extfieldsofstudy: Medicine Biology

text:
Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

Background Neurons are highly polarized cells consisting of three distinct functional domains: the cell body (and associated dendrites), the axon and the synapse. Previously, it was believed that the clinical phenotypes of neurodegenerative diseases were caused by the loss of entire neurons, however it has recently become apparent that these neuronal sub-compartments can degenerate independently, with synapses being particularly vulnerable to a broad range of stimuli. Whilst the properties governing the differential degenerative mechanisms remain unknown, mitochondria consistently appear in the literature, suggesting these somewhat promiscuous organelles may play a role in affecting synaptic stability. Synaptic and non-synaptic mitochondrial subpools are known to have different enzymatic properties (first demonstrated by Lai et al., 1977). However, the molecular basis underpinning these alterations, and their effects on morphology, has not been well documented. Methods The current study has employed electron microscopy, label-free proteomics and in silico analyses to characterize the morphological and biochemical properties of discrete sub-populations of mitochondria. The physiological relevance of these findings was confirmed in-vivo using a molecular genetic approach at the Drosophila neuromuscular junction. Results Here, we demonstrate that mitochondria at the synaptic terminal are indeed morphologically different to non-synaptic mitochondria, in both rodents and human patients. Furthermore, generation of proteomic profiles reveals distinct molecular fingerprints – highlighting that the properties of complex I may represent an important specialisation of synaptic mitochondria. Evidence also suggests that at least 30% of the mitochondrial enzymatic activity differences previously reported can be accounted for by protein abundance. Finally, we demonstrate that the molecular differences between discrete mitochondrial sub-populations are capable of selectively influencing synaptic morphology in-vivo. We offer several novel mitochondrial candidates that have the propensity to significantly alter the synaptic architecture in-vivo. Conclusions Our study demonstrates discrete proteomic profiles exist dependent upon mitochondrial subcellular localization and selective alteration of intrinsic mitochondrial proteins alters synaptic morphology in-vivo. Electronic supplementary material The online version of this article (10.1186/s13024-017-0221-9) contains supplementary material, which is available to authorized users.

Background
Ageing is a fundamental risk factor for the development of a large range of neurodegenerative diseases, which are characterized by the selective death of neuronal subpopulations [1,2]. Neurons are highly polarized cells consisting of three distinct functional domains: the cell body, axon and the synapse. Previously, it was believed that the clinical phenotypes of neurodegenerative diseases were caused by the loss of entire neurons [3], however it has recently become apparent that these neuronal sub-compartments can degenerate independently of one another [4,5], with synapses being particularly vulnerable to a broad range of stimuli. Whilst the properties of the potential differential degenerative mechanisms remain largely unknown, numerous themes have consistently appeared in the literature, suggesting that proteins regulating the ubiquitin-proteasome system [6][7][8], oxidative stress [9][10][11][12] and mitochondria [1,6,10,[12][13][14][15] may all play a role in regulating the stability of the synaptic compartment.
Synaptic compartments constantly demand ATP to maintain ionic gradients and neurotransmission events [16]. In addition synapses also demonstrate a substantial need for calcium buffering machinery [13]. Accordingly, sub-populations of mitochondria are enriched pre-and post-synaptically [1,6,10]. Such synaptic mitochondria are reportedly distinguishable from non-synaptic mitochondria, displaying unique enzymatic [17], calcium buffering [18,19] and antioxidant properties [10]. However, if and how these distinctive subpopulations of synaptic mitochondria influence the vulnerability of synaptic compartments remains largely unknown.
In an attempt to address this, we have used label-free proteomics to characterise the proteomes of synaptic and non-synaptic mitochondria following established biochemical isolation methods [17]. By utilizing this methodology, we have generated proteomic profiles that reveal consistent molecular fingerprints for synaptic and nonsynaptic mitochondria. Quantitative fluorescent western blotting was used to confirm these proteomic and fractionation data in a range of species, including rat and ovine tissues. These results were consistent with the existence of distinct mitochondrial sub-populations containing patterns of relative protein abundances that are conserved between different mammalian species. To determine if the molecular differences between these mitochondrial subpopulations may be capable of influencing the vulnerability of synapses, we manipulated the expression of mitochondrial candidates in vivo to assess synaptic stability in the Drosophila neuromuscular system. Our data demonstrate that selective knock-down of intrinsic mitochondrial proteins identified in this manner have the potential to alter synaptic morphology and the area of the presynaptic active zone in vivo. Thus, changes in mitochondrial protein expression may contribute to increased synaptic vulnerability and dysfunctional neurotransmission in early molecular pathological processes during ageing and/or disease.

Ethics
In line with the 3Rs no animals were bred or sacrificed specifically for this project.

Rats
Nine male wild-type Sprague Dawley rats aged ca. 24 weeks (actual range 168-171 days) were used. Rats were group housed (3-4 per cage) in a SPF facility in open-top cages and maintained on a 12-12 h light-dark cycle (lights on at 07:00 h), under controlled temperature (22 ± 2°C) and humidity (55 ± 5%) with free access to drinking water and standard 14% protein rodent diet (Harlan Teklad). Rats were killed by conscious decapitation and brains were immediately excised. Brain stem and cerebellum were removed and discarded. Fresh forebrains were weighed and pooled for homogenization before mitochondrial preparations.

Sheep
Three Scottish blackface female sheep aged 5 years were utilized. Sheep were sacrificed at the Farm Animal Teaching Hospital, Royal (Dick) School of Veterinary Studies, University of Edinburgh by anaesthetisation and exsanguination. Brains were excised, bisected at the sagittal midline and the brain stem and cerebellum were removed and discarded. Tissues were submerged in ice cold high magnesium artificial cerebral spinal fluid (NaCl 125 mM; NaHCO 3 26 mM; glucose 25 mM; KCl 2.5 mM; NaH 2 PO 4 (2H 2 O) 1.25 mM; CaCl 2 1 mM; MgCl 2 4 mM) to maintain brain structural integrity before cortical mitochondrial preparations.

Mitochondrial preparations
Methodology comprehensively reported in Lai et al. [17] was employed to produce proteomic data comparable to early biochemical reports. All procedures were carried out at 4°C. Forebrains were homogenized by hand in isolation medium (0.32 M sucrose, 1 mM K-EDTA, 10 mM, Tris HCl) to give a 1:10 homogenate. The homogenate was centrifuged at 1300 g for 3 min and the resulting pellet was manually resuspended in 15 ml isolation medium before recentrifugation as before. Supernatants were pooled and centrifuged at 17,000 g for 10 min to give the crude mitochondrial pellet (CM). The CM pellet was resuspended in 15 ml isolation medium and 5 ml of the suspension was layered into 3 tubes containing 7 ml of 7.5% Ficoll-sucrose medium (7% Ficoll, 0.32 M sucrose, 50 μM K-EDTA, 10 mM Tris HCl) on top of 7 ml of 10% Ficollsucrose medium (10% Ficoll, 0.32 M sucrose, 50 μM K-EDTA, 10 mM Tris HCl) and centrifuged at 99,000 g for 30 min on a swing-out Beckman ultracentrifuge. This resulted in a myelin (My) fraction banded at the top, a synaptosomal (Syn) fraction at the second interphase and a 'free' (non-synaptic) mitochondrial pellet (M) at the bottom of the tube. The myelin fraction was aspirated and the synaptic fraction collected, without disturbing the pellet. This was then diluted in 3× isolation medium and centrifuged at 18,500 g for 10 min. The non-synaptic mitochondrial pellet was frozen immediately on dry ice before storage at −80°C. The resulting synaptosomal pellet was lysed and resuspended in 30 ml 6 mM Tris-HCl (pH 8.1) and centrifuged at 11,800 g for 10 min. The supernatant was removed and the pellet was again resuspended in 10 ml of 6 mM Tris-HCl (pH 8.1) and recentrifuged at 8300 g for 10 min. The supernatant was discarded and the pellet resuspended in 10 ml of 3% Ficoll medium (3% Ficoll, 0.12 M mannitol, 30 mM sucrose, 25 μM K-EDTA, 5 mM Tris-HCl).
One third of this suspension was layered into 3 tubes containing 5 ml 4.5% Ficoll medium (4.5% Ficoll, 0.24 M mannitol, 60 mM sucrose, 50 μM K-EDTA, 10 mM Tris-HCl) on top of 10 ml 6% Ficoll (6% Ficoll, 0.24 M mannitol, 60 mM sucrose, 50 μM K-EDTA, 10 mM Tris-HCl) and centrifuged at 11300 g for 30 min in a swing-out Beckman ultracentrifuge. After centrifugation, the top band was removed and the intermediate band decanted and diluted with an equal volume of isolation medium. This lysate was then centrifuged at 17,000 g for 10 min providing synaptosomally derived mitochondria (SM population) and an SM2 fraction (pellet). The SM and SM2 fractions were pooled to give a greater yield for proteomic experiments and validation, frozen on dry ice and stored at −80°C.

Protein concentration assay
Samples were homogenized in label-free or RIPA buffer +1% protease cocktail inhibitor (Thermo Scientific). After homogenization, samples were centrifuged at 20,000 g for 20 min at 4°C. The supernatant containing the solubilized protein was removed and pellets discarded. Protein concentration of samples was determined using a Pierce Micro BCA assay kit according to the manufacturers instructions.

Label-free proteomics
Synaptic and non-synaptic mitochondrial preparations were extracted in SDT lysis buffer containing 100 mM Tris-HCl (pH 7.6), 4% (W/V) sodium dodecyl sulfate (VWR) and 0.1 M d/l-dithiothreitol (Sigma). For efficient protein extraction, lysates were freeze-thawed and homogenized in SDT buffer several times. Protein concentration was then determined using BCA assay. Aliquots (2 mg) of each mitochondrial preparation were processed through FASP (filteraided sample preparation) involving buffer exchange to 8 M urea and alkylation with 50 mM iodoacetamide prior to a double digestion with trypsin (Roche, sequencing grade), initially overnight, then for an additional 5 h at 30°C . Resulting peptides were desalted then separated using an Ultimate 3000 RSLC (Thermo Scientific) nanoflow LC system. Using an ESI Easy Spray source at 50°C, technical replicates (3 × 0.75 μg) of each sample were loaded with a constant flow of 5 μL/min onto an Acclaim PepMap100 nanoViper C18 trap column (100 μm inner diameter, 2 cm length; Thermo Scientific). After trap enrichment for 3 min, peptides were eluted onto an Acclaim PepMap RSLC nanoViper, C18 column (75 μm, 50 cm; Thermo Scientific) with a linear gradient of 2-40% solvent B (80% acetonitrile with 0.08% formic acid) over 90 min with a constant flow of 300 nL/min. The HPLC system was coupled to a linear ion trap Orbitrap hybrid mass spectrometer (LTQ-Orbitrap Velos Pro, Thermo Scientific) via a nanoelectrospray ion source (Thermo Scientific). The spray voltage was set to 1.6 kV, and the temperature of the heated capillary was set to 250°C. Full-scan MS survey spectra (m/z 335-1800) in profile mode were acquired in the Orbitrap with a resolution of 60,000 after accumulation of 1,000,000 ions. The 15 most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision-induced dissociation (normalized collision energy, 35%; activation Q, 0.250; and activation time, 10 ms) in the LTQ after the accumulation of 10,000 ions. Dynamic exclusion parameters were set as follows: repeat count, 1; repeat duration, 30 s; exclusion list size, 500; exclusion duration, 45 s; exclusion mass width, plus/minus 10 ppm (relative to reference mass). Maximal filling times were 1000 ms for the full scans and 150 ms for the MS/ MS scans. Precursor ion charge state screening was enabled, and all unassigned charge states as well as singly charged species were rejected. The lock mass (445.120024), option was enabled for survey scans to improve mass accuracy. Data were acquired using the Xcalibur software (for all raw data see PRIDE project accession: PXD005537).
Raw proteomic data were imported into Progenesis for characterization and analysis of relative ion abundance. 2D representations of MS/MS output were created for each sample and these were aligned to determine similar features (average alignment score > 90%). Following alignment, data was filtered by retention time with features detected below 5 min and above 110 min discarded to correct for elution variability and peptides with charge state between 2 and 5 only included in the search. The runs were grouped according to subcellular localization (synaptic and non-synaptic) and Statistical P values were automatically generated in Progenesis software through a 1 way ANOVA on the ArcSinh transform of the normalized data.
Peptides were filtered by the following criteria: power < 0.8, fold change >2, p<0.05 and the remaining data were exported from Progenesis for identification of individual peptide sequences using the Uniprot Swall subspecies Rattus norvegicus via Mascot Search Engine (V2.3.2). Enzyme specificity was set to that of trypsin, allowing for cleavage Nterminal to proline residues. Other parameters used were as follows. (i) Variable modifications: methionine oxidation, methionine dioxidation, protein N-acetylation, gln → pyro-glu. A cutoff score of >29 was used based on Mascot probability threshold of 0.05 that the observed hit is a random event. As an indication of identification certainty, the false discovery rate for peptide matches above identity threshold was set at 1%.
Identified proteins were re-imported into Progenesis for further processing. Proteins were subject to stringent filtering parameters to eliminate those which had <2 unique peptides, <2-fold change between subpopulations and p>0.05 to obtain the proteins which demonstrated the largest significant variation in expression between synaptic and non-synaptic mitochondria. See Additional file 1: Table S1 and Additional file 2: Table S2 for raw unfiltered and filtered data respectively.

Ingenuity pathway analysis
IPA was performed as previously described [20] with the interaction data limited as follows: direct and indirect interactions; experimentally observed data only; 35 molecules per network; 10 networks per dataset. Prediction activation scores (z-score) were calculated in IPA. The z-score is a statistical measure of the match between an expected relationship direction and the observed protein expression. Positive z-scores indicate activation (orange) and negative z-scores indicate inhibition (blue) [21].

Transmission electron microscopy
Human mixed sex cortical samples (n = 4) from patients without neurological phenotypes were prepared for electron microscopy as previously outlined in detail [23]. Briefly, fresh post-mortem samples, stored in 0.1 M PB were trimmed into small cortical blocks and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PB for 48 h. Mouse brain samples were prepared as follows: anesthetized WT male C57BL/6 J mice ((n = 4) intraperitoneal injection of Ketanest (100 mg/kg) and Rompun (5 mg/kg)) were killed by perfusion fixation with 0.1 M phosphate buffer containing 4% paraformaldehyde and 2.5% glutaraldehyde before removing the brain and immersing it in fixative for a further 12 h. Following fixation, both human and mouse brain samples were washed in 0.1 m phosphate buffer before cutting free floating 70-μm-thick coronal sections on a Vibratome. Sections were postfixed in 1% osmium tetroxide in 0.1 m phosphate buffer for 45 min. Following dehydration through an ascending series of ethanol solutions and propylene oxide, all sections were embedded on glass slides in Durcupan resin. Regions of interest were glued onto a resin block for sectioning. Ultrathin sections (60-70 nm) were cut and collected on Formvarcoated grids (Agar Scientific), stained with uranyl acetate and lead citrate in an LKB "Ultrostainer", and then quantitatively assessed in a Philips CM12 transmission electron microscope. Negatives taken in the microscope were scanned into an Apple Macintosh G5 computer using an Epson 4870 Photo flat-bed scanner at 600dpi and subsequently processed using Adobe Photoshop. Images from 232 human and 270 murine slices were analysed using the ImageJ measurements function.

Drosophila stocks
Flies were raised on standard cornmeal food at room temperature. Homology of rat gene of interest and Drosophila orthologue was determined by input into DIOPT (DRSC Integrative Ortholog Prediction Tool [24]). Offtarget effects of candidate lines were assessed using E-RNAi software [25]. Candidate lines exhibited no off-target effects based on genetic mapping data and all displayed 97.21-100% efficiency for targeting the intended gene (Ndufb8 = 97.75%; mitofilin = 97.21%; Vdac1 = 100%; Aldh = 100%). The elav-Gal4 driver strain (Bloomington Drosophila stock center) was used for all experiments. Stocks were obtained from the VDRC (IDs: 21,707, 47,615, 101,336, 30,413) and Bloomington Drosophila stock center (Canton-S). Crosses were maintained at 22°C for 24 h before removal of adults and embryos were incubated in a water bath at 30°C to increase levels and activity of the Gal4 proteins.

Immunohistochemistry
Third instar larvae were selected and dissected in PBS (n = 8). The dissected larval neuromuscular junctions (NMJs) were fixed in Bouin's fixative (15:5:1 picric acid, 37% formaldehyde and acetic acid) for 10 min and washed thoroughly in PBT (PBS + 0.1% TritonX-100). Preparations were blocked in PBT + 10% normal goat serum for 2 h then incubated in primary antibody overnight at 4°C. NMJs were again washed extensively in PBT and incubated in secondary antibody at room temperature for 2 h. Samples were mounted on microscope slides using Vectashield mounting medium (Vector Laboratories) and imaged on a Zeiss confocal microscope. Images were quantified using the Andlauer & Sigrist, 2012 protocol [26] in ImageJ. Briefly, segmented masks of the fluorescent signal were generated for each channel and the dimension and intensity of each particle was quantified. Primary antibody: Horseradish peroxidase (HRP (1:200)); secondary antibody: anti-HRP goat biotinylated anti-rabbit (1:400). All antibodies from Jackson ImmunoResearch.

Statistical analysis
Data were collected in Microsoft Excel and statistical tests were performed in GraphPad Prism 6 software. For all analyses p<0.05 was considered statistically significant. Statistical tests used are detailed in the results or figure legends where appropriate.

Mitochondrial subcellular localisation dictates organelle morphology in rodents and humans
Enzymatic activity differences between neuronal mitochondrial sub-populations were first comprehensively described in the 1970s [17]. However, the molecular underpinnings of these biochemical differences and their morphological or physiological consequences were never elucidated. We therefore began by investigating whether mitochondria residing at synaptic terminals were morphologically different from those in non-synaptic compartments. It has previously been suggested that the size of mitochondria may provide indications of biochemical processes as well as neuronal integrity and survival. Numerous studies have begun to describe mitochondrial morphology in the cell body [10,[27][28][29] but few demonstrate characterisation of those organelles in the axon and synapsethe compartments that show heightened vulnerability to degenerative insult. To establish whether size differences existed between synaptic and non-synaptic mitochondria, we examined transmission electron microscopy images from human and mouse cortical tissue. Mitochondria were classified as 'synaptic' if they were <1 μm from the post-synaptic density and vesicles were clearly present within the presynaptic terminal. Mitochondria outside of the pre-and postsynaptic boutons were classed as non-synaptic; anything ambiguous was excluded from the analysis. The results indicate that in both mouse and human cortex, non-synaptic mitochondria appear significantly larger than the organelles present within synaptic terminals (( Fig. 1) mouse: p<0.0001; human: p=0.0099, respectively). Non-synaptic populations in both species demonstrated elongation versus that of synaptic mitochondria, which displayed a spherical morphology (data not shown). Although these morphological differences between mitochondrial sub-populations may solely reflect adaptations to the particular size and structure of cellular compartments, we hypothesised that biochemical adaptations would also likely exist due to the dynamic interplay between protein expression and organelle configuration (see Picard et al., 2013 for a comprehensive review on mitochondrial morphology and function [30]).

Label-free proteomics reveals significant mitochondrial heterogeneity
To identify molecular differences between mitochondria derived from synaptic and non-synaptic neuronal compartments, we performed label-free proteomics on mitochondrial-enriched fractions isolated from rat forebrain. Quantitative label-free proteomic analyses identified >1500 proteins associated with both synaptic and non-synaptic mitochondria, revealing dynamic variations in protein expression dependent upon subcellular localisation ( Fig. 2a and b). Strikingly, over 400 proteins were altered by greater than 2-fold between mitochondrial subpopulations (Fig. 2c), demonstrating significant compartment-dependent biochemical adaptations.
Relative purity of mitochondrial isolates was verified using bioinformatics (Table 1), quantitative enrichment analyses utilizing the raw proteomic data and quantitative fluorescent western blotting (QFWB) on ovine forebrain. The mitochondrial yield obtained from rat forebrain was not sufficient for validation purposes (see methods). The mitochondrial markers VDAC1 and COXIV indicated significant enrichment in synaptic and non-synaptic mitochondrial fractions versus whole brain lysate suggesting purification of the mitochondria from their respective subcellular compartments (Fig. 3a). To demonstrate the purification of mitochondria from discrete neuronal compartments, we calculated the normalised average abundance of the wellestablished synaptic markers Snap25, Synaptopodin (Synpo), Synaptojanin-1 (Synj1) and Psd3 for both the synaptic and non-synaptic mitochondrial preparations. Indeed, expression levels of Snap25, Synpo, Synj1 and Psd3 indicated significant enrichment in synaptic mitochondrial preparations versus non-synaptic suggesting purification of synaptically derived mitochondria (Fig. 3c). In addition to demonstrating purification of mitochondrial subpopulations, we again employed QFWB to determine the veracity of the proteomic data. We observed corresponding protein expression trends in each tissue preparation, as indicated by the proteomics, for multiple proteins (Fig. 3d-f). Taken together, these results indicate the relative purity of the mitochondrial isolates and suggest the proteomic data is representative of the molecular alterations occurring in the tissue samples. Furthermore, the data indicate that the observed heterogeneity in mitochondrial protein expression may be conserved between mammalian species.

Expression profiling highlights bioenergetic alterations between synaptic and non-synaptic mitochondrial proteomes
Having confirmed the veracity of the proteomic data, we next sought to further dissect the proteomic data using Biolayout Express 3D [31]. The software applies unbiased Markov clustering algorithms to the input data and groups proteins displaying similar expression trends. This allows visualisation of spatial profiles promoting the identification of physiological cascades altered within the dataset. The nodes, shown as spheres with identical dimensions, signify individual proteins and the edges, represented by connecting lines, are indicative of the correlation of protein expression within the data. Graphs were constructed in 3D space utilizing the 1511 identified proteins from the mitochondrial proteomic analysis (see Fig. 2b and c), generating 10 protein clusters. In agreement with the principal component analysis correlation graphs (Fig. 2d and e), network clustering of the data displayed similar trends with regards to fragmentation of the graph into two localisationdependent networks (Fig. 4). The fragmentation of the dataset into 2 distinct groups suggests that significant heterogeneity in protein expression exists between the mitochondrial populations derived from discrete neuronal sub-compartments. Indeed, with further examination we exhibited that proteins with increased abundance in synaptic mitochondria cluster into the distinct network on the left of the graph whereas those with increased abundance in non-synaptic mitochondria are observed in the right network (Fig. 4). To identify the functional cascades associated    that appear to demonstrate differential expression between the two mitochondrial subpopulations, the top 3 protein clusters were entered into Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 to determine the basic functions of the clustered proteins ( Fig. 4). Interestingly, the proteins that exhibit upregulation in synaptic mitochondria (clusters 1 and 2) are associated with the inner membrane bioenergetic complexes, which suggests that distinct synaptic and non-synaptic mitochondrial protein expression may reflect altered local requirements for energetic expenditure. Interestingly, these data correlate with the original biochemical study by Lai et al. [17], which reported variations in mitochondrial enzymatic activities dependent upon the subcellular localisation of the organelles. Here, we can account for approximately 35% of those reported enzymatic activity alterations, which are likely due to the differing abundance of particular enzymes in the discrete sub-pools of mitochondria.
Pathway analysis highlights mitochondrial complex I expression differences as potentially conserved regulators in a range of neurodegenerative conditions In order to determine how differential protein expression in mitochondrial subpopulations may synergistically regulate downstream molecular pathways and cellular processes, we performed an in silico analysis using Ingenuity Pathway Analysis (IPA) software. The software promotes the identification of statistically significant functional groups of proteins, based on known protein interactions and biological functions reported in the published literature [20]. Functional networks generated by IPA software are statistically ranked according to a score calculated via a righttailed Fischer's exact test, taking into account the number of original input proteins and the size of the resulting network. With input of the 411 differentially expressed proteins from the mitochondrial proteomic data (>2-fold change), the analysis revealed that 154 (37%) candidates have previously been associated with neurological diseases in the published literature (Fig. 5a), suggesting that a multitude of these proteins may play a role in regulating synaptic stability. Further examination into the molecular cascades that may be associated the pathogenesis of neurodegenerative diseases highlights that mitochondrial complex I is an upstream regulator of degenerative processes (Fig. 5b). The particular expression of proteins downstream of complex I in synaptic, but not non-synaptic, mitochondria have previously been implicated in numerous neurodegenerative diseases including Huntington's disease, Parkinson's disease and Lewy body dementia -all of which demonstrate synaptic perturbations early during pathogenesis (for a comprehensive review see [1]). These results suggest that modulation of the synaptic mitochondrial proteome may synergistically drive the demise of the synapse in age-related neuropathologies. In conjunction, assessment of the downstream physiological cascades modulated by synaptic mitochondrial protein expression identified significant inhibition of synaptic transmission cascades (Fig 5c). The increased expression of alpha-synuclein, GAD1, SH3GL1, SH3GL2, S100B PPP3R1 and SYT1 in the non-synaptic mitochondrial proteome appears to indirectly inhibit synaptic transmission. Conversely, the enhanced expression of SNPH, PRNP, GNAI1 and GNAI2 in synaptic mitochondria inhibits neurotransmission cascades in the synaptic compartment. This suggests that heterogeneity in discrete mitochondrial proteomes and their associated downstream pathways has the propensity to promote functional alterations at the synaptic terminal.

Mitochondrial proteins regulate synaptic morphology in vivo
Although we had generated a compendium of the molecular differences present between synaptic and nonsynaptic mitochondrial populations, it remained unclear if any of the identified proteins were capable of actively modulating synaptic stability in vivo. To assess this, we employed a molecular genetic approach using the Drosophila larval neuromuscular junction (NMJ) to screen individual proteins to examine their potential role in regulating synaptic morphology. The larval NMJ is an excellent model system to unravel the molecular mechanisms underpinning synaptic structure, function and plasticity [32]. Fundamental mammalian biological and neurological pathways demonstrate conservation in Drosophila and recent evidence has indicated that up to 75% of human disease related genes exhibit a functional orthologue in the fruit fly [33]. Larval NMJs harbour glutamatergic synaptic boutons with contiguous invaginated post-synaptic membranes [34,35], which display significant homology to those found in the mammalian central nervous system. Neurotransmission between these connections has the propensity to invoke plastic modifications [32,34,36], providing physiologically relevant mechanistic insights into proteins regulating the structure and function of the synapse in mammalian cognition [36].
In order to assess whether the expression of particular proteins in synaptic mitochondria may be regulating the morphology of the synapse, we utilised the Drosophila UAS/Gal4 system, which allows tissue specific expression of a particular transgene with use of selected drivers [37]. We selected candidates based on magnitude of change, mitochondrial localisation and availability of Drosophila orthologues (see Table 2). Interestingly, the short-listed candidates displayed increased protein expression in synaptic versus non-synaptic mitochondria. Thus, we aimed to selectively knock-down the expression of these mitochondrial proteins in third instar larva neurons for assessment of associated phenotypes. Panneural expression of the RNAi constructs (v30413; v47616; v10136; v21707) under control of the elav-Gal4 driver resulted in viable larva from all crosses. To assess synaptic morphology and potential mitochondrial-dependent alterations in synaptic transmission, we examined the larval muscle 12/13 NMJ by immunohistochemistry with antibodies against the presynaptic marker horseradish peroxidase (HRP) and active zone marker bruchpilot (BRP). Here, we found that the selective RNAi-mediated knock-down of single candidates produced striking synaptic phenotypes at the NMJ (Fig. 6). NDUFB8, mitofilin, VDAC1 and ALDH demonstrated varied phenotypes at the NMJ with alterations in distinct synaptic parameters including bouton diameter and total bouton area (Fig. 6). The most severe phenotype was associated with selective knock-down of aldehyde dehydrogenase (ALDH), which promoted significant reductions (p<0.05) in synaptic bouton area with concomitant loss of distinguishable Ib and Is boutons (Fig.  6a). In conjunction, there was a significant decrease (p<0.0001) in synaptic bouton active zone staining (Fig. 7a) relative to control NMJs. Similarly, knock-down of neuronal NDUFB8 expression promoted aberrant NMJ morphology ( Fig. 6a and b) with significant increases in bouton diameter. Despite this, the NMJs demonstrated a corresponding reduction in active zone area (p<0.0001) versus controls ( Fig. 7a and c). Interestingly, we did not observe any obvious perturbations in axonal branching or morphology in our elav-Gal4/RNAi lines ( Fig. 6f and g), suggesting that selective knock-down of these candidates promotes selective alterations in synaptic morphology, mediated by mitochondrial protein expression. Significant reductions in the area of active zone staining in both the ALDH and NDUFB8 RNAi lines suggests that mitochondria may have a direct functional impact on vesicular release and bouton firing properties. Although the mechanistic pathways modulating these alterations remain elusive, regulation of the mitochondrial proteome appears to be significant mediator of synaptic-specific structural and biochemical properties.

Discussion
The current study has demonstrated novel insights into how a 'top-down' approach may be utilised for identifying individual proteins that may modulate the stability of synaptic compartments in vivo. By using a range of techniques we have shown that neuronal mitochondria derived from synaptic and non-synaptic cellular compartments display significant morphological and biochemical differences. Electron microscopy analyses from mouse and human cortical tissue suggest that non-synaptic mitochondria are significantly larger than those found in the synaptic terminal. Alongside these morphological alterations, we have also demonstrated that upwards of 400 mitochondrial associated proteins identified from our label-free proteomic experiments display a ≥ 2-fold expression difference between sub-populations. Using molecular genetic tools at the Drosophila neuromuscular junction, we were able to manipulate the expression of 4 of the identified candidates that displayed increased abundance in synaptic mitochondria, resulting in selective alterations in synaptic, but not obvious axonal morphology in vivo. Our data promote further understanding of the basic biology of the mitochondria and how these organelles may regulate the vulnerability of the synapse in a physiologically relevant model. In addition, the results presented may provide increased insight into how mitochondrial perturbations influence the synaptic alterations commonly occurring during ageing and disease. Our initial proteomic screen revealed a remarkably large and heterogeneous list of candidates that significantly differed in expression dependent upon mitochondrial subcellular localisation. Despite this heterogeneity, upon further analysis of the data utilising in silico tools, it became apparent that many of these proteins have previously been associated with perturbations in synaptic transmission. Experimental manipulation of 4 of these candidates produced varied synaptic phenotypes. The differing morphological phenotypes likely reflect the functional specificities, biological roles and downstream cascades associated with the proteins. NDUFB8, a mitochondrial complex I subunit, produced a relatively mild phenotype at the NMJ with morphological overgrowth but retention of branching patterns seen in controls. The preservation of numerous morphological characteristics despite NDUFB8 knock-down may reflect a compensatory mechanism occurring within the elaborate multi-subunit complex I. Conversely, reductions in ALDH expression resulted in the most severe phenotype, with significant alterations in synaptic bouton morphology and striking alterations within the NMJ architecture. This is likely due to its primary role in protecting mitochondria from endogenous aldehydes generated by lipid peroxidation [38]. With reductions in ALDH, it is probable that an accumulation of noxious metabolites occurs, resulting in cytotoxicity. In support of our observations, a previous study from our laboratory [39] reported that Drosophila harbouring mutant ALDH displayed spontaneous degeneration of axons in the olfactory receptor neuron, however the presence of a synaptic phenotype was not morphologically assessed in that system. Furthermore, there have been several recent reports documenting a potential detoxification role for ALDH specifically in populations of dopaminergic neurons [40]. Notably, sub-populations of dopaminergic cells from the brains of Parkinson's disease patients demonstrated a significant reduction in ALDH [41], which may contribute to alterations in synaptic stability resulting in the activation of degenerative cascades. Despite NDUFB8 and ALDH demonstrating varied synaptic bouton phenotypes, both candidates promoted a significant reduction in the active zone area. Mitochondrial morphology and function has been associated with alterations in the efficacy of the presynaptic active zone in numerous investigations, however the molecular mediators regulating such events remain elusive. Synaptic transmission is a bioenergetically demanding process requiring the persistent aerobic production of ATP and buffering of intracellular Ca 2+ concentrations. Evidence indicates that mitochondria redistribute and bind to the active zone in response to synaptic transmission for homeostatic regulation at the synapse [42,43], promoting the controlled exocytosis underpinning neurotransmission. Disruptions in the mitochondrial proteome may have the propensity to alter the functional organisation and stability of the active zone by reductions in calcium buffering, ATP availability and/or vesicular endocytosis. Although we directly manipulated the expression of NDUFB8 and ALDH, it is likely that the NMJ mitochondria harboured further disruptions to the proteome that were hierarchically driven by alterations in the candidates, which may have synergistically promoted aberrations at the active zone.
Although a conventional RNAi screen would require multiple RNAi and point mutations to be certain of the importance of specific candidates, we have presented several novel synaptic specific phenotypes with selective knock-down of single mitochondrial candidates. However, it is unlikely that the differential expression of all of these proteins can solely regulate synaptic morphology and stability. Instead, it is probable that multiple cellular and molecular pathways, up-and downstream of the mitochondria, converge to modulate compartmental stability in mammalian systems. Thus, differentially expressed mitochondrial candidates and/or their downstream cascades warrant further in vivo investigation to examine the nature and extent of their role in the modulation of synaptic architecture. Despite this, protein expression within the mitochondria, as well as the subcellular localisation of the organelle, are clearly important variables that must be considered when characterising cascades negatively affecting synaptic morphology, transmission and physiology.
A next logical step in determining how the mitochondrial proteome may be contributing to synaptic vulnerability in vivo, is to perform an ageing study using the same tools and techniques presented in the current investigation. It has been well documented that mitochondria dynamically alter dependent on their environment [10,[17][18][19], and we have provided further evidence for this hypothesis here. However, few studies have attempted to investigate how the synaptic mitochondrial proteome alters during ageing and how variations in protein expression may impact on the function and architecture of the synapse in a physiologically relevant model. An examination of such processes provides scope to potentially identify mitochondrial proteins that may be contributing to synaptic demise during normal healthy and pathological ageing. Furthermore, these data may indicate why such large disparities appear to exist with regards to the vulnerability of synaptic and neuronal populations with different pathogenic insults.
Although the use of label-free proteomics has provided a fairly comprehensive coverage of the mitochondrial proteome (this study yields the highest identification of mitochondrial proteins in a single in vivo analysis to date (1511)), for improved detection of low molecular weight proteins other techniques such as tandem mass tagging with high fractionation may be employed for future experiments. It is highly likely that numerous mitochondrial proteins that were not detected or removed from the data due to stringent filtering parameters, may influence synaptic stability in vivo. Despite this, it is abundantly clear that proteomics and molecular genetics are powerful tools to identify candidates that may impact upon synaptic structure and function.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Engineering

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Green biologics: The algal chloroplast as a platform for making biopharmaceuticals

ABSTRACT Most commercial production of recombinant pharmaceutical proteins involves the use of mammalian cell lines, E. coli or yeast as the expression host. However, recent work has demonstrated the potential of eukaryotic microalgae as platforms for light-driven synthesis of such proteins. Expression in the algal chloroplast is particularly attractive since this organelle contains a minimal genome suitable for rapid engineering using synthetic biology approaches; with transgenes precisely targeted to specific genomic loci and amenable to high-level, regulated and stable expression. Furthermore, proteins can be tightly contained and bio-encapsulated in the chloroplast allowing accumulation of proteins otherwise toxic to the host, and opening up possibilities for low-cost, oral delivery of biologics. In this commentary we illustrate the technology with recent examples of hormones, protein antibiotics and immunotoxins successfully produced in the algal chloroplast, and highlight possible future applications.

Introduction
Biopharmaceuticals (protein biologics) is an industry estimated to be worth in excess of $140 billion 1 and encompasses products such as monoclonal antibodies, immunotoxins, antigens, hormones, enzymes, clotting factors and bioactive peptides. 2 These recombinant proteins are produced mainly using heterotrophic fermentation technologies with the biological platforms being either mammalian cell lines such as Chinese Hamster Ovary cells, or microorganisms such as bacteria or yeasts. 3 Whilst these are highly advanced and successful technologies, there is a need for additional platforms that offer new opportunities for the production of therapeutic proteins. Emerging technologies include virus-mediate transient expression in insect cell lines 4 or in tobacco plants, 5 and stable expression in the chloroplasts of plants and algae. 6,7 The use of unicellular algae as cell factories is particularly attractive as a low-cost, low-tech and sustainable approach, especially for countries lacking advanced fermentation infrastructures. As illustrated in Fig. 1, efficient production of algal biomass can be achieved in a cheap, sterile and disposable polythene tubing system that is easily scaled and managed. Each »40 litre 'hanging bag' is bubbled with CO 2 -enriched air and illuminated directly with sunlight, or indirectly using artificial lighting provided by LEDs powered by sunlight captured using photovoltaic devices. Whilst the latter adds to the capital costs, superior daily biomass productivities are obtained through 24 hour illumination using light of optimal intensity and wavelength, and tight control of the culture temperature. Cultivation of the algae uses a simple medium of basic nutrients, thereby keeping media costs as low as $0.002 per liter. 8 Importantly, algal species grown commercially for the food ingredients and healthfood markets (e.g. species of Chlorella, Dunaliella and Haematococcus) already have GRAS (generally recognized as safe) status. The safety of these species offers the possibility of topical application of a biopharmaceutical such as an anti-microbial protein as a crude cell lysate (e.g. formulated into a cream or spray), and therefore avoiding costly investment in purification. Alternatively, it might be possible to use the whole algae for oral delivery (to animals, if not to humans) of vaccines, anti-microbials or hormoneswith the dried cells exploited as a natural method of encapsulation and storage that overcomes the need for a costly cold chain, and the components of the algal cell possibly acting as an effective adjuvant. 9 Recent surveys of the literature show that over 50 different biopharmaceuticals have been successfully produced in microalgae. 9,10 Although production using nuclear genetic engineering is reported for several freshwater and marine species of eukaryotic microalgae, the majority of the research has focused instead on chloroplast engineering using the freshwater green alga Chlamydomonas reinhardtii. Insertion of transgenes into the small chloroplast genome rather than the nuclear genome offers several clear advantages: not least the ability to do precise and predictable 'DNA surgery' in which transgenes are integrated into specific, neutral loci within the genome via homologous recombination, and stable, high-level, stable expression is readily achieved. 11 Furthermore, protein folding and disulphide bond formation occurs readily in the chloroplast allowing the correct assembly of complex therapeutic proteins with multiple domains or multiple subunits, as discussed below. Finally, the chloroplast compartment can serve as a safe sub-cellular site for hyper-accumulation of recombinant protein without affecting the biology of the rest of the cell. 12 The growing interest in exploitation of the algal chloroplast is now driving the development of synthetic biology tools by ourselves and other groups that allow a rapid and efficient pipeline for design and production of engineered strains. Below we highlight this technology and give three examples of applications in the field of biopharmaceuticals.
The C. reinhardtii chloroplast as an emerging synbio platform Chloroplast genomes (or 'plastomes') are polyploid circular molecules possessing 100-200 genes, with most encoding core components of the photosynthetic apparatus or the organelle's transcriptiontranslation machinery (Fig. 2). Gene structure and expression is essentially prokaryotic in nature, reflecting the evolution of the chloroplast from a cyanobacterial ancestor. Hence, genes are often arranged as operons, transcribed by a eubacterialtype RNA polymerase and the mRNA translated on 70S ribosomes. 13 Chloroplast transformation was first achieved using C. reinhardtii whereby a photosynthetic mutant carrying a chloroplast gene deletion was restored to phototrophy by microparticle bombardment with a plasmid carrying the wild-type gene. Molecular analysis showed that the mutant locus had been repaired through efficient homologous recombination (HR) between sequences on the plastome and the introduced DNA. Since then, C. reinhardtii has been used extensively as a laboratory model for reverse-genetic studies of chloroplast gene expression and photosynthetic function, with Figure 1. A low-cost, single-use photobioreactor system for commercial production of algal biomass. This 'hanging bag' system was developed by the Cawthron Institute, New Zealand for production of microalgae as aquaculture feed and for cultivation of Haematococcus pluvialisa natural source of the high-value nutraceutical astaxanthin. We have successfully adapted the system for endolysin and vaccine production in C. reinhardtii (L. Stoffels, B. Parker and S. Purton, submitted). The 40 litre bags are optimally illuminated and sterile 5% CO 2 /95% air supplied at the base of each bag for phototrophic growth and for mixing. Both batch and continuous operation is possible. ©Supreme Health, New Zealand. Reproduced by permission of Supreme Health, New Zealand. Permission to reuse must be obtained from the rightsholder. specific gene knockouts or site-directed changes introduced into the plastome through HR-mediated engineering. 14 More recently, the focus has shifted to biotechnological applications and the development of the C. reinhardtii chloroplast as a protein factory through the addition of novel genes into the plastome to make valuable recombinant products. 11 Improvements in the transformation technology have helped to advance this field and we now are beginning to see the application of synthetic biology (synbio) principles. These include gene design in silico using dedicated codon optimization software and validated cis elements such as promoters and untranslated regions. 15,16,17 Building the designed constructs in vitro is then aided by rapid assembly of standardized DNA parts using methods such as Golden Gate 18 that ensure the 'one-step' assembly of multiple parts in the correct order and orientation (Fig. 3). Accompanying this are methods for large-scale refactoring of the plastome and for regulating the expression of the transgenes. 19,20,21 Finally, the development of strategies for 'marker-free' generation of transgenic lines that avoid the use of antibiotic resistance markers, 22 and a technique for bio-containment of the transgene through codon reassignment 23 will help to address regulatory issues and public concerns regarding commercial cultivation of transgenic microalgae. Further details of these tools are given in Fig. 3.
Three case studies: Human growth hormone, endolysins and an immunotoxin Human growth hormone (hGH) is a 22 kDa protein that is produced naturally in the pituitary gland. Deficiency of the hormone results in growth defects, but can be successfully treated by administration of recombinant hGH. 24 As the only post-translational steps required for biological activity are removal of the N-terminal methionine and formation of two intrapeptide disulphide bonds, then recombinant production is feasible using a prokaryotic host. E. coli is currently the preferred platform, although correct folding and bond formation requires export into the periplasm. 25 However, the demand for recombinant hGH is huge and growing, with a predicted global market of $4.5 billion by 2022, thereby justifying the exploration of alternative production platforms including chloroplasts. Recent work by our group has demonstrated that functional hGH can be produced in the C. reinhardtii chloroplast by expression of a codon-optimized synthetic gene fused to the promoter and 5 0 untranslated region of the endogenous psaA gene. 22 Yields of hGH in the transformant were approximately 500 mg per liter of culture, so there is a need to increase this significantly before we can compete with bacterial platforms. Nevertheless, biological activity could be demonstrated even in crude cell lysates using a standard assay where addition of the lysate specifically stimulated growth of a rat lymphoma cell line. This work highlights the potential of the algal chloroplast as a future platform for making simple biopharmaceuticals such as hGH, insulin and bioactive peptides.
In a second study from our group, the production of endolysins in the C. reinhardtii chloroplast was investigated. 26 Endolysins are antibacterial proteins produced during bacteriophage infection that digest the bacterial cell wall for phage progeny release at the end of the lytic cycle. These enzymes typically show a high degree of specificity for the target bacterium of the phage. Furthermore, the emergence of resistance to endolysins appears to be extremely rare. Consequently, endolysins have potential as protein antibiotics, with recombinant forms shown to be highly effective when added to bacterial cultures or biofilms. 27 The chloroplast is a particularly attractive platform for recombinant production since it mimics the prokaryotic environment where endolysins are produced naturally, but unlike bacterial hosts, it lacks any peptidoglycan cell wall that might be compromised during over-expression of an endolysin gene. Using  the same strategy as for hGH production, two different endolysins -Pal (36 kDa) and Cpl1 (40 kDa)that target the major human pathogen Streptococcus pneumoniae were successfully produced in C. reinhardtii. Each enzyme showed a high lytic activity against cultures of S. pneumoniae even when presented as crude cell lysates, suggesting that the algal platform could be used for simple, low-cost formulations of anti-bacterial creams or sprays targeting topical bacterial infections, or infections of the nasal pharynx.
The third case study illustrates a possible niche for the algal chloroplast platform that addresses issues encountered with existing prokaryotic and eukaryotic hosts. Tran et al. 12 investigated the synthesis of immunotoxins in the C. reinhardtii chloroplast. These chimeric proteins are targeted therapeutics that have applications in cancer treatment, and consist of an antibody domain for binding to the target cell and a cytotoxic enzyme that inhibits proliferation of the cell. As shown in Fig. 4, the immunotoxin is a complex multi-domain protein that requires correct folding and disulphide bond formation to generate the active homodimer. Production of such proteins within bacterial hosts is challenging because these expression platforms often fail to fold proteins with multiple domains efficiently and are unable to form disulphide bonds. Conversely, production of such cytotoxic proteins in eukaryotic hosts such as CHO cells or yeast is not feasible because of the lethal effect of the toxin on the cytosolic translation apparatus. The work of Tran et al. 12 demonstrates that the algal chloroplast not only possesses the machinery necessary to fold and assemble complex eukaryotic proteins, but that the 70S ribosomes are unaffected by the toxic protein and the organelle is able to contain the protein preventing any inhibitory effect on the host's cytosolic ribosomes. The chloroplast therefore offers an attractive platform for efficient production of these highly complex therapeutics.

Resources and future applications
Advances in the genetic engineering of the C. reinhardtii plastome, in particular the application of synbio strategies, have simplified and accelerated the process of creating designer strains expressing therapeutic proteins. In our lab, we have sought to develop a simple, low-cost pipeline that can readily be adopted by other groups, including those in developing countries. Chassis strains and DNA parts are available through the Chlamydomonas Resource Center (www.chlamycollection.org) and our software for codon optimization is free to download (github.com/khai-/CUO). Our chloroplast transformation protocol simply involves agitation of a cell/ DNA suspension in the presence of glass beads, rather than the use of expensive microparticle bombardment equipment, and we have developed a simple PCRbased method for confirming transgene insertion and homoplasmy of the plastome. 22 Our on-going work on scale-up using the hanging bag system shows that this is a cost-effective and easily manageable cultivation method; and biomass productivity could be further improved through optimization of key parameters such as light, CO 2 delivery, mixing and media composition. 28 Currently, recombinant protein yields are low (typically 0.5-5% of total soluble protein) compared to established microbial platforms, but better understanding of chloroplast gene regulation and the use of orthogonal mechanisms to induce and drive transgene expression, 29 Figure 4. Illustration of a designer immunotoxin produced in the C. reinhardtii chloroplast showing the multi-domain structure. The human CD22-scFv domain was fused to the hinge and constant domains of a human IgG1 and to exotoxin A from Pseudomonas aeruginosa lacking domain 1a. This created an immunotoxin that formed a homodimer through disulphide bonds between the hinge regions. Redrawn from 12 . chloroplasts of tobacco have been reported as high as 70% of total soluble protein. 30 In addition, protein productivity could be improved through genetic enhancement ("domestication") of the chassis strains to improve their performance in photobioreactors. 31 Alternatively, the chloroplast engineering technology could eventually be transferred to faster-growing and more robust native species of green algae such as Chlorella that are better suited to intensive commercial cultivation. 32 Possible applications of the algal chloroplast platform extend beyond human therapeutics, and are particularly attractive where the cost of production and storage are key issues. For example, microalgae are a natural part of the diet for insect larvae, juvenile shellfish, fish fry, etc. Consequently, engineered C. reinhardtii strains have been proposed for oral delivery of toxins to insect pests such as mosquitoes, 33,34 or delivery of vaccines and growth hormones to farmed fish and shellfish. 35 Similarly, the GRAS status and nutritional value of various green algal species opens up the possibility of "functionalized feed" for poultry and livestock whereby dried algae formulated into the feed also contains beneficial vaccines, anti-microbials or dietary enzymes. 36 To date, no biopharmaceuticals produced in microalgae has been approved for commercial production and only a handful have been tested in animal experiments. Significant further research and development of microalgal platforms is therefore required. However, conventional production of protein biologics is expensive and technically demandingrequiring capital-intensive fermentation facilities, and costly downstream processing, cold storage and transportation, and sterile delivery methods. To meet the future needs of the global population, alternative low-tech, low-cost and sustainable production systems such as microalgae must be considered.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.


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The Roles of Reactive Oxygen Species and Nitric Oxide in Perfluorooctanoic Acid-Induced Developmental Cardiotoxicity and l-Carnitine Mediated Protection

Perfluorooctanoic acid (PFOA) is an environmental contaminant that could induce developmental cardiotoxicity in a chicken embryo, which may be alleviated by l-carnitine. To explore the roles of reactive oxygen species (ROS) and nitric oxide (NO) in such changes and the potential effects of l-carnitine, fertile chicken eggs were exposed to PFOA via an air cell injection, with or without l-carnitine co-treatment. The ROS and NO levels in chicken embryo hearts were determined with electron spin resonance (ESR), and the protein levels of the nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) p65 and inducible nitric oxide synthase (iNOS) in chicken embryo hearts were assessed with western blotting. The results of ESR indicated that PFOA exposure induced an elevation in the ROS levels in ED19 chicken embryo hearts and hatchling chicken hearts, while l-carnitine could alleviate such changes. Meanwhile, increased NO levels were observed in ED19 embryo hearts and hatchling hearts following PFOA exposure, while l-carnitine co-treatment exerted modulatory effects. Western blotting revealed that p65 translocation in ED19 embryo hearts and hatchling hearts was enhanced by PFOA, while l-carnitine co-treatment alleviated such changes. iNOS expression levels in ED19 embryo hearts followed the same pattern as NO levels, while a suppression of expression was observed in hatchling hearts exposed to PFOA. ROS/NF-κB p65 and iNOS/NO seem to be involved in the late stage (ED19 and post hatch) of PFOA-induced developmental cardiotoxicity in a chicken embryo. l-carnitine could exert anti-oxidant and NO modulatory effects in the developing chicken embryo hearts, which likely contribute to its cardioprotective effects.

Heart Rates of Hatchling Chickens
The heart rates of the hatchling chickens treated with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + L-carnitine 100 mg/kg were assessed with electrocardiography. Consistent with previous results, exposure to PFOA 2 mg/kg resulted in a significantly elevated heart rate relative to the control animals, while co-treatment with 100 mg/kg L-carnitine effectively reverted such changes ( Figure 1L). Figure 1. The general parameters of the embryonic day 6 (ED6), 10, 15, and 19 chicken embryos and hatchling chickens and the heart rates of the hatchling chickens. Fertile chicken eggs were air cell injected with vehicle, perfluorooctanoic acid (PFOA)2 mg/kg, or PFOA 2 mg/kg + L-carnitine 100 mg/kg, and then incubated to ED6, 10, 15, 19, or hatch. (A) heart weight of ED6 chicken embryos (N = 10 per group); (B) heart weight of ED10 chicken embryos (N = 9-10 per group); (C) heart weight of ED15 chicken embryos (N = 9-11 per group); (D) liver weight of ED15 chicken embryos (N = 9-11 per group);(E) slim body weight of ED19 chicken embryos (N = 4-5 per group); (F) heart index of ED19 chicken embryos (N = 4-5 per group); (G) liver index of ED19 chicken embryos (N = 4-5 per group); (H) slim body weight of hatchling chickens (N = 5-8 per group); (I) heart index of hatchling chickens (N = 5-8 per group); (J) liver index of hatchling chickens (N = 5-8 per group); (K) hatchability of hatchling chickens (total number hatched/total number reached hatchling size) (N = 6-9 per group); (L) heart rates of hatchling chickens (N = 5 per group). * Statistically different from control group (p < 0.05). # statistically different from PFOA 2 mg/kg group (p < 0.05). + Specified treatment is present at the indicated group; − specified treatment is not present at the indicated group. Error bars represent standard derivation (SD).

Heart Rates of Hatchling Chickens
The heart rates of the hatchling chickens treated with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + L-carnitine 100 mg/kg were assessed with electrocardiography. Consistent with previous results, exposure to PFOA 2 mg/kg resulted in a significantly elevated heart rate relative to the control animals, while co-treatment with 100 mg/kg L-carnitine effectively reverted such changes ( Figure 1L). With the ESR method, the ROS levels of chicken embryo hearts from different stages (ED6, 10, 15, and 19), as well as hatchling chicken hearts, were assessed. The results indicated that no significant changes to ROS levels were present in ED6, 10, or 15 chicken embryo hearts (Figure 2A-C), while a significant increase in the ROS levels was detected in ED19 chicken embryo hearts and hatchling chicken hearts ( Figure 2D,E). Meanwhile, L-carnitine co-treatment effectively reverted the ROS surge in ED19 chicken embryo hearts ( Figure 2D). In hatching chickens, a trend of decrease was observed in the L-carnitine co-treatment group hearts compared to the PFOA treated group hearts, but this was not statistically significant (p = 0.10, Figure 2E).

Electron Spin Resonance (ESR)Results of the Reactive Oxygen Species (ROS)Levels in ED6, 10, 15, and 19 Chicken Embryo Hearts and Hatchling Chicken Hearts
With the ESR method, the ROS levels of chicken embryo hearts from different stages (ED6, 10, 15, and 19), as well as hatchling chicken hearts, were assessed. The results indicated that no significant changes to ROS levels were present in ED6, 10, or 15 chicken embryo hearts (Figure 2A-C), while a significant increase in the ROS levels was detected in ED19 chicken embryo hearts and hatchling chicken hearts ( Figure 2D,E). Meanwhile, L-carnitine co-treatment effectively reverted the ROS surge in ED19 chicken embryo hearts ( Figure 2D). In hatching chickens, a trend of decrease was observed in the L-carnitine co-treatment group hearts compared to the PFOA treated group hearts, but this was not statistically significant (p = 0.10, Figure 2E).

ESR Results of the Nitric Oxide (NO)Levels in ED6, 10, 15, and 19 Chicken Embryo Hearts and Hatchling Chicken Hearts
As described in Section 2.3, the NO levels of the chicken embryo and hatchling chicken hearts were assessed with the ESR method. Similar to the ROS results, no remarkable changes were observed in the NO levels in ED6, 10, or 15 chicken embryo hearts ( Figure 3A-C). On the other hand, a significant increase in the NO level was found in the ED19 chicken embryo hearts following 2 mg/kg PFOA exposure. Moreover, co-treatment with 100 mg/kg L-carnitine significantly further increased the NO levels in ED19 hearts ( Figure 3D). In the hatchling chicken hearts, however, PFOA still significantly elevated the NO levels, while L-carnitine significantly decreased the NO levels relative to PFOA-exposed animals ( Figure 3E).

ESR Results of the Nitric Oxide (NO)Levels in ED6, 10, 15, and 19 Chicken Embryo Hearts and Hatchling Chicken Hearts
As described in Section 2.3, the NO levels of the chicken embryo and hatchling chicken hearts were assessed with the ESR method. Similar to the ROS results, no remarkable changes were observed in the NO levels in ED6, 10, or 15 chicken embryo hearts ( Figure 3A-C). On the other hand, a significant increase in the NO level was found in the ED19 chicken embryo hearts following 2 mg/kg PFOA exposure. Moreover, co-treatment with 100 mg/kg L-carnitine significantly further increased the NO levels in ED19 hearts ( Figure 3D). In the hatchling chicken hearts, however, PFOA still significantly elevated the NO levels, while L-carnitine significantly decreased the NO levels relative to PFOA-exposed animals ( Figure 3E).

Western Blotting for p65 Translocation in ED10, 15, and 19 Chicken Embryo Hearts and Hatchling Chicken Hearts
Western blotting was used to examine the translocation of the NF-κB p65 protein in the chicken embryo and hatching chicken hearts. Different trends in different embryonic stages were revealed. At ED10 and ED15, no statistical changes were observed among the groups ( Figure 4A,B). At ED19, a significant increase of p65 translocation was observed in PFOA treated animals, while L-carnitine effectively returned the p65 translocation level back to the control level ( Figure 4C). In hatchling chickens, however, a significant decrease in the p65 translocation level was observed in both PFOA treated animals and PFOA + L-carnitine treated animals ( Figure 4D).

Western Blotting for p65 Translocation in ED10, 15, and 19 Chicken Embryo Hearts and Hatchling Chicken Hearts
Western blotting was used to examine the translocation of the NF-κB p65 protein in the chicken embryo and hatching chicken hearts. Different trends in different embryonic stages were revealed. At ED10 and ED15, no statistical changes were observed among the groups ( Figure 4A,B). At ED19, a significant increase of p65 translocation was observed in PFOA treated animals, while L-carnitine effectively returned the p65 translocation level back to the control level ( Figure 4C). In hatchling chickens, however, a significant decrease in the p65 translocation level was observed in both PFOA treated animals and PFOA + L-carnitine treated animals ( Figure 4D).

Western Blotting for Inducible Nitric Oxide Synthase (iNOS) in ED19 Chicken Embryo Hearts and Hatchling Chicken Hearts
Since significant differences were observed for the levels of NO in ED19 chicken embryos and hatchling chickens, as indicated by the ESR results, the inducible nitric oxide synthase (iNOS) levels in these animals were assessed with western blotting. As shown in Figure 5, the protein levels of iNOS significantly increased in the ED19 hearts from PFOA/L-carnitine co-treated animals, relative to both the control and PFOA treated animals ( Figure 5A). On the other hand, the expression of iNOS was suppressed in PFOA treated animals relative to the control group, while L-carnitine co-treatment alleviated such changes ( Figure 5B).

Western Blotting for Inducible Nitric Oxide Synthase (iNOS) in ED19 Chicken Embryo Hearts and Hatchling Chicken Hearts
Since significant differences were observed for the levels of NO in ED19 chicken embryos and hatchling chickens, as indicated by the ESR results, the inducible nitric oxide synthase (iNOS) levels in these animals were assessed with western blotting. As shown in Figure 5, the protein levels of iNOS significantly increased in the ED19 hearts from PFOA/L-carnitine co-treated animals, relative to both the control and PFOA treated animals ( Figure 5A). On the other hand, the expression of iNOS was suppressed in PFOA treated animals relative to the control group, while L-carnitine co-treatment alleviated such changes ( Figure 5B).

Figure 5.
Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + L-carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with RIPA buffer supplemented with PMSF and then assessed with western blotting, and the expressions were normalized to control animals. (A) iNOS relative expression levels of ED19 chicken embryo hearts; (B) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group (p < 0.05). # statistically different from PFOA 2 mg/kg group (p < 0.05). + Specified treatment is present at the indicated group; − specified treatment is not present at the indicated group. Error bars represent standard derivation (SD).

Discussion
In this study, the ROS and NO levels in PFOA-exposed chicken embryo hearts at different stages were evaluated with the ESR method, and ROS/NF-κB p65 and iNOS/NO were investigated as potential underlying mechanisms. The effects of L-carnitine were also investigated, whose protective effects have been reported previously [14]. In the current study, no significant differences in the ROS and NO levels were observed in the early stage embryo hearts, but our results indicated that ROS and NO might play important roles in late stage embryo hearts, which were associated with NF-κB p65 and iNOS.

Perfluorooctanoic Acid (PFOA)Induced Developmental Cardiotoxicity in a Chicken Embryo
It has been well-established that PFOA could induce developmental toxicity, with endpoints such as embryo loss, a low birth weight, and high post birth mortality [11,27]. No definitive explanations were found for these changes. One of the vital organs during embryo development is Western blotting for inducible nitric oxide synthase (iNOS) in ED19 chicken embryo hearts and hatchling chicken hearts. Fertile chicken eggs were air cell injected with vehicle, PFOA 2 mg/kg, or PFOA 2 mg/kg + L-carnitine 100 mg/kg, and then incubated to ED19 or hatch. At the desired stage, hearts were collected, protein was extracted with RIPA buffer supplemented with PMSF and then assessed with western blotting, and the expressions were normalized to control animals. (A) iNOS relative expression levels of ED19 chicken embryo hearts; (B) iNOS relative expression levels of hatchling chicken embryo hearts. N = 3 per group. * Statistically different from control group (p < 0.05). # statistically different from PFOA 2 mg/kg group (p < 0.05). + Specified treatment is present at the indicated group; − specified treatment is not present at the indicated group. Error bars represent standard derivation (SD).

Discussion
In this study, the ROS and NO levels in PFOA-exposed chicken embryo hearts at different stages were evaluated with the ESR method, and ROS/NF-κB p65 and iNOS/NO were investigated as potential underlying mechanisms. The effects of L-carnitine were also investigated, whose protective effects have been reported previously [14]. In the current study, no significant differences in the ROS and NO levels were observed in the early stage embryo hearts, but our results indicated that ROS and NO might play important roles in late stage embryo hearts, which were associated with NF-κB p65 and iNOS.

Perfluorooctanoic Acid (PFOA)Induced Developmental Cardiotoxicity in a Chicken Embryo
It has been well-established that PFOA could induce developmental toxicity, with endpoints such as embryo loss, a low birth weight, and high post birth mortality [11,27]. No definitive explanations were found for these changes. One of the vital organs during embryo development is the heart, which starts to develop at a very early stage [28], making it vulnerable to extragenous disruptions. A chicken embryo is an established model for the study of development and various diseases, including tumor and cardiovascular diseases [29]. In previous studies, PFOA has been associated with developmental cardiotoxicity in chicken embryos, in terms of an altered morphology and function, which was achieved at an exposure level comparable to those of an occupationally exposed human [12], suggesting the human health relevancy and need for further mechanistic studies. Interestingly, the commonly-accepted mechanism of action for PFOA, PPAR α activation, failed to explain most of the observed effects [13,14]. Meanwhile, a nutrient, L-carnitine, was found to be quite effective against such changes [14], which was consistent with various reports that L-carnitine may protect the heart [17,18]. In the current study, such protection was once again confirmed, as L-carnitine co-treatment reverted the heart rate elevation induced by PFOA in hatchling chickens, thus suggesting its potential to be utilized in cardiovascular development protection.

L-Carnitine, PFOA, Reactive Oxygen Species (ROS), and Nuclear Factor K-Light Chain-Enhancer of Activated B cells (NF-κB)
Both L-carnitine and PFOA are involved in fatty acid β-oxidation, which is a process closely coupled to ROS generation [30]. It has been reported that PFOA could induce ROS in various cell types and organisms [22,31], which might contribute to its toxicity. L-carnitine has been reported to possess anti-oxidant effects in multiple studies [20,32]; however, it may also promote ROS generation since it facilitates β-oxidation [23]. In the current study, the ROS levels were determined in ED6, 10, 15, 19, and hatchling chicken hearts. While no remarkable changes were observed in earlier stage hearts, a significant increase in the ROS level was observed in ED19 chicken embryo hearts exposed to PFOA, suggesting the role of ROS in PFOA-induced developmental cardiotoxicity. This result is consistent with previous studies with PFOA [22,31]. Meanwhile, co-treatment with L-carnitine significantly decreased the ROS level in ED19 hearts, suggesting the anti-oxidant effects of L-carnitine, which may help to explain the protective effects exerted by L-carnitine. The observed effects make good sense in terms of chicken embryo development, in which ED19 is the stage with relative hypoxia [33]. Similarly, in hatchling chicken hearts, the elevated ROS levels are still present in PFOA-exposed animals, while L-carnitine co-treatment decreased the ROS level to the point that it was not significantly different from that of the control group. However, the extent of anti-oxidant effects of L-carnitine in those hatchlings seems to be somewhat decreased, as the ROS level in the co-treatment group did not statistically differ from the level in the PFOA treated group. This might be explained by the extensive labor required for the hatching process [33], in which β-oxidation serves as an important resource of energy [34]. An abundant oxygen supply post hatch might be another reason for this result.
Several signaling pathways could be activated by ROS, and one of the most important signaling pathways is the NF-κB [35]. The elevation of p65 is associated with an increased ROS level [36]. In the current study, increased p65 translocation was observed in ED19 chicken embryos, while L-carnitine returned it to the normal level, suggesting that L-carnitine exerted anti-oxidant effects in ED19 chicken embryo hearts, consequently decreasing the activation of p65, which is consistent with the ESR data of ROS levels. Interestingly, different results were observed in hatchling chicken hearts: PFOA and/or L-carnitine both suppressed p65 translocation. Notably, in ED15 embryo hearts, p65 translocation also showed a similar pattern to the hatchling hearts, although this was not statistically significant. This contradiction suggests that PFOA might be able to modulate the p65 signaling pathway by other means than ROS generation in hatchling chickens. The "normal" pattern of p65 translocation observed at ED19 might actually be a combination of ROS generation induced changes, along with the modulation by PFOA via other routes. This could help to explain the relatively small percentage change inp65 translocation in ED19 hearts. Considering the fact that PFOA is involved in immunotoxicity and tumor formation [9,10], both of which involve the NF-κB pathway, further study focusing on the interactions between PFOA and NF-κB in developing organisms is required.

L-Carnitine, PFOA, NO, and iNOS
Nitric oxide (NO) is an important signaling molecule in the cardiovascular system. Besides its well known effect of vasodilatation, it also has roles in inflammation and cardiovascular diseases [37]. In the developing heart, NO is known to alleviate hypoxia damage and vascular modeling [38,39], and thus, the NO levels in developing chicken embryo hearts were assessed as a potential mechanism of action for PFOA and/or L-carnitine. While no remarkable changes were detected for ED6, 10, and 15 hearts, a significant elevation of NO in ED19 in PFOA exposed hearts was revealed. Interestingly, co-treatment with L-carnitine further increased the NO levels. The elevated NO levels in PFOA-exposed hearts might represent compensation, while L-carnitine mediated a further increase in the NO level that may contribute to its protective effects. In hatchling hearts, however, such changes were no longer present; while PFOA still elevated the NO levels, co-treatment with L-carnitine exerted the reverse effects. The very different oxygen supply condition and labor required during hatching might contribute to such pheromones. To address such intriguing results, the expression levels of iNOS in ED19 and hatchling hearts were assessed. iNOS is one of the major resources of NO in living organisms, and is likely to be associated with vascular dysfunction [40]. While many existing studies correlated iNOS to negative outcomes in the cardiovascular system [40,41], our data have suggested its protective potential against developmental cardiotoxicity in ED19 chicken embryos, which seems to be a transient effect as such changes disappear after hatching, within a period of 48-72 h. In hatchling chicken hearts, iNOS expression seems to be slightly suppressed by PFOA exposure, while co-treatment with L-carnitine abolished such changes, suggesting a role of iNOS and NO in PFOA-induced developmental cardiotoxicity. Moreover, the inconsistent results of the NO and iNOS levels in PFOA treated animals suggested that other enzymes may contribute to the NO level elevation. Possible enzymes include endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) [40], and xanthine oxidase [42]. The exact role of iNOS and NO in the hatching process and PFOA-induced developmental cardiotoxicity will be further pursued.

Materials
PFOA, L-carnitine, and the chemicals used for ESR were purchased from Sigma (St. Louis, MO, USA). Fertile chicken eggs (Gallus gallus) were purchased from Linwen Trading Co. Ltd. (Jining, China). The antibody against p65 was purchased from Abcam (Cambridge, MA, USA). The antibody against GAPDH was purchased from ZSbio (Beijing, China). Other chemicals and consumables were of the highest grade obtainable.

Chicken Embryo Treatment and Incubation
Upon arrival, fertile chicken eggs were cleaned with povidone iodide and candled in a dark room to mark the air cell with pencil. The eggs were then air cell injected with vehicle or pre-made dosing solutions to achieve the doses of PFOA 2 mg/kg or PFOA 2 mg/kg + L-carnitine 100 mg/kg. For the detailed injection method, please refer to Jiang et al. [12]. The dose of PFOA was based on the results of the hatchling chicken serum PFOA concentration [12], while the dose of L-carnitine was determined according to the reference dose set by the US Food and Drug Administration (USFDA) [43]. The doses were calculated so that a 0.1 µL/g injection volume resulted in the desired doses to the whole egg. To facilitate the homogenization of L-carnitine in the dosing suspension, 10% ethanol and 20% distilled water were included in the dosing solution. Injected eggs were initially incubated in a Keyu incubator (Dezhou, China) at 37.9 • C Celsius and 50% humidity, and the conditions were gradually changed to 37.1 • C Celsius and 70% humidity (temperature and humidity were controlled automatically by the incubator). Eggs were turned every 3 h until ED19. Hatched chickens were kept in a warmed brood box with water provided until the heart function assessment and sample collection (within 24 h post hatch). All the procedures for the handling of chicken embryos and hatchling chickens were approved by the Institutional Animal Care and Use Committee (IACUC) of Qingdao University (Approved protocol 2015018, 5 March 2015).

Chicken Embryo Sample Collection
At the desired time points, the eggs were opened up, the embryos were carefully removed from the eggs and quickly decapitated, and the hearts were carefully dissected out and immediately frozen in a −80 • C freezer until use.

Hatchling Chicken Heart Rate Assessment and Sample Collection
Hatchling chickens (approximately 12-24 h post hatch) were anesthetized with 33 mg/kg pentobarbital via an intraperitoneal injection and the heart rate was assessed with BL-420 E+ (Taimeng, China), as described in Jiang et al. [14], before being quickly decapitated while still under anesthesia, at which point the hearts were collected and frozen in a −80 • C freezer until further use.

ESR
ED6, 10, 15, 19, and D1 hatchling chicken hearts were assessed with ESR for reactive oxygen species (ROS) and nitric oxide (NO). Briefly, the tissues were homogenized with 5× volume ESR buffer (10 mM N-tert-butyl-a phenylnitrone, 2 mM diethylene-triaminepentacetic acid, 0.1 mM ethylene diaminetetraacetic acid, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in phosphate buffered saline, pH 7.4) and centrifuged at 12,000 rpm for 20 min at 4 • C Celsius. The resulting supernatant was isolated and mixed with 10 µL each of 0.5 M Sodium dithionite, 10 mM L-argentine, 0.3 M ferrous sulfate, 10 mM calcium chloride, and 0.6 M sodium diethyldithiocarbamate. Samples were then incubated in dark at 37 • C Celsius for 1 h, and were then put on ice and mixed with 200 µL ethyl acetate thoroughly. The samples were then centrifuged at 1100 rpm for 10 min, and 140 µL of ethyl acetate phase was used for ESR detection. The ESR parameters used were: central field: 3480 G, sweep width: 250 G, static field: 3355 G. Microwave bridge frequency: 9.86 GHz, power 12.91 mW, step 2 dB. The signal for nitric oxide was read at 3437 G, while the signal for ROS was read at 3493 G.

Western Blotting
ED10, 15, 19, and D1 hatchling chicken hearts were investigated with western blotting for the translocation status of NF-κB p65 and the protein expression levels of iNOS. For p65, the cytoplasmic and nuclear protein extractions were prepared with the nuclear and cytoplasm protein extraction kit (KGP1100, KeyGenBioTECH, Nanjing, China) following the manufacturer provided protocol. For iNOS, independent samples were extracted with a radio immunoprecipitation assay buffer (RIPA) supplemented with 1:100 PMSF (Beyotime, Beijing, China). Western blotting was performed as described in Wang et al. [42], with slight modifications. P65 antibody (ab3523, dilution 1:2000) was purchased from Abcam (Cambridge, UK). iNOS antibody (bs-2072R, dilution 1:1000) was purchased from Bioss (Beijing, China). GADPH antibody (TA-09, dilution 1:3000) was purchased from Nobleryder (Beijing, China). Blots were visualized with an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA), along with a UVP 810 gel imager (UVP, Upland, CA, USA), and then analyzed with ImageJ (NIH, Bethesda, MD, USA). The results were normalized to the internal control (GAPDH) of the corresponding samples. For the p65 translocation status assay, the ratio of nuclear p65 to cytoplasmic p65 from the same animal was calculated, and semi-quantified as a ratio to the control sample on the same gel (the cytoplasmic extraction and nuclear extraction of one animal were always assessed on the same gel). Three independent samples were used in each group.

Statistical Analysis
Statistical analysis was performed with SPSS 17.0 (SPSS Inc. Chicago, IL, USA). One-way analysis of variance (ANOVA) was performed to detect differences. When ANOVA returned significant results, post-hoc least significant difference (LSD) tests were used to compare the results among groups. The results were considered statistically significant when p < 0.05.