Introduction
Parkinson’s Disease (PD) is the most prevalent neurodegenerative movement disorder associated with progressive dopaminergic neuronal loss in the
substantia nigra pars compacta (
SNpc). Despite extensive research efforts, the underlying cause of PD largely remains unknown [
18]. However, a number of cellular processes, including mitochondrial function, have been implicated in the aetiology of PD [
21,
25,
36]. First insights into the role of mitochondria in PD came from the observation that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces dopamine neuron death through inhibition of the complex I of the mitochondrial respiratory chain, resulting in parkinsonism [
51]. Later, a number of genes associated with familial forms of PD were identified to play a role in the maintenance of mitochondrial function, further suggesting its involvement in PD pathogenesis [
1,
52]. Moreover, mitochondrial DNA (mtDNA) variations and depletion in PD have been extensively investigated, although without definitive conclusions [
48]. Notably, higher levels of mtDNA deletions in
SNpc of both PD and aged brains have been reported [
9,
40], and reduced mtDNA copy number has been suggested as a biomarker of PD [
19,
54].
The mitochondrial DNA polymerase gamma encoded by the nuclear POLG1 gene, is responsible for the synthesis of mtDNA [
26]. Association of variants of
POLG1 with parkinsonism was first reported in 2004, a study of seven families with
POLG1-related progressive external ophthalmoplegia (PEO) revealed a co-segregation of parkinsonism with
POLG1 variations, with the age of onset of parkinsonism varying between 36 and 75 years; post-mortem examination of two patients showed loss of pigmented dopaminergic neurons in the
SNpc, but no presence of Lewy Body pathology [
44]. In the same year, another study described a family with PEO, neuropathy and late-onset parkinsonism [
45].
POLG1-related PEO can occur in either autosomal recessive (arPEO) form, characterized by ptosis and ophthalmoparesis, or autosomal dominant (adPEO) form that can include symptoms of myopathy, hearing loss, cataracts, ovarian failure, axonal neuropathy, ataxia, depression and parkinsonism [
55]. Since then, additional cases of
POLG1-associated parkinsonism have been described, often secondary to PEO and ataxia, with late age-of-onset, complete or partial L- DOPA response, and Lewy body pathology, but only in some cases [
10,
56,
63]. However, early-onset
POLG1 variant-related parkinsonism has also been observed in patients without PEO [
17,
47]. Interestingly, a study of eleven patients with
POLG1 variant-related encephalopathy revealed severe nigral neuronal loss and nigrostriatal depletion through DAT imaging, without any clinical signs of parkinsonism [
74].
Here, we present a case study of a female patient with a novel variation in
POLG1 (p.Q811R), who was diagnosed with early-onset parkinsonism, and subsequently, adPEO. We employed a recently developed protocol to differentiate patient-derived induced pluripotent stem cells (iPSCs) into 3D midbrain dopaminergic neuron-containing spheroids (MDNS) [
35] to examine POLG1
Q811R cellular alterations.
Discussion
Neurodegeneration of midbrain dopaminergic neurons is a key hallmark of PD. Using reprogramming technologies, it is possible to generate patient-specific iPSCs, and further differentiate them into brain cell types, opening up the possibility to assess early cellular alterations associated with a patient specific neuropathology. Here, we report a case study of a female PD patient with a p.Q811R (c.2432A > G) variation in the polymerase domain of the POLG1 gene. The patient presented cataract, early-onset parkinsonism, premature ovarian failure and sign of fatigue and myopathy, resulting in the final diagnosis of adPEO.
In order to investigate her parkinsonian phenotype on a cellular level, we employed a recently developed protocol to obtain iPSCs-derived pigmented dopaminergic neurons-containing MDNS. The MDNS were predominantly composed of MAP2-positive neurons. They had a midbrain dopaminergic identity, confirmed by co-expression of VMAT2, FOXA2 and TH (Fig.
2b) [
41]. Astrocytes (GFAP-positive), whose role in PD has recently emerged [
12], were also detected. Prior to the read-outs, the MDNS were differentiated for 100 DIV. Several recent studies showed that maintaining iPSC-derived neural cultures beyond 100 days allows cells to fully mature [
67], and leads to the emergence of PD phenotypes including DA oxidation mediating mitochondrial and lysosomal dysfunction [
14,
16,
46].
Quantitative proteomic analysis of MDNS yielded 61 differentially expressed proteins between POLG1
Q811R and control MDNS (Fig.
4b and c). Interestingly, the IPA analysis identified several key pathways in POLG1
Q811R linked to increased neuroprotection and neuronal survival, which could explain the higher number of surviving dopaminergic neurons following dissociation of the MDNS. The MAPK11 protein that was upregulated in POLG1
Q811R MDNS compared to control MDNS has been linked to several canonical pathways by IPA, involving p38 MAPK signaling (Fig.
4g). P38 MAPK activation has been shown to contribute to PD pathology by driving neuroinflammation and neurodegeneration through activation of microglia and induction of NO production [
75]. Interestingly, extracellular NM activates p38 MAPK pathway, also leading to microglial activation [
75]. Mitochondrial enzyme ARG2 was also upregulated in POLG1
Q811R MDNS, implicating urea cycle-related pathways of arginine degradation, citrulline biosynthesis and metabolism (Fig.
4g). A recent study identified increased ARG2 in AD patient brain [
27]. ARG2 is thought to have a neuroprotective function due to its negative effect on NO production [
3]. However, NO, known to lead to neurotoxicity when at high levels, is also important for synaptic plasticity, and the observations of altered arginine metabolism in AD brain also opens the question whether ARG2 increase is a consequence or a cause in AD pathogenesis, and its implication in the progression of the disease [
43]. The role of the urea cycle and arginine metabolism in PD have not been investigated yet, and could not be ruled out as potential new mechanisms involved in PD pathogenesis.
Addition of DA during maturation stages of the differentiation, triggered the darkening of the neurons that composed the MDNS (Fig.
5a), suggesting higher levels of NM in POLG1
Q811R neurons [
35]. NM is an insoluble pigment found in catecholaminergic neurons of the SN and some other brain regions, believed to protect neurons from neurotoxic DA quinones and iron, but that may also increase neuronal vulnerability during neurodegeneration [
82]. However, the exact function and involvement of NM in PD is still not fully understood. The proteomic data together with the stronger pigmentation observed was suggestive of higher NM production/accumulation or defect in NM degradation in the POLG1
Q811R MDNS compared to controls (Fig.
5a). Indeed, proteomic analysis revealed a trend towards decreased levels of DA degrading enzymes, MAO-A and MAO-B, and a significant decrease of MAO-B measured by Western blotting (Fig.
5d and e). However, PD pathology is normally associated with increased MAO-B activity, and MAO-B inhibitors are effective in the initial treatment of PD [
38]. Our data therefore raises the question whether PD dopaminergic neurons are prone to accumulate NM very early in the disease, compare to control dopaminergic neurons. A possible explanation could be that decrease in MAO-A and MAO-B levels was prompted by the supplementation of the differentiation media with DA. It has been reported that chronic treatment with L-DOPA leads to inhibition of MAO activity, an effect opposite of that of acute L-DOPA treatment [
57]. However, a similar inhibitory effect would then be expected in the control MDNS. It is also possible that the decrease in MAO-B is a protective mechanism against mitochondrial pathology in POLG1
Q811R MDNS, as the activity of the enzyme has been linked to increased ROS and mitochondrial impairment [
37,
70]. Notably, despite PD therapies focusing predominantly on inhibition of MAO-B to reduce DA breakdown and, potentially, to decrease formation of toxic DA oxidation products [
77,
78], the MAO-B isoform is predominantly expressed in glial cells, while the DA breakdown in neurons occurs mainly via the MAO-A isoform [
23]. A recent study assessed expression levels of MAO-A and MAO-B in different brain regions in patient with PD and PD-associated disorders, postulating that elevated MAO-B could be a marker of astrogliosis [
73]. Reportedly, elevated MAO-B was observed in the
SNpc of patients with progressive supranuclear palsy and, to some extent, MSA, but no significant changes were detected in PD patients, consistent with previous reports of limited astrogliosis in PD [
72]. On the other hand, transcriptional upregulation of MAO-A and MAO-B have been reported in patients with PARK2 and GBA variants in iPSC-derived neurons-based studies [
34,
76]. Employing a differentiation protocol that includes addition of L-DOPA or DA may help in the future to examine NM formation and DA metabolism in PD neurons, in vitro.
Alteration in POLG1
Q811R MDNS energy metabolism was identified by the proteomic analysis and IPA (Fig.
4), and confirmed by the increased glycolysis rate, but not the oxidative phosphorylation in POLG1
Q811R MDNS, compared to controls (Fig.
7). Elevated glycolysis rate was in line with the upregulation of PFKM essential for the drive of glycolysis and LDHA that is responsible for the conversion of pyruvate to lactate. Upregulation of genes encoding LDHA, have been observed in animal models of PD and samples from PD patients, and has been associated with aging and increased mtDNA mutations, thought to upregulate glycolysis as a compensatory mechanism for a decrease in ATP production [
39,
58,
60]. A study in mice carrying mtDNA mutations expressing a proofreading-deficient version of
POLG1 reported elevated brain levels of lactate due to an increase in LDHA/LDHB ratio [
58]. Additionally, a recent study in a transgenic PS1/APP AD mouse model revealed decreased content of lactate as well as downregulation of LDHA and LDHB in PS1/APP mice, but an increase in LDHA/LDHB ratio postulated to compensate for neuronal lactate deficit and increase lactate production [
80]. It has also been shown that increase in LDHA can mediate resistance to Aβ toxicity through upregulation of aerobic glycolysis as a protective mechanism, but it is possible that this effect is present in the prodromal stages of AD [
50], while at a later stages of the disease aerobic glycolysis and elevated lactate production may contribute to the cognitive decline associated with AD [
28]. Notably, the elevated levels of LDHA in skeletal muscle have also been reported in cohorts of patients with PEO and mitochondrial encephalopathy lactic acidosis and stroke-like episodes syndrome (MELAS) [
61], suggesting implications for not only PD, but also mitochondrial diseases.
The implications of elevated glycolysis in PD is somewhat controversial. Mitochondrial dysfunction and increased glycolysis has been observed in the peripheral blood of prodromal and early-stage PD patients [
68]. In addition to that, PARK2 and PINK1, loss-of-function variations of which lead to familial PD, have recently been shown to negatively regulate the Warburg effect, a switch from mitochondrial respiration to aerobic glycolysis when oxygen supply is normal in cancer [
2,
79]. These results implicate increased glycolysis in the pathogenesis of PD. At the same time, two recent studies showed neuroprotective effect of glycolysis-enhancing drugs Meclizine and Terazosin, suggesting that glycolysis can provide beneficial effect when energy metabolism and ATP production is compromised [
15,
32].
Our iPSCs-based model, for investigating neuronal phenotype and pathology in POLGQ811R variant with early-onset PD, is a valuable tool to gain insights into cellular mechanisms affected in this patient (catecholamine degradation, aSYN aggregation, energy metabolism) and to establish a link between clinical and cellular phenotypes.
Materials and methods
Generation and maintenance of iPSCs
Human dermal fibroblasts were obtained by punch skin biopsy from a PD patient carrying heterozygous p.Q811R variation in the POLG1 gene (POLG1
Q811R/WT; referred to as POLG1
Q811R in the text and figures), after written informed consent. Fibroblasts were cultured and expanded in culture medium containing DMEM media (Thermo Fisher Scientific), 10% fetal bovine serum and 1% Penicillin-Streptomycin. For reprogramming, fibroblasts were transduced using CytoTune™-iPS 2.0 Sendai reprogramming kit (Thermo Fisher Scientific) according to manufacturer’s instructions. The cells were maintained in fibroblast media with daily media changes until day 6, when the cells were re-seeded onto a layer of irradiated mouse embryonic fibroblasts (MEF) feeder cells in WiCell medium composed of advanced DMEM/F12, 10% Knock-Out Serum Replacement, 2 mM L-glutamine, 1% non-essential amino acids (all from Thermo Fisher Scientific), 50 μM β-mercaptoethanol (Sigma-Aldrich) and 20 ng/ml FGF2 (Thermo Fisher Scientific). Three to four weeks later, individual colonies were picked and expanded, before being bio-banked and characterized, similarly to our previous lines [
31].
Differentiation of iPSCs towards midbrain dopaminergic identity
To differentiate the iPSC into midbrain dopaminergic neurons, we modified previously established protocols [
35] was used. Briefly, following expansion, iPSCs were detached with dispase II (Thermo Fisher Scientific) and seeded in ultra-low attachment flasks in WiCell media supplemented with 10 mM ROCK inhibitor Y27632 (Sellekchem) and 20 ng/ml FGF2 (Thermo Fisher Scientific). The following day (denoted as day 0), the media was changed to neural induction medium (NIM) composed of advanced DMEM/F12, 2 mM L-glutamine, 1% non-essential amino acids, 1% N2 supplement, 1% Penicillin-Streptomycin (all Thermo Fisher Scientific). Media was supplemented with 0.1 μM LDN (Stemgent), 10 μM SB 431542 (Sigma-Aldrich), 200 ng/ml SHH-C (Thermo Fisher Scientific), 1 μM SAG (Sellekchem) and 0.8 μM CHIR (Sigma-Aldrich) (day 0–4) and replaced every other day. On day 6 SB and SHH-C were removed from the media and SAG was increased to 2 μM, and on day 10 LDN was removed also. From day 12 onwards the cells were grown in NIM supplemented with 100 ng/ml FGF8 (Thermo Fisher Scientific) and 2 μM SAG, 10 ng/ml BDNF (R&D Systems) and 200 μM AA (Sigma-Aldrich). From day 22, the media was replaced with neural differentiation medium (NDM) containing Neurobasal media with 2 mM L-glutamine, 1% non-essential amino acids, 1% N2 supplement, 1% B27 without vitamin A, 1% Penicillin-Streptomycin (all from Thermo Fisher Scientific), supplemented with 100 ng/ml FGF8 and 2 μM SAG, 10 ng/ml BDNF, 10 ng/ml GDNF (R&D Systems), 200 μM AA, 500 μM db-cAMP (Sigma-Aldrich) and 1 ng/ml TGFb (Peprotech). From day 30 onwards, FGF8 and SAG were removed from the media and 50 μM DA (Sigma-Aldrich) was added to the media to promote formation of neuromelanin. For the readouts, differentiated spheroids at 100 DIV were either collected, washed and snap-frozen or fixed, for analysis of the proteome, and immunocytochemistry (ICC), respectively, or dissociated using 0.05% Trypsin-EDTA (Thermo Fisher Scientific) and re-seeded on plates coated with 40 μg/ml poly-ornithine (Sigma-Aldrich) and 15 μg/ml laminin (Thermo Fisher Scientific), ICC, amperometry and SeaHorse analysis.
Differentiation of iPSCs towards astrocytes
The differentiation of healthy and POLGQ811R iPSCs into astrocytes followed the same protocol as for midbrain dopaminergic identity, for the first 10 days. From day 12, cells were differentiated in NIM media supplemented with 100 ng/ml FGF8 and 2 μM SAG until day 30. Then, cells were dissociated, filter-strained (100 μm), and transferred to new ultra-low-adherent flasks with neural expansion medium (NEM) containing DMEM-F12, 2 mM L-glutamine, 1% non-essential amino acids, 1% N2 supplement, 1% B27 without vitamin A, 1% Penicillin-Streptomycin, and 0.2 μg/ml heparin (Sigma-Aldrich), supplemented with 20 ng/ml FGF2 and 20 ng/ml EGF (Peprotech). On day 60, spheroids were washed and dissociated into single cells, and seeded to adherent culture flasks coated with Poly-L-ornithine/laminin, in NEM containing 20 ng/ml CNTF (R&D Systems). Finally, from day 80 onwards, astrocytes were cultured in NDM with 20 ng/ml CNTF. Cells were passaged at confluency.
Amperometry
In vitro high-speed chronoamperometric measurements (2 HZ) of DA release, were performed according to previously described protocol [
4,
29], on dissociated spheroids seeded onto 4 well-plates at a density of about 80,000 cells/cm
2. Briefly, Recording was done using FAST-16 mk III hardware (Quanteon) coupled to Nafion®-coated carbon fiber electrodes (30 μm diameter, 150 μm length). A square wave potential of 0.55 V; 0.0 V resting potential was applied vs an Ag/AgCl reference. Prior to recording the electrode was calibrated at room temperature in stirred 0.1 M PBS by on addition of Ascorbic Acid (20 mM) followed by three additions of DA (2 μM). The electrodes used displayed a linearity correlation of > 0.99, a selectivity of DA over ascorbic acid of > 1000:1, and a limit of detection below 0.01 μM.
Immunocytochemisty
For ICC differentiated spheroids were either collected or dissociated at 100 DIV and seeded onto poly-ornithine/ laminin-coated clear bottom 96-well microplates (Greiner Bio One) at 200,000 cells/cm2 and fixed in 4% paraformaldehyde (Sigma-Aldrich). After fixation cells were washed in PBS, and fixed spheroids were equilibrated in 30% sucrose overnight. Fixed spheroids were then mounted in OCT (Sigma-Aldrich) and cut using a cryostat into 20 μM sections. Dissociated cells and spheroid sections were blocked in 10% donkey serum in PBS with 0.1% (dissociated cells) PBS-Tween 20 with or 0.3% (sections) Triton-X (Sigma-Aldrich). Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C: mouse anti-AFP (Sigma-Aldrich, A8452, 1:500), mouse anti-SMA (Sigma-Aldrich, A2547, 1:500), rabbit anti-TUJ1 (Covance, PRB-435P, 1:500), mouse anti-Oct4 (Millipore, MAB4401, 1:200), mouse anti-Nanog (BD Biosciences, 560,483, 1:200), mouse anti-TRA-1-81 (Thermo Fisher Scientific, 411,100, 1:200), rabbit anti-TH (Millipore, AB152, 1:500), mouse anti-TH (Millipore, MAB318, 1:250), sheep anti-TH (Abcam, ab113, 1:500), goat anti-FOXA2(Santa-Cruz, sc11415, 1:250), rabbit anti-VMAT2 (Immunostar, 20,042, 1:500), mouse anti-aSYN (Santa-Cruz, sc12767, 1:200), rabbit anti-GFAP (DAKO, Z0334, 1:2000), chicken anti-MAP2 (Abcam, ab92434, 1:2000). AlexaFluor-488, AlexaFluor-555 and AlexaFluor-647-labelled secondary antibodies (Thermo Fisher Scientific) were used at 1:400 in PBS or PBS-Tween 20 at RT for 1 h. DAPI (1:10,000) was used for nuclei counterstaining. Images were acquired using an inverted epifluorescence microscope LRI-Olympus IX-73, Metamorph and ImageJ software were used for image analysis and quantification.
Western blot
Protein extraction was performed using M-PER (Thermo Fisher Scientific) following manufacturer’s instructions. Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to quantify the protein. 13 μg of protein from each sample was loaded on Bolt 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific) and then transferred to nitrocellulose membranes using iBlot transfer device (Thermo Fisher Scientific). Membranes were blocked in with either 5% skim milk (Sigma-Aldrich) or 5% BSA (for phosphorylated protein) (VWR) diluted in PBS-Tween 20. The membranes were incubated with primary antibodies diluted in blocking solution at 4 °C, overnight: mouse anti-Actin (Sigma-Aldrich, A5441, 1:20000), rabbit anti-ASYN (Cell Signaling, 2642S, 1:1000), mouse anti-ASyO5 (Agriseria, AS132718, 1:1000), rabbit anti-phosphoS129 (Abcam, ab168381, 1:500), mouse anti-DRP1 (Abcam, ab56788, 1:500), rabbit anti-MAO-B (Abcam, ab175136, 1:2000), mouse anti-MFN2 (Abcam, ab56889, 1:500), anti-rabbit anti-OPA1 (Abcam, ab157457, 1:500), rabbit anti-TH (Millipore, AB152, 1:2000), mouse anti-TH (Millipore, MAB318, 1:1000), mouse anti-TOMM20 (Abcam, ab56783), rabbit anti-TOMM20 (Santa Cruz, sc11415, 1:200), rabbit anti-TFAM (Abcam, ab131607, 1.200), rabbit anti-VDAC1 (Abcam, ab15895, 1:1000), rabbit anti-LDHA (Cell Signaling, #2012, 1:1000), rabbit anti-PFKM (Abcam, ab97353, 1:1000). Washed membranes were incubated with peroxidase-conjugated secondary antibody (R&D Systems) at 1:2000 and developed using ChemiDoc gel imaging system (Bio-Rad). Analysis of the blots was performed using Bio-Rad Image Lab software and the protein levels were normalized to beta actin.
Oxygen consumption rate
Control and POLGQ811R MDNS dissociated cultures, astrocytes and fibroblasts were seeded onto poly-ornithine/laminin-coated Seahorse 96-well plates (Agilent Technologies) at 200,000 to 300,000 cells/cm2. Empty wells were used as background controls. Oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) analysis was performed 2 days later, on the Seahorse XFe 96 analyzer using the Seahorse XF Mito Stress Test Kit (Agilent Technologies), according to manufacturer’s instructions. Briefly, cells were washed three times and incubated in XF-Base medium supplemented with 5 mM pyruvate, 2 mM L-Glutamine and 10 mM glucose in non-ventilated, non-CO2 incubator at 37 °C for 1 h. For measurements, three cycles for MDNS dissociated cultures and 5 cycles for astrocytes and fibroblasts of 30 s mix, followed by 2 min measurement were used for the baseline and sequential treatments of 1 μg/mL Oligomycin, 1 μM FCCP, 2uM Rotenone (for MDNS, rotenone was combined with 500 μM succinate prodrug NV118), 1 μg/mL Antimycin. Background-corrected measurements of OCR and ECAR were normalized to the total protein level using Protein Assay Kit II (Bio-Rad).
LC MS/MS proteomics
Cell pellets were homogenized using a FastPrep®-24 instrument (MP Biomedicals,
www.mpbio.com) with Lysing Matrix D for five repeated cycles (speed 6.5 m/s, 40 s/cycle) in 200 μl of the buffer containing 2% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (TEAB). Samples were centrifuged at 16000 g for 10 min and the supernatants were transferred to clean tubes. The lysis tubes were washed with 100 μl of the lysis buffer, centrifuged at 16000 g for 10 min, the supernatants were combined with the corresponding lysates from the previous step. Protein concentration in the combined lysates was determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific) and the Benchmark™ Plus microplate reader (BIO-RAD) with bovine serum albumin (BSA) solutions as standards. A representative reference sample was prepared, containing equal amounts from the 8 individual samples.
Tryptic digestion and tandem mass tag (TMT) labelling
Aliquots containing 30 μg of each sample including the reference were reduced with 100 mM DL-dithiothreitol (DTT) at 56 °C for 30 min. The reduced samples were processed using the modified filter-aided sample preparation (FASP) method (Wisniewski JR et al. Nat Methods. 2009 May;6(5):359–62). In short, reduced samples were transferred to 30 kDa MWCO Pall Nanosep centrifugation filters (Sigma-Aldrich) and washed twice with 8 M urea. Additional washes with digestion buffer (1% sodium deoxycholate in 50 mM TEAB) was performed before and after alkylation with 10 mM methyl methanethiosulfonate for 30 min at room temperature. Protein digestions were performed using Trypsin (Pierce MS grade) in digestion buffer, first with 0.3 μg Trypsin at 37 °C overnight followed by new addition of 0.3 μg trypsin and incubation t at 37 °C for 2 h. Produced tryptic peptides were collected by centrifugation and labelled using TMT 10-plex isobaric mass tagging reagents (Thermo Scientific) according to the manufacturer instructions. Labelled samples were combined and sodium deoxycholate was removed by acidification with 10% TFA.
The combined TMT-labeled sample was fractionated into 40 primary fractions by basic reversed-phase chromatography (bRP-LC) using a Dionex Ultimate 3000 UPLC system (Thermo Fischer Scientific). Peptide separations were performed using a reversed-phase XBridge BEH C18 column (3.5 μm, 3.0 × 150 mm, Waters Corporation) and a linear gradient from 3 to 40% solvent B over 17 min followed by an increase to 100% B over 5 min. Solvent A was 10 mM ammonium formate buffer at pH 10.00 and solvent B was 90% acetonitrile, 10% 10 mM ammonium formate at pH 10.00. The primary fractions were concatenated into 20 fractions (1 + 21, 2 + 22, … 20 + 40), evaporated and reconstituted in 15 μl of 3% acetonitrile, 0.2% formic acid for nLC-MS/MS analysis.
nLC-MS/MS
The fractions were analyzed on an orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer interfaced with Easy-nLC1200 liquid chromatography system (Thermo Fisher Scientific). Peptides were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm × 2 cm, particle size 5 μm, Thermo Fischer Scientific) and separated on an in-house packed analytical column (75 μm × 30 cm, particle size 3 μm, Reprosil-Pur C18, Dr. Maisch) using a linear gradient from 5 to 33% B over 77 min followed by an increase to 100% B for 3 min, and 100% B for 10 min at a flow of 300 nL/min. Solvent A was 0.2% formic acid and solvent B was 80% acetonitrile, 0.2% formic acid. MS scans were performed at 120000 resolution, m/z range 375–1375. MS/MS analysis was performed in a data-dependent, with top speed cycle of 3 s for the most intense doubly or multiply charged precursor ions. Precursor ions were isolated in the quadrupole with a 0.7 m/z isolation window, with dynamic exclusion set to 10 ppm and duration of 45 s. Isolated precursor ions were subjected to collision induced dissociation (CID) at 35 collision energy with a maximum injection time of 50 ms. Produced MS2 fragment ions were detected in the ion trap followed by multinotch (simultaneous) isolation of the top 10 most abundant fragment ions for further fragmentation (MS3) by higher-energy collision dissociation (HCD) at 65% and detection in the Orbitrap at 50000 resolutions, m/z range 100–500.
Proteomic data analysis
Identification and relative quantification were performed using Proteome Discoverer version 2.2 (Thermo Fisher Scientific). The database search was performed using the Mascot search engine v. 2.5.1 (Matrix Science, London, UK) with MS peptide tolerance of 5 ppm and fragment ion tolerance of 0.6 Da. Tryptic peptides were accepted with 1 missed cleavage; methionine oxidation was set as a variable modification, cysteine methylthiolation, TMT-6 on lysine and peptide N-termini were set as fixed modifications. Percolator was used for PSM validation with the strict FDR threshold of 1%.
Quantification was performed in Proteome Discoverer 2.2. TMT reporter ions were identified in the MS3 HCD spectra with 3 mmu mass tolerance, and the TMT reporter intensity values for each sample were normalized within Proteome Discoverer 2.2 on the total peptide amount. Only the unique identified peptides were considered for the relative quantification.
Differential expression analysis
Differentially expressed proteins were identified by using unpaired t-test analysis with a P-value cut-off of 0.001.
The differentially expressed proteins identified from unpaired t-tests were visualized using a volcano plot, that displays log2-fold-change against -log(10) (p-value) from the t-test.
To graphically present the distribution of the differentially expressed proteins identified for the samples a heatmap plot was used after non-supervised hierarchical clustering, with a P-value cut-off of 0.001 log2 transformed data, using the R-package gplots.
Protein expression variances between groups are displayed as principal component analysis (PCA) to show similarities or differences among the samples belonging to the two different groups using the R-packages prcomp and ggplot2 on log2 transformed data.
Ingenuity pathway analysis
To identify significant canonical pathways in which differentially expressed proteins were enriched, pathway enrichment analysis was conducted with the web-based pathway analysis tool IPA (Ingenuity, Systems,
www.ingenuity.com, Redwood City, CA). Differentially expressed proteins were uploaded into IPA along with the protein identifiers,
p-values and fold change values. A cut-off of a 0.001 P-value was used for the proteins to be included in the analyses. Each identifier was mapped to the Ingenuity knowledge base. Canonical pathway analysis identified the pathways, from the IPA library of canonical pathways, which were most significant to the input data set firstly using a ratio of the number of proteins from the dataset in a given pathway divided by the total number of molecules that make up the canonical pathway and secondly a Fisher’s exact test to assess the probability of the association to the canonical pathway.
Statistics
Statistical analysis was performed using Prism 7 software (GraphPad). Data are presented as mean ± standard error of the mean. Comparisons between control and diseased groups (N = 4 per group) were analyzed using an unpaired t-test. A value of p < 0.05 was considered to be statistically significant for all measurements, except for the proteomic analysis where p < 0.001 was considered to be statistically significant. To calculate the intensity of pigmentation of the MDNS, we measured the pixels value ranging from 0 (black) to 255 (white) on a grey scale, and presented the results as pixel values subtracted from the white value (255); up to 80 MDNS counted per group.
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