanz, E., Quintana, A., Deem, J. D., Steiner, R. A., Palmiter, R. D., and McKnight, is cited, in accordance with accepted academic practice. No use, distribution or G. S. (2015). Fertility-regulating Kiss1 neurons arise from hypothalamic reproduction is permitted which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 July 2020 | Volume 8 | Article 660